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The development of non-viral gene-activated matrices for bone regeneration using polyethyleneimine (PEI) and collagen-based scaffolds
Abstract
The healing potential of scaffolds for tissue engineering can be enhanced by combining them with genes to produce gene-activated matrices (GAMs) for tissue regeneration. We examined the potential of using polyethyleneimine (PEI) as a vector for transfection of mesenchymal stem cells (MSCs) in monolayer culture and in 3D collagen-based GAMs. PEI-pDNA polyplexes were fabricated at a range of N/P ratios and their optimal transfection parameters (N/P 7 ratio, 2μg dose) and transfection efficiencies (30±8%) determined in monolayer culture. The polyplexes were then loaded onto collagen, collagen-glycosaminoglycan and collagen-nanohydroxyapatite scaffolds where gene expression was observed up to 21 days with a polyplex dose as low as 2μg. Transient expression profiles indicated that the GAMs act as a polyplex depot system whereby infiltrating cells become transfected over time as they migrate throughout the scaffold. The collagen-nHa GAM exhibited the most prolonged and elevated levels of transgene expression. This research has thus demonstrated that PEI is a highly efficient pDNA transfection agent for both MSC monolayer cultures and in the 3D GAM environment. By combining therapeutic gene therapy with highly engineered scaffolds, it is proposed that these GAMs might have immense capability to promote tissue regeneration.
... All mRNA nanoparticles were formed with each of the nonviral vectors according to the manufacturer's instructions or according to protocols previously described by our group such as in the case of Superfect and branched PEI nanoparticles. 29,30 As the ratio of vector:nucleic acid can have a significant impact on gene delivery efficiency, three different vector:mRNA (v/w) ratios or nitrogen/phosphate (N/P) ratios (in the case of PEI) were screened for each vector to identify optimal conditions for MSC transfection. The N/P ratio refers to the ratio of positively charged nitrogen (N) groups in a polymer-based vector to negatively charged phosphate groups (P) in the nucleic acid with which it is complexed with. ...
... 38,39 The range of N/P or mass ratios chosen for each vector was guided by the manufacturer's recommendations or previously published work on the vector systems. 29,30 For polymeric materials (branched PEI, Superfect, and jetPEI), all mRNA nanoparticles formed were positively charged, indicating complete complexation of the nucleic acid within the cationic materials ( Figure 1A). Superfect-mRNA nanoparticles showed the highest overall charge with a range of 40− 45.9 mV, whereas jetPEI-mRNA nanoparticles were much less cationic with a range of 2.9−7.7 mV. ...
... It has been well-documented that varying scaffold compositions can alter protein expression kinetics in the case of DNA-loaded scaffolds. 30,37 Therefore, it would be reasonable to assume that similar observations would be expected in mRNA-loaded scaffolds. For example, within our own group, Walsh et al. ...
Messenger RNA (mRNA) represents a promising therapeutic tool in the field of tissue engineering for the fast and transient production of growth factors to support new tissue regeneration. However, one of the main challenges to optimizing its use is achieving efficient uptake and delivery to mesenchymal stem cells (MSCs), which have been long reported as difficult-to-transfect. The aim of this study was to systematically screen a range of nonviral vectors to identify optimal transfection conditions for mRNA delivery to MSCs. Furthermore, for the first time, we wanted to directly compare the protein expression profile from three different types of mRNA, namely, unmodified mRNA (uRNA), base-modified mRNA (modRNA), and self-amplifying mRNA (saRNA) in MSCs. A range of polymer-and lipid-based vectors were used to encapsulate mRNA and directly compared in terms of physicochemical properties as well as transfection efficiency and cytotoxicity in MSCs. We found that both lipid-and polymer-based materials were able to successfully condense and encapsulate mRNA into nanosized particles (<200 nm). The overall charge and encapsulation efficiency of the nanoparticles was dependent on the vector type as well as the vector:mRNA ratio. When screened in vitro, lipid-based vectors proved to be superior in terms of mRNA delivery to MSCs cultured in a 2D monolayer and from a 3D collagen-based scaffold with minimal effects on cell viability, thus opening the potential for scaffold-based mRNA delivery. Modified mRNA consistently showed the highest levels of protein expression in MSCs, demonstrating 1.2-fold and 5.6-fold increases versus uRNA and saRNA, respectively. In summary, we have fully optimized the nonviral delivery of mRNA to MSCs, determined the importance of careful selection of the mRNA type used, and highlighted the strong potential of mRNA for tissue engineering applications.
... Our lab has pioneered the use of gene-activated scaffolds, by incorporating non-viral gene delivery vectors including chitosan [38], polyethyleneimine (PEI) [39], hydroxyapatite [40,41], and the GET peptide [42] nanoparticles into collagen-based scaffolds, facilitating the effective delivery of therapeutic nucleic acids to cells as they infiltrate scaffolds. The gene-activated scaffold approach has proven effective for bone repair in vivo, significantly enhancing bone formation [38,[43][44][45][46]. The GET peptide is capable of delivering a multitude of different cargoes, and consists of a 16-residue heparan sulphate-GAG binding peptide derived from the fibroblast growth factor 2 (FGF2) protein (FGF2B domain); LK15, an amphiphilic pan-nucleic acid interaction sequence; and8 arginine (8R) (named FLR) [47][48][49]. ...
... Rat mesenchymal stem and endothelial progenitor cells were isolated from 6-8 week old male Sprague Dawley rats, as approved by the Research Ethics Committee of the Royal College of Surgeons in Ireland under application number TH017, according to protocols described in [46]. The femurs of both hindlimbs were clipped at both ends in order to expose the bone marrow. ...
... rMSCs were transfected at a seeding density of 2.1 × 10 4 cells per well in 12 well cell culture plates, 24 h after seeding as per the seeding densities described in previous work carried out in the lab [46]. rMSCs were transfected with pGLuc, or pSDF-1α using the protocol described in Section 4.1.2. ...
Non-viral gene delivery has become a popular approach in tissue engineering, as it permits the transient delivery of a therapeutic gene, in order to stimulate tissue repair. However, the efficacy of non-viral delivery vectors remains an issue. Our lab has created gene-activated scaffolds by incorporating various non-viral delivery vectors, including the glycosaminoglycan-binding enhanced transduction (GET) peptide into collagen-based scaffolds with proven osteogenic potential. A modification to the GET peptide (FLR) by substitution of arginine residues with histidine (FLH) has been designed to enhance plasmid DNA (pDNA) delivery. In this study, we complexed pDNA with combinations of FLR and FLH peptides, termed GET* nanoparticles. We sought to enhance our gene-activated scaffold platform by incorporating GET* nanoparticles into collagen–nanohydroxyapatite scaffolds with proven osteogenic capacity. GET* N/P 8 was shown to be the most effective formulation for delivery to MSCs in 2D. Furthermore, GET* N/P 8 nanoparticles incorporated into collagen–nanohydroxyapatite (coll–nHA) scaffolds at a 1:1 ratio of collagen:nanohydroxyapatite was shown to be the optimal gene-activated scaffold. pDNA encoding stromal-derived factor 1α (pSDF-1α), an angiogenic chemokine which plays a role in BMP mediated differentiation of MSCs, was then delivered to MSCs using our optimised gene-activated scaffold platform, with the aim of significantly increasing angiogenesis as an important precursor to bone repair. The GET* N/P 8 coll–nHA scaffolds successfully delivered pSDF-1α to MSCs, resulting in a significant, sustained increase in SDF-1α protein production and an enhanced angiogenic effect, a key precursor in the early stages of bone repair.
... This method was first used by Fang and colleagues in 1996 [27] and since then many more iterations have been developed for different tissue engineering applications including in our laboratory where pDNA, siRNA and miRNAs complexed with polyethyleneimine (PEI), nanohydroxyapatite (nHA), chitosan or dendrimers have been delivered on collagenbased scaffolds for a number of different therapeutic indications. [28][29][30][31][32][33][34][35][36][37] The aim of this study was to develop and optimize a scaffold-based system for the localized delivery of the GET peptide allowing for safe, efficient therapeutic gene delivery. Specifically, the first aim was to determine the optimal GET-pDNA nanoparticle formulation involving physicochemical characterization, in vitro efficacy and biocompatibility testing, and in vivo safety and efficacy testing. ...
... Previously, polymeric and polysaccharide-based gene delivery vectors have fully complexed pDNA into nanoparticles. [28,30,36] The gel electrophoresis study ( Figure 1D) demonstrated that the pDNA is safely complexed with GET as the negatively charged pDNA is immobilized within the gel when complexed to GET at CR3, The GET peptide contains three segments, a heparan sulfate-GAG binding domain, an amphiphilic region which binds to pDNA, and a cell penetrating peptide domain to facilitate cell entry. The GET-pDNA complexes (2 µg pDNA) had an average diameter of 100-160nm as shown by DLS (A). ...
... An MTT assay was used to measure cell metabolic activity post-transfection and compared to untransfected cells serving as the 100% viable control. As shown in Figure 2C [52][53][54] PEI, [28,55] chitosan, [30,56] as well as CPPs, [57,58] and other novel peptide-based macromolecular structures such as PAMAM dendrimers and star-shaped and hyperbranched polymers, [36,59] by up to 20%. Transfection peaked at day 3 at all CR and the transfection was transient with a gradual decrease to approximately 40 ± 5% by day 7 and 25 ± 6% by day 14. ...
... Nanoparticles were prepared by mixing pDNA or mRNA with PEI and incubating for 30 minutes. For DNA delivery, 2.08 x 10 2 ng/cm 2 of pDNA was complexed with PEI at N:P of 7 [60]. For RNA delivery, 1.3 x 10 2 ng/cm 2 of mRNA at a 1:2 molar ratio of mRNA:gRNA was complexed with PEI at N:P 15. ...
... We characterized RALA's transfection efficiency of pDNA in comparison to commercial vectors Lipofectamine 3000 and polyethyleneimine (PEI). PEI was used at N:P = 7 as optimized in previous reports [7,60], and Lipofectamine was used according to manufacturer's protocol at a ratio of 3 μL of Lipofectamine per 1 μg of pDNA. Each of these vectors were used for Cas9-T2A-GFP pDNA transfection and flow cytometry supported by fluorescent imaging were used to assess the transfection rates and cell viability. ...
CRISPR gene editing offers unprecedented genomic and transcriptomic control for precise regulation of cell function and phenotype. However, delivering the necessary CRISPR components to therapeutically relevant cell types without cytotoxicity or unexpected side effects remains challenging. Viral vectors risk genomic integration and immunogenicity while non-viral delivery systems are challenging to adapt to different CRISPR cargos, and many are highly cytotoxic. The arginine-alanine-leucine-alanine (RALA) cell penetrating peptide is an amphiphilic peptide that self-assembles into nanoparticles through electrostatic interactions with negatively charged molecules before delivering them across the cell membrane. This system has been used to deliver DNAs, RNAs, and small anionic molecules to primary cells with lower cytotoxicity compared to alternative non-viral approaches. Given the low cytotoxicity, versatility, and competitive transfection rates of RALA, we aimed to establish this peptide as a new CRISPR delivery system in a wide range of molecular formats across different editing modalities. We report that RALA was able to effectively encapsulate and deliver CRISPR in DNA, RNA, and ribonucleic protein (RNP) formats to primary mesenchymal stem cells (MSCs). Comparisons between RALA and commercially available reagents revealed superior cell viability leading to higher numbers of transfected cells and the maintenance of cell proliferative capacity. We then used the RALA peptide for the knock-in and knock-out of reporter genes into the MSC genome as well as for the transcriptional activation of therapeutically relevant genes. In summary, we establish RALA as a powerful tool for safer and effective delivery of CRISPR machinery in multiple cargo formats for a wide range of gene editing strategies.
... Several efforts have been devoted to reducing cytotoxicity during the past decades, and one of the simplest ways is altering the ratio of PEI nitrogen to DNA phosphate (N/P ratio) [31,39]. It has been shown that reducing the N/P ratio of PEI/DNA polyplexes from 10 to 7 significantly affects the cytotoxicity of MSCs [40]. However, the gene transfection efficiencies are also accordingly decreased in most situations [25,40]. ...
... It has been shown that reducing the N/P ratio of PEI/DNA polyplexes from 10 to 7 significantly affects the cytotoxicity of MSCs [40]. However, the gene transfection efficiencies are also accordingly decreased in most situations [25,40]. Thus, one of the major challenges of using PEI to deliver genes to MSCs is how to maintain the high gene delivery capability of PEI while minimizing its cytotoxicity. ...
Cationic polyethylenimine (PEI) is regarded as the “golden standard” of non-viral gene vectors. However, the superiority of PEI with high positive charge density also induces its major drawback of cytotoxicity, which restricts its application for an effective and safe gene delivery to stem cells. To redress this shortcoming, herein, a magnetic gene complex containing uniform iron oxide nanoparticles (UIONPs), plasmid DNA, and free PEI is prepared through electrostatic interactions for the gene delivery to bone marrow-derived mesenchymal stem cells (BM-MSCs). Results show that UIONPs dramatically promote the gene delivery to BM-MSCs using the assistance of magnetic force. In addition, decreasing the free PEI nitrogen to DNA phosphate (N/P) ratio from 10 to 6 has little adverse impact on the transgene expression levels (over 300 times than that of PEI alone at the N/P ratio of 6) and significantly reduces the cytotoxicity to BM-MSCs. Further investigations confirmed that the decrease of free PEI has little influence on the cellular uptake after applying external magnetic forces, but that the reduced positive charge density decreases the cytotoxicity. The present study demonstrates that the magnetic gene delivery not only contributes to the enhanced gene delivery efficiency but also helps to reduce required amount of PEI, providing a potential strategy for an efficient and safe gene delivery to stem cells.
... Solid scaffolds have been also prepared to deliver non-viral [194,242e260], adenoviral [261], and rAAV vectors [262,263] using chitosan [243], collagen [194,242,244,246,247,251,252] with HAp [255,256,259] or with PGA [248e250], gelatin with collagen, PGA, or both [245], b-TCP [261], PCL [257,262], poly-L-lactide acid (PLLA) [263] or PLGA [258] or both [253], or titanium and derivatives [254,260] as means to transfer sequences for reporter genes [243,246,251,253,256] or for therapeutic TGF-b [258], BMP-2 [245,247e250,254,255,259,260,262,263] and BMP-4 [242], RUNX2 [257,261], parathyroid hormone (amino acids 1e34) (PTH [1e34]) [194,242], and vascular endothelial growth factor (VEGF) [244,252,259] in order to activate MSC osteogenesis in vitro [243,245,248e251,253e260,262,263] and to heal bone defects in vivo [194,242,244,246,247,252,258,261,262]. Hosseinkhani et al. [245] impregnated a complex of cationized gelatin and plasmid DNA coding for BMP-2 in PGA fiber fabrics, collagen sponges, or collagen sponges reinforced by the incorporation of PGA fibers. ...
... Solid scaffolds have been also prepared to deliver non-viral [194,242e260], adenoviral [261], and rAAV vectors [262,263] using chitosan [243], collagen [194,242,244,246,247,251,252] with HAp [255,256,259] or with PGA [248e250], gelatin with collagen, PGA, or both [245], b-TCP [261], PCL [257,262], poly-L-lactide acid (PLLA) [263] or PLGA [258] or both [253], or titanium and derivatives [254,260] as means to transfer sequences for reporter genes [243,246,251,253,256] or for therapeutic TGF-b [258], BMP-2 [245,247e250,254,255,259,260,262,263] and BMP-4 [242], RUNX2 [257,261], parathyroid hormone (amino acids 1e34) (PTH [1e34]) [194,242], and vascular endothelial growth factor (VEGF) [244,252,259] in order to activate MSC osteogenesis in vitro [243,245,248e251,253e260,262,263] and to heal bone defects in vivo [194,242,244,246,247,252,258,261,262]. Hosseinkhani et al. [245] impregnated a complex of cationized gelatin and plasmid DNA coding for BMP-2 in PGA fiber fabrics, collagen sponges, or collagen sponges reinforced by the incorporation of PGA fibers. ...
Biomaterial-assisted gene therapy is a promising strategy for the treatment of various musculoskeletal disorders such as those concerning the articular cartilage, bones, tendons and ligaments, and meniscus as it can deliver candidate gene sequences in a spatially and temporally controlled manner in sites of tissue damage over prolonged periods of time that may be required to durably enhance the specific, natural repair mechanisms in vivo in direct, non-invasive procedures that avoid the arduous manipulation and implantation of patient-dependent cells genetically modified in vitro. In the present work, we provide an overview of the most up-to-date approaches and outcomes in experimental, relevant models of such disorders in vivo using biomaterial-guided gene transfer that may be employed in a near future to treat patients during a clinical intervention as a means to achieve an effective, safe, and persistent translational healing of musculoskeletal injuries.
... Solid scaffolds have been also prepared to deliver non-viral [194,242e260], adenoviral [261], and rAAV vectors [262,263] using chitosan [243], collagen [194,242,244,246,247,251,252] with HAp [255,256,259] or with PGA [248e250], gelatin with collagen, PGA, or both [245], b-TCP [261], PCL [257,262], poly-L-lactide acid (PLLA) [263] or PLGA [258] or both [253], or titanium and derivatives [254,260] as means to transfer sequences for reporter genes [243,246,251,253,256] or for therapeutic TGF-b [258], BMP-2 [245,247e250,254,255,259,260,262,263] and BMP-4 [242], RUNX2 [257,261], parathyroid hormone (amino acids 1e34) (PTH [1e34]) [194,242], and vascular endothelial growth factor (VEGF) [244,252,259] in order to activate MSC osteogenesis in vitro [243,245,248e251,253e260,262,263] and to heal bone defects in vivo [194,242,244,246,247,252,258,261,262]. Hosseinkhani et al. [245] impregnated a complex of cationized gelatin and plasmid DNA coding for BMP-2 in PGA fiber fabrics, collagen sponges, or collagen sponges reinforced by the incorporation of PGA fibers. ...
