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Bioplotting of a bioactive alginate dialdehyde-gelatin composite hydrogel containing bioactive glass nanoparticles
Abstract
Alginate dialdehyde-gelatin (ADA-GEL) constructs incorporating bioactive glass nanoparticles (BGNPs) were produced by biofabrication to obtain a grid-like highly-hydrated composite. The material could induce the deposition of an apatite layer upon immersion in a biological-like environment to sustain cell attachment and proliferation. Composites were formulated with different concentrations of BGNPs synthetized from a sol-gel route, namely 0.1% and 0.5% (w/v). Strontium doped BGNPs were also used. EDS analysis suggested that the BGNPs loading promoted the growth of bone-like apatite layer on the surface when the constructs were immersed in a simulated body fluid. Moreover, the composite constructs could incorporate with high efficiency ibuprofen as a drug model. Furthermore, the biofabrication process allowed the successful incorporation of MG-63 cells into the composite material. Cells were distributed homogeneously within the hydrogel composite, and no differences were found in cell viability between ADA-GEL and the composite constructs, proving that the addition of BGNPs did not influence cell fate. Overall, the composite material showed potential for future applications in bone tissue engineering.
... Bioactive glasses (BGs), a family of bioreactive inorganic materials, are highly investigated biomaterials in bone tissue engineering due to their outstanding features, e.g. regulation of cell behavior via the release of biologically active ions, osteoconductivity and osteoinductivity (Leite et al., 2016;Xynos, Edgar, Buttery, Hench, & Polak, 2001). For this reason, recently, increasing attention has been paid to the development of composites by incorporation of BGs in suitable hydrogels, which are proposed for applications in TE (Venkatesan, Bhatnagar, Manivasagan, Kang, & Kim, 2015). ...
... Icariin is a natural based drug which is extracted from Epimedium species and is known for the promotion of osteoblast cells and the inhibition of osteoclast cells, being species thus relevant for the intended applications in bone tissue engineering (Shen et al., 2020;Zhang, Liu, Huang, Wismeijer, & Liu, 2014;Zhao et al., 2010). The present study differs from our previous work (Leite et al., 2016) in that here nBG synthesized by flame spraying technology (and not sol-gel derived BG particles) were used and microcapsules were developed. In addition, we investigated the release of a natural drug in contrast to a synthetic drug (ibuprofen) used in the previous study (Leite et al., 2016). ...
... The present study differs from our previous work (Leite et al., 2016) in that here nBG synthesized by flame spraying technology (and not sol-gel derived BG particles) were used and microcapsules were developed. In addition, we investigated the release of a natural drug in contrast to a synthetic drug (ibuprofen) used in the previous study (Leite et al., 2016). ...
Sodium alginate is a natural biocompatible polymer obtained from brown algae which has found numerous biomedical applications. Inorganic fillers, such as nanosized bioactive glass (nBG) particles, are well known for their outstanding properties in terms of being osteoconductive and osteoinductive and therefore finding application in bone tissue engineering. In this study, the impact of nBG particles on alginate hydrogels was investigated for applications of the composite hydrogel in biofabrication and drug delivery. The influence of nBG particles on properties such as printability, drug release ability and bioactivity (capability to form a hydroxyapatite (HAp) layer on the surface of nBG-alginate capsules) was studied. In vitro cell studies proved high cell viability of all inks. Due to the presence of nBG particles, more precise printed grids and pore sizes were achieved. Moreover, a decrease of the release of the model drug icariin in the presence of nBG particles was determined. The formation of a HAp layer on the surface of nBG-alginate capsules was assessed by FTIR, XRD and SEM. Overall, the addition of nBG particles into alginate hydrogels led to the improvement of the relevant properties investigated. Therefore, nBG-alginate systems should gain more attention for 3 D bioprinting and drug delivery approaches.
Supplemental data for this article is available online at https://doi.org/10.1080/26889277.2022.2039078 .
... [20][21][22][23] Natural bone is highly vascularized, thus the osteogenesis-angiogenesis coupling process is pivotal in designing strategies for bone repair. [24] Previous work has shown the possibility of incorporating bioactive glass nanoparticles (BGNPs) into hydrogels for promoting the mineralization of the resulting constructs, [25] and for inducing early osteogenesis/chondrogenic differentiation. [26] Besides, copperdoped mesoporous BGNPs (CuMBGNs) are well-known for their ability to promote pro-angiogenic and osteo/odontogenic functions in bone and wound healing. ...
... [27,28] On the other hand, alginate dialdehyde-gelatin (ADA-GEL or AG) hydrogels have been proven to be cytocompatible offering a versatile biomaterial for cell encapsulation and for 3D bioprinting. [25,26,29,30] Therefore, an advanced nanocomposite bioink is expected to be developed here, introducing aminated CuMBGNs (ACuMBGNs) with free amino groups (-NH 2 ) to act as nanocrosslinkers inside the AG system with free aldehyde groups. Associated multiple functions are expected to be realized through this approach, which are presented in this study. ...
... [87,88] This result is consistent with previous reports on BG-containing polymer systems. [25] Nanoparticle-hydrogel composites exhibit multifunctional and self-healing properties, which make them attractive to develop "smart" materials. [89] Such materials can potentially be used to engineer tissue-implant interactions and to develop advanced therapeutic approaches, especially for tissue reconstruction and regeneration. ...
Bioprinting has seen significant progress in recent years for the fabrication of bionic tissues with high complexity. However, it remains challenging to develop cell-laden bioinks exhibiting superior physiochemical properties and bio-functionality. In this study, a multifunctional nanocomposite bioink is developed based on amine-functionalized copper (Cu)-doped mesoporous bioactive glass nanoparticles (ACuMBGNs) and a hydrogel formulation relying on dynamic covalent chemistry composed of alginate dialdehyde (oxidized alginate) and gelatin, with favorable rheological properties, improved shape fidelity, and structural stability for extrusion-based bioprinting. The reversible dynamic microenvironment in combination with the impact of cell-adhesive ligands introduced by aminated particles enables the rapid spreading (within 3 days) and high survival (>90%) of embedded human osteosarcoma cells and immortalized mouse bone marrow-derived stroma cells. Osteogenic differentiation of primary mouse bone marrow stromal stem cells (BMSCs) and angiogenesis are promoted in the bioprinted alginate dialdehyde-gelatin (ADA-GEL or AG)-ACuMBGN scaffolds without additional growth factors in vitro, which is likely due to ion stimulation from the incorporated nanoparticles and possibly due to cell mechanosensing in the dynamic matrix. In conclusion, it is envisioned that these nanocomposite bioinks can serve as promising platforms for bioprinting complex 3D matrix environments providing superior physiochemical and biological performance for bone tissue engineering.
... respectively, presumably because substituting Sr for Ca enhanced the rate of glass dissolution and ion release which can subsequently accelerate the crosslinking rate of the hydrogel [ 100 , 127 ]. It is worth noting that the gel viscosity increased in line with BG content and in a time-dependent manner likely due to the enhanced interaction between the free L-guluronic acid units of polymers and the released Ca 2 + and/or Sr 2 + from BG particles [100] . It is common knowledge that mechanical properties of hydrogels increase by addition of BG particles, often up to a threshold concentration depending on the hydrogel matrix, because of the stiffening effect of these particles [ 128 , 129 ]. ...
... The biocompatibility of ALG/GEL hydrogel with a variety of mammalian cells and its tunable rheological properties have made it a potential candidate for use as a bioink to fabricate 3D printed cell-laden constructs for use in bone regeneration [ 177 , 178 ]. In a recent study, Leite and co-workers investigated the influence of BG nanoparticles with and without Sr (i.e., 55 SiO 2 -40 CaO-5 P 2 O 5 and 55 SiO 2 -30 CaO-5 P 2 O 5 -10% SrO, mol.%) at two different concentrations of 0.1 and 0.5 % (w/v) on the ability of ADA/GEL hydrogel to preserve the viability of embedded MG-63 cells during printing [100] . Cell viability in both BG-loaded and unloaded hydrogels was comparable, suggesting that the nano-BGs, irrespective of the composition, had no adverse impact on the viability of encapsulated cells. ...
... What is more, all formulations containing BG nanoparticles promoted the formation of an apatite layer on their surface after immersion in SBF and indicated high potential for sustained release of ibuprofen. As the amount of nano-BG increased, the release of ibuprofen became slower, likely owing to the formation of a hydrogen bonding between the carboxylic acid moieties of the ibuprofen and silanol groups of the BG particles [100] . ...
Successful tissue regeneration requires a scaffold with tailorable biodegradability, tissue-like mechanical properties, structural similarity to extracellular matrix (ECM), relevant bioactivity, and cytocompatibility. In recent years, injectable hydrogels have spurred increasing attention in translational medicine as a result of their tunable physicochemical properties in response to the surrounding environment. Furthermore, they have the potential to be implanted via minimally invasive procedures while enabling deep penetration, which is considered a feasible alternative to traditional open surgical procedures. However, polymeric hydrogels may lack sufficient stability and bioactivity in physiological environments. Composite hydrogels containing bioactive glass (BG) particulates, synergistically combining the advantages of their constituents, have emerged as multifunctional biomaterials with tailored mechanical properties and biological functionalities. This review paper highlights the recent advances in injectable composite hydrogel systems based on biodegradable polymers and BGs. The influence of BG particle geometry, composition, and concentration on gel formation, rheological and mechanical behavior as well as hydration and biodegradation of injectable hydrogels have been discussed. The applications of these composite hydrogels in tissue engineering are additionally described, with particular attention to bone and skin. Finally, the prospects and current challenges in the development of desirable injectable bioactive hydrogels for tissue regeneration are discussed to outline a roadmap for future research.
Statement of Significance
Developing a biomaterial that can be readily available for surgery, implantable via minimally invasive procedures, and be able to effectively stimulate tissue regeneration is one of the grand challenges in modern biomedicine. This review summarizes the state-of-the-art of injectable bioactive glass-polymer composite hydrogels to address several challenges in bone and soft tissue repair. The current limitations and the latest evolutions of these composite biomaterials are critically examined, and the roles of design parameters, such as composition, concentration, and size of the bioactive phase, and polymer-glass interactions on the rheological, mechanical, biological, and overall functional performance of hydrogels are detailed. Existing results and new horizons are discussed to provide a state-of-the-art review that may be useful for both experienced and early-stage researchers in the biomaterials community.
... Biofabrication is described as the generation of complex biological products from raw materials such as matrices, biomaterials, and molecules [1] . Currently, three-dimensional (3D)-bioprinting has gained increased attention in biofabrication, as it could endow precise control on the positions of biopolymers and encapsulated cells to mimic the structure and function of the living tissues [2,3] . Hence, 3D printed scaffolds are successfully used in bone and cartilage cement [4,5] , catheters [4], drug delivery [6] , tissues and organs [7] due to their appropriate porosity, good mechanical properties, and biocompatibility [5,8] . ...
... Hence, 3D printed scaffolds are successfully used in bone and cartilage cement [4,5] , catheters [4], drug delivery [6] , tissues and organs [7] due to their appropriate porosity, good mechanical properties, and biocompatibility [5,8] . Meanwhile, 3D printed scaffolds could achieve enormous interactions between cells and materials that allow homogeneous cell distribution, cell loading efficiency, and also high cellular viability [2,9] . It is known that for favorable cell adhesion and growth, 3D scaffolds must support cellular proliferation, maintain their differentiation functions, and mimic the physiological extracellular matrix (ECM) [8] . ...
... Controlling the biodegradability of the scaffolds is an important feature in tissue engineering applications [2,10,51] . An ideal scaffold should protect its shape fidelity during the cell proliferation, and degrade along with new tissue regeneration preventing further impurities [53] . ...
In the last decade, 3D-bioprinting has attracted attention due to its capability to produce complex scaffolds. The selection of suitable biomaterials for the bioink design is very important for the success
of 3D-bioprinting. In this study, chitosan and gelatin were chemically modified into methacrylated chitosan (ChiMA) and methacrylated gelatin (GelMA) with the methacrylic anhydride in order to obtain crosslinking points on the polymeric backbone. The eligible bioinks were formulated with the layered double hydroxide nanoparticles (LDHs). The effect of changing the amount of LDHs on the printability of the bioinks was evaluated by using rheological analysis and printability test with the extrusion-based 3D-bioprinting. The bioinks were crosslinked under UV light. Mechanical, swelling, degradation properties, and cell-adhesion behaviors of the obtained ChiMA/
GelMA nanohybrid scaffolds containing LDHs were investigated. Based on the rheology and the printing results, ChiMA/GelMA nanohybrid scaffold containing 5% LDHs (ChiMA-G5) was found to
be the optimal bioink. Notably, compression strength, elongation at break, and elastic modulus of ChiMA-G5 scaffold were higher than neat and other ChiMA/GelMA scaffolds. In vitro cell culture studies showed that LDHs do not have any negative effects. These findings indicate that the developed ChiMA-G5 bioink has great potential as a bioink to utilize for tissue engineering applications.
... Cell viability is an essential feature considering the application of the developed devices in tissue engineering and regeneration. 150 This could be because ADA-GEL hydrogel possesses a highly porous structure. 27 Moreover, during the degradation of ADA-GEL, gelatin partially released from the matrix, creating space for the encapsulated cells in the hydrogel. ...
... This result suggests that ADA-GEL based hydrogels containing different Chapter 4: Composite capsules of alginate dialdehyde-gelatin-bioactive glass for cell delivery application 93 concentrations of BG used in this study are compatible with MG-63 cells and the fabricationprocess did not compromise cell viability. This is in agreement with the report of Leite et al.150 They found that after 24 h of cultivation, the viability of MG-63 cells encapsulated in printed scaffolds made from ADA-GEL containing sol-gel derived bioactive glass nanoparticles (nBG) was similar to that in samples without nBG.150 ...
... This is in agreement with the report of Leite et al.150 They found that after 24 h of cultivation, the viability of MG-63 cells encapsulated in printed scaffolds made from ADA-GEL containing sol-gel derived bioactive glass nanoparticles (nBG) was similar to that in samples without nBG.150 ...
Alginate-based hydrogels are being widely used in tissue engineering due to their biocompatible character. However, alginate hydrogels degrade very slowly and exhibit poor cell adhesion capability because of their lack of specific biomolecular anchoring sites for mammalian cells. The oxidation of alginate leading to alginate dialdehyde (ADA) can enhance its biodegradability. The incorporation of specific proteins such as gelatin into alginate based hydrogels can improve the cell-matrix interaction of the hydrogel, gelatin, for example, contains the cell adhesive peptide sequence, namely RGD (arginine-glycine-aspartic acid). In this research project, alginate dialdehyde-gelatin (ADA-GEL) based hydrogels were utilized for scaffold development and biofabrication for tissue engineering applications. The main goal was to develop new technologies to expand the applications of ADA-GEL in a variety of form. Porous ADA-GEL scaffolds were produced via freeze-drying process as a standard scaffold fabrication method. The scaffolds were coated with mesoporous bioactive glass nanoparticles (MBGN) using dip coating method. The compressive strength and elastic modulus of the scaffolds coated with MBGN were higher than that of non-coated samples but the difference was not significant. The coating process did not change the microstructure of the scaffolds. The presence of MBGN diminished the degradability of the samples. The formation of HA layer on the scaffolds coated with MBGN was observed, indicating that MBGN coating could enhance the bioactivity of ADA-GEL based freeze-dried scaffolds, intended for bone tissue engineering. The viability of MCT3T-E1 cells cultured on uncoated scaffolds was higher than that on the MBGN-coated samples over 21 days of cultivation. Nevertheless, the positive effect of MBGN on osteogenic differentiation of MCT3T-E1 cells was observed. A second technology investigated involved the development of mineralizing ADA-GEL composite microcapsules. Composite microcapsules based on ADA, gelatin, and 45S5 BG microparticles were fabricated using a pressure driven extrusion for cell encapsulation. The shorter gelation time of the ADA-GEL mixture was observed when the BG particles were added in the mixture, leading to a larger average size of microcapsules (>1500 µm) compared to that of microcapsules without BG particles (~1000 µm). Degradation rate of the samples decreased with increasing BG content. The incorporation of BG particles improved the bioactivity of ADA-GEL based microcapsules. The formation of a HA layer on the surface of the capsules containing BG particles after incubation in both DMEM and SBF was observed. Although the viability of MG-63 cells encapsulated in samples containing BG particles was lower than that in samples without BG particles, the ALP activity of the cells slightly increased with increasing BG content. The third technology investigated involved 3D printing of ADA-GEL based structures. In order to improve the printability of ADA-GEL based bioink, methylcellulose (MC) was blended with the ADA-GEL mixture. The bioink consisting of 2.5% ADA, 2.5% gelatin, and 9% MC exhibited good printability and capability to maintain shape fidelity. 10-layer printed scaffolds with pores in both vertical and horizontal directions could be attained when the bioink was printed in the XXYY pattern. The samples crosslinked with Ba2+ were more stable and stiffer than those crosslinked with Ca2+. The viability of ST2 cells entrapped in Ba2+ crosslinked samples increased after 21 days of cultivation while it decreased for the case of Ca2+ crosslinked samples. However, the mixing of cells into the bioink led to death and damage of many cells. Only 2% of the cells survived after they were mixed with the bioink. Finally, a sacrificial ink containing 5% gelatin and 9% MC was developed. It showed good printability and it was applied as sacrificial supporting structures for the fabrication of ADA-GEL 3D constructs. ADA-GEL mixtures were casted in the sacrificial molds and were then crosslinked with divalent cations. The sacrificial structures were dissolved during incubation under cell culture conditions. The 3D constructs of cell-laden ADA-GEL were used as a model for an in vitro cell study. The effects of different cations (Ca2+ and Ba2+) on ADA-GEL properties and on the behavior of MG-63 cells encapsulated in ADA-GEL constructs were investigated. In line with previous finding, the Ba2+ crosslinked samples were stiffer and degraded slower; nevertheless, both Ba2+ and Ca2+ crosslinked samples exhibited similar gelatin release profile. In the first 2 weeks, the viability of MG-63 cells embedded in the Ba2+ crosslinked samples was higher than that in Ca2+ crosslinked samples. Then, cell viability in both groups was comparable after 3 weeks of cultivation. The fabrication of grid-like structures of cell-laden ADA-GEL by printing the gel in the gap between strands of the sacrificial ink was also carried out. It was found that the strands in different layers of the obtained ADA-GEL scaffold did not merge into each other, suggesting that this technique might be potentially applicable to fabricate larger and more complex 3D constructs based on ADA-GEL. Overall, the outcomes of these studies expand the application potential of ADA-GEL based materials in various tissue engineering strategies, the present results represent thus a contribution to the continuous efforts to develop versatile, biocompatible hydrogels for tissue engineering, highlighting the capability of ADA-GEL system, also combined with second phases such as BG, MBGN and MC, for innovative scaffold, microcapsule and 3D printed (biofabricated) constructs.
... Bioactive glass/alginate-based hydrogel composites for bioprinting have already been studied, but they contained a low amount of glass up to 1 wt.% only [29,30]. Hence, we investigated various combinations of algMC blends containing a higher amount of MBG than studied by now (up to 7 wt.%) with incorporated Zn 2+ ions in the glass network. ...
... Starting with the 3-9 algMC blend which showed promising results in previous work by our group as a versatile bioink for bioprinting of different cell types [17], we added to this ink different amounts of MBG particles (3, 5, 7 or 10 wt.%), which was much more than the amount of added bioactive glass in previous studies of alginate-based composites, dealing with amounts up to 1 wt.%. In one of them, the addition of 0.1 and 0.5 wt.% of bioactive glass into alginate dialdehyde-gelatin ink did not affect viability of MG-63 cells in printed scaffolds [29]. In another study, 1 wt.% of bioactive glass in gelatin-alginate ink slightly decreased viability and proliferation of hMSC in correlation to the increased viscosity induced by the addition of the glass, but stimulated early osteogenic differentiation [30]. ...