... Solid scaffolds have been also prepared to deliver non-viral [194,242e260], adenoviral [261], and rAAV vectors [262,263] using chitosan [243], collagen [194,242,244,246,247,251,252] with HAp [255,256,259] or with PGA [248e250], gelatin with collagen, PGA, or both [245], b-TCP [261], PCL [257,262], poly-L-lactide acid (PLLA) [263] or PLGA [258] or both [253], or titanium and derivatives [254,260] as means to transfer sequences for reporter genes [243,246,251,253,256] or for therapeutic TGF-b [258], BMP-2 [245,247e250,254,255,259,260,262,263] and BMP-4 [242], RUNX2 [257,261], parathyroid hormone (amino acids 1e34) (PTH [1e34]) [194,242], and vascular endothelial growth factor (VEGF) [244,252,259] in order to activate MSC osteogenesis in vitro [243,245,248e251,253e260,262,263] and to heal bone defects in vivo [194,242,244,246,247,252,258,261,262]. Hosseinkhani et al. [245] impregnated a complex of cationized gelatin and plasmid DNA coding for BMP-2 in PGA fiber fabrics, collagen sponges, or collagen sponges reinforced by the incorporation of PGA fibers. ...
... Later on this scaffold had been used in clinical trials for DFU healing [9] and was approved by the FDA for clinical application in 2016. Our group has tailored the Col/GAG scaffold for many different indications including bone and cartilage repair applications as well as for nucleic acid delivery [29][30][31][32][33][34][35]. The aim of this study was to use the Col/GAG scaffold as both a regenerative scaffold for skin wound healing and also as a delivery device for siMMP-9. ...
... Our group has been applying collagen based scaffolds for tissue regeneration for many years, and to date, two collagen-based products have been successfully translated to the clinic for bone (CE mark received) and cartilage repair. Recently, the group have focused on the use of these scaffolds as effective platforms for gene delivery in order to further improve tissue regeneration [32,34,46,[51][52][53][54]. These studies verified that the collagen based scaffolds can serve as a depot for effectively localized delivery of pDNA or microRNA. ...
Impaired wound healing of diabetic foot ulcers has been linked to high MMP-9 levels at the wound site. Strategies aimed at the simultaneous downregulation of the MMP-9 level in situ and the regeneration of impaired tissue are critical for improved diabetic foot ulcer (DFU) healing. To fulfil this aim, collagen/GAG (Col/GAG) scaffolds activated by MMP-9-targeting siRNA (siMMP-9) were developed in this study. The siMMP-9 complexes were successfully formed by mixing the RALA cell penetrating peptide with siMMP-9. The complexes formulated at N:P ratios of 6 to 15 had a diameter around 100 nm and a positive zeta potential about 40 mV, making them ideal for cellular uptake. In 2 dimensional (2D) culture of human fibroblasts, the cellular uptake of the complexes surpassed 60% and corresponded to a 60% reduction in MMP-9 gene expression in low glucose culture. In high glucose culture, which induces over-expression of MMP-9 and therefore serves as an in vitro model mimicking conditions in DFU, the MMP-9 gene could be downregulated by around 90%. In the 3D culture of fibroblasts, the siMMP-9 activated Col/GAG scaffolds displayed excellent cytocompatibility and ~60% and 40% MMP-9 gene downregulation in low and high glucose culture, respectively. When the siMMP-9 complexes were applied to THP-1 macrophages, the primary cell type producing MMP-9 in DFU, MMP-9 gene expression was significantly reduced by 70% and 50% for M0 and M1 subsets, in 2D culture. In the scaffolds, the MMP-9 gene and protein level of M1 macrophages decreased by around 50% and 30% respectively. Taken together, this study demonstrates that the RALA-siMMP-9 activated Col/GAG scaffolds possess high potential as a promising regenerative platform for improved DFU healing.
... Preparation of star-PLL-pDNA, PEI-pDNA and L-PLL-pDNA nanomedicines 32-& 64-star-PLL-pDNA nanomedicines were formulated at various N/P ratios (the molar ratio of positively charged nitrogen arms present in the star-PLL to negatively charged phosphate groups present in the pDNA molecule) from N/P 2-10 and pDNA doses (2 μg-20 μg) via the dropwise addition of star-PLL to pDNA in molecular biology grade H 2 0. Nanomedicines were allowed to equilibrate for 5-10 min at room temperature (RT) prior to use. PEI-pDNA N/P 10 (2 μg) nanomedicines have been previously optimized within our laboratory for gene delivery to MSCs [39]. Briefly, pDNA was added to sterile, molecular biology grade H 2 0 followed by the dropwise addition of 25 kDa branched PEI and 30-min equilibration at RT. LPLL-pDNA (1 μg) nanomedicines were used at a mass ratio (M:R) 2:1 (L-PLL: pDNA) as previously optimized by us for MSC transfection [26]. ...
... The high transgene expression levels facilitated in this difficult to transfect cell type on a 3D scaffold highlights the advantage of the star-PLL vector over others for TE applications [61]. Furthermore, their enhanced transfection efficiency over the PEI-pDNA gene activated scaffold that we have previously demonstrated could support the healing of critical sized bone defects in vivo [39] demonstrates the significant potential of the star-PLL vector in forming a functional gene activated scaffold with therapeutic in vivo potential. Reporter transgene expression from the 64-star-PLL-pGLuc scaffold was transient and prolonged over 28 days which is widely accepted within the TE field as optimal for encouraging tissue repair [62] and supports the above theory that the scaffold acts as a depot for the retention of nanomedicines over time. ...
It is increasingly being recognised within the field of tissue engineering that the regenerative capacity of biomaterial scaffolds can be augmented via the incorporation of gene therapeutics. However, the field still lacks a biocompatible gene delivery vector which is capable of functionalizing scaffolds for tailored nucleic acid delivery. Herein, we describe a versatile, collagen based, gene-activated scaffold platform which can transfect autologous host cells in vivo via incorporation of star-shaped poly(˪-lysine) polypeptides (star-PLLs) and a plasmid DNA (pDNA) cargo. Two star-PLL vectors with varying number and length of poly(˪-lysine) arms were assessed. In vitro, the functionalization of a range of collagen based scaffolds containing either glycosaminoglycans (chondroitin sulfate or hyaluronic acid) or ceramics (hydroxyapatite or nano-hydroxyapatite) with star-PLL-pDNA nanomedicines facilitated prolonged, non-toxic transgene expression by mesenchymal stem cells (MSCs). We demonstrate that the star-PLL structure confers enhanced spatiotemporal control of nanomedicine release from functionalized scaffolds over a 28-day period compared to naked pDNA. Furthermore, we identify a star-PLL composition with 64 poly(˪-lysine) arms and 5 (˪-lysine) subunits per arm as a particularly effective vector, capable of facilitating a 2-fold increase in reporter transgene expression compared to the widely used vector polyethylenimine (PEI), a 44-fold increase compared to a 32 poly(˪-lysine) armed star-PLL and a 130-fold increase compared to its linear analogue, linear poly(˪-lysine) (L-PLL) from a collagen-chondroitin sulfate gene activated scaffold. In an in vivo subcutaneous implant model, star-PLL-pDNA gene activated scaffolds which were implanted cell-free exhibited extensive infiltration of autologous host cells, nanomedicine retention within the implanted construct and successful host cell transfection at the very early time point of just seven days. Overall, this article illustrates for the first time the significant ability of the star-PLL polymeric structure to transfect autologous host cells in vivo from an implanted biomaterial scaffold thereby forming a versatile platform with potential in numerous tissue engineering applications.
... Additionally, cells transfected with the viral vector maintained a healthier morphology than those treated with the PEI non-viral vector after 2 days of transfection. These observations indicate that polycationic PEI, at higher N/P ratios such as 5 and 7, was toxic to hMSCs, even though these ratios were safe for HEK293T cells, as shown in Figure 2. The optimal N/P ratios (around 5-7) of PEI/plasmid complexes for gene transfection of hMSCs are consistent with the published accounts [25]. Consequently, lentiviral vectors were chosen as the TP gene delivery method for hMSCs in subsequent studies. ...
Gene-directed enzyme prodrug therapy (GDEPT) has been developed over several decades as a targeted cancer treatment aimed at minimizing toxicity to healthy cells. This approach involves three key components: a non-toxic prodrug, a gene encoding an enzyme that converts the prodrug into an active chemotherapy drug, and a gene carrier to target cancer cells. In this study, the prodrug doxifluridine was enzymatically converted into the chemotherapy drug 5-fluorouracil via thymidine phosphorylase, using human mesenchymal stem cells (hMSCs) as delivery vehicles. The hMSCs were first transduced with thymidine phosphorylase-encoded lentiviral vectors produced by HEK293T cells, then co-cultured with A549 adenocarcinoma cells in the presence of doxifluridine. The results showed that after 3 days of prodrug treatment, cell viability in both A549 cancer cells and hMSCs dropped by about 50%, and by day 5, viability had decreased to 10%. In summary, exogenous thymidine phosphorylase expressed in hMSCs successfully converted the non-toxic prodrug doxifluridine into the chemotherapy agent 5-fluorouracil, effectively eliminating both cancer cells and hMSCs within a short period.
... In vitro toxicity. Rat mesenchymal stem cells (rMSCs) were isolated from 6-8 week old Sprague Dawley rats as approved by the RCSI Research Ethics Committee using methods previously described [43]. Briefly, the femora of both hind limbs of the rats were clipped to expose the bone marrow, which was flushed out using expansion media using an 18 G needle and 12 mL syringe into a 65 mm Petri dish. ...
Hydrogels with low toxicity, antimicrobial potency and shear-thinning behavior are promising materials to combat the modern challenges of increased infections. Here, we report on 8-arm star block copolypeptides based on poly(L-lysine), poly(L-tyrosine) and poly(S-benzyl-L-cysteine) blocks. Three star block copolypeptides were synthesized with poly(S-benzyl-L-cysteine) always forming the outer block. The inner block comprised either two individual blocks of poly(L-lysine) and poly(L-tyrosine) or a statistical block copolypeptide from both amino acids. The star block copolypeptides were synthesized by the Ring Opening Polymerization (ROP) of the protected amino acid N-carboxyanhydrides (NCAs), keeping the overall ratio of monomers constant. All star block copolypeptides formed hydrogels and Scanning Electron Microscopy (SEM) confirmed a porous morphology. The investigation of their viscoelastic characteristics, water uptake and syringe extrudability revealed superior properties of the star polypeptide with a statistical inner block of L-lysine and L-tyrosine. Further testing of this sample confirmed no cytotoxicity and demonstrated antimicrobial activity of 1.5-log and 2.6-log reduction in colony-forming units, CFU/mL, against colony-forming reference laboratory strains of Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus, respectively. The results underline the importance of controlling structural arrangements in polypeptides to optimize their physical and biological properties.
... The cross-linked scaffolds were then washed with PBS (Gibco, London, UK) before soak-loading polyplex nanoparticles on to the scaffolds for 40 min. Based on previous studies [35,36,39], polyplex particles were formulated by mixing a specified amount of branched cationic 25 kDa polyethyleneimine (PEI) (Sigma-Aldrich, Dublin, Ireland) for anionic pDNA (fixed at a dose of 2 µg) delivery [28,36,40] and given an N/P ratio of 10. Three scaffold groups are prepared: (1) gene-free scaffold (GFS, no pDNA), (2) β-klotho gene-activated scaffold and (3) Glyoxalase-1/β-Klotho gene-activated scaffold (dual GAS). ...
Tissue engineering approaches aim to provide biocompatible scaffold supports that allow healing to progress often in healthy tissue. In diabetic foot ulcers (DFUs), hyperglycemia impedes ulcer regeneration, due to complications involving accumulations of cellular methylglyoxal (MG), a key component of oxidated stress and premature cellular aging which further limits repair. In this study, we aim to reduce MG using a collagen-chondroitin sulfate gene-activated scaffold (GAS) containing the glyoxalase-1 gene (GLO-1) to scavenge MG and anti-fibrotic β-klotho to restore stem cell activity in diabetic adipose-derived stem cells (dADSCs). dADSCs were cultured on dual GAS constructs for 21 days in high-glucose media in vitro. Our results show that dADSCs cultured on dual GAS significantly reduced MG accumulation (−84%; p < 0.05) compared to the gene-free controls. Similar reductions in profibrotic proteins α-smooth muscle actin (−65%) and fibronectin (−76%; p < 0.05) were identified in dual GAS groups. Similar findings were observed in the expression of pro-scarring structural proteins collagen I (−62%), collagen IV (−70%) and collagen VII (−86%). A non-significant decrease in the expression of basement membrane protein E-cadherin (−59%) was noted; however, the dual GAS showed a significant increase in the expression of laminin (+300%). We conclude that dual GAS-containing Glo-1 and β-klotho had a synergistic MG detoxification and anti-fibrotic role in dADSC's. This may be beneficial to provide better wound healing in DFUs by controlling the diabetic environment and rejuvenating the diabetic stem cells towards improved wound healing.
... Apart from the above-mentioned examples that mainly concern viral vector therapies in regenerative medicine, non-viral gene therapies can also be applied in the regeneration of tissues. For example, O'Brien and colleagues report the efficacy of PEI and collagen-based scaffolds for bone regenerations through non-viral GAM technology [328]. When seeded with rMSCs, PEI polyplex GAMs, synthesised from 2 µg of pDNA per scaffold, can achieve sustained release of gene expression for 21 days. ...
The development of biological methods over the past decade has stimulated great interest in the possibility to regenerate human tissues. Advances in stem cell research, gene therapy, and tissue engineering have accelerated the technology in tissue and organ regeneration. However, despite significant progress in this area, there are still several technical issues that must be addressed, especially in the clinical use of gene therapy. The aims of gene therapy include utilising cells to produce a suitable protein, silencing over-producing proteins, and genetically modifying and repairing cell functions that may affect disease conditions. While most current gene therapy clinical trials are based on cell- and viral-mediated approaches, non-viral gene transfection agents are emerging as potentially safe and effective in the treatment of a wide variety of genetic and acquired diseases. Gene therapy based on viral vectors may induce pathogenicity and immunogenicity. Therefore, significant efforts are being invested in non-viral vectors to enhance their efficiency to a level comparable to the viral vector. Non-viral technologies consist of plasmid-based expression systems containing a gene encoding, a therapeutic protein, and synthetic gene delivery systems. One possible approach to enhance non-viral vector ability or to be an alternative to viral vectors would be to use tissue engineering technology for regenerative medicine therapy. This review provides a critical view of gene therapy with a major focus on the development of regenerative medicine technologies to control the in vivo location and function of administered genes.
... Once the scaffolds were ready, polyplexes at an N/P10 ratio (nitrogen to phosphate ratio) were prepared by mixing a predetermined volume of branched polyethyleneimine (PEI) solution with plasmid DNA (pDNA) encoding for the human beta-Klotho gene (β-Klotho), obtained from SinoBiological, Beijing, China. The N/P10 ratio was chosen based on our previous studies that found that polyplexes formulated at an N/P 10 ratio could effectively form small, stable cationic nanoparticles with plasmids as large as GLuc (5.76 kb) [21,23]. Prior to use, the plasmids were diluted in endotoxin free water to obtain a working concentration of 0.5 µg/µL. ...
Wound healing requires a tight orchestration of complex cellular events. Disruption in the cell-signaling events can severely impair healing. The application of biomaterial scaffolds has shown healing potential; however, the potential is insufficient for optimal wound maturation. This study explored the functional impact of a collagen-chondroitin sulfate scaffold functionalized with nanoparticles carrying an anti-aging gene β-Klotho on human adipose-derived stem cells (ADSCs) for rejuvenative healing applications. We studied the response in the ADSCs in three phases: (1) transcriptional activities of pluripotency factors (Oct-4, Nanog and Sox-2), proliferation marker (Ki-67), wound healing regulators (TGF-β3 and TGF-β1); (2) paracrine bioactivity of the secretome generated by the ADSCs; and (3) regeneration of basement membrane (fibronectin, laminin, and collagen IV proteins) and expression of scar-associated proteins (α-SMA and elastin proteins) towards maturation. Overall, we found that the β-Klotho gene-activated scaffold offers controlled activation of ADSCs’ regenerative abilities. On day 3, the ADSCs on the gene-activated scaffold showed enhanced (2.5-fold) activation of transcription factor Oct-4 that was regulated transiently. This response was accompanied by a 3.6-fold increase in the expression of the anti-fibrotic gene TGF-β3. Through paracrine signaling, the ADSCs-laden gene-activated scaffold also controlled human endothelial angiogenesis and pro-fibrotic response in dermal fibroblasts. Towards maturation, the ADSCs-laden gene-activated scaffold further showed an enhanced regeneration of the basement membrane through increases in laminin (2.1-fold) and collagen IV (8.8-fold) deposition. The ADSCs also expressed 2-fold lower amounts of the scar-associated α-SMA protein with improved qualitative elastin matrix deposition. Collectively, we determined that the β-Klotho gene-activated scaffold possesses tremendous potential for wound healing and could advance stem cell-based therapy for rejuvenative healing applications.