... The same effect was observed in the case of magnetite microparticle-containing algMC inks, where with an increased amount of these particles the overall viscosity was elevated, resulting in solid state of the ink when more than 50% of particles was added, making it not printable [44]. A similar effect was observed with lower amounts of glass (0.1 and 0.5%) where crosslinking time was shorter when more glass was added [29]. Additionally, higher viscosity can be caused by a pre-crosslinking effect due to sudden release of calcium ions from the MBG. ...
Bioactive glasses have been used for bone regeneration applications thanks to their excellent osteoconductivity, an osteostimulatory effect, and high degradation rate, releasing biologically active ions. Besides these properties, mesoporous bioactive glasses (MBG) are specific for their highly ordered mesoporous channel structure and high specific surface area, making them suitable for drug and growth factor delivery. In the present study, calcium (Ca) (15 mol%) in MBG was partially and fully substituted with zinc (Zn), known for its osteogenic and antimicrobial properties. Different MBG were synthesized, containing 0, 5, 10, or 15 mol% of Zn. Up to 7 wt.% of Zn-containing MBG could be mixed into an alginate-methylcellulose blend (algMC) while maintaining rheological properties suitable for 3D printing of scaffolds with sufficient shape fidelity. The suitability of these composites for bioprinting applications has been demonstrated with immortalized human mesenchymal stem cells. Uptake of Ca and phosphorus (P) (phosphate) ions by composite scaffolds was observed, while the released concentration of Zn2+ corresponded to the initial amount of this ion in prepared glasses, suggesting that it can be controlled at the MBG synthesis step. The study introduces a tailorable bioprintable material system suitable for bone tissue engineering applications.
... 211 Similar enhanced interactions have been observed between BGs and alginate due to the presence of Ca in BGs. 220,221 In addition to their incorporation in injectable hydrogels, PBGSs can also be included in 3D printed hydrogels for bone regeneration application, which is introduced in the following section. 4.1.2. ...
... 32 However, it should be noted that the presence of BGs may cause cytotoxicity towards loaded cells as the local pH may increase due to the excessive release of ions during dissolution. 220 The employment of PBGSs as fillers in 3D printed hydrogels has not been widely investigated, though these porous particles are expected to have higher surface reactivity and greater interactions with hydrogel matrices and the embedded cells in comparison to non-porous and irregularly shaped BGs. Furthermore, their porous structure offers PBGSs the possibility to deliver therapeutic biomolecules and release them in a sustained and controlled way close to cells in bioinks. ...
... 232 In addition, the released ions from PBGSs can also affect the behavior of embedded cells. 220,233 Recent studies have shown the promising potential of PBGSs as multifunctional rigid fillers to formulate composite bioinks for 3D bioprinting. 32 However, this topic has not been extensively investigated and challenges remain for future research. ...
In recent years, porous bioactive glass micro/nanospheres (PBGSs) have emerged as attractive biomaterials in various biomedical applications where such engineered particles provide suitable functions, from tissue engineering to drug delivery. The design and synthesis of PBGSs with controllable particle size and pore structure are critical for such applications. PBGSs have been successfully synthesized using melt-quenching and sol–gel based methods. The morphology of PBGSs is controllable by tuning the processing parameters and precursor characteristics during the synthesis. In this comprehensive review on PBGSs, we first overview the synthesis approaches for PBGSs, including both melt-quenching and sol–gel based strategies. Sol–gel processing is the primary technology used to produce PBGSs, allowing for control over the chemical compositions and pore structure of particles. Particularly, the influence of pore-forming templates on the morphology of PBGSs is highlighted. Recent progress in the sol–gel synthesis of PBGSs with sophisticated pore structures (e.g., hollow mesoporous, dendritic fibrous mesoporous) is also covered. The challenges regarding the control of particle morphology, including the influence of metal ion precursors and pore expansion, are discussed in detail. We also highlight the recent achievements of PBGSs in a number of biomedical applications, including bone tissue regeneration, wound healing, therapeutic agent delivery, bioimaging, and cancer therapy. Finally, we conclude with our perspectives on the directions of future research based on identified challenges and potential new developments and applications of PBGSs.
... Unlike the behavior of SaOS-2 cells, hBMSCs incorporated in hydrogel containing BG and CNF maintained good viability but also limited proliferation over 14 d [81] . Leite et al. [82] focused on the integration of MG-63 cells in alginate-dialdehyde/ gelatin (ADA-GEL) hydrogel combined with bioactive glass nanoparticles. In previous studies, ADA-GEL already had been proven to be cytocompatible in combination with cell seeding, encapsulation or as 3D printed constructs [69 , 113-117] . ...
... As ADA-GEL itself lacks bioactive behavior (mineralization ability), bioactive glass nanoparticles (BGNPs) were integrated to promote the deposition of a calcium phosphate layer, which should lead to enhanced osseointegration of the construct [118] . ADA-GEL was compounded with ternary formulations of 0.1% and 0.5% (w/v) BGNPs (SiO 2 -CaO-P 2 O 5 ) or strontium doped BGNPs (SiO 2 -CaO-P 2 O 5 -SrO), respectively [82] . Strontium was used due to its favorable effects on osteogenic stimulation as well as in vivo bone formation ability [119 , 120] . ...
... The special characteristic of BG fillers, being dissolvable or bioreactive, remains to be exploited in future bioink strategies, not only for bone regeneration (bioprinted) scaffolds. An interesting approach is the incorporation of mesoporous BG particles in the hydrogel, where the particles can act as local carrier for growth factors or therapeutic drugs, thus exploiting the dual effect of biologically active ions and drug release in the proximity of cells [82] . ...
The growing demand for personalized implants and tissue scaffolds requires advanced biomaterials and processing strategies for the fabrication of three-dimensional (3D) structures mimicking the complexity of the extracellular matrix. During the last years, biofabrication approaches like 3D printing of cell laden (soft) hydrogels have been gaining increasing attention to design such 3D functional environments which resemble natural tissues (and organs). However, most of these polymeric hydrogels show poor stability and low printing fidelity and hence various approaches in terms of multi-material mixtures are being developed to enhance pre- and post-printing features as well as cytocompatibility and post-printing cellular development. Additionally, bioactive properties improve the binding to the surrounding (host) tissue at the implantation site. In this review we focus on the state-of-the-art of a particular type of heterogeneous bioinks, which are composed of polymeric hydrogels incorporating inorganic bioactive fillers. Such systems include isotropic and anisotropic silicates like bioactive glasses and nanoclays or calcium-phosphates like hydroxyapatite (HAp) which provide in-situ crosslinking effects and add extra functionality to the matrix, for example mineralization capability. The present review paper discusses in detail such bioactive composite bioink systems based on the available literature, revealing that a great variety has been developed with substantially improved bioprinting characteristics, in comparison to the pure hydrogel counterparts, and enabling high viability of printed cells. The analysis of the results of the published studies demonstrates that bioactive fillers are a promising addition to hydrogels to print stable 3D constructs for regeneration of tissues. Progress and challenges of the development and applications of such composite bioink approaches are discussed and avenues for future research in the field are presented.
Statement of significance
Biofabrication, involving the processing of biocompatible hydrogels including cells (bioinks), is being increasingly applied for developing complex tissue and organ mimicking structures. A variety of multi-material bioinks is being investigated to bioprint 3D constructs showing shape stability and long-term biological performance. Composite hydrogel bioinks incorporating inorganic bioreactive fillers for 3D bioprinting are the subject of this review paper. Results reported in the literature highlight the effect of bioactive fillers on bioink properties, printability and on cell behavior during and after printing and provide important information for optimizing the design of future bioinks for biofabrication, exploiting the extra functionalities provided by inorganic fillers. Further functionalization with drugs/growth factors can target enhanced printability and local drug release for more specialized biomedical therapies.
... As such it has been used as doping materials for HAp to enhance their osteogenic properties both in classical tissue engineering 129 and in bioprinting. 89 In a recent work, strontium carbonate (SrCO3) nanoparticles (≈15 nm) were also directly used in bioink for bone printing. In this context, it was reported to increase the viscosity as well as to enhance MSCs viability and osteogenic differentiation leading to rapid mineralized nodule formation. ...
... These somehow contradictory results underline the difficulty of properly assessing the cytoxicity of colloid materials which is a crucial challenge for the development of composite bioinks (as discussed in the section 4 of this manuscript).Silicate-based colloids. There are two main types of silicate-based materials used as additives in bioinks for bone printing : bioactive glass particles54,88,89 and clay nanoplatelets.[90][91][92] Bioactive glasses (Na2O-CaO-SiO2-P2O5) are glass-ceramic nanomaterials consisting of various proportions of silica (SiO2), calcium oxide (CaO), sodium oxide (Na2O) and phosphorus pentoxyde (P2O5). ...
... These nanoparticles form a strong chemical bond to bone mineral and their dissolution products (Si, Ca, P and Na ions) stimulates osteoprogenitor cells at the genetic level to promote rapid bone formation.122,123 In printed composite bioinks, bioactive glass was reported to enhance the osteogenic activities of osteoblast-like cells (MG-63 and SaOS-2 cells) as well as bone marrow-derived mesenchymal stem cells (BMSCs) and led to rapid mineralization of the bioprinted construct.54,88,89 However, it is important to note that the increase in viscosity due to the presence of bioactive glass particles yielded, in some cases, a lower post-printing viability of SaOS-2 cells.54 ...