... Owing to the short half-lives of recombinant human growth factors and the needed for clinical effectiveness, supraphysiological dosages were used, and their production was expensive. 265,266 The supraphysiological dosages of recombinant human growth factors could also cause several side effects, such as ectopic formation of bone tissue and soft tissue swelling. 267 Therefore, the non-viral gene therapy then became a promising alternative strategy for bone tissue repair. ...
DNA, a genetic material, has been employed in different scientific directions for various biological applications as driven by DNA nanotechnology in the past decades, including tissue regeneration, disease prevention, inflammation inhibition, bioimaging, biosensing, diagnosis, antitumor drug delivery, and therapeutics. With the rapid progress in DNA nanotechnology, multitudinous DNA nanomaterials have been designed with different shape and size based on the classic Watson–Crick base-pairing for molecular self-assembly. Some DNA materials could functionally change cell biological behaviors, such as cell migration, cell proliferation, cell differentiation, autophagy, and anti-inflammatory effects. Some single-stranded DNAs (ssDNAs) or RNAs with secondary structures via self-pairing, named aptamer, possess the ability of targeting, which are selected by systematic evolution of ligands by exponential enrichment (SELEX) and applied for tumor targeted diagnosis and treatment. Some DNA nanomaterials with three-dimensional (3D) nanostructures and stable structures are investigated as drug carrier systems to delivery multiple antitumor medicine or gene therapeutic agents. While the functional DNA nanostructures have promoted the development of the DNA nanotechnology with innovative designs and preparation strategies, and also proved with great potential in the biological and medical use, there is still a long way to go for the eventual application of DNA materials in real life. Here in this review, we conducted a comprehensive survey of the structural development history of various DNA nanomaterials, introduced the principles of different DNA nanomaterials, summarized their biological applications in different fields, and discussed the current challenges and further directions that could help to achieve their applications in the future.
... The stability of the gene complexes during scaffold fabrication as well as the controlled release rate during scaffold degradation in situ are additional considerations to be addressed [6,7]. A variety of GAM systems, including nanoparticles, hydrogels, freeze dried scaffolds, and electrospun membranes have been investigated with the aim of the successful delivery of complexes to cells of interest [8][9][10][11]. Most GAMs are fabricated by preparing the scaffolds and gene complexes separately and formulating them together before administration in a biological system. Electrospun fibers have become increasingly attractive as a scaffold due to their structural similarities to that of extracellular matrix (ECM) [12]. ...
The fabrication of fiber mats via electrospinning has been adopted in the last decades to produce high quality scaffolds for tissue engineering. However, an effective combination of electrospinning methods with gene delivery therapies remains a challenge. In this study, we describe how the delivery of gene complexes via electrospun mats that contain different volumes of gelatin (Gel), collagen (Col), and polyethylene glycol (PEG) can affect gene expression by transfected cells. Non-viral complexes were formulated by using lipid modified polyethylenimine (PEI) polymer and plasmid DNAs (pDNA) like the reporter Green Fluorescent Protein (GFP) and the therapeutically relevant Bone Morphogenetic Protein-2 (BMP-2) and electrospuned after being mixed with different volumes of Gel-Col-PEG mats and delivered to human myoblast (C2C12) and mouse osteoblast cells (MC3T3). The entrapment of GFP complexes via different homogeneous electrospun fiber mats revealed that a high fraction of collagen in the mats affected the quality of the fibers and led to reduced transfection efficiency on target cells. On the other hand, the fabrication of double-layered mats that contained collagen without complexes as a first layer and gelatin-collagen-PEG with complexes as a second layer successfully induced GFP expression and ALP activity in C2C12 cells. We conclude that this study has established the advantage of formulating multilayered bioactive collagen-based mats for gene delivery applications.
... MSCs were transfected with MR75 nanoparticles (determined after physicochemical assessment), delivering 0.5 µg, 1 µg, and 2 µg of pLuc. These doses were chosen based on previous work carried out in the literature [58]. ...
Non-viral vectors offer a safe alternative to viral vectors for gene therapy applications, albeit
typically exhibiting lower transfection efficacies. As a result, there remains a significant need for
the development of a non-viral delivery system with low cytotoxicity and high transfection efficacy as
a tool for safe and transient gene delivery. This study assesses MgAl-NO3 layered double hydroxide
(LDH) as a non-viral vector to deliver nucleic acids (pDNA, miRNA and siRNA) to mesenchymal
stromal cells (MSCs) in 2D culture and using a 3D tissue engineering scaffold approach. Nanoparticles
were formulated by complexing LDH with pDNA, microRNA (miRNA) mimics and inhibitors, and
siRNA at varying mass ratios of LDH:nucleic acid. In 2D monolayer, pDNA delivery demonstrated
significant cytotoxicity issues, and low cellular transfection was deemed to be a result of the poor
physicochemical properties of the LDH–pDNA nanoparticles. However, the lower mass ratios
required to successfully complex with miRNA and siRNA cargo allowed for efficient delivery to
MSCs. Furthermore, incorporation of LDH–miRNA nanoparticles into collagen-nanohydroxyapatite
scaffolds resulted in successful overexpression of miRNA in MSCs, demonstrating the development
of an efficacious miRNA delivery platform for gene therapy applications in regenerative medicine.
... A large range of biomaterial scaffolds have been applied to design nonviral gene delivery systems capable to release, in a sustainable and controlled way, therapeutic genes in desired tissues [76,104] (Table 3) including articular cartilage [105,106] and bone [107][108][109][110][111][112][113][114]. In the field of bone regeneration, which is of high relevance to restore the subchondral bone defect, collagen-based scaffolds have been widely used either alone [107,109,111,112,115] or combined with ceramic particles [109,113,114] to deliver pDNAs encoding for BMP-2 [108,110,[112][113][114], BMP-7 [114], TGF-β1 [105,106], PDGF [107,109,111], VEGF [108,109,113], or FGF-2 [112]. Chitosan has been widely used as a scaffolding material in different cartilage tissue engineering approaches due to its cationic nature, acting as healing accelerator and exhibiting antimicrobial activities [116]. ...
Osteochondral defects involve both the articular cartilage and the underlying subchondral bone. If left untreated, they may lead to osteoarthritis. Advanced biomaterial-guided delivery of gene vectors has recently emerged as an attractive therapeutic concept for osteochondral repair. The goal of this review is to provide an overview of the variety of biomaterials employed as nonviral or viral gene carriers for osteochondral repair approaches both in vitro and in vivo, including hydrogels, solid scaffolds, and hybrid materials. The data show that a site-specific delivery of therapeutic gene vectors in the context of acellular or cellular strategies allows for a spatial and temporal control of osteochondral neotissue composition in vitro. In vivo, implantation of acellular hydrogels loaded with nonviral or viral vectors has been reported to significantly improve osteochondral repair in translational defect models. These advances support the concept of scaffold-mediated gene delivery for osteochondral repair.
... One study reported that the expression of OCN and ALP, as well as the activity of ALP, was reduced in a 3D model to approximately half with respect to that detected in a two-dimensional (2D) model. However, this reduction from 2D to 3D did not prevent the treatment from enhancing bone repair when tested in vivo [179], highlighting the importance of moving from in vitro models to in vivo ones. ...
Recently, a large spectrum of biomaterials emerged, with emphasis on various pure, blended, or doped calcium phosphates (CaPs). Although basic cytocompatibility testing protocols are referred by International Organization for Standardization (ISO) 10993 (parts 1–22), rigorous in vitro testing using cutting-edge technologies should be carried out in order to fully understand the behavior of various biomaterials (whether in bulk or low-dimensional object form) and to better gauge their outcome when implanted. In this review, current molecular techniques are assessed for the in-depth characterization of angiogenic potential, osteogenic capability, and the modulation of oxidative stress and inflammation properties of CaPs and their cation- and/or anion-substituted derivatives. Using such techniques, mechanisms of action of these compounds can be deciphered, highlighting the signaling pathway activation, cross-talk, and modulation by microRNA expression, which in turn can safely pave the road toward a better filtering of the truly functional, application-ready innovative therapeutic bioceramic-based solutions.
... 67 However, non-viral vectors are typically hindered by low transfection efficiency and therefore efficacy. As such, a body of research is currently being carried out to enhance their efficiency through their incorporation into biomaterial scaffolds (Fig. 1).Com-).Combining non-viral vectors complexed with pDNA within a biomaterial scaffold facilitates enhanced gene transfer 68 and advancements in delivery methods over the last two decades have resulted in a reduction in the dose required from the milligram (mg) range, reported in earlier studies, to the microgram (µg) range (Fig. 2). The incorporation of a calcium phosphate-pDNA (40 µg) precipitate mixed with collagen demonstrated enhanced efficiency of reporter plasmids in vivo. ...
Recent advances in tissue engineering have made progress towards the development of biomaterials capable of the delivery of growth factors, such as BMPs, in order to promote enhanced tissue repair. However, controlling the release of these growth factors on demand and within the desired localised area is a significant challenge and the associated high costs and side effects of uncontrolled delivery have proven increasingly problematic in clinical orthopaedics. Gene therapy may be a valuable tool to avoid the limitations of local delivery of growth factors. Following a series of setbacks in the 1990′s, the field of gene therapy is now seeing improvements in safety and efficacy resulting in substantial clinical progress and a resurgence in confidence. Biomaterial scaffold‐mediated gene therapy provides a template for cell infiltration and tissue formation while promoting transfection of cells to engineer therapeutic proteins in a sustained but ultimately transient fashion. Additionally, scaffold‐mediated delivery of RNA‐based therapeutics can silence specific genes associated with orthopaedic pathological states. This review will provide an overview of the current state‐of‐the‐art in the field of gene‐activated scaffolds and their use within orthopaedic tissue engineering applications.
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Osteomyelitis, a severe bone infection, is an extremely challenging complication in the repair of traumatic bone defects. Furthermore, the use of long-term high-dose antibiotics in standard treatment increases the risks of antibiotic resistance. Herein, an antibiotic-free, collagen silver-doped hydroxyapatite (coll-AgHA) scaffold reinforced with a 3D printed polycaprolactone (PCL) framework was developed with enhanced mechanical properties to be used in the repair of load-bearing defects with antimicrobial properties as a preventative measure against osteomyelitis. The AgHA particles were fabricated in varying Ag doses and loaded within freeze-dried collagen scaffolds at two concentrations. The optimised Ag dose (1.5 mol% Ag) and AgHA concentration (200 wt%) within the collagen scaffold demonstrated in vitro osteogenic and antibacterial properties against S. aureus (S. aureus), the main causative pathogen of osteomyelitis. The addition of the PCL framework to the coll-AgHA scaffolds significantly enhanced the compressive modulus from 4 to 12 MPa while maintaining high porosity as well as both pro-osteogenic and antibacterial properties. The reinforced coll-AgHA scaffolds were implanted in vivo and demonstrated enhanced bone repair, significantly greater vessel formation, and calcified tissue in a load-bearing critical sized defect in rats. Taken together, these results confirm the capacity of this novel biomaterial scaffold as a preventative measure against infection in bone repair for use in load-bearing defects, without the use of antibiotics.
Stem cells are an essential consideration in the fields of tissue engineering and regenerative medicine, understanding how nanoengineered biomaterials and mesenchymal stem cells (MSCs) interact is crucial for their role in bone regeneration. Utilising structural stability of Selenium nanoparticles (Se-NPs) and biological properties of natural polymers, Se-NPs functionalized, injectable, thermoresponsive hydrogels with interconnected molecular structure were prepared to identify their role in osteogenic differentiation of different types of meschymal stem cells. The study provides comprehensive characterization of their structural and biological properties. Results showed that hydrogels go from sol to gel transition with the help of β-glycerophosphate; functionalization with Se-NPs significantly enhanced the biological response as it as it stabilized the polymeric structure by forming Se-O covalent bonds. Further results suggest that Se-NPs enhance the differentiation of MSCs toward osteogenic lineage in 2D as well as in 3D. We demonstrated that Se-NPs functionalized hydrogels enhance the differentiation of osteoporotic bone-derived MSCs. We have also focused on specific cell surface marker expression (CD105, CD90, CD73, CD45, CD34) based on exposure to Se-NP functionalized hydrogel in healthy rats BMSC. This study provides essential evidence for pre-clinical/clinical applications, highlighting the potential of nanoengineered biocompatible elastic hydrogels for bone regeneration in diseased bone.
Back pain is a global epidemiological and socioeconomic problem affecting up to 80% of people at some stage during their life and is often due to degeneration of the intervertebral disc (IVD). Therapies aimed at restoring the intradiscal space have predominantly focused on delivery of biomaterials, cells, or growth factors, among others, with variable degrees of success. While viral gene delivery strategies have emerged as promising therapeutic options in recent years, these approaches often have off‐target effects and are associated with immunogenicity risks and other comorbidities. Consequently, nonviral methods have gained traction as potential avenues for gene delivery. Herein, enhanced cell‐penetrating peptide (CPP) systems are used to deliver microRNAs in an in vitro and ex vivo model of disc degeneration. The data suggest that nanoparticle complexation of CPPs with (miR‐221‐inhibitor + miR‐149‐mimic) promotes protective effects in nucleus pulposus cells challenged with inflammatory cytokines TNF‐α and IL‐1β. Specifically, increases in matrix deposition, significant decreases in the secretion of an array of inflammatory cytokines, and decreased expression of matrix degradation enzymes MMP13 and ADAMTS5 are observed. These miR‐CPP nanocomplexes can be further employed for targeting of the pericellular matrix space through homing, thus providing a promising approach for therapies of the intradiscal space.
Mobilizing endogenous progenitor cells to repair damaged tissue in situ has the potential to revolutionize the field of regenerative medicine, while the early establishment of a vascular network will ensure survival of newly generated tissue. In this study, a gene‐activated scaffold containing a stromal derived factor 1α plasmid (pSDF1α), a pro‐angiogenic gene that is also thought to be involved in the recruitment of mesenchymal stromal cells (MSCs) to sites of injury is described. It is shown that over‐expression of SDF1α protein enhanced MSC recruitment and induced vessel‐like structure formation by endothelial cells in vitro. When implanted subcutaneously, transcriptomic analysis reveals that endogenous MSCs are recruited and significant angiogenesis is stimulated. Just 1‐week after implantation into a calvarial critical‐sized bone defect, pSDF1α‐activated scaffolds are recruited MSCs and rapidly activate angiogenic and osteogenic programs, upregulating Runx2, Dlx5, and Sp7. At the same time‐point, pVEGF‐activated scaffolds are recruited a variety of cell types, activating endochondral ossification. The early response induced by both scaffolds leads to complete bridging of the critical‐sized bone defects within 4‐weeks. The versatile cell‐free gene‐activated scaffold described in this study is capable of harnessing and enhancing the body's own regenerative capacity and has immense potential in a myriad of applications.
RNA delivery has been demonstrated to be a potent method of vaccine delivery, as demonstrated by the recent success of the COVID-19 vaccines. Polymers have been shown to be effective vehicles for RNA delivery, with poly(ethylene imine) (PEI) being the current gold standard for delivery. Nonetheless, PEI has toxicity concerns, and so finding alternatives is desirable. Poly(2-oxazoline)s are a promising alternative to PEI, as they are generally biocompatible and offer a high degree of control over the polymer structure. Here, we have synthesized an ionizable primary amine 2-oxazoline and combined it with a double bond containing oxazoline to synthesize a small library of charged statistical and block copolymers. The pendant double bonds were reacted further to decorate the polymers with glucose via a thiol–ene click reaction. All polymers were shown to have excellent cell viability, and the synthesized block polymers showed promising complexation efficiencies for the saRNA, demonstrating a clear structure–property relationship. The polymer transfection potential was tested in various cell lines, and a polymer composition with an amine/glucose ratio of 9:27 has demonstrated the best transfection potential across all cell lines tested. Overall, the results suggest that block polymers with a cationic segment and high levels of glycosylation have the best complexation efficiency and RNA expression levels.
The advancement of tissue engineering for regenerating injured tissues and organs has progressed significantly in recent years. Various techniques have been used to modify the cells' microenvironments in the targeted tissue via their extracellular environment for achieving these aims. The 3D structured scaffolds alone or combined with bioactive molecules or genes and cells hold great promise for the development of functional engineered tissues. As an emerging and state-of-the-art technology in this field, integrating tissue engineering and gene therapy, known as gene-activated matrix (GAM), has gained immense attention as a promising approach for restoring damaged or dysfunctional tissues' function and structure. Nonetheless, fabricating GAMs with low cytotoxicity, high transfection efficiency, and long-term gene delivery efficiency is still challenging. Here we provide a complete overview of different tissue engineering approaches and their ongoing preclinical research trials. Moreover, the GAM strategy with a focus on gene-activated matrix development, faithful application, and future prospects as a tissue repair and regeneration replacement is assayed. The challenges and future research prospects in regenerative medicine are also presented. Eventually, we propose that GAMs offer a basic mechanistic infrastructure for “tissue engineering” to pave the way for clinical translation and achieve personalized regenerative medicine.
In recent decades, human mesenchymal stem cells (hMSCs) have gained momentum in the field of cell therapy for treating cartilage and bone injuries. Despite the tri-lineage multipotency, proliferative properties, and potent immunomodulatory effects of hMSCs, their clinical potential is hindered by donor variations, limiting their use in medical settings. To address this challenge, gene delivery technologies have emerged as a promising approach to modulate the phenotype and commitment of hMSCs towards specific cell lineages, thereby enhancing osteochondral repair strategies. This review provides a comprehensive overview of current non-viral gene delivery approaches used to engineer MSCs, highlighting key factors such as the choice of nucleic acid or delivery vector, transfection strategies, and experimental parameters. Additionally, it outlines various protocols and methods for qualitative and quantitative evaluation of their therapeutic potential as a delivery system in osteochondral regenerative applications. In summary, this technical review offers a practical guide for optimizing non-viral systems in osteochondral regenerative approaches.
Graphical Abstract
hMSCs constitute a key target population for gene therapy techniques. Nevertheless, there is a long way to go for their translation into clinical treatments. In this review, we remind the most relevant transfection conditions to be optimized, such as the type of nucleic acid or delivery vector, the transfection strategy, and the experimental parameters to accurately evaluate a delivery system. This survey provides a practical guide to optimizing non-viral systems for osteochondral regenerative approaches.
Various research fields use the transfection of mammalian cells with genetic material to induce the expression of a target transgene or gene silencing. It is a tool widely used in biological research, bioproduction, and therapy. Current transfection protocols are usually performed on 2D adherent cells or suspension cultures. The important rise of new gene therapies and regenerative medicine in the last decade raises the need for new tools to empower the in situ transfection of tissues and 3D cell cultures. This review will present novel in situ transfection methods based on a chemical or physical non-viral transfection of cells in tissues and 3D cultures, discuss the advantages and remaining gaps, and propose future developments and applications.
Pathological hair loss (also known as alopecia) and shortage of hair follicle (HF) donors have posed an urgent requirement for HF regeneration. With the revelation of mechanisms in tissue engineering, the proliferation of HFs in vitro has achieved more promising trust for the treatments of alopecia and other skin impairments. Theoretically, HF organoids have great potential to develop into native HFs and attachments such as sweat glands after transplantation. However, since the rich extracellular matrix (ECM) deficiency, the induction characteristics of skin-derived cells gradually fade away along with their trichogenic capacity after continuous cell passaging in vitro. Therefore, ECM-mimicking support is an essential prelude before HF transplantation is implemented. This review summarizes the status of providing various epidermal and dermal cells with a three-dimensional (3D) scaffold to support the cell homeostasis and better mimic in vivo environments for the sake of HF regeneration. HF-relevant cells including dermal papilla cells (DPCs), hair follicle stem cells (HFSCs), and mesenchymal stem cells (MSCs) are able to be induced to form HF organoids in the vitro culture system. The niche microenvironment simulated by different forms of biomaterial scaffold can offer the cells a network of ordered growth environment to alleviate inductivity loss and promote the expression of functional proteins. The scaffolds often play the role of ECM substrates and bring about epithelial-mesenchymal interaction (EMI) through coculture to ensure the functional preservation of HF cells during in vitro passage. Functional HF organoids can be formed either before or after transplantation into the dermis layer. Here, we review and emphasize the importance of 3D culture in HF regeneration in vitro. Finally, the latest progress in treatment trials and critical analysis of the properties and benefits of different emerging biomaterials for HF regeneration along with the main challenges and prospects of HF regenerative approaches are discussed.
The incorporation of pro-osteogenic growth factors into bone graft materials to enhance bone regeneration is a key research area within the field of bone tissue engineering and regenerative medicine. However, growth factors directly incorporated in protein form are easily degraded, and have a limited active half-life, which cannot exert long-term and stable osteoinductive and oteoconductive effects. The combination of gene therapy and tissue engineering through gene-activated matrix (GAM) may provide a good alternative solution to overcome such limitations. Scaffold materials can be combined together with plasmid DNA and a chemical-based transfection agent to form GAM, through which transfected cells could secrete growth factors in a sustained manner over a longer time duration; thereby enabling bone graft materials to act as a repository of therapeutic genes, while providing structural support and a scaffold matrix for new bone tissue ingrowth. In this study, we prepared hydroxyapatite/chitosan-microspheres (HA/CS-MS) with microfabrication technology and emulsification method, and loaded the polyethylene imine/bone morphogenetic protein 2 plasmid (PEI/pBMP2) complexes with high transfection capacity (transfection efficiency up to 54.79% ± 4.95%), thus forming a novel GAM system with superior bone regeneration capacity—PEI/pBMP2-HA/CS-MS. The in vitro experiments in this study demonstrated that our GAM had excellent biocompatibility (with cell viability over 95%), and that the as-fabricated microsphere material possessed a nano-network fibrous structure similar to natural extracellular matrix (ECM), together with a higher surface area that can provide more cell adhesion sites. The sizes of the prepared microspheres were mainly distributed in the 160–180 µm range, while the maximal loading rate of PEI-pBMP2 complexes was 59.79% ± 1.85%. As a loaded complexes system, the GAM can release plasmids in a slow controlled manner, effectively transfecting surrounding target cells (release effect for up to 21 days), while cells adherent to the material can also take up plasmids, resulting in sustained secretion of the target protein, thereby effectively promoting bone regeneration. In vivo data from micro-computed tomography (micro-CT) and histological staining showed that the use of the composite materials effectively enhanced bone regeneration in defect areas. These findings thus demonstrated that the novel GAM system had excellent osteoinductivity with significant clinical potential.
In the human body, articular cartilage facilitates the frictionless movement of synovial joints. However, due to its avascular and aneural nature, it has a limited ability to self-repair when damaged due to injury or wear and tear over time. Current surgical treatment options for cartilage defects often lead to the formation of fibrous, non-durable tissue and thus a new solution is required. Nature is the best innovator and so recent advances in the field of tissue engineering have aimed to recreate the microenvironment of native articular cartilage using biomaterial scaffolds. However, the inability to mirror the complexity of native tissue has hindered the clinical translation of many products thus far. Fortunately, the advent of 3D printing has provided a potential solution. 3D printed scaffolds, fabricated using biomimetic biomaterials, can be designed to mimic the complex zonal architecture and composition of articular cartilage. The bioinks used to fabricate these scaffolds can also be further functionalised with cells and/or bioactive factors or gene therapeutics to mirror the cellular composition of the native tissue. Thus, this review investigates how the architecture and composition of native articular cartilage is inspiring the design of biomimetic bioinks for 3D printing of scaffolds for cartilage repair. Subsequently, we discuss how these 3D printed scaffolds can be further functionalised with cells and bioactive factors, as well as looking at future prospects in this field.
The materials used nowadays for bone replacement do not fully meet the requirements for complete regeneration, which is why new ones are being tested. Background/Aim. Despite numerous attempts to improve bone tissue regeneration, no fulfilling material has been found yet. This study investigated the influence of poly-lactide-co-glycolide (PLGA) and poly-ethylene-imine (PEI) as coatings for hydroxyapatite (HAP) bone carrier onto bone tissue regenerative potential in rabbit's calvarial defect. Methods. Calvarial defects measuring 6 mm in diameter were made in 19 skeletally mature rabbits. Defects were filled with one of the following materials: PLGA coated HAP (HAP+PLGA), PEI coated HAP (HAP+PEI) and bovine HAP - Bio-Oss (positive control). Unfilled defects represented negative control. Histological analysis was performed in order to determine the inflammatory response of the host tissue. Formation of the new bone was evaluated by means of histomorphometric analysis. All analysis have been conducted in samples obtained 3, 6 and 9 weeks after implantation. Results. Three weeks post-implantation, a trend toward increased healing in HAP+PLGA group compared to other investigated materials was noticed, with no statistically significant difference between study groups (p>0.05). However, after six and 9 weeks, significant healing was observed in favour of the HAP coated with PLGA compared to other groups (p
The unique physico-chemical properties of cationic polymers and their ability to be easily modified make them attractive for many biological applications. As a result there is a vast amount of research focussed on designing novel natural or synthetic cationic polymers with specific biological functionality.
Cationic Polymers in Regenerative Medicine brings together the expertise of leading experts in the field to provide a comprehensive overview of the recent advances in cationic polymer synthesis, modification and the design of biomaterials with different structures for therapeutic applications. Chapters cover recent developments in novel cationic polymer based systems including poly(L-lysine), Poly(N,N-dimethylaminoethyl methacrylate) and cationic triazine dendrimers as well as cationic polymer-coated micro- and nanoparticles and cationic cellulose and chitin nanocrystals. Applications discussed in the book include drug and gene delivery, therapeutics in thrombosis and inflammation as well as gene therapy.
Suitable both for an educational perspective for those new to the field and those already active in the field, the book will appeal to postgraduates and researchers. The broad aspects of the topics covered are suitable for polymer chemists interested in the fundamentals of the materials systems as well as pharmaceutical chemists, bioengineering and medical professionals interested in their applications.
To improve the encapsulation of GO to CL-20, PEI was utilized as a linker bridge to self-assemble into composite CL-20/PEI/GO in this study. The self-assembly was in-depth characterized, including Zeta potential, SEM, DLS, XPS, Raman spectra, HPLC and power-XRD. The possible self-assembled mechanism was also proposed, which indicates the electrostatic interaction introduced by PEI should be responsible for the self-assembly. The performance of a self-assembled composite was characterized by BAM and DSC analysis. And the results indicate the enhanced interaction promotes GO more efficiently encapsulate CL-20 and improve the safety of the energetic material. Therefore, this work demonstrates a novel and facile method to solve the deprived interaction and corresponding weak desensitization between desensitizer and energetic material.
Vascularisation is important in nerve tissue engineering to provide blood supply and nutrients for long-term survival of implanted cells. Furthermore, blood vessels in regenerating nerves have been shown to serve as tracks for Schwann cells to migrate along and thus form Bands of Büngner which promote axonal regeneration. In this study, we have developed tissue-engineered constructs containing aligned endothelial cells, or co-cultures of both endothelial cells and Schwann cells to test whether these structures could promote regeneration across peripheral nerve gaps. Type I rat tail collagen gels containing HUVECs (Human Umbilical Vein Endothelial Cells, 4 × 10^6 cells/ml) were cast in perforated tethering silicone conduits to facilitate cellular self-alignment and tube formation for 4 days of culture. For co-culture constructs, optimal tube formation and cellular alignment was achieved with a ratio of 4:0.5 × 10^6 cells/ml (HUVECs:Schwann cells). An in vivo test of the engineered constructs to bridge a 10 mm gap in rat sciatic nerves for 4 weeks revealed that constructs containing only HUVECs significantly promoted axonal regeneration and vascularisation across the gap, as compared to conventional aligned Schwann cell constructs and those containing co-cultured HUVECs and Schwann cells. Our results suggest that tissue-engineered constructs containing aligned endothelial cells within collagen matrix could be good candidates to treat peripheral nerve injury.
Vascularisation is important in nerve tissue engineering to provide blood supply and nutrients for long-term survival of implanted cells. Furthermore, blood vessels in regenerating nerves have been shown to serve as tracks for Schwann cells to migrate along and thus form Bands of Büngner which promote axonal regeneration. In this study, we have developed tissue-engineered constructs containing aligned endothelial cells, or co-cultures of both endothelial cells and Schwann cells to test whether these structures could promote regeneration across peripheral nerve gaps. Type I rat tail collagen gels containing HUVECs (Human Umbilical Vein Endothelial Cells, 4×10⁶ cells/ml) were cast in perforated tethering silicone tubes to facilitate cellular self-alignment and tube formation for 4 days of culture. For co-culture constructs, optimal tube formation and cellular alignment was achieved with a ratio of 4:0.5×10⁶ cells/ml (HUVECs:Schwann cells). An in vivo test of the engineered constructs to bridge a 10 mm gap in rat sciatic nerves for 4 weeks revealed that constructs containing only HUVECs significantly promoted axonal regeneration and vascularisation across the gap, as compared to conventional aligned Schwann cell constructs and those containing co-cultured HUVECs and Schwann cells. Our results suggest that tissue-engineered constructs containing aligned endothelial cells within collagen matrix could be good candidates to treat peripheral nerve injury.
Statement of Significance
Nerve tissue engineering provides a potential way to overcome the limitations associated with current clinical grafting techniques for the repair of severe peripheral nerve injuries. However, the therapeutic cells within engineered nerve tissue require effective vascularisation in order to survive. This work therefore aimed to develop engineered nerve constructs containing aligned tube-like structures made from endothelial cells. Not only did this provide a method to improve vascularisation, it demonstrated for the first time that aligned endothelial cells can outperform Schwann cells in promoting nerve regeneration in the rat sciatic nerve model. This has introduced the concept of developing pre-vascularised engineered nerve tissues, and indicated the potential usefulness of endothelial cell structures in tissue engineering for peripheral nerve repair.
Novel biomaterials can be used to provide a better environment for cross talk between vessel forming endothelial cells and wound healing instructor stem cells for tissue regeneration. This study seeks to investigate if a collagen scaffold containing a proangiogenic gene encoding for the chemokine stromal-derived factor-1 alpha (SDF-1α GAS) could be used to enhance functional responses in a coculture of human umbilical vein endothelial cells (HUVECs) and human adipose-derived stem/stromal cells (ADSCs). Functional responses were determined by (1) monitoring the amount of junctional adhesion molecule VE-cadherin released during 14 days culture, (2) expression of provasculogenic genes on the 14th day, and (3) the bioactivity of secreted factors on neurogenic human Schwann cells. When we compared our SDF-1α GAS with a gene-free scaffold, the results showed positive proangiogenic determination characterized by a transient yet controlled release of the VE-cadherin. On the 14th day, the coculture on the SDF-1α GAS showed enhanced maturation than its gene-free equivalent through the elevation of provasculogenic genes (SDF-1α—7.4-fold, CXCR4—1.5-fold, eNOS—1.5-fold). Furthermore, we also found that the coculture on SDF-1α GAS secretes bioactive factors that significantly (p < 0.01) enhanced human Schwann cells’ clustering to develop toward Bünger band-like structures. Conclusively, this study reports that SDF-1α GAS could be used to produce a bioactive vascularized construct through the enhancement of the cooperative effects between endothelial cells and ADSCs.
Vascularization is still one of the major challenges in tissue engineering. In the context of tissue regeneration, the formation of capillary-like structures is often triggered by the addition of growth...
Enhancing angiogenesis is the prime target of current biomaterial‐based wound healing strategies. However, these approaches largely overlook the angiogenic role of the cells of the nervous system. Therefore, we explored the role of a collagen‐chondroitin sulfate scaffold functionalized with a proangiogenic gene stromal‐derived factor‐1α (SDF‐1α)—an SDF‐1α gene‐activated scaffold on the functional regulation of human Schwann cells (SCs). A preliminary 2D study was conducted by delivering plasmids encoding for the SDF‐1α gene into a monolayer of SCs using polyethyleneimine‐based nanoparticles. The delivery of the SDF‐1α gene into the SCs enhanced the production of proangiogenic vascular endothelial growth factor (VEGF). Subsequently, we investigated the impact of SDF‐1α gene‐activated scaffold (3D) on the SCs for 2 weeks, using a gene‐free scaffold as control. The transfection of the SCs within the gene‐activated scaffold resulted in transient overexpression of SDF‐1α transcripts and triggered the production of bioactive VEGF that enhanced endothelial angiogenesis. The overexpression of SDF‐1α also caused transient activation of the transcription factor c‐Jun and supported the differentiation of SCs towards a repair phenotype. This was characterized by elevated expression of neurotrophin receptor p75NGFR. During this developmental stage, the SCs also performed an extensive remodelling of the basement matrix (fibronectin, collagen IV, and laminin) to enrich their environment with the pro‐neurogenic matrix protein laminin, revealing an enhanced pro‐neurogenic behavior. Together, this study shows that SDF‐1α gene‐activated scaffold is a highly bioinstructive scaffold capable of enhancing proangiogenic regenerative response in human SCs for improved wound healing.
Although most bone fractures typically heal without complications, a small proportion of patients (≤10%) experience delayed healing or potential progression to non-union. Interleukin-1 (IL-1β) plays a crucial role in fracture healing as an early driver of inflammation. However, the effects of IL-1β can impede the healing process if they persist long after the establishment of a fracture hematoma, making it a promising target for novel therapies. Accordingly, the overall objective of this study was to develop a novel gene-based therapy that mitigates the negative effects of IL-1β-driven inflammation while providing a structural template for new bone formation. A collagen-hydroxyapatite scaffold (CHA) was used as a platform for the delivery of nanoparticles composed of pDNA, encoding for IL-1 receptor antagonist (IL-1Ra), complexed to the robust non-viral gene delivery vector, polyethyleneimine (PEI). Utilizing pDNA encoding for Gaussia luciferase and GFP as reporter genes, we found that PEI-pDNA nanoparticles induced a transient gene expression profile in rat bone marrow-derived mesenchymal stromal cells (BM-MSCs), with a transfection efficiency of 14.8 ± 1.8% in 2D. BM-MSC viability was significantly affected by PEI-pDNA nanoparticles as evaluated using CellTiter Blue; however, after 10 days in culture this effect was negligible. Transfection with PEI-pIL-1Ra nanoparticles led to functional IL-1Ra production, capable of antagonizing IL-1β-induced expression of secreted embryonic alkaline phosphatase from HEK-Blue-IL-1β reporter cells. Sustained treatment with IL-1β (0.1, 1, and 10 ng/ml) had a dose-dependent negative effect on BM-MSC osteogenesis, both in terms of gene expression (Alpl and Ibsp) and calcium deposition. BM-MSCs transfected with PEI-IL-1Ra nanoparticles were found to be capable of overcoming the inhibitory effects of sustained IL-1β (1 ng/ml) treatments on in vitro osteogenesis. Ultimately, IL-1Ra gene-activated CHA scaffolds supported mineralization of BM-MSCs under chronic inflammatory conditions in vitro, demonstrating potential for future therapeutic applications in vivo.