The development of extrusion-based bioprinting for tissue engineering is conditioned by the design of bioinks displaying adequate printability, shape stability, and postprinting bioactivity. In this context, simple bioink formulations, made of cells supported by a polymer matrix, often lack the necessary versatility. To address this issue, intense research work has been focused on introducing colloidal particles into the ink formulation. By creating weak cross-links between polymer chains, added particles modify the rheology and mechanical behavior of bioinks to improve their printability and structural integrity. Additionally, nano- and microscopic particles display composition- and structure-specific properties that can affect the cellular behavior and enhance the formation of tissue within the printed material. This Review offers a comprehensive picture of the role of colloids in bioprinting from a physicochemical and biological perspective. As such, it provides guidance on devising adaptable bioinks for the fabrication of biomimetic tissues.
... Furthermore, the interaction of bioactive rigid fillers with the surrounding hydrogel matrix during and after the bioprinting process has been characterized only to a limited extent in the published literature. Also, quite often the actual shape fidelity and dimensions of printed 3D composite structures are not specified, for example, the height of printed scaffolds is not considered as only planar grid-like structures have been usually produced for such composite hydrogels [36][37][38][39][40]. Hence, in our approach, we aimed to design an ADA-GEL-based composite bioink for printing cell-laden porous 3D structures with tunable degradation properties through exploiting the bivalent ion release from BIF and the in-situ crosslinking of the hydrogel network. ...
... Most publications incorporating BIF in bioinks focus on bone tissue engineering applications due to the bioactivity of the applied bioactive glass fillers in body fluid-like media and the resulting improved adhesiveness of bone cells [29,37,39,52,64,90]. In this study, the intended application of the investigated composite hydrogels is in soft tissue engineering. ...
The development of hydrogels suitable for biofabrication is essential to enable advanced approaches for tissue engineering and regenerative medicine. Applications in both hard and soft tissues require tailor-made bioinks to guide cellular behavior in a 3D matrix. In this study we aimed to enhance the stability and adjust the degradation behavior of alginate dialdehyde-gelatine (ADA-GEL) hydrogels using an in-situ crosslinking mechanism additionally to external crosslinking. To test this approach, we added 0.1 and 0.5% (w/v) bioactive inorganic fillers (BIF) based on sub-micrometric calcium-silicate particles to ADA-GEL hydrogels. Such BIF release bivalent Ca²⁺ ions that can internally crosslink alginate chains and tune the degradation of the hydrogel over 28 days of incubation. It was found that pure ADA-GEL dissolved quickly after the first day while the composite hydrogels remained stable exhibiting reduced degradation (20–50% of the initial weight). 3D (bio)printing of composite bioinks revealed improved printing accuracy, 3D shape fidelity as well as cell spreading throughout the entire matrix during 14 days of evaluation. Chemically, BIF did not seem to have an influence on ADA-GEL Schiff's base bonds and also the mechanical properties of the composite hydrogels were comparable to those of pure ADA-GEL with Young's moduli of about 4 kPa. Comprehensive rheology measurements were carried out to determine printing parameters and the influence of BIF on the bioprinting process. We conclude that the novel hydrogel composition based on ADA-GEL system incorporating calcium-silicate BIF exhibits tunable behavior in vitro up to at least 28 days of incubation leading to improved stability and time-dependent cell behavior in 3D bioprinted constructs.
... Frontiers in Bioengineering and Biotechnology | www.frontiersin.org January 2022 | Volume 9 | Article 767256 10 MBG (0.1 and 0.5 wt%) (Leite et al., 2016;Tavares et al., 2021). However, they did not present a negative control to exclude possible signals of the glass. ...
... This effect is especially strong with higher amounts of MBG, as used in the present study (7 wt %). It happens probably due to the mesoporous structure, specific for high binding capacity (Migneco et al., 2020), knowing that this effect was not observed when bioactive glass (not MBG) was included in a hydrogel (Leite et al., 2016). This is most likely the reason for the lack of studies presenting results based on fluorescence imaging of cells embedded in MBG-containing bioinks. ...
Besides osteoconductivity and a high degradation rate, mesoporous bioactive glasses (MBGs) are specific for their highly ordered channel structure and high specific surface area, making them suitable as drug and/or growth factor delivery systems. On the other hand, the mesoporous channel structure and MBG composition can have an effect on common cell evaluation assays, leading to inconclusive results. This effect is especially important when MBG is mixed in composite bioinks, together with cells. Additionally, the hydrogel component of the ink can influence the degradation of MBG, leading to different ion releases, which can additionally affect the analyses. Hence, our aim here was to show how the MBG structure and composition influence common cell viability and differentiation assays when calcium (Ca)-or magnesium (Mg)-containing glass is part of an alginate-based composite bioink. We suggested pre-labeling of cells with DiI prior to bioprinting and staining with calcein-AM to allow identification of metabolically active cells expressing signals in both green and red channels, allowing the use of fluorescence imaging for cell viability evaluations in the presence of high amounts (7 wt %) of MBGs. The release and uptake of ions during degradation of CaMBG and MgMBG were significantly changed by alginate in the composite bioinks, as confirmed by higher release and uptake from bulk glasses. Additionally, we detected a burst release of Mg 2+ from composites only after 24 h of incubation. Furthermore, we demonstrated that released ions and the mesoporous channel structure affect the measurement of lactate dehydrogenase (LDH) and alkaline phosphatase activity (ALP) in bioprinted composite scaffolds. Measured LDH activity was significantly decreased in the presence of CaMBG. On the other hand, the presence of MgMBG induced increased signal measured for the ALP. Taken together, our findings show how composite bioinks containing MBGs can interfere with common analyses, obtaining misleading results.
... 12,13 However, disadvantages such as low viscosity at concentrations within the limit of biocompatibility, lack of cell adhesion motifs and uncontrolled degradation kinetics in physiological conditions usually lead to low printing accuracy and poor cell-material interaction in alginate. 12,14 Gelatin (GEL), produced by denaturation of collagen, which involves breaking of the triple helix structure of collagen into random coils, is a highly biocompatible and biodegradable protein widely used in biomedical applications such as TE, wound dressing, gene therapy, and drug delivery. 4,11,13 Furthermore, gelatin exposes cell adhesion peptides with the RGD (Arg-Gly-Asp) sequence of collagen, which enhances cellmaterial interactions and promotes cell adhesion and proliferation for tissue regeneration. ...
... 4,11,13 Furthermore, gelatin exposes cell adhesion peptides with the RGD (Arg-Gly-Asp) sequence of collagen, which enhances cellmaterial interactions and promotes cell adhesion and proliferation for tissue regeneration. 4,11,13,14 The insufficient mechanical properties and rapid degradation hinder the application of pure gelatin in TE scaffolds. 11,12 The drawbacks of alginate and gelatin can be overcome by combining gelatin with alginate di-aldehyde (ADA) through covalent crosslinking leading to ADA-GEL. ...
The concept of adding inorganic fillers into hydrogels to form hydrogel nanocomposites often provides advantageous properties which can be exploited for successful 3D biofabrication. In this study, a new composite hydrogel combining oxidized alginate–gelatin (ADA‐GEL) hydrogel and laponite nanoclay as inorganic nanofiller was successfully developed and characterized. The results showed that the addition of 0.5% (wt/vol) laponite nanoplatelets improved the printability of ADA‐GEL hydrogels enabling the fabrication of detailed structures since a low effect of material spreading and reduced tendency to pore closure appeared. Furthermore, a comparison of different needle types (cylindrical and conical; same inner diameter of 250 μm) in filament fusion test showed that the pattern dispensed by cylindrical tip has enhanced printing accuracy and pattern fidelity when compared with the pattern from conical tip. A glass flip test determined a processing window of 1–2 h after composite ink preparation. Overall, laponite/ADA‐GEL hydrogel composites are confirmed as promising inks for 3D bioprinting.
... This result suggests that ADA-GEL based hydrogels containing different concentrations of BG used in this study are compatible with MG-63 cells and the fabrication process did not compromise cell viability. This is in agreement with the report of Leite et al. [47] They found that after 24 h of cultivation, the viability of MG-63 cells encapsulated in printed scaffolds made from ADA-GEL containing sol-gel derived bioactive glass nanoparticles (nBG) was similar to that in samples without nBG. [47] To quantify the viability of encapsulated cells in ADA-GEL based capsules containing different concentrations of BG over the incubation time, WST-8 assay was performed after 3, 7, and 21 d of culture. ...
... This is in agreement with the report of Leite et al. [47] They found that after 24 h of cultivation, the viability of MG-63 cells encapsulated in printed scaffolds made from ADA-GEL containing sol-gel derived bioactive glass nanoparticles (nBG) was similar to that in samples without nBG. [47] To quantify the viability of encapsulated cells in ADA-GEL based capsules containing different concentrations of BG over the incubation time, WST-8 assay was performed after 3, 7, and 21 d of culture. As seen in Figure 7, the viability of MG-63 cells increased significantly in all samples from day 3 to day 7. ...
The effect of the incorporation of 45S5 bioactive glass (BG) microparticles (mean particle size ≈ 2 µm) on the fabrication and physicochemical properties of alginate dialdehyde‐gelatin hydrogel capsules is investigated. The addition of BG particles decreases the hydrogel gelation time by ≈79% and 91% for the samples containing 0.1% w/v and 0.5% w/v BG, respectively. Moreover, it results in increasing average diameter of hydrogel capsules produced via a pressure‐driven extrusion technique from about 1000 µm for the samples without BG to about 1700 and 1900 µm for the samples containing BG at concentrations of 0.1% w/v and 0.5% w/v, respectively. The presence of BG particles in the capsules decreases the degradation rate and improves the bioactivity of the materials. The viability of MG‐63 cells encapsulated in all samples increases during the first 7 d of cultivation and maintains the same level during 21 d of cultivation. The early cell viability in samples containing BG is lower than that in samples without BG. The results show that 45S5 BG can positively regulate the osteogenic activity of cells incorporated in hydrogel capsules. The fabricated composite capsules exhibit promising potential for cell delivery in bone regeneration applications.
... In addition, gelatin, unlike alginate, has a positive charge that ensures cell and protein binding [17] . Alginate-gelatin bioink is commonly used as a basis for bone and cartilage tissue engineering [18][19][20] . ...
The necessity to preserve meniscal function prompts the research and development of novel treatment options, like three-dimensional (3D) bioprinting. However, bioinks for meniscal 3D bioprinting have not been extensively explored. Therefore, in this study, a bioink composed of alginate, gelatin, and carboxymethylated cellulose nanocrystal (CCNC) was formulated and evaluated. Firstly, bioinks with varying concentrations of the aforementioned components were subjected to rheological analysis (amplitude sweep test, temperature sweep test, and rotation). The optimal bioink formulation of 4.0% gelatin, 0.75% alginate, and 1.4% CCNC dissolved in 4.6% D-mannitol was further used for printing accuracy analysis, followed by 3D bioprinting with normal human knee articular chondrocytes (NHAC-kn). The encapsulated cells' viability was > 98%, and collagen II expression was stimulated by the bioink. The formulated bioink is printable, stable under cell culture conditions, biocompatible, and able to maintain the native phenotype of chondrocytes. Aside from meniscal tissue bioprinting, it is believed that this bioink could serve as a basis for the development of bioinks for various tissues.
... An alginate solution of 2.0% w v -1 (Sigma-Aldrich, Germany) was prepared in PBS (Gibco Life Technologies, Thermofisher), following published methods [30]. The hydrogel was then filtrated through 0.45 lm millipore filters (Rotilabo Ò -syringe filters, Carl Roth, Germany) to keep it sterile (i.e. ...
The efficacy of chemotherapeutic procedures relies on delivering proper concentrations of anti-cancer drugs in the tumor surroundings, so as to prevent potential side effects on healthy tissues. Novel drug carrier platforms should not just be able to deliver anticancer molecules, but also allow for adjustements in the way these drugs are administered to the patients. We developed a system for delivering water-insoluble drugs, based on the use of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), or bis(2-ethylhexyl) sulfosuccinate benzyl-n-hexadecyldimethylammonium (BHD-AOT), embedded into oxidized alginate-gelatin (ADA/Gel) hydrogel, emulating a patch for topic applications. After being loaded with curcumin, cancer cells such as human colorectal adenocarcinoma (HCT116 and DLD-1) and melanoma cell lines (MEL501), and non-malignant cells such as mammary epithelial cell lines (NMuMG) and embryonal fibroblasts (NIH 3T3 or NEO cells) were analyzed for biocompatibility and cytotoxic effects. The results show that the proposed system can load comparatively higher concentrations of the drug (with respect to other nano/microcarriers in the literature), and that it can enhance the likelihood of the drug being uptaken by cancer cells instead of non-malignant cells. These assays were complemented by diffusion studies across the stratum corneum of rat skin, with the aim of determining the system’s efficiency during topical application. Finally, the stability of the patch was tested after lyophilization to determine its potential pharmaceutical use. As a whole, the combined system represents a highly reliable and robust method for embedding and delivering complex insoluble chemotherapeutical molecules, and it is less invasive than other alternative methods in the literature.
... For example, the study performed by Wang et al., where BAG polyphosphate was added to an alginate and gelatin-based hydrogel ink for 3D bioprinting of SaOS-2 cells, enhancing their proliferation and mineralization. [147] Similarly, in a study by Ojansivu et [150] The composite ink allowed for the successful incorporation of MG-63 cells and was further functionalised by the addition of ibuprofen. Cells were homogeneously distributed throughout the composite ink, and no differences were shown in cell viability between hydrogel alone and the composite constructs, indicating that the addition of BAGs did not influence cell viability. ...
In most cases, bone injuries heal without complications, however, there is an increasing number of instances where bone healing needs major clinical intervention. Available treatment options have severe drawbacks, such as donor site morbidity and limited availability for autografting. Bone graft substitutes containing growth factors would be a viable alternative, however they have been associated with dose-related safety concerns and lack control over spatial architecture to anatomically match bone defect sites. 3D printing offers a solution to produce patient specific bone graft substitutes that are customized to the patient bone defect with temporal control over the incorporated therapeutics to maximize their efficacy. Inspired by the natural constitution of bone tissue, composites made of inorganic phases, such as nanosilicate particles, calcium phosphate, and bioactive glasses, combined with biopolymer matrices have been investigated as building blocks for the biofabrication of bone constructs. Besides capturing elements of the bone physiological structure, these inorganic/organic composites can be designed for specific cohesivity, rheological and mechanical properties, while both inorganic and organic constituents contribute to the composite bioactivity. This review provides an overview of 3D printed composite biomaterial-inks for bone tissue engineering. Furthermore, key aspects in biomaterial-ink design, 3D printing techniques, and the building blocks for composite biomaterial-inks are summarized.
... [62][63][64] Compared to other biomaterials, BGs can also act as vehicles for the controlled release of ions that can regulate gene expression of cells, which makes them multifunctional candidates in cancer treatment. [65][66][67][68][69] These carriers show slow and continuous in vitro sustained drug release due to the dissolution of the glass matrix, which is accompanied by ion release as well. 70 MBGs, first synthesized two decades ago, have become an ideal option in topical and targeted tumor therapies due to their ability to deliver drugs along with various therapeutic elements. ...
There is an ongoing profound shift in using glass as a primarily passive material to one that instills active properties. We believe and demonstrate that bioactive glasses (BGs) and glass-ceramics (BGCs) as functional biomaterials for cancer therapy can transform the world of healthcare in the 21st century. Melt/gel-derived BGs and BGCs can carry many exotic elements, including less common rare-earth, and trigger highly efficient anticancer properties via the combination of radiotherapy, photothermal therapy, magnetic hyperthermia, along with drug or therapeutic ions delivery. The addition of these dopants modifies the bioactivity, imparts novel functionalities, and induces specific biological effects that are not achievable using other classes of biomaterials. In this paper, we have briefly reviewed and discussed the current knowledge on promising compositions, processing parameters, and applications of BGs and BGCs in treating cancer. We also envisage the need for further research on this particular, unique class of BGs and BGCs.
... Moreover, nanostructures can strengthen the structure of network hydrogels and give advanced mechanical and thermal attributes. So far, there are a few inorganic components, for instance, hydroxyapatite [10,11], graphene oxide [18,19], carbon nitride quantum dots [20], layered double hydroxides [21][22][23], clay minerals [24][25][26], zirconium oxide nanoparticles (NPs) [27], bioactive glass nanoparticles [28], metal oxide NPs [29], silica nanoparticles [30], carbon nanotubes [31], and nitrogen-doped carbon dots [32] have been studied. Amongst these nanomaterials, zinc oxide nanoparticles (ZnO NPs) are very attractive to scientists for various biomedical applications due to numerous benefits, including long-term use, the crucial role of zinc in physiological manners, biodegradability, biocompatibility, easier functionalization because of possessing -OH group rich surface, low cost, several loading of drugs/therapeutic molecules, and simple fabrication [33][34][35]. ...
There has been a great interest in injectable hydrogels for cartilage tissue regeneration since it can simply fill damaged defects within minimum invasive surgical therapies. Injectable nanocomposite hydrogels provide a three-dimensional (3D) scaffold that fits perfectly with the defects and combines the structure of an extracellular matrix to mimic cartilage cells. In this research, we present novel thermoresponsive injectable gelatin (GEL)/oxidized alginate (OA) hydrogels reinforced by zinc oxide nanoparticles (ZnO NPs) and N-hydroxysuccinimide (NHS)/1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as chemical cross-linkers of polymer chains in the 3D scaffold system. The effect of ZnO NPs concentrations was investigated on the mechanical properties of scaffolds, and the outcomes revealed that boosting ZnO NPs content improved the storage modulus of the hydrogel by more than five folds. Comprehension of the injectable hydrogel viscoelasticity is essential in the formulation of suitable GEL/OA/ZnO nanocomposite scaffolds for cartilage tissue engineering. The morphology of the scaffolds possesses porous structures. The scaffold containing 0.05% ZnO NPs has a denser structure compared with uncross-linked hydrogel, and the pore size reduces by increasing the amount ZnO NPs. The in vitro swelling ratio has decreased at higher quantity of ZnO, due to the smaller pore sizes in the scaffolds, which minimizes the water absorption. The in vitro biodegradation reveals that the scaffold containing a higher amount of ZnO NPs (0.05%) is more durable than the one without ZnO NPs. The nanocomposite scaffold exposed biocompatibility >90% for MTT assay and excellent cell adhesion to MG-63 cells.
... High viscosity bioinks can easily be processed in combination with other bioinks or biomaterial inks by multichannel plotting and they enable the fabrication of constructs with defined macropores, which might be beneficial for nutrient and oxygen supply in a volumetric biofabricated construct. In the last few years, many groups developed bioinks of the second category which show good biological response despite their high viscosity (in non-flowing state), mainly for bone and cartilage regeneration, but also in soft tissue applications or as wound filler for deep skin burns [13][14][15]. Highly viscous bioinks possess a potentially high polymer content, preventing the crucial problem of sedimentation of cells or particles due to a closer polymer net compared to low viscous bioinks which are impaired by cell sedimentation [16]. While for low viscosity bioinks, the timeframe of cell sedimentation lies within a range of a few minutes, being relevant for bioprinting; these times are significantly higher for highly viscous bioinks [16]. ...
Highly viscous bioinks offer great advantages for the three-dimensional fabrication of cell-laden constructs by microextrusion printing. However, no standardised method of mixing a high viscosity biomaterial ink and a cell suspension has been established so far, leading to non-reproducible printing results. A novel method for the homogeneous and reproducible mixing of the two components using a mixing unit connecting two syringes is developed and investigated. Several static mixing units, based on established mixing designs, were adapted and their functionality was determined by analysing specific features of the resulting bioink. As a model system, we selected a highly viscous ink consisting of fresh frozen human blood plasma, alginate, and methylcellulose, and a cell suspension containing immortalized human mesenchymal stem cells. This bioink is crosslinked after fabrication. A pre-crosslinked gellan gum-based bioink providing a different extrusion behaviour was introduced to validate the conclusions drawn from the model system. For characterisation, bioink from different zones within the mixing device was analysed by measurement of its viscosity, shape fidelity after printing and visual homogeneity. When taking all three parameters into account, a comprehensive and reliable comparison of the mixing quality was possible. In comparison to the established method of manual mixing inside a beaker using a spatula, a significantly higher proportion of viable cells was detected directly after mixing and plotting for both bioinks when the mixing unit was used. A screw-like mixing unit, termed “HighVisc”, was found to result in a homogenous bioink after a low number of mixing cycles while achieving high cell viability rates.