Viral carrier transport efficiency of gene delivery is high, depending on the type of vector. However, viral delivery poses significant safety concerns such as inefficient/unpredictable reprogramming outcomes, genomic integration, as well as unwarranted immune responses and toxicity. Thus, non-viral gene delivery methods are more feasible for translation as these allow safer delivery of genes and can modulate gene expression transiently both in vivo, ex vivo, and in vitro. Based on current studies, the efficiency of these technologies appears to be more limited, but they are appealing for clinical translation. This review presents a summary of recent advancements in orthopedics, where primarily bone and joints from the musculoskeletal apparatus were targeted. In connective tissues, which are known to have a poor healing capacity, and have a relatively low cell-density, i.e., articular cartilage, bone, and the intervertebral disk (IVD) several approaches have recently been undertaken. We provide a brief overview of the existing technologies, using nano-spheres/engineered vesicles, lipofection, and in vivo electroporation. Here, delivery for microRNA (miRNA), and silencing RNA (siRNA) and DNA plasmids will be discussed. Recent studies will be summarized that aimed to improve regeneration of these tissues, involving the delivery of bone morphogenic proteins (BMPs), such as BMP2 for improvement of bone healing. For articular cartilage/osteochondral junction, non-viral methods concentrate on targeted delivery to chondrocytes or MSCs for tissue engineering-based approaches. For the IVD, growth factors such as GDF5 or GDF6 or developmental transcription factors such as Brachyury or FOXF1 seem to be of high clinical interest. However, the most efficient method of gene transfer is still elusive, as several preclinical studies have reported many different non-viral methods and clinical translation of these techniques still needs to be validated. Here we discuss the non-viral methods applied for bone and joint and propose methods that can be promising in clinical use.
In orthopedics as well as in dentistry, there is an increasing need for novel biomaterials and clinical strategies to achieve predictable bone regeneration. These novel molecular strategies have the potential to eliminate the limitations of currently available approaches. Specifically, they have the potential to reduce or eliminate the need to harvest autogenous bone, and the overall complexity of the clinical procedures. In this review, emerging tissue engineering strategies that have been or are being currently developed based on current understanding of bone biology, development and wound healing will be discussed. In particular, protein/peptide based approaches, DNA/RNA therapeutics, cell therapy and use of exosomes will be briefly covered. The review ends with a summary on the current status of these approaches, their clinical translational potentials and challenges.
Demineralized bone matrix (DBM) scaffold has good biocompatibility, low antigenicity, natural porous structure and no cytotoxicity and is an appropriate material for bone regeneration. However, osteoinductive growth factors are often removed during preparation, which will destroy the osteoinductive capacity of DBM scaffold. Biomaterial combined with gene therapy is a promising approach to effectively avoid this adverse side effect. This study developed a human bone morphogenetic protein 2 (hBMP2) gene-activated DBM scaffold to enhance the osteoinductive of DBM and improve bone repair. Bone marrow mesenchymal stem cells (MSCs) derived microvesicles (MVs) was obtained and polyethyleneimine (PEI) and hBMP2 plasmids (phBMP2) were sequentially coated on MVs by layer-by-layer (LBL) self-assembly to form a MVs-PEI/phBMP2 non-viral gene vector. Finally, gene-activated scaffold (DBM/MVs-PEI/phBMP2) was prepared by loading MVs-PEI/phBMP2 onto DBM scaffold. The experimental results showed that the MVs-PEI/phBMP2 exhibited higher transfection efficiency and lower cytotoxicity to MSCs when MVs/PEI weight ratio=5, and could enhance the osteogenic differentiation of MSCs in vitro. Rat subcutaneous implantation showed that DBM/MVs-PEI/phBMP2 scaffold could efficiently enhance the deposition of collagen fibers, positive osteocalcin, osteopntin and CD34 of endogenous. Rabbit femoral condylar defect experiments proved that DBM/MVs-PEI/phBMP2 scaffold could significantly promote bone repair. This study presented a novel, highly efficient and low cytotoxicity gene delivery vector based on MVs. The gene-activated DBM scaffold based on MVs not only could promote bone formation but also could promote angiogenesis, implying that this kind of gene-activated scaffold can be a promising bone substitute material.
Current treatments for articular cartilage defects relieve symptoms but often only delay cartilage degeneration. Mesenchymal stem cells (MSCs) have shown chondrogenic potential but tend to undergo endochondral ossification when implanted in vivo. Harnessing factors governing joint development to functionalize biomaterial scaffolds, termed developmental engineering, might allow to prime host MSCs to regenerate mature articular cartilage in situ without requiring cell isolation or ex vivo expansion. Therefore, the aim of this study is to develop a gene‐activated scaffold capable of delivering developmental cues to host MSCs, thus priming MSCs for articular cartilage differentiation and inhibiting endochondral ossification. It is shown that delivery of the SOX‐Trio induced MSCs to over‐express COL2A1 and ACAN and deposit a sulfated and collagen type II rich extracellular matrix while hypertrophic gene expression and collagen type X deposition is inhibited. When cell‐free SOX‐Trio‐activated scaffolds are implanted ectopically in vivo, they induced spontaneous chondrogenesis without evidence of hypertrophy. MSCs pre‐cultured on SOX‐Trio‐activated scaffolds prior to implantation differentiate into phenotypically stable chondrocytes as evidenced by a lack of collagen X expression or vascular invasion. This SOX‐trio‐activated scaffold represents a potent, single treatment, developmentally inspired strategy to prime MSCs in situ for articular cartilage defect repair.
Bone is a hard yet dynamic tissue with remarkable healing capacities. Research to date has greatly advanced the understanding of how bone heals and has led to marked success in the treatment of bone injuries. Nevertheless, the effective treatment of nonunions and large bone defects continues to present a challenge for orthopedic surgeons. Biomaterials provide researchers with a powerful instrument to potentially guide effective bone tissue regeneration in challenging healing environments. However, the most appropriate biomaterial for bone tissue engineering continues to be an area of intense debate. Indeed, the mechanical properties of real bone can be reproduced in vitro but the development of a functional bone substitute goes beyond the sole mechanical properties. The faithful reproduction of bone as a functional organ requires the combination of different cell types and temporal regulation of the molecular signaling involved during the different stages of bone formation and regeneration. This is not at all a trivial task. Herein, critical aspects of bone healing/regeneration including mechanical loading, inflammation, vascularization, and innervation are described. The success and the challenges behind the development of biomaterials, and the technologies used to functionalize them, in order to support the underlying cellular mechanisms are also highlighted. Regeneration of large bone defects is a major clinical challenge. While biomaterials hold great promise and can potentially help many patients, translating this promise into the clinic is still proving problematic. Herein, the fundamental biological issues are highlighted together with examples of biomaterial manipulation of the underlying biology.
Corneal blindness is one of the most common causes of vision loss worldwide, affecting millions of people. To treat these patients, researchers have been examining different approaches to engineer corneal scaffolds suitable for transplantation. Scaffolds have been developed to replace part or all of the cornea depending on the patient requirements. Both acellular and cell‐seeded scaffolds have been tested in animal models. Materials that have been under investigation for manufacturing scaffolds include collagen, silk fibroin, amniotic membrane, decellularized cornea, fibrin, chitosan, gelatin, agarose, alginate, and hyaluronic acid in addition to several synthetic polymers. Different combinations of materials, fiber crosslinking techniques, and incorporation of bioactive molecules have also been examined. Factors such as the physical properties, cytocompatibility, degradation behavior, and optical characteristics have to be considered when selecting a suitable scaffold material. Recent advancements in materials fabrication techniques such as bioprinting, electrospinning, and different collagen alignment techniques, allow scaffolds to be generated that more accurately mimic the structure of the corneal stroma. A number of scaffolds have commenced clinical trials to determine their suitability for corneal regeneration. This review examines different design parameters, biomaterials, and fabrication processes used to develop scaffolds for corneal tissue engineering. Different state‐of‐the‐art approaches for generating scaffolds to replace the different layers of the cornea are discussed, and the advantages and limitations of different biomaterials and manufacturing techniques are highlighted.
Background:
Calcium ions (Ca2+ ) influence natural bone healing, and calcium is frequently used in bone tissue engineering scaffolds and cements. Scaffolds can also incorporate gene delivery systems to further promote osteoblast differentiation. Thus, our goal was to identify if Ca2+ concentration affects the transfection of bone marrow stromal cells since these cells play a major role in bone healing and can infiltrate gene-activated scaffolds designed to promote bone growth.
Methods:
Bone marrow-derived mesenchymal stem cells (BMSCs) and were cultured in media with Ca2+ concentrations ranging from 0 mM to 20 mM and transfected with polyethyleneimine-plasmid DNA (PEI-pDNA) complexes. Cell viability and transfection efficiency were determined using MTS assays and flow cytometry, respectively. PEI-pDNA complex localization in BMSCs was assessed using fluorescence microscopy. To determine BMSC differentiation, mRNA for osteocalcin and CBFA1 was quantified using real time-PCR. Calcium deposition was qualitatively assessed after 3 and 14 days using Alizarin Red staining.
Results:
Our results indicate that Ca2+ levels between 8-12 mM positively impacted transfection of BMSCs with PEI-pDNA complexes in terms of cell viability and transfection efficiency. A Ca2+ concentration of 10 mM also increased the expression of an osteogenic gene, osteocalcin, when the cells were transfected with pDNA encoding BMP-2.
Conclusion:
Ca2+ at a 10 mM concentration can significantly reduce toxicity and enhance transfection efficiency when combined with PEI-pDNA complexes, and this combination can be specifically applied to further enhance the differentiation of BMSCs by using the combination of PEI-pBMP-2 and 10 mM Ca2+ as compared to PEI-pBMP-2 alone.
Gene-activated matrices (GAM), a combination of scaffolds and plasmid delivery systems, are widely used in regenerative medicine, but few compared plasmid BMP-2 in hemostatic gelatin sponge. We compared the osteogenic differentiation ability of bone morphogenetic protein-2 (BMP-2)–transfected preosteoblasts cultured into a hemostatic gelatin sponge with that of non-transfected cells cultured into BMP-2–activated hemostatic gelatin sponge. GAM was prepared from hemostatic gelatin sponge embedded with complexes (plasmid BMP-2 mixed with TransIT-2020® transfection reagent). The standard transfection method using mouse preosteoblasts (MC3T3-E1) mixed with the complex loaded onto the scaffold was compared with the GAM group. Non-transfected cells were loaded onto the scaffold as the control group. Transmission and scanning electron microscopy results revealed that TransIT-2020®/plasmid BMP-2 complexes formed homogeneous non-aggregated structures, which were round in shape and 200–250 nm in size. Alkaline phosphatase activity, the marker of bone differentiation, was significantly higher in GAM group compared to that in standard and control groups on differentiation days 14, 21, and 28. The concentration of released BMP-2 was significantly higher in GAM group than that in standard group on days 3 and 7, consistent with confocal microscopy results.
3D printing is a versatile technique widely applied in tissue engineering due to its ability to manufacture large quantities of scaffolds or constructs with various desired architectures. In this study, we demonstrated that poly (lactic acid) (PLA) scaffolds fabricated via fused deposition not only retained the original interconnected microporous architectures, the scaffolds also exhibited lower lactic acid dissolution as compared to the freeze-PLA scaffold. The 3D-printed scaffolds were then grafted with human bone morphogenetic protein-2 (BMP-2) via the actions of polydopamine (PDA) coatings. The loading and release rate of BMP-2 were monitored for a period of 35 days. Cellular behaviors and osteogenic activities of co-cultured human mesenchymal stem cells (hMSCs) were assessed to determine for efficacies of scaffolds. In addition, we demonstrated that our fabricated scaffolds were homogenously coated with PDA and well grafted with BMP-2 (219.1 ± 20.4 ng) when treated with 250 ng/mL of BMP-2 and 741.4 ± 127.3 ng when treated with 1000 ng/mL of BMP-2. This grafting enables BMP-2 to be released in a sustained profile. From the osteogenic assay, it was shown that the ALP activity and osteocalcin of hMSCs cultured on BMP-2/PDA/PLA were significantly higher when compared with PLA and PDA/PLA scaffolds. The methodology of PDA coating employed in this study can be used as a simple model to immobilize multiple growth factors onto different 3D-printed scaffold substrates. Therefore, there is potential for generation of scaffolds with different unique modifications with different capabilities in regulating physiochemical and biological properties for future applications in bone tissue engineering.
The angiogenesis of an implanted construct is among the most important issues in tissue engineering. In this study, spermine was used to modify Bombyx mori silk fibroin (BSF) to synthesize cationized BSF (CBSF). CBSF and BSF were coated on the surface of polyethyleneimine (PEI)/vascular endothelial growth factor 165/angiopoietin-1 coexpression plasmid DNA (pDNA) complexes to form CBSF/BSF/PEI/pDNA quaternary complexes. BSF scaffolds loaded with carrier/pDNA complexes were prepared as dermal regeneration scaffolds by freeze-drying. In one set of experiments, scaffolds were covered on chick embryo chorioallantoic membrane (CAM) to investigate the influence of carrier/pDNA complexes on angiogenesis; in another set of experiments, scaffolds were implanted into dorsal full-thickness wounds in Sprague-Dawley rats to evaluate the effect of carrier/pDNA complex-loaded BSF scaffolds on neovascularization and dermal tissue regeneration. After modification with spermine, the surface zeta potential value of BSF rose to +11 mV from an initial value of -9 mV, and the isoelectric point of BSF increased from 4.20 to 9.04. The in vitro transfection of human umbilical vein endothelial cells (EA.hy926) with quaternary complexes revealed that the CBSF/BSF/PEI/pDNA complexes clearly exhibited lower cytotoxicity and higher transfection efficiency than the PEI/pDNA complexes. The CAM assay showed a more abundant branching pattern of blood vessels in BSF scaffolds loaded with CBSF/BSF/PEI/pDNA complexes than in BSF scaffolds without complexes or loaded with PEI/pDNA complexes. The in vivo experimental results demonstrated that the incorporation of CBSF/BSF/PEI/pDNA complexes could effectively enhance angiogenesis in the implanted BSF scaffolds, thereby promoting the regeneration of dermal tissue, providing a new scaffold for the regeneration of dermal tissue and other tissues containing blood vessels.
Several non-permanent polycations possessing substantial buffering capacity below physiological pH, such as lipopolyamines and polyethylenimines, are efficient transfection agents per se, i.e. without the addition of lysosomotropic bases, or cell targeting, or membrane disruption
agents. These vectors have been shown to deliver genes as well as oligonucleotides both in vitro and in vivo. Our hypothesis is that their efficiency relies on extensive endosome swelling and rupture that provides an escape mechanism for the polycation/DNA particles.
MSCs are nonhematopoietic stromal cells that are capable of differentiating into, and contribute to the regeneration of, mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, and adipose. MSCs are rare in bone marrow, representing ∼1 in 10,000 nucleated cells. Although not immortal, they have the ability to expand manyfold in culture while retaining their growth and multilineage potential. MSCs are identified by the expression of many molecules including CD105 (SH2) and CD73 (SH3/4) and are negative for the hematopoietic markers CD34, CD45, and CD14. The properties of MSCs make these cells potentially ideal candidates for tissue engineering. It has been shown that MSCs, when transplanted systemically, are able to migrate to sites of injury in animals, suggesting that MSCs possess migratory capacity. However, the mechanisms underlying the migration of these cells remain unclear. Chemokine receptors and their ligands and adhesion molecules play an important role in tissue-specific homing of leukocytes and have also been implicated in trafficking of hematopoietic precursors into and through tissue. Several studies have reported the functional expression of various chemokine receptors and adhesion molecules on human MSCs. Harnessing the migratory potential of MSCs by modulating their chemokine-chemokine receptor interactions may be a powerful way to increase their ability to correct inherited disorders of mesenchymal tissues or facilitate tissue repair in vivo. The current review describes what is known about MSCs and their capacity to home to tissues together with the associated molecular mechanisms involving chemokine receptors and adhesion molecules.
Disclosure of potential conflicts of interest is found at the end of this article.
In the literature there are conflicting reports on the optimal scaffold mean pore size required for successful bone tissue engineering. This study set out to investigate the effect of mean pore size, in a series of collagen–glycosaminoglycan (CG) scaffolds with mean pore sizes ranging from 85 μm to 325 μm, on osteoblast adhesion and early stage proliferation up to 7 days post-seeding. The results show that cell number was highest in scaffolds with the largest pore size of 325 μm. However, an early additional peak in cell number was also seen in scaffolds with a mean pore size of 120 μm at time points up to 48 h post-seeding. This is consistent with previous studies from our laboratory which suggest that scaffold specific surface area plays an important role on initial cell adhesion. This early peak disappears following cell proliferation indicating that while specific surface area may be important for initial cell adhesion, improved cell migration provided by scaffolds with pores above 300 μm overcomes this effect. An added advantage of the larger pores is a reduction in cell aggregations that develop along the edges of the scaffolds. Ultimately scaffolds with a mean pore size of 325 μm were deemed optimal for bone tissue engineering.