... Overview hydrogel extrusion processes have high potential also for Bioprinting (Biofabrication). In this context, Bioprinting of cellladen hydrogels incorporating BG particles as bioreactive fillers is an area of increasing interest in the biofabrication field [75,76]. Finally, different 3D-printing techniques have been applied successfully for obtaining polymer-BG composites using PCL [77], PLA [78], silk [79], and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (commonly known as PHBV) [80]. ...
3D printing, or additive manufacturing, is a transformative technology platform impacting various disciplines, including biomaterials and biomedical devices. We present scientists, engineers, and medical professionals' perspectives about 3D printing of biomaterials and biomedical devices in this special issue. This issue is geared towards understanding the structure–process–property relationships involving different materials in vitro, in vivo, and in silico environments. The focus issue covers polymer, ceramic, glass, metallic and composite biomaterials involving various 3D-printing processes such as fused deposition, powder bed fusion, Bioprinting, directed energy deposition, and binder jetting; addressing research topics including tissue engineering, drug delivery, porous metal implants, bioink development, cell–materials interactions, wear degradation, nano-bio materials, and challenges in point-of-care delivery. Such diversity of research topics is an accurate representation of what is happening in this field globally. This article presents some of the success stories, challenges, and future directions in the 3D printing of biomaterials and devices.Graphic abstract
... Since the ECM is mostly composed of polysaccharides, glycosaminoglycans, and various proteins, ADA-GEL, based on a polysaccharide as well as the collagen derived component gelatine ( Fig. 1), provides a promising matrix for biomimetic tissue engineering applications [11]. The potential of ADA-GEL for bioprinting approaches has been demonstrated [12][13][14]. However, mechanical demands in physiological conditions remain challenging for hydrogels with thermoresponsive characteristics. ...
This work explored 3D bioplotting to mimic the intrinsic hierarchical structure of natural articular cartilage. Alginate dialdehyde-gelatine (ADA-GEL) was used as a hydrogel ink to create hierarchically ordered scaffolds. In comparison to previously reported ADA-GEL compositions, we introduce a modified formulation featuring increased amounts of thermally modified gelatine. Gelatine was degraded by hydrolysis which resulted in tailorable printability characteristics further substantiated by rheological analysis. ADA(3.75%w/v)-GEL(7.5%w/v) with gelatine modified at 80 °C for 3 h could be printed in hierarchical complex structures reaching scaffold heights of over 1 cm. The hierarchical structure of the scaffolds was confirmed via µ-CT analysis. To examine mechanical properties as well as the suitability of the hydrogel as a proper matrix for cell seeding and encapsulation, nanoindentation was performed. Elastic moduli in the range of ∼ 5 kPa were measured. Gelatine heat pre-treatment resulted in modifiable mechanical and rheological characteristics of ADA-GEL. In summary, this study demonstrates the possibility to enhance the printability of ADA-GEL hydrogels to fabricate hierarchical scaffold structures with shape stability and fidelity, without the necessity to change the initial hydrogel chemistry by the use of additives or crosslinkers, providing a valuable approach for fabrication of designed scaffolds for cartilage tissue engineering.
... Besides, nanostructures can reinforce the network structure of OA-GEL, providing enhanced thermal and mechanical attributes. Until now, a small number of inorganic components such as layered double hydroxides (Fu et al., 2010;Hu and Chen, 2014;Nourafkan et al., 2017), hydroxyapatite (Gheysari et al., 2020;Karvandian et al., 2020), clay minerals (Chen et al., 2004;Okay and Oppermann, 2007;Tjong, 2006), graphene oxide (Fan et al., 2013;Liu et al., 2013a), metal oxide nanoparticles (NPs) (Bhardwaj et al., 2008), bioactive glass nanoparticles (Leite et al., 2016), and carbon nanotubes (Zhang et al., 2009) have been studied. Recently, it has been shown that substances with great features like nanosheets are desirable reinforcers because of greater stress distribution across layers and charged layer-layer interplays . ...
Stem cell treatment is promising in the various disorders treatment, but its effect is confined by the adverse conditions in the damaged tissues. The utilization of hydrogels has been suggested as a procedure to defeat this issue by developing the engraftment and survival of injected stem cells. Specifically, injectable hydrogels have drawn much attention due to their shape adaptability, ease of use, and the capability to reach body parts that are hard to access. In this study, the thermosensitive injectable hydrogels based on oxidized alginate, gelatin, and carbon nitride quantum dots (CNQDs) have been fabricated for tissue engineering. The mechanical characteristics of the nanocomposite hydrogels were investigated by rheology analysis. The results show that increasing the amount of CNQDs improve the mechanical strength of the nanocomposite hydrogels. The Cross-section morphology of freeze dried hydrogels comprising 0.25, 1.5, and 3.0% CNQDs indicate porous structure with interrelated pores. Besides, the result of in vitro degradation reveals that the hydrogels comprising CNQDs are more durable than the one without CNQDs. A reduction in the biodegradation and swelling ratio is perceived with the addition of CNQDs. The cell viability and attachment show that the nanocomposite hydrogels are biocompatible (>88%) with great cell adhesion to osteosarcoma cell line MG63 depending on the presence of CNQDs.
... ADA with different GEL contents has been reported to have advantages in several biomedical applications such as wound dressing [15], improvement of cartilage tissue formation [16], development of cell adhesive surfaces [17] and as injectable cell delivery vehicle for adipose tissue engineering [18]. However, although several previous studies showed good cytocompatibility of ADA-GEL with several cell types in 2D [19][20][21][22] and 3D [23][24][25][26][27], cytotoxic effects on endothelial cells were also observed [28]. In some reports [29,30], the cells remained in the growth arrest phase, or their viability decreased for up to several days before regaining the proliferative stage in 3D. ...
A hydrogel system based on oxidized alginate covalently crosslinked with gelatin (ADA-GEL) has been utilized for different biofabrication approaches to design constructs, in which cell growth, proliferation and migration have been observed. However, cell–bioink interactions are not completely understood and the potential effects of free aldehyde groups on the living cells have not been investigated. In this study, alginate, ADA and ADA-GEL were characterized via FTIR and NMR, and their effect on cell viability was investigated. In the tested cell lines, there was a concentration-dependent effect of oxidation degree on cell viability, with the strongest cytotoxicity observed after 72 h of culture. Subsequently, primary human cells, namely fibroblasts and endothelial cells (ECs) were grown in ADA and ADA-GEL hydrogels to investigate the molecular effects of oxidized material. In ADA, an extremely strong ROS generation resulting in a rapid depletion of cellular thiols was observed in ECs, leading to rapid necrotic cell death. In contrast, less pronounced cytotoxic effects of ADA were noted on human fibroblasts. Human fibroblasts had higher cellular thiol content than primary ECs and entered apoptosis under strong oxidative stress. The presence of gelatin in the hydrogel improved the primary cell survival, likely by reducing the oxidative stress via binding to the CHO groups. Consequently, ADA-GEL was better tolerated than ADA alone. Fibroblasts were able to survive the oxidative stress in ADA-GEL and re-entered the proliferative phase. To the best of our knowledge, this is the first report that shows in detail the relationship between oxidative stress-induced intracellular processes and alginate di-aldehyde-based bioinks.
... Alginate [13][14][15][16], alginate with the combination of bi-calcium phosphate and BMP-2 [17,18], alginategelatin [19][20][21], alginate-bioglass-gelatin [22], alginate-gelatin-carboxymethyl chitosan [23], alginate-HA [24][25][26][27], sodium alginate-bioactive glass-gelatin [28], alginate-nano-silica-gelatin [29], alginate-poly(amino acid) [30], alginate-gelatin-agarose-calcium phosphate-biosilica [31,32], alginate-gelatin-HA [33], atsttrin-HA [34], alginatepolycaprolactone-arginylglycylaspartic acid-gelatin-methaacrylamide [35], alginate-polycaprolactone-collagen [36], alginate dialdehyde-gelatin-bioactive glass [37] alginate-polyvinyl alcohol-HA [38], alginate-polyvinyl alcoholhydroxyapatite-collagen [39], RGD-g-alginate and nHA-plasmid DNA [40], alginate-HA-chitosan [41], alginatemethylcellulose blend-CPC [42], alginate-poly(ethyeneimine)-silica [43], alginate-calcium phosphate cementgellan gum-VEGF [44], alginate-graphene oxide [45], alginate-collagen-fibrin gel [46], alginate-gelatin methacryloylβ-tricalcium phosphate (TCP) [47], alginate-nanocellulose and bioactive glass modified gelatin [48], alginate-SiO 2 -gelatin [49], alginate-plasma-methylcellulose calcium phosphate cement [50], alginate-poly(lactic-co-glycolic acid)/β-tricalcium phosphate [51], alginate-TEMPO-oxidized cellulose [52], sodium alginate-nHA-cellulose nanofiber-polyvinyl alcohol [53], alginate-gelatine-β-tricalcium phosphate [54] and alginate-decellularized extracellular matrix [55] were developed to improve properties of the 3D printed scaffolding system toward bone tissue construction. ...
Millions of bone graft procedures are performed by clinicians to treat defective and diseased bone to improve the quality of a patient's life. Bone is a hierarchically structured tissue that is composed of nano-hydroxyapatite (nHA), collagen and other ingredients. Autograft, allograft and synthetic graft are widely used techniques to treat bone-related problems. However, the problem persists in the insufficient donor site and transmissible disease which eventually demands alternative substitutes to these techniques. A synthetic graft is a better alternative where the development of 3D synthetic artificial bone tissue for the repair and replacement of diseased and non-functional tissue is an emerging area of research for the last three decades. Tissue engineering is a relatively new field of research that aims to develop 3D structured tissues by the combination of materials, cells and growth factors and so on [1]. In the last three decades, different methods and techniques have been utilized for the preparation of scaffolds in tissue engineering. Solvent casting and particle leaching, freeze-drying, thermal-induced phase separation, gas foaming, electrospinning, rapid prototyping, additive manufacturing, stereolithography, fused deposition melting, selective laser sintering and 3D bioprinting methods [2] are among the methods that often provide a 3D structured extracellular cellular matrix to support the native tissue. 3D bioprinting 3D bioprinting is commonly used for tissue engineering applications that aim to develop 3D structured artificial tissue by a combination of biomaterials, cells and growth factors that can mimic the natural structure and function of native tissue. Similarly, bio-inks also play an important role in the construction of artificial tissue. Several reviews have been published in the prospect of the application of 3D bioprinting for bone tissue engineering [3-7]. Both natural and synthetic polymeric substances are often utilized for the 3D printing applications. Some of the well-known natural polymeric substances are collagen, chitosan, alginate, carrageenan, gelatin, etc. and synthetic polymeric substances are polycaprolactone, polylactic acid, polyethylene terephthalate, etc. Alginate Alginate is an anionic polysaccharide that is commonly isolated from brown seaweed. The structure of alginate is mainly composed of mannuronic (M) and guluronic acids (G). The unique property of alginate to easily crosslink with divalent cationic materials to form a hydrogel makes it an ideal candidate for the construction of artificial tissue using 3D bioprinting. Several artificial tissues have been made by utilizing alginate including skin, bone, etc. [8]. Recently, a review was published that mainly focuses on the construction of artificial bone tissue with alginate injectable hydrogel and alginate composites [9,10]. However, alginate has an issue with cell adhesion and J. 3D Print. Med. (Epub ahead of print)
... The inclusion of bioactive NPs in such systems could be used in the context of mineralized tissues. [226] Dai et al. 3D bioprinted a mixture of alginate, gelatin, and fibrinogen, crosslinked by Ca 2+ , TG, and thrombin respectively to generate GBM models. Interestingly, in this matrix, glioma stem cells were able to maintain key stemness biomarkers (e.g., Nestin), and at the same time, cells exhibited some glial differentiation and increased VEGF secretion over time. ...
The establishment of tumor microenvironment using biomimetic in vitro models that recapitulate key tumor hallmarks including the tumor supporting extracellular matrix (ECM) is in high demand for accelerating the discovery and preclinical validation of more effective anticancer therapeutics. To date, ECM‐mimetic hydrogels have been widely explored for 3D in vitro disease modeling owing to their bioactive properties that can be further adapted to the biochemical and biophysical properties of native tumors. Gathering on this momentum, herein the current landscape of intrinsically bioactive protein and peptide hydrogels that have been employed for 3D tumor modeling are discussed. Initially, the importance of recreating such microenvironment and the main considerations for generating ECM‐mimetic 3D hydrogel in vitro tumor models are showcased. A comprehensive discussion focusing protein, peptide, or hybrid ECM‐mimetic platforms employed for modeling cancer cells/stroma cross‐talk and for the preclinical evaluation of candidate anticancer therapies is also provided. Further development of tumor‐tunable, proteinaceous or peptide 3D microtesting platforms with microenvironment‐specific biophysical and biomolecular cues will contribute to better mimic the in vivo scenario, and improve the predictability of preclinical screening of generalized or personalized therapeutics.
... In 2016, alginate dialdehyde-gelatin (ADA-GEL) constructs incorporating Sr-doped BG nanoparticles (BGNPs) were produced by biofabrication in order to obtain a grid-like composite for future application in bone tissue engineering [188]. However, to the best of the authors' knowledge, no studies about bioinks using Cu-doped glasses as inorganic fillers are currently available in the literature. ...
Copper is one of the most used therapeutic metallic elements in biomedicine, ranging from antibacterial approaches to cancer theranostics. This element could be easily incorporated into different types of biomaterials; specifically, copper-doped bioactive glasses (BGs) provide great opportunities for biomedical engineers and clinicians as regards their excellent biocompatibility and regenerative potential. Although copper-incorporated BGs are mostly used in bone tissue engineering, accelerated soft tissue healing is achievable, too, with interesting potentials in wound treatment and skin repair. Copper can modulate the physico-chemical properties of BGs (e.g., reactivity with bio-fluids) and improve their therapeutic potential. Improving cell proliferation, promoting angiogenesis, reducing or even prohibiting bacterial growth are counted as prominent biological features of copper-doped BGs. Recent studies have also suggested the suitability of copper-doped BGs in cancer photothermal therapy (PTT). However, more research is needed to determine the extent to which copper-doped BGs are actually applicable for tissue engineering and regenerative medicine strategies in the clinic. Moreover, copper-doped BGs in combination with polymers may be considered in the future to produce relatively soft, pliable composites and printable inks for use in biofabrication.
... Incorporation of nanomaterials has been used to add new functionalities to bioinks, for the fact that even small concentrations can significantly impact the properties of a hydrogel network. BG is an attractive additive for mechanical reinforcement of Alg-Gel bioinks because of their high specific surface area [26,27]. The reinforcement effects were varied depending on the size and solid concentration of BG. ...
Alginate–gelatin (Alg–Gel) composite hydrogel is extensively used in extrusion-based bioprinting. Although Alg–Gel blends possess excellent biocompatibility and printability, poor mechanical properties have hindered its further clinical applications. In this study, a series of design by incorporating bioactive glass nanoparticles (BG) (particle size of 12 and 25 nm) into Alg–Gel hydrogel have been considered for optimizing the mechanical and biological properties. The composite Alg–Gel–BG bioink was biophysically characterized by mechanical tests and bioprinting practice. Biocompatibility of Alg–Gel–BG bioink was then investigated by bioprinting mouse dermal fibroblasts. Mechanical tests showed enhanced stiffness with increasing concentration of incorporated BG. But the maximum concentration of BG was determined 1.0 wt% before blends became too viscous to print. Meanwhile, the incorporation of BG did not affect the highly porous structure and biodegradation of Alg–Gel hydrogel, while the mechanical strength and printability were enhanced. In addition, the cellular proliferation and adhesion in the bioprinted constructs were significantly enhanced by BG (12 nm), while extension was not affected. Therefore, our strategy of incorporating BG in Alg–Gel composite hydrogel represents an easy-to-use approach to the mechanical reinforcement of cell-laden bioink, thus demonstrating their suitability for future applications in extrusion-based bioprinting.
... The primary disadvantage of these bioactive ceramics is their brittle nature, which limits their clinical applications at load bearing sites. To overcome this, a variety of polymer/bioceramic composites have been proposed more recently to fabricate nanofibers, foams, hydrogels and additively manufactured scaffolds for tissue engineering [18][19][20][21][22]. The presence of bioactive ceramic nanoparticles in the composite scaffolds could possibly substitute the use of bone stimulating growth factors, which are expensive and labile thereby limiting the processability. ...
The focus of research in diamine functionalised graphene oxide (GO) has been limited to the use of diamines either as crosslinker or to achieve simultaneous functionalisation, reduction and stitching of GO sheets, especially in the case of ethylene diamine (EDA). Controlling the extent of stitching and functionalisation has to date remained a challenge. In particular, synthesis of colloidally stable monofunctionalised GO-NH2 with dangling amine groups using diamines has remained elusive. This has been the limiting factor towards the utility of EDA functionalised GO (GO-NH2) in the field of polymer-based nanocomposites. We have synthesised colloidally stable GO-NH2 with dangling amine groups and subsequently demonstrated its utility as a surfactant to synthesize colloidally stable waterborne polymer nanoparticles with innate affinity to undergo film formation at room temperature. Thermally annealed dropcast polymer/GO-NH2 nanocomposite films exhibited low surface roughness (~1 µm) due to the homogeneous distribution of functionalised GO sheets within the polymer matrix as observed from confocal laser scanning microscopy, scanning electron microscopy and transmission electron microscopy. The films exhibited considerable electrical conductivity (~0.8 S m-1), demonstrating the potential of GO-NH2/polymer nanocomposite for a wide range of applications.
... In order to adapt the mechanical properties and printability of alginate based bioinks, mostly (1) multi-material bioinks [16][17][18][19], (2) non-degradable particle or fiber filled systems [20][21][22][23] or (3) printing with support (sacrificial) material [24,25] are utilized. All those approaches have the common goal of overcoming the lack of shape fidelity of pure alginate solutions, which is due to the mostly viscous characteristics dominating over the elastic behavior of non-crosslinked polymer solutions. ...
Many different biofabrication approaches as well as a variety of bioinks have been developed by researchers working in the field of tissue engineering. A main challenge for bioinks often remains the difficulty to achieve shape fidelity after printing. In order to overcome this issue, a homogeneous pre-crosslinking technique, which is generally applicable to all alginate-based materials, was developed in this study. With this technique it was possible to markedly enhance the printability of a 2 % (w/v) alginate solution, without using a higher polymer content, fillers or support structures. It was possible to print 3D porous scaffolds with a height of around 5 mm. Furthermore, the rheological behavior of different pre-crosslinking degrees was studied. Shear forces on cells as well as the flow profile of the bioink inside the printing nozzle during the process were estimated. A high cell viability of printed NIH/3T3 cells embedded in the novel bioink of more than 85 % over a time period of two weeks could be observed. Furthermore, also the Young's Modulus of selected hydrogels, as well as the chemical characterization of alginate in terms of M/G ratio and molecular weight, were determined.
... Alginate dialdehyde-gelatin (ADA-GEL) hydrogel has been used for cell encapsulation and it has been shown to exhibit good cell adhesion, proliferation and migration properties [7][8][9]. The benefit of using such hydrogel is that two advantages-mild ADA crosslinking with divalent ions and cell adhesion to GEL-can be combined as the two components (ADA and GEL) form a covalent bond via Schiff's base reaction [9,10]. ...