Crosslinking and the resultant changes in mechanical properties have been shown to influence cellular activity within collagen biomaterials. With this in mind, we sought to determine the effects of crosslinking on both the compressive modulus of collagen-glycosaminoglycan scaffolds and the activity of osteoblasts seeded within them. Dehydrothermal, 1-ethyl-3-3-dimethyl aminopropyl carbodiimide and glutaraldehyde crosslinking treatments were first investigated for their effect on the compressive modulus of the scaffolds. After this, the most promising treatments were used to study the effects of crosslinking on cellular attachment, proliferation, and infiltration. Our experiments have demonstrated that a wide range of scaffold compressive moduli can be attained by varying the parameters of the crosslinking treatments. 1-Ethyl-3-3-dimethyl aminopropyl carbodiimide and glutaraldehyde treatments produced the stiffest scaffolds (fourfold increase when compared to dehydrothermal crosslinking). When cells were seeded onto the scaffolds, the stiffest scaffolds also showed increased cell number and enhanced cellular distribution when compared to the other groups. Taken together, these results indicate that crosslinking can be used to produce collagen-glycosaminoglycan scaffolds with a range of compressive moduli, and that increased stiffness enhances cellular activity within the scaffolds.
There is an enduring and unmet need for a bioactive, load-bearing tissue-engineering scaffold, which is biocompatible, biodegradable and capable of facilitating and promoting osteogenesis when implanted in vivo. This study set out to develop a biomimetic scaffold by incorporating osteoinductive hydroxyapatite (HA) particles into a highly porous and extremely biocompatible collagen-based scaffold developed within our laboratory over the last number of years to improve osteogenic performance. Specifically we investigated how the addition of discrete quantities of HA affected scaffold porosity, interconnectivity, mechanical properties, in vitro mineralisation and in vivo bone healing potential. The results show that the addition of HA up to a 200 weight percentage (wt%) relative to collagen content led to significantly increased scaffold stiffness and pore interconnectivity (approximately 10 fold) while achieving a scaffold porosity of 99%. In addition, this biomimetic collagen-HA scaffold exhibited significantly improved bioactivity, in vitro mineralisation after 28 days in culture, and in vivo healing of a critical-sized bone defect. These findings demonstrate the regenerative potential of these biodegradable scaffolds as viable bone graft substitute materials, comprised only of bone’s natural constituent materials, and capable of promoting osteogenesis in vitro and in vivo repair of critical-sized bone defects.
Tissue engineering using cell-seeded biodegradable scaffolds offers a new bone regenerative approach that might circumvent many of the limitations of current therapeutic modalities. The aim of this experiment was to study the early healing events around mesenchymal stem cell-seeded collagen-glycosaminoglycan scaffolds.
The 5-mm critical size defects were created in the calvarial bones of 41 Wistar rats. The defects were either left empty to serve as controls (n = 11), filled with cell-free scaffolds (n = 12), cell-seeded scaffolds that were maintained in standard culture medium (n = 9), or cell-seeded scaffolds that were maintained in osteoinductive factor-supplemented medium (n = 9). The animals were sacrificed at 7 days after surgery, and specimens were prepared for histological analysis. Early healing events such as host cell penetration, blood vessel in-growth, and scaffold integration were observed. The degree of inflammatory cell infiltrate was assessed.
While defects in the control group healed with a thin fibrous tissue, the collagen-glycosaminoglycan scaffold in the test groups preserved the three-dimensional form of the defects. After 7 days in vivo, the scaffold maintained its integrity and appeared populated with host cells. The cell-seeded scaffold induced more inflammatory response compared to the cell-free scaffolds. New blood vessels and areas of early bone formation were also evident in the cell-seeded scaffolds.
In conclusion, the findings show that mesenchymal stem cell-seeded collagen-glycosaminoglycan scaffolds have good tissue tolerance and exhibit an osteoinductive effect as indicated by early stage healing.
Bone regeneration requires scaffolds that possess suitable mechanical and biological properties. This study sought to develop a novel collagen-nHA biocomposite scaffold via two new methods. Firstly a stable nHA suspension was produced and added to a collagen slurry (suspension method), and secondly, porous collagen scaffolds were immersed in nHA suspension after freeze-drying (immersion method). Significantly stronger constructs were produced using both methods compared to collagen only scaffolds, with a high porosity maintained (>98.9%). It was found that Coll-nHA composite scaffolds produced by the suspension method were up to 18 times stiffer than the collagen control (5.50 +/- 1.70 kPa vs. 0.30 +/- 0.09 kPa). The suspension method was also more reproducible, and the quantity of nHA incorporated could be varied with greater ease than with the immersion technique. In addition, Coll-nHA composites display excellent biological activity, demonstrating their potential as bone graft substitutes in orthopaedic regenerative medicine.
The pore structure of three-dimensional scaffolds used in tissue engineering has been shown to significantly influence cellular activity. As the optimal pore size is dependant on the specifics of the tissue engineering application, the ability to alter the pore size over a wide range is essential for a particular scaffold to be suitable for multiple applications. With this in mind, the aim of this study was to develop methodologies to produce a range of collagen-glycosaminoglycan (CG) scaffolds with tailored mean pore sizes. The pore size of CG scaffolds is established during the freeze-drying fabrication process. In this study, freezing temperature was varied (−10 degrees C to −70 degrees C) and an annealing step was introduced to the process to determine their effects on pore size. Annealing is an additional step in the freeze-drying cycle that involves raising the temperature of the frozen suspension to increase the rate of ice crystal growth. The results show that the pore size of the scaffolds decreased as the freezing temperature was reduced. Additionally, the introduction of an annealing step during freeze-drying was found to result in a significant increase (40%) in pore size. Taken together, these results demonstrate that the methodologies developed in this study can be used to produce a range of CG scaffolds with mean pore sizes from 85 to 325 microm. This is a substantial improvement on the range of pore sizes that were possible to produce previously (96-150 microm). The methods developed in this study provide a basis for the investigation of the effects of pore size on both in vitro and in vivo performance and for the determination of the optimal pore structure for specific tissue engineering applications.
The commercially available branched polyethyleneimine (PEI) with a molar mass of 25 kD (PEI-25K) is an effective in vitro vector to transfer genes, but its cytotoxicity limits its applications in bio-related research. To solve such an efficiency-versus-cytotoxicity catch-22 problem, the disulfide bond has been previously used to link less toxic short PEI chains (2 kD), but previous literature results are controversial. Recently, we found that it is vitally important to remove both carbon dioxide and water in the linking reaction as well as to control the structure of the resultant chains linked by dithiobis(succinimidyl propionate) (DSP). Under a programmable mixing of PEI and DSP, we can use laser light scattering (LLS) to in-situ monitor the linking reaction kinetics in DMSO in terms of the change of the average molar mass (M(w)). Therefore, we were able to withdraw a series of linked PEI chains with different molar masses from one reaction mixture. Two such linked PEI samples (M(w) approximately 7 kD, PEI-7K-L and approximately 400 kD, PEI-400K-L) were used to illustrate the effect of the sample preparation and the chain structure on the in vitro gene transfection and cytotoxicity. Our results reveal that PEI-7K-L is less cytotoxic and more effective in the gene transfection than both PEI-25K and Lipofectamine 2000 in the in vitro gene transfection. However, PEI-400K-L has no gene transfection efficiency even though it is non-toxic.
Several polycations possessing substantial buffering capacity below physiological pH, such as lipopolyamines and polyamidoamine polymers, are efficient transfection agents per se--i.e., without the addition of cell targeting or membrane-disruption agents. This observation led us to test the cationic polymer polyethylenimine (PEI) for its gene-delivery potential. Indeed, every third atom of PEI is a protonable amino nitrogen atom, which makes the polymeric network an effective "proton sponge" at virtually any pH. Luciferase reporter gene transfer with this polycation into a variety of cell lines and primary cells gave results comparable to, or even better than, lipopolyamines. Cytotoxicity was low and seen only at concentrations well above those required for optimal transfection. Delivery of oligonucleotides into embryonic neurons was followed by using a fluorescent probe. Virtually all neurons showed nuclear labeling, with no toxic effects. The optimal PEI cation/anion balance for in vitro transfection is only slightly on the cationic side, which is advantageous for in vivo delivery. Indeed, intracerebral luciferase gene transfer into newborn mice gave results comparable (for a given amount of DNA) to the in vitro transfection of primary rat brain endothelial cells or chicken embryonic neurons. Together, these properties make PEI a promising vector for gene therapy and an outstanding core for the design of more sophisticated devices. Our hypothesis is that its efficiency relies on extensive lysosome buffering that protects DNA from nuclease degradation, and consequent lysosomal swelling and rupture that provide an escape mechanism for the PEI/DNA particles.
Since the first published examination of poly(ethylenimine) (PEI) as a gene delivery vehicle, there has been a flurry of research aimed at this polycation and its role in gene therapy. Here we will briefly review PEI chemistry and the characterization of PEI/DNA complexes used for gene delivery. Additionally, we will note various PEI transfection considerations and examine findings involving other polycationic gene delivery vehicles used with cellular targeting ligands. The current state of our knowledge regarding the mechanism of PEI/DNA transfection will also be discussed. Finally, we will survey toxicity issues related to PEI transfection.
Non-viral gene vectors are commonly used for gene therapy owing to safety concerns with viral vectors. However, non-viral vectors are plagued by low levels of gene transfection and cellular expression. Current efforts to improve the efficiency of non-viral gene delivery are focused on manipulations of the delivery vector, whereas the influence of the cellular environment in DNA uptake is often ignored. The mechanical properties (for example, rigidity) of the substrate to which a cell adheres have been found to mediate many aspects of cell function including proliferation, migration and differentiation, and this suggests that the mechanics of the adhesion substrate may regulate a cell's ability to uptake exogeneous signalling molecules. In this report, we present a critical role for the rigidity of the cell adhesion substrate on the level of gene transfer and expression. The mechanism relates to material control over cell proliferation, and was investigated using a fluorescent resonance energy transfer (FRET) technique. This study provides a new material-based control point for non-viral gene therapy.
Nonviral delivery vectors are attractive for gene therapy approaches in tissue engineering, but suffer from low transfection efficiency and short-term gene expression. We hypothesized that the sustained delivery of poly(ethylenimine) (PEI)-condensed DNA from three-dimensional biodegradable scaffolds that encourage cell infiltration could greatly enhance gene expression. To test this hypothesis, a PEI-condensed plasmid encoding beta-galactosidase was incorporated into porous poly(lactide-co-glycolide) (PLG) scaffolds, using a gas foaming process. Four conditions were examined: condensed DNA and uncondensed DNA encapsulated into PLG scaffolds, blank scaffolds, and bolus delivery of condensed DNA in combination with implantation of PLG scaffolds. Implantation of scaffolds incorporating condensed beta-galactosidase plasmid into the subcutaneous tissue of rats resulted in a high level of gene expression for the entire 15-week duration of the experiment, as exemplified by extensive positive staining for beta-galactosidase gene expression observed on the exterior surface and throughout the cross-sections of the explanted scaffolds. No positive staining could be observed for the control conditions either on the exterior surface or in the cross-section at 8- and 15-week time points. In addition, a high percentage (55-60%) of cells within scaffolds incorporating condensed DNA at 15 weeks demonstrated expression of the DNA, confirming the sustained uptake and expression of the encapsulated plasmid DNA. Quantitative analysis of beta-galactosidase gene expression revealed that expression levels in scaffolds incorporating condensed DNA were one order of magnitude higher than those of other conditions at the 2- week time point and nearly two orders of magnitude higher than those of the control conditions at the 8- and 15-week time points. This study demonstrated that the sustained delivery of PEI-condensed plasmid DNA from PLG scaffolds led to an in vivo long-term and high level of gene expression, and this system may find application in areas such as bone tissue engineering.
Non-viral gene delivery is emerging as a realistic alternative to the use of viral vectors with the potential to have a significant impact on clinical therapies. The documented dangers of using the efficient recombinant viruses as carriers have led many to explore the possible advantages of using polymer-based non-viral vectors. To date there is no gene delivery vehicle that contains all the desirable characteristics but they do exist individually in a variety of non-viral carriers, e.g. degradable, low toxicity, cell specific, relatively efficient and capable of delivering multiple genes. Polymers may not be as effective as the viral vehicles; however, the continued focus and growth of knowledge in this field has already resulted in improved delivery. Over the past 10 years, significant progress has been made through the design of specific polymers for this application. Another interesting development in this field is the influx of research on combination approaches to non-viral gene delivery. Scaffolds made of both natural and synthetic materials are being utilized to aid in sustained delivery of the polymer vectors. While the non-viral gene therapy field is currently receiving a large degree of dedicated research there is now the realistic potential of a clinically relevant output. This review presents a summary of combinatorial delivery systems of non-viral polyplexes delivered via tissue engineered scaffolds. For polyplexes to move into the clinical arena, it is important that we uncover and understand the technical hurdles that need to be overcome so that the efficacy of this promising technology can be established.
The cellular structure of collagen-glycosaminoglycan (CG) scaffolds used in tissue engineering must be designed to meet a number of constraints with respect to biocompatibility, degradability, pore size, pore structure, and specific surface area. The conventional freeze-drying process for fabricating CG scaffolds creates variable cooling rates throughout the scaffold during freezing, producing a heterogeneous matrix pore structure with a large variation in average pore diameter at different locations throughout the scaffold. In this study, the scaffold synthesis process was modified to produce more homogeneous freezing by controlling of the rate of freezing during fabrication and obtaining more uniform contact between the pan containing the CG suspension and the freezing shelf through the use of smaller, less warped pans. The modified fabrication technique has allowed production of CG scaffolds with a more homogeneous structure characterized by less variation in mean pore size throughout the scaffold (mean: 95.9 μm, CV: 0.128) compared to the original scaffold (mean: 132.4 μm, CV: 0.185). The pores produced using the new technique appear to be more equiaxed, compared with those in scaffolds produced using the original technique.
The synthesis of nanophase hydroxyapatite (nHA) is of importance in the field of biomaterials and bone tissue engineering. The bioactive and osteoconductive properties of nHA are of much benefit to a wide range of biomedical applications such as producing bone tissue engineered constructs, coating medical implants, or as a carrier for plasmid DNA in gene delivery. This study aimed to develop a novel low-temperature dispersant-aided precipitation reaction to produce nHA particles (<100 nm), which are regarded as being preferable to micron-sized agglomerates of nHA. The variables investigated and optimized include the reaction pH, the rate of reactant mixing, use of sonication, order of addition, and concentration of the primary reactants, in addition, the effect of using poly(vinyl alcohol) (PVA) surfactant and Darvan 821A® dispersing agent during the reaction was also examined. It was found that by fine-tuning the synthesis parameters and incorporating the dispersing agent, monodisperse, phase-pure nano-sized particles under 100 nm were attained, suitable for clinical applications in bone regeneration.
One of the key challenges in tissue engineering is to understand the host response to scaffolds and engineered constructs. We present a study in which two collagen-based scaffolds developed for bone repair: a collagen-glycosaminoglycan (CG) and biomimetic collagen-calcium phosphate (CCP) scaffold, are evaluated in rat cranial defects, both cell-free and when cultured with MSCs prior to implantation. The results demonstrate that both cell-free scaffolds showed excellent healing relative to the empty defect controls and somewhat surprisingly, to the tissue engineered (MSC-seeded) constructs. Immunological analysis of the healing response showed higher M1 macrophage activity in the cell-seeded scaffolds. However, when the M2 macrophage response was analysed, both groups (MSC-seeded and non-seeded scaffolds) showed significant activity of these cells which are associated with an immunomodulatory and tissue remodelling response. Interestingly, the location of this response was confined to the construct periphery, where a capsule had formed, in the MSC-seeded groups as opposed to areas of new bone formation in the non-seeded groups. This suggests that matrix deposited by MSCs during in vitro culture may adversely affect healing by acting as a barrier to macrophage-led remodelling when implanted in vivo. This study thus improves our understanding of host response in bone tissue engineering.
Transplantation of mesenchymal stem cells (MSCs) derived from adult bone marrow has been proposed as a potential therapeutic approach for post-infarction left ventricular (LV) dysfunction. However, age-related functional decline of stem cells has restricted their clinical benefits after transplantation into the infarcted myocardium. The limitations imposed on patient cells could be addressed by genetic modification of stem cells. This study was designed to improve our understanding of genetic modification of human bone marrow derived mesenchymal stem cells (hMSCs) by polyethylenimine (PEI, branched with Mw 25 kD), one of non-viral vectors that show promise in stem cell genetic modification, in the context of cardiac regeneration for patients. We optimized the PEI-mediated reporter gene transfection into hMSCs, evaluated whether transfection efficiency is associated with gender or age of the cell donors, analysed the influence of cell cycle on transfection and investigated the transfer of therapeutic vascular endothelial growth factor gene (VEGF). hMSCs were isolated from patients with cardiovascular disease aged from 41 to 85 years. Optimization of gene delivery to hMSCs was carried out based on the particle size of the PEI/DNA complexes, N/P ratio of complexes, DNA dosage and cell viability. The highest efficiency with the cell viability near 60% was achieved at N/P ratio 2 and 6.0 μg DNA/cm(2) . The average transfection efficiency for all tested samples, middle-age group (<65 years), old-age group (>65 years), female group and male group was 4.32%, 3.85%, 4.52%, 4.14% and 4.38%, respectively. The transfection efficiency did not show any correlation either with the age or the gender of the donors. Statistically, there were two subpopulations in the donors; and transfection efficiency in each subpopulation was linearly related to the cell percentage in S phase. No significant phenotypic differences were observed between these two subpopulations. Furthermore, PEI-mediated therapeutic gene VEGF transfer could significantly enhance the expression level.
The efficient delivery of nucleic acids into cells is critical for successful gene therapy or gene knockdown. Polyethylenimines (PEIs) are positively charged polymers which complex and deliver DNA for gene transfection or small interfering RNAs (siRNAs) for the induction of RNA interference (RNAi), and mediate their endosomal release. Likewise, various liposomes act as transfection reagents, with some lipids showing increased endocytosis and influencing endosomal escape. This study combines the favourable properties of PEI and lipid systems for DNA and siRNA delivery. Various lipids and lipid combinations, which cover a broad range of physicochemical properties and form optimal liposomes, are assessed. By addition of the liposomes to pre-formed polyplexes, based on the low molecular weight PEI F25-LMW, we establish liposome-PEI complexes (lipopolyplexes) and characterise them in comparison to their 'parent' polyplexes and liposomes regarding size, shape and zeta-potential. Furthermore, while the lipidation of polyplexes generally decreases their toxicity, our studies on DNA transfection and siRNA-mediated knockdown also establish certain lipopolyplexes based on rigid, negatively charged lipids as particularly efficient vehicles for nucleic acid delivery, further improving DNA transfection. The analysis of their mechanism and kinetics of cellular uptake confirms that the biological properties of lipopolyplexes are mainly determined by the liposome shell. We conclude that certain lipopolyplexes show improved biological properties over PEI complexes, thus representing potentially attractive non-viral vectors for gene therapy and RNAi.
Inspired by physiological events that accompany the "wound healing cascade", the concept of developing a tissue either in vitro or in vivo has led to the integration of a wide variety of growth factors (GFs) in tissue engineering strategies in an effort to mimic the natural microenvironments of tissue formation and repair. Localised delivery of exogenous GFs is believed to be therapeutically effective for replication of cellular components involved in tissue development and the healing process, thus making them important factors for tissue regeneration. However, any treatment aiming to mimic the critical aspects of the natural biological process should not be limited to the provision of a single GF, but rather should release multiple therapeutic agents at an optimised ratio, each at a physiological dose, in a specific spatiotemporal pattern. Despite several obstacles, delivery of more than one GF at rates mimicking an in vivo situation has promising potential for the clinical management of severely diseased tissues. This article summarises the concept of and early approaches toward the delivery of dual or multiple GFs, as well as current efforts to develop sophisticated delivery platforms for this ambitious purpose, with an emphasis on the application of biomaterials-based deployment technologies that allow for controlled spatial presentation and release kinetics of key biological cues. Additionally, the use of platelet-rich plasma or gene therapy is addressed as alternative, easy, cost-effective and controllable strategies for the release of high concentrations of multiple endogenous GFs, followed by an update of the current progress and future directions of research utilising release technologies in tissue engineering and regenerative medicine.
Nucleic acids can be complexed with cationic polymer to form DNA nanoparticles (polyplex) which are then immobilized on the surface coated extracellular matrix protein (ECM), the process termed as reverse transfection. ECM-containing proteins provide a surface for cell attachment and sustain the release of polyplexes from their surface, thereby inducing transgene expression for prolonged period of time. Consequently, long-term expression of the desired protein can be achieved with the smaller amount of required DNA, as compared to bolus delivery. First of all, we investigated the different ECM components as a coating material and the range of optimal coating density in different ECM was examined for enhanced transfection to neighboring cells. Reporter genes such as luciferase (luc) and enhanced green fluorescent protein (eGFP) were initially used to quantitate transfection efficiencies from polyplex from the coated ECMs of Collagen type I (Col I), fetal bovine serum protein (FBS), bovine serum albumin (BSA). DNA was complexed with positively charged polyethyleneimine (PEI) at N/P ratio 9. Our initial work exhibited that, in the case of both NIH/3T3 cell line and bone marrow stromal (D1) cell line, Col I facilitated the greatest cell adhesion compared to the other coating proteins and 0.5 microg/cm2 of Col I coating density resulted in highest transfection efficiency. On the other hand, comparison of reverse delivery system with atelocollagen-I have shown that reverse delivery system to yield ten times higher transfection efficiency than atelocollagen-PEI/DNA delivery system and one hundred times higher than atelocollagen-naked plasmid delivery system. Moreover, the amount of DNA used for reverse delivery system was much lower than the other systems. This methodology would be applied to induce cellular differentiation in 3-dimensional scaffold after coating scaffolds with genes inducing the differentiation in the nanoparticle formulation. Our final goal is to search for the optimal conditions for the differentiation of stem cells to specific cell types.
Current strategies to enhance gene transfer have focused on the development of vectors to increase the efficiency of DNA delivery. However, the extracellular matrix and microenvironment have a profound impact on numerous cellular activities including spreading and proliferation; two processes that have been associated with gene transfer efficiency. This study was designed to test the hypothesis that the presence of a biomineralized coating on biodegradable substrates would affect transgene expression following nonviral gene delivery. Thin films were prepared from polymeric microspheres, while biomineralized films were fabricated from microspheres previously soaked in modified simulated body fluid. Mineralized films were significantly more rigid and had widespread mineral coverage compared with nonmineralized substrates. Human mesenchymal stem cells (MSCs) were cultured on biomineralized or nonmineralized films and transfected with plasmid DNA condensed with linear polyethyleneimine (PEI). Compared with cells transfected on nonmineralized films, increases in gene expression were detected in the presence of biomineral at all charge ratios examined. We observed increased uptake of both PEI and DNA by cells on mineralized films. The results of these studies offer an approach to modulate gene delivery and improve the potential benefit of nonviral gene delivery approaches.
It has been established that for orthopedic‐related research, nanomaterials (materials defined as those with constituent dimensions less than 100 nm in at least one direction) have superior properties compared to conventional counterparts. This review summarizes studies that have demonstrated enhanced in vitro and in vivo osteoblast (bone‐forming cells) functions (such as adhesion, proliferation, synthesis of bone‐related proteins, and deposition of calcium‐containing mineral) on nanostructured metals, ceramics, polymers, and composites thereof compared to currently used implants. These results strongly imply that nanomaterials may improve osseointegration, which is crucial for long‐term implant efficacy. This review also focuses on novel drug‐carrying magnetic nanoparticles designed to treat various bone diseases (such as osteoporosis). Although further investigation of the in vivo responses and toxicity of these novel nanomaterials pertinent for orthopedic applications are needed, nanotechnology clearly has already demonstrated the ability to produce better bone implants and therefore should be further investigated. Copyright © 2009 John Wiley & Sons, Inc.
This article is categorized under: Implantable Materials and Surgical Technologies > Nanotechnology in Tissue Repair and Replacement
Collagen/calcium phosphate scaffolds have been used for bone reconstruction due to their inherent similarities to the bone extracellular matrix. Calcium phosphate alone has also been used as a non-viral vector for gene delivery. The aim of this study was to determine the capability of a collagen/calcium phosphate scaffold to deliver naked plasmid DNA and mediate transfection in vivo. The second goal of the study was to deliver a plasmid encoding vascular endothelial growth factor(165) (pVEGF(165)) to promote angiogenesis, and hence bone formation, in a mouse intra-femoral model. The delivery of naked plasmid DNA resulted in a 7.6-fold increase in mRNA levels of beta-Galactosidase compared to the delivery of plasmid DNA complexed with a partially degraded PAMAM dendrimer (dPAMAM) in a subcutaneous murine model. When implanted in a muirne intra-femoral model, the delivery of pVEGF(165) resulted in a 2-fold increase in bone volume at the defect site relative to control scaffolds without pVEGF(165). It was concluded that a collagen/calcium phosphate scaffold can mediate transfection without the use of additional transfection vectors and can promote bone formation in a mouse model via the delivery of pVEGF(165).
The two growth factors VEGF and PDGF are involved in the process of bone regeneration. For this reason, we developed a brushite-chitosan system which controls the release kinetics of incorporated VEGF and PDGF to enhance bone healing. PDGF (250 ng) was incorporated in the liquid phase. Alginate microsphere-encapsulated VEGF (350 ng) was pre-included in small cylindrical chitosan sponges. VEGF and PDGF release kinetics and tissue distribution were determined using iodinated ((125)I) growth factor. In vivo, PDGF was more rapidly delivered from these systems implanted in rabbit femurs than VEGF. 80% of PDGF was released by the end of two weeks while only 70% of VEGF was delivered after a period of three weeks. Both GFs released from the brushite-chitosan constructs remained located around the implantation site (5 cm) with negligible systemic exposure. A PDGF bone peak concentration of approximately 5 ng/g was achieved on the 4th day. Thereafter, PDGF concentrations stayed higher than 2 ng/g during the first week. These scaffolds also provided a local VEGF bone concentration above 3 ng/g during a total of 4weeks, with a peak concentration of 5.5 ng/g on the 7th day. The present work demonstrates that our brushite-chitosan system is capable of controlling the release rate and localization of both GFs within a bone defect. The effect on bone formation was considerably enhanced with PDGF loaded brushite-chitosan scaffolds as well as with the PDGF/VEGF combination.
Tissue engineering scaffolds capable of gene delivery can provide a structure that supports tissue formation while also inducing the expression of inductive factors. Sustained release strategies are hypothesized to maintain elevated plasmid concentrations locally that can enhance gene transfer. In this report, we investigate the relationship between plasmid release kinetics and the extent and duration of transgene expression. Scaffolds were fabricated from polymer microspheres modified with cationic polymers (polyethylenimine, poly(L-lysine), poly(allylamine hydrochloride), polydiallyldimethylammonium) or polydopamine (PD), with PD enhancing incorporation and slowing release. In vivo implantation of scaffolds into the peritoneal fat pad had no significant changes in the level and duration of transgene expression between PD and unmodified scaffolds. Control studies with plasmid dried onto scaffolds, which exhibited a rapid release, and scaffolds with extended leaching to reduce initial quantities released had similar levels and duration of expression. Changing the plasmid design, from a cytomegalovirus (CMV) to an ubiquitin C (UbC) promoter substantially altered the duration of expression. These studies suggest that the initial dose released and vector design affect the extent and duration of transgene expression, which may be sustained over several weeks, potentially leading to numerous applications in cell transplantation and regenerative medicine.
Realization of systems able to both recruit cells and influence their fate (affecting their processes) represents a new approach for tissue regeneration. We investigated the potency of gene activated matrix (GAM) and implemented the GAM strategy in order to achieve a control of gene expression, as well as a specific cell recruitment. To this aim we developed a 3D DNA bio-activated collagen matrix by Poly (ethylenimine) (PEI)/DNA complex immobilization in the matrix through biotin/avidin bond. Moreover, we realised a serum based chemotactic gradient within the matrix in order to directionally attract NIH3T3 cells. In this system, cells are recruited and forced to migrate through the matrix where they find the bound PEI/DNA complexes and are transfected. The transfected cells can act as local in vivo bioreactors, secreting plasmid encoded proteins that augment tissue repair and regeneration. 3D cell migration and cell transfection were monitored through time-lapse video microscopy and fluorescence microscopy. Cell transfection was also quantified through FACS analysis. Results show that our engineered matrix is able to recruit external cells and transfect them once internalized, therefore it could help in tissue repairing strategy.
Human mesenchymal stem cells offer a potential alternative to embryonic stem cells in clinical applications. The ability of these cells to self-renew and differentiate into multiple tissues, including bone, cartilage, fat, and other tissues of mesenchymal origin, makes them an attractive candidate for clinical applications. Patients who experience fracture nonunion and metabolic bone diseases, such as osteogenesis imperfecta and hypophosphatasia, have benefited from human mesenchymal stem cell therapy. Because of their ability to modulate immune responses, allogeneic transplant of these cells may be feasible without a substantial risk of immune rejection. The field of regenerative medicine is still facing considerable challenges; however, with the progress achieved thus far, the promise of stem cell therapy as a viable option for fracture nonunion and metabolic bone diseases is closer to reality. In this review, we update the biology and clinical applicability of human mesenchymal stem cells for bone repair and metabolic bone diseases.
In this study, we examined the effects of varying collagen concentration and crosslink density on the biological, structural and mechanical properties of collagen-GAG scaffolds for bone tissue engineering. Three different collagen contents (0.25%, 0.5% and 1% collagen) and two different dehydrothermal (DHT) crosslinking processes [1] 105 degrees C for 24 h and [2] 150 degrees C for 48 h were investigated. These scaffolds were assessed for (1) pore size, (2) permeability (3) compressive strength and (4) cell viability. The largest pore size, permeability rate, compressive modulus, cell number and cell metabolic activity was all found to occur on the 1% collagen scaffold due to its increased collagen composition and the DHT treatment at 150 degrees C was found to significantly improve the mechanical properties and not to affect cellular number or metabolic activity. These results indicate that doubling the collagen content to 1% and dehydrothermally crosslinking the scaffold at 150 degrees C for 48 h has enhanced mechanical and biological properties of the scaffold making it highly attractive for use in bone tissue engineering.
Lyophilised collagen scaffolds have shown enormous potential in tissue engineering in a number of areas due to their excellent biological performance. However, they are limited for use in bone tissue engineering due to poor mechanical properties. This paper discusses the development of a calcium-phosphate coating for collagen scaffolds in order to improve their mechanical properties for bone tissue engineering. Pure collagen scaffolds produced in a lyophilization process were coated by immersing them in sodium ammonium hydrogen phosphate (NaNH(4)HPO(4)) followed by calcium chloride (CaCl(2)). The optimal immersing sequence, duration, as well as the optimal solution concentration which facilitated improved mechanical properties of the scaffolds was investigated. The influence of the coating on composition, structural and material properties was analysed. This investigation successfully developed a novel collagen/calcium-phosphate composite scaffold. An increase in the mechanical properties of the scaffolds from 0.3 kPa to up to 90 kPa was found relative to a pure collagen scaffold, while the porosity was maintained as high as 92%, indicating the potential of the scaffold for bone tissue engineering or as a bone graft substitute.
Non-viral gene delivery suffers from a number of limitations including short transgene expression times and low transfection efficiency. Collagen scaffolds have previously been investigated as in vitro DNA reservoirs, which allow sustained release of genetic information. Efficient viral gene-transfer from these scaffolds has previously been demonstrated. However, due to concerns about the safety of viral gene therapy, the use of non-viral vectors may be preferable. In this study a DNA-dendrimer complex embedded in a cross-linked collagen scaffold was investigated as a reservoir for non-viral delivery. Elution from the scaffolds and transfection of seeded rat mesenchymal stem cells were used to evaluate the scaffold's ability to act as a reservoir for the complexes. Elution from the scaffolds was minimal after 2 days with a total of 25% of the complexes released after 7 days. Extended transgene expression after DNA-dendrimer complex delivery from the scaffolds in comparison to direct delivery to cells was observed. The elongated transfection period and relatively high levels of reporter gene expression are significant advantages over other non-viral gene therapy techniques. This platform has the potential to be an effective method of scaffold-mediated gene delivery suitable for in vitro and in vivo applications.
The objective of this study was to develop a biomimetic, highly porous collagen-hydroxyapatite (HA) composite scaffold for bone tissue engineering (TE), combining the biological performance and the high porosity of a collagen scaffold with the high mechanical stiffness of a HA scaffold. Pure collagen scaffolds were produced using a lyophilization process and immersed in simulated body fluid (SBF) to provide a biomimetic coating. Pure collagen scaffolds served as a control. The mechanical, material, and structural properties of the scaffolds were analyzed and the biological performance of the scaffolds was evaluated by monitoring the cellular metabolic activity and cell number at 1, 2, and 7 days post seeding. The SBF-treated scaffolds exhibited a significantly increased stiffness compared to the pure collagen group (4-fold increase), while a highly interconnected structure (95%) was retained. FTIR indicated that the SBF coating exhibited similar characteristics to pure HA. Micro-CT showed a homogeneous distribution of HA. Scanning electron microscopy also indicated a mineralization of the collagen combined with a precipitation of HA onto the collagen. The excellent biological performance of the collagen scaffolds was maintained in the collagen-HA scaffolds as demonstrated from cellular metabolic activity and total cell number. This investigation has successfully developed a biomimetic collagen-HA composite scaffold. An increase in the mechanical properties combined with an excellent biological performance in vitro was observed, indicating the high potential of the scaffold for bone TE.
Reparation of bone defects remains a major clinical and economic concern, with more than 3 million bone grafts performed annually only in the United States and the EU. The search for alternatives to autologous bone grafting led to the approval by the FDA of an absorbable collagen carrier combined with rhBMP-2 for the treatment of certain bone diseases and fractures. The present work is focused on the production of a collagen-targeted rhBMP-2 based system to improve bone formation. We produced a modified rhBMP-2 with only an additional collagen-binding decapeptide derived from the von Willebrand factor and tested its affinity to collagen and its ability to induce ectopic bone formation in vivo when implanted in combination with absorbable collagen sponges or hydroxyapatite. The results showed not only that the rhBMP2-CBD had an increased affinity to collagen, but also that this binding was very stable during a prolonged period of time. In vivo experiments demonstrated that this rhBMP2-CBD maintained its osteoinductive activity, being capable of inducing new bone formation even at lower concentrations than native rhBMP-2. These results indicate that the combination of the fusion protein with absorbable collagen may be a suitable and safer alternative to rhBMP-2 for bone repair purposes.
This study investigated the effects of dual delivery of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2) for bone regeneration in a rat cranial critical size defect. Four groups of scaffolds were generated with VEGF (12 microg), BMP-2 (2 mug), both VEGF (12 microg) and BMP-2 (2 microg), or no growth factor released from gelatin microparticles incorporated within the scaffold pores. These scaffolds were implanted within an 8 mm rat cranial critical size defect (n=8-9 for each group). At 4 and 12 weeks, implants were retrieved and evaluated by microcomputed tomography (microCT) and histological scoring analysis. Additionally, 4 week animals were perfused with a radiopaque material to visualize and quantify blood vessel formation. Histological analysis revealed that for all groups at 4 weeks, a majority of the porous scaffold volume was filled with vascularized fibrous tissue; however, bone formation appeared most abundant in the dual release group at this time. At 12 weeks, both dual release and BMP-2 groups showed large amounts of bone formation within the scaffold pores and along the outer surfaces of the scaffold; osteoid secretion and mineralization were apparent, and new bone was often in close or direct contact with the scaffold interface. MicroCT results showed no significant difference among groups for blood vessel formation at 4 weeks (<4% blood vessel volume); however, the dual release group showed significantly higher bone formation (16.1+/-9.2% bone volume) than other groups at this time. At 12 weeks, dual release and BMP-2 groups exhibited significantly higher bone formation (39.7+/-14.1% and 37.4+/-18.8% bone volume, respectively) than either the VEGF group or blank scaffolds (6.3+/-4.8% and 7.8+/-7.1% bone volume, respectively). This work indicates a synergistic effect of the dual delivery of VEGF and BMP-2 on bone formation at 4 weeks and suggests an interplay between these growth factors for early bone regeneration. For the doses investigated, the results show that the addition of VEGF does not affect the amount of bone formation achieved by BMP-2 at 12 weeks; however, they also indicate that delivery of both growth factors may enhance bone bridging and union of the critical size defect compared to delivery of BMP-2 alone.
Current techniques to educate dendritic cells (DCs) ex vivo for immunotherapy are plagued by inefficient protocols and DC modifications are often transient and lost upon transplantation. This study investigated the role of sustained presentation of GM-CSF and PEI condensed pDNA (PEI-DNA) on gene transfer and long-term gene expression. Appropriate GM-CSF signaling during DC transfection promoted PEI-DNA uptake, although high cytokine concentrations induced intercellular DNA degradation, indicating the need for controlled presentation. Poly(lactide-co-glycolide) scaffolds that continuously stimulated DCs with both GM-CSF and PEI-DNA led to a 20-fold increase in gene expression, and high levels of expression persisted for a period of 10 days, in vitro. These results encourage the exploitation of biomaterials and GM-CSF to develop novel delivery vectors for genetically modified DCs or to genetically program host DCs in situ for vaccination and the treatment of autoimmunity.
The induction of a calcifying matrix was studied in vitro and compared for three biomaterials (collagen sponge, hydroxyapatite material and a mixture of both (Biostite)) cultured with human osteoblast-like cells. The influence of biomaterials on organic matrix synthesis and the calcification process was analysed at the ultrastructural level (transmission electron microscopy and X-ray microanalysis). Biomaterials were well tolerated by bone cells. Whichever biomaterial was used, osteoblasts proliferated and synthesized a new matrix constituted of fibrillar and non-fibrillar elements. This activity appeared earlier and was more intense with Biostite than with collagen sponge alone. A deposition of a mineral substance in this newly formed matrix was observed with the collagen sponge and Biostite, but never with hydroxyapatite alone. The mineral deposits were identified as hydroxyapatite crystals, similar to those observed and analysed in bone tissue. These in vitro observations clearly demonstrated the property of Biostite to produce a calcified collagenous matrix similar to bone tissue. However, in vivo confirmation is required before extending the use of this biomaterial to periodontology.
Nonviral gene transfer into the central nervous system (CNS) offers the prospect of providing safe therapies for neurological disorders and manipulating gene expression for studying neuronal function. However, results reported so far have been disappointing. We show that the cationic polymer polyethylenimine (PEI) provides unprecedentedly high levels of transgene expression in the mature mouse brain. Three different preparations of PEI (25-, 50-, and 800-kD) were compared for their transfection efficiencies in the brains of adult mice. The highest levels of transfection were obtained with the 25-kD polymer. With this preparation, DNA/PEI complexes bearing mean ionic charge ratios closest to neutrality gave the best results. Under such conditions, and using a cytomegalovirus (CMV)-luciferase construction, we obtained up to 0.4 10(6) RLU/microgram DNA (equivalent to 0.4 ng of luciferase), which is close to the values obtained using PEI to transfect neuronal cultures and the more easily transfected newborn mouse brain (10(6) RLU/microgram DNA). Widespread expression (over 6 mm3) of marker (luciferase) or functional genes (bcl2) was obtained in neurons and glia after injection into the cerebral cortex, hippocampus, and hypothalamus. Transgene expression was found more than 3 months post-injection in cortical neurons. No morbidity was observed with any of the preparations used. Thus, PEI, a low-toxicity vector, appears to have potential for fundamental research and genetic therapy of the brain.
Complexes of DNA with cationic lipids and cationic polymers are frequently used for gene transfer. Extracellular interactions of the complexes with anionic glycosaminoglycans (GAGs) may interfere with gene transfer. Interactions of GAGs with the carrier-DNA complexes were studied using tests for DNA relaxation (ethidium bromide intercalation), DNA release (electrophoresis), and transfection (pCMVbetaGal transfer into RAA smooth muscle cells). Several cationic lipid formulations (DOTAP, DOTAP/Chol, DOTAP/DOPE, DOTMA/DOPE, DOGS) and cationic polymers (fractured dendrimer, polyethylene imines 25 kDa and 800 kDa, polylysines 20 kDa and 200 kDa) were tested. Polycations condensed DNA more effectively than the monovalent lipids. Hyaluronic acid did not release or relax DNA in any complex, but it inhibited the transfection by some polyvalent systems (PEI, dendrimers, DOGS). Gene transfer by the other carriers was not affected by hyaluronic acid. Sulfated GAGs (heparan sulfate, chondroitin sulfates B and C) completely blocked transfection, except in the case of the liposomes with DOPE. Sulfated GAGs relaxed and released DNA from some complexes, but these events were not prerequisites for the inhibition of transfection. In conclusion, polyvalent delivery systems with endosomal buffering capacity (DOGS, PEI, dendrimer) were most sensitive to the inhibitory effects of GAGs on gene transfer, while fusogenic liposomes (with DOPE) were the most resistant systems.
The inability to deliver growth factors locally in a transient but sustained manner is a substantial barrier to tissue regeneration. Systems capable of localized plasmid gene delivery for prolonged times may offer lower toxicity and should be well-suited for growth factor therapeutics. We investigated the potency of plasmid gene delivery from genes physically entrapped in a polymer matrix (gene activated matrix) using bone regeneration as the endpoint in vivo. Implantation of gene activated matrices at sites of bone injury was associated with retention and expression of plasmid DNA for at least 6 weeks, and with the induction of centimeters of normal new bone in a stable, reproducible, dose- and time-dependent manner.
Transfection of primary cells and stem cells is a problem in the laboratory routine and further in tissue engineering and gene therapy. Most methods working effectively for cell lines in culture fail to transfect primary cells. Here we describe the use of the Nucleofector technology developed by amaxa biosystems. We were able to transfect primary human melanocytes, human coronary smooth muscle cells, human chondrocytes, and human mesenchymal stem cells with high efficiencies (28.9-45.3%). All primary cell types failed to be transfected satisfactorily by methods based on liposome-mediated transfection in our hands. The viability of the transfected cells varied between 11.2% and 75% in comparison to untreated cells. Only 200,000 cells per transfection sample were needed. In summary, this method presents an effective and fast mean for transfection of primary and stem cells demonstrated by four cell types which are only transfected with low efficiency by other methods.
Polyethylenimine (PEI) was combined with plasmid DNA and freeze dried following the addition of sucrose as a lyoprotectant and pore-forming agent. Freeze-dried PEI DNA condensates were dry mixed with granular polylactideglycolic acid (PLGA) then compression molded and sponged to encapsulated PEI DNA. A measurement of the elastic modulus indicated that 91 wt% sucrose substituted for 95 wt% sodium chloride as a porogen, resulting in PLGA sponges with a mechanical modulus of 100 kPa. The PEI DNA was retained (80%) within PLGA sponges prepared with sucrose during the leaching and subsequent 2-week release studies, whereas sodium chloride PLGA sponges caused the premature release (100%) of PEI DNA within 2 days. In vitro gene transfer studies with PEI DNA PLGA sponges established that adherent and infiltrating fibroblasts expressed reporter gene for 15 days compared with the short, 3-day expression mediated by direct gene of PEI DNA on cells in culture. The results demonstrate an approach to encapsulate condensed DNA in a PLGA sponge for the purpose of retaining DNA within the matrices and creating efficient gene transfer during tissue engineering.
Safe and efficient gene delivery would have great potential in gene therapy and tissue engineering, but synthetic biomaterial surfaces endowed with efficient gene-transferring functions do not yet exist. Inspired by naturally occurring biomineralization processes, we co-precipitated DNA with inorganic minerals onto cell-culture surfaces. The DNA/mineral nanocomposite surfaces obtained not only supported cell growth but also provided high concentrations of DNA in the immediate microenvironment of the cultured cells. Gene transfer from the engineered surfaces was as efficient as an optimized commercial lipid transfection reagent; in addition, the extent of gene transfer was adjustable by varying the mineral composition. DNA/mineral nanocomposite surfaces represent a promising system for enhancing gene transfer and controlling the extent of gene transfer for various biomedical applications, including tissue engineering or gene therapy of bone.
Supraphysiological concentrations of exogenous growth factors are typically required to obtain bone regeneration, and it is unclear why lower levels are not effective. We hypothesized that delivery of bone progenitor cells along with appropriate combinations of growth factors and scaffold characteristics would allow physiological doses of proteins to be used for therapeutic bone regeneration. We tested this hypothesis by measuring bone formation by rat bone marrow stromal cells (BMSCs) transplanted ectopically in SCID mice using alginate hydrogels. The alginate was gamma-irradiated to vary the degradation rate and then covalently modified with RGD-containing peptides to control cell behavior. In the same delivery vehicle, we incorporated bone morphogenetic protein-2 (BMP2) and transforming growth factor-beta3 (TGF-beta3), either individually or in combination. Individual delivery of BMP2 or TGF-beta3 resulted in negligible bone tissue formation up to 22 weeks, regardless of the implant degradation rate. In contrast, when growth factors were delivered together from readily degradable hydrogels, there was significant bone formation by the transplanted BMSCs as early as 6 weeks after implantation. Furthermore, bone formation, which appeared to occur by endochondral ossification, was achieved with the dual growth factor condition at protein concentrations that were more than an order of magnitude less than those reported previously to be necessary for bone formation. These data demonstrate that appropriate combinations of soluble and biomaterial-mediated regulatory signals in cell-based tissue engineering systems can result in both more efficient and more effective tissue regeneration.
Bone mineral contains hydroxyapatite (HA). This is the surface that mature osteoblasts and osteocytes interact with. Synthetic HA is widely used in orthopedic surgeries as an implant or implant coating. The bone-like HA surfaces increase implant union and bone formation; however, the mechanisms accounting for this effect on osteoblasts are not known. In this study, we compared gene expression profiles of osteoblasts responding to HA or plastic surfaces for 24 h. Expression profiles were also compared between HA discs processed with gravity-sieved compared with combined gravity and air-jet-sieved HA powders. The latter, composed of smaller HA particles, exhibits an increase in grain boundary surface area. Discs made with either HA powder similarly up-regulated osteoblast expression of 10 genes (including proliferin 3, Glvr-1, DMP-1, and tenascin C) and down-regulated 15 genes (such as osteoglycin) by more than 2-fold compared with plastic surfaces. The overall changes are indicative of an immediate (24-h) response to the HA surface and a trend toward osteoblast differentiation. In addition, subsets of modulated genes exist that are unique to each HA subtype. Taken together, we identified HA responsive genes evident within 24 h of surface contact, indicating a critical role for extracellular mineral surfaces in the regulation of osteoblast gene expression and phenotype.
Glycosaminoglycans (GAGs) are negatively charged polysaccharides that are found, e.g. on cell surface. GAGs have been reported to influence gene transfection. We have previously reported that cationic lipid-mediated gene transfection can be improved by combining a small polyethylenimine (PEI) with cationic lipids. In the present study, we examined if GAGs have any effect on the synergism of small PEIs and other cationic carriers. We used wild-type CHO (GAG+) and pgsB-618 cells (GAG-). Transfection efficiency was studied using lacZ and GFP reporter genes. We found that GAGs decreased the overall level of transgene expression in a reagent-dependent manner, but the synergism caused by low-MW PEIs was less affected. There were no major differences between cell lines in cellular uptake or intracellular localization of plasmids when measured with flow cytometry and confocal microscopy, respectively. In conclusion, cell-surface GAGs interfere with transfection efficiency of different cationic reagents, but that is not necessarily related to the synergy of small PEIs and cationic lipids.
The relatively high transfection efficiency of polyethylenimine (PEI) vectors has been hypothesized to be due to their ability to avoid trafficking to degradative lysosomes. According to the proton sponge hypothesis, the buffering capacity of PEI leads to osmotic swelling and rupture of endosomes, resulting in the release of the vector into the cytoplasm.
The mechanism of PEI-mediated DNA transfer was investigated using quantitative methods to study individual steps in the overall transfection process. In addition to transfection efficiency, the cellular uptake, local pH environment, and stability of vectors were analyzed. N-Quaternized (and therefore non-proton sponge) versions of PEI and specific cell function inhibitors were used to further probe the proton sponge hypothesis.
Both N-quaternization and the use of bafilomycin A1 (a vacuolar proton pump inhibitor) reduced the transfection efficiency of PEI by approximately two orders of magnitude. Chloroquine, which buffers lysosomes, enhanced the transfection efficiency of N-quaternized PEIs and polylysine by 2-3-fold. In contrast, chloroquine did not improve the transfection efficiency of PEI. The measured average pH environment of PEI vectors was 6.1, indicating that they successfully avoid trafficking to acidic lysosomes. Significantly lower average pH environments were observed for permethyl-PEI (pH 5.4), perethyl-PEI (pH 5.1), and polylysine (pH 4.6) vectors. Cellular uptake levels of permethyl-PEI and perethyl-PEI vectors were found to be 20 and 90% higher, respectively, than that of parent PEI vectors, indicating that the reduction in transfection activity of the N-quaternized PEIs is due to a barrier downstream of cellular uptake. A polycation/DNA-binding affinity assessment showed that the more charge dense N-quaternized PEIs bind DNA less tightly than PEI, demonstrating that poor vector unpackaging was not responsible for the reduced transfection activity of the N-quaternized PEIs.
The results obtained are consistent with the proton sponge hypothesis and strongly suggest that the transfection activity of PEI vectors is due to their unique ability to avoid acidic lysosomes.
Gene therapy approaches to bone tissue engineering have been widely explored. While localized delivery of plasmid DNA encoding for osteogenic factors is attractive for promoting bone regeneration, the low transfection efficiency inherent with plasmid delivery may limit this approach. We hypothesized that this limitation could be overcome by condensing plasmid DNA with nonviral vectors such as poly(ethylenimine) (PEI), and delivering the plasmid DNA in a sustained and localized manner from poly(lactic-co-glycolic acid) (PLGA) scaffolds. To address this possibility, scaffolds delivering plasmid DNA encoding for bone morphogenetic protein-4 (BMP-4) were implanted into a cranial critical-sized defect for time periods up to 15 weeks. The control conditions included no scaffold (defect left empty), blank scaffolds (no delivered DNA), and scaffolds encapsulating plasmid DNA (non-condensed). Histological and microcomputed tomography analysis of the defect sites over time demonstrated that bone regeneration was significant at the defect edges and within the defect site when scaffolds encapsulating condensed DNA were placed in the defect. In contrast, bone formation was mainly confined to the defect edges within scaffolds encapsulating plasmid DNA, and when blank scaffolds were used to fill the defect. Histomorphometric analysis revealed a significant increase in total bone formation (at least 4.5-fold) within scaffolds incorporating condensed DNA, relative to blank scaffolds and scaffolds incorporating uncondensed DNA at each time point. In addition, there was a significant increase both in osteoid and mineralized tissue density within scaffolds incorporating condensed DNA, when compared with blank scaffolds and scaffolds incorporating uncondensed DNA, suggesting that delivery of condensed DNA led to more complete mineralized tissue regeneration within the defect area. This study demonstrated that the scaffold delivery system encapsulating PEI-condensed DNA encoding for BMP-4 was capable of enhancing bone formation and may find applications in other tissue types.
The lack of safe and efficient gene-delivery methods is a limiting obstacle to human gene therapy. Synthetic gene-delivery agents, although safer than recombinant viruses, generally do not possess the required efficacy. In recent years, a variety of effective polymers have been designed specifically for gene delivery, and much has been learned about their structure-function relationships. With the growing understanding of polymer gene-delivery mechanisms and continued efforts of creative polymer chemists, it is likely that polymer-based gene-delivery systems will become an important tool for human gene therapy.