Alginate dialdehyde–gelatin (ADA–GEL) hydrogels have been reported to be suitable matrices for cell encapsulation. In general, application of ADA–GEL as bioink has been limited to planar structures due to its low viscosity. In this work, ring shaped constructs of ADA–GEL hydrogel were fabricated by casting the hydrogel into sacrificial molds which were 3D printed from 9% methylcellulose and 5% gelatin. Dissolution of the supporting structure was observed during the 1st week of sample incubation. In addition, the effect of different crosslinkers (Ba2+ and Ca2+) on the physicochemical properties of ADA–GEL and on the behavior of encapsulated MG-63 cells was investigated. It was found that Ba2+ crosslinked network had more than twice higher storage modulus, and mass decrease to 70% during incubation compared to 42% in case of hydrogels crosslinked with Ca2+. In addition, faster increase in cell viability during incubation and earlier cell network formation were observed after Ba2+ crosslinking. No negative effects on cell activity due to the use of sacrificial materials were observed. The approach presented here could be further developed for cell-laden ADA–GEL bioink printing into complex 3D structures.
Bioactive glass (BG) has recently been proposed to be used for wound healing with Vaseline®. To accelerate the wound healing by incorporation of the Zinc (Zn) into BG, undoped and 5 wt% zinc-doped 45S5 BG (16 wt%) were prepared with Vaseline® to create the ointments for wound healing as a VBG and VBG-Zn, respectively. The results showed that desired composition, irregular form, and no porous surface have obtained for BGs. FT-IR spectrum of Zn-doped BG demonstrates increasing Network Connectivity due to Zn incorporation. Ointments exhibited pseudoplastic behaviour and viscoelastic characteristic, indicating that ointments have good topical application. Ointments did not toxic effect towards L929 cells in terms of biocompatibility for topical applications. In vitro wound healing assays revealed that VBG-Zn promotes wound healing thanks to incorporation of the Zn accelerates skin wound closure. All findings point to VBG-Zn ointment having a greater potential for wound healing applications.
Graphical abstract
Volume 23A provides a comprehensive review of established and emerging 3D printing and bioprinting approaches for biomedical applications, and expansive coverage of various feedstock materials for 3D printing. The Volume includes articles on 3D printing and bioprinting of surgical models, surgical implants, and other medical devices. The introductory section considers developments and trends in additively manufactured medical devices and material aspects of additively manufactured medical devices. The polymer section considers vat polymerization and powder-bed fusion of polymers. The ceramics section contains articles on binder jet additive manufacturing and selective laser sintering of ceramics for medical applications. The metals section includes articles on additive manufacturing of stainless steel, titanium alloy, and cobalt-chromium alloy biomedical devices. The bioprinting section considers laser-induced forward transfer, piezoelectric jetting, microvalve jetting, plotting, pneumatic extrusion, and electrospinning of biomaterials. Finally, the applications section includes articles on additive manufacturing of personalized surgical instruments, orthotics, dentures, crowns and bridges, implantable energy harvesting devices, and pharmaceuticals. For information on the print version of Volume 23A, ISBN: 978-1-62708-390-4, follow this link.
Three-dimensional (3D) bioprinting is an advanced technology to fabricate artificial 3D tissue constructs containing cells and hydrogels for tissue engineering and regenerative medicine. Nanocomposite reinforcement endows hydrogels with superior properties and tailored functionalities. A broad range of nanomaterials, including silicon-based, ceramic-based, cellulose-based, metal-based, and carbon-based nanomaterials, have been incorporated into hydrogel networks with encapsulated cells for improved performances. This review emphasizes the recent developments of cell-laden nanocomposite bioinks for 3D bioprinting, focusing on their reinforcement effects and mechanisms, including viscosity, shear-thinning property, printability, mechanical properties, structural integrity, and biocompatibility. The cell-material interactions were discussed to elaborate on the underlying mechanisms between the cells and the nanomaterials. The biomedical applications of cell-laden nanocomposite bioinks are summarized with a focus on bone and cartilage tissue engineering. Finally, the limitations and challenges of current cell-laden nanocomposite bioinks are identified. The prospects are concluded in designing multi-component bioinks with multi-functionality for various biomedical applications.
Statement of Significance
3D bioprinting, an emerging technology of additive manufacturing, has been one of the most innovative tools for tissue engineering and regenerative medicine. Recent developments of cell-laden nanocomposite bioinks for 3D bioprinting, and cell-materials interactions are the subject of this review paper. The reinforcement effects and mechanisms of nanocomposites on the viscosity, printability and biocompatibility of bioinks and 3D printed scaffolds are addressed mainly for bone and cartilage tissue engineering. It provides detailed information for further designing and optimizing multi-component bioinks with multi-functionality for specialized biomedical applications.
A novel biomaterial comprising alginate dialdehyde‐gelatine (ADA‐GEL) hydrogel augmented by lysozyme loaded mesoporous cerium doped silica‐calcia nanoparticles (Lys‐Ce‐MSNs) was 3D printed to create bioactive scaffolds. Lys‐Ce‐MSNs raised the mechanical stiffness of the hydrogel composite scaffold and induced surface apatite mineralization, when the scaffold was immersed in simulated body fluid (SBF). Moreover, the scaffolds could co‐deliver bone healing (Ca and Si) and antioxidant ions (Ce), and Lys to achieve antibacterial (and potentially anticancer) properties. The nanocomposite hydrogel scaffolds could hold and deliver Lys steadily. Based on the in vitro results, the hydrogel nanocomposite containing Lys assured improved pre‐osteoblast cell (MC3T3‐E1) proliferation, adhesion, and differentiation, thanks to the biocompatibility of ADA‐GEL, bioactivity of Ce‐MSNs, and the stabilizing effect of Lys on the scaffold structure. On the other hand, the proliferation level of MG63 osteosarcoma cells decreased, likely due to the anticancer effect of Lys. Last but not least, cooperatively, alongside gentamicin (GEN), Lys brought about a proper antibacterial efficiency to the hydrogel nanocomposite scaffold against gram‐positive and gram‐negative bacteria. Taken together, ADA‐GEL/Lys‐Ce‐MSN nanocomposite holds great promise for 3D printing of multifunctional hydrogel BTE scaffolds, able to induce bone regeneration, address infection, and potentially inhibit tumor formation and growth. This article is protected by copyright. All rights reserved
Silicate‐based bioactive glasses have shown potential applications for orthopedic and dental applications due to its versatile chemical composition, and its suitable osteoconductive and osteoinductive properties. These bioactive glasses were initially obtained by melt‐derived method and recently it is widely synthesized by the sol–gel method. The method of synthesizing these materials by sol–gel method opens the possibility of homogenous composition through the glass structure, low sintering temperature, and can alter the gel structure to desired morphology. For fulfilling the concept of “Lab‐to‐product” development, the sol–gel method is highly flexible to develop it as nanoparticles, mesoporous structures, electrospun fibers, 3D printable scaffolds, and template‐assisted 3D structure formation. And hence when modifying the structure of the bioactive glass to the possible products, quick real‐time applications will be possible. The sol–gel bioactive glass in combination with 3D printing hydrogel techniques made possible for futuristic customized scaffolds. In this review, we discuss about history of sol–gel bioactive glass, phase diagrams of bioactive glass systems, the major bioactive glass composition used for sol–gel synthesis, methods for fabricating nano‐ and microstructures, and futuristic applications of 3D customized scaffolds.
The application of inorganic biomaterials in regenerative medicine is increasingly expanded. Taking advantages of attractive properties of the inorganic biomaterials, sorts of functional bioinks have been developed based on inorganic biomaterials and were applied to construct inorganic/organic/cell‐laden systems for tissue regeneration via 3D bioprinting technology. In this review, we aim to summarize the existing inorganic biomaterials‐based bioinks (referred to as “inorganic‐bioinks”) for 3D bioprinting regenerative scaffolds. We introduce the recently developed inorganic‐bioinks from the perspective of the function of bioinks, and especially highlight the incorporation of inorganic biomaterials improving the printability, mechanical strength, and bioactivity of the bioinks for different tissue regeneration. Subsequently, the current applications of the inorganic‐bioinks in constructing 3D cell‐laden scaffolds for tissue regeneration are presented. Finally, challenges and prospects for the inorganic biomaterials‐based bioprinting strategy are discussed. Taking advantages of attractive properties of the inorganic biomaterials, sorts of functional bioinks have been developed based on inorganic biomaterials and were applied to construct inorganic/organic/cell‐laden systems for tissue regeneration via 3D bioprinting technology. In this review, we aim to summarize the existing inorganic biomaterials‐based bioinks (referred to as “inorganic‐bioinks”) for 3D bioprinting regenerative scaffolds.
Biomimetic hydrogels composed of natural polysaccharides have invariably blossomed as niche biomaterials in tissue engineering applications. The prospects of creating an extracellular matrix (ECM)-like milieu from such hydrogels has garnered considerable importance. In this study, we have fabricated bioscaffolds comprising dialdehyde alginate and xanthan gum and explored their potential use in tissue regeneration. The fabricated scaffolds displayed an interconnected porous network structure that is highly desirable for the aforesaid application. The scaffolds were endowed with good mechanical properties, thermostability, protein adsorption efficacy and degradability. Curcumin-loaded hydrogels exhibited appreciable antibacterial activity against E. coli. In vitro cytocompatibility studies revealed that the scaffolds promoted adhesion and proliferation of 3T3 fibroblast cells. The Western blot analysis of p53 gene indicated no growth arrest or apoptosis in 3T3 cells thus, signifying the non-toxic nature of the scaffolds. Furthermore, the ECM formation was confirmed via SDS-PAGE analysis. The overall results clearly validated these scaffolds as effectual biomaterials for tissue engineering applications.
A limiting factor in large bone defect regeneration is the slow and disorganized formation of a functional vascular network in the defect area, often resulting in delayed healing or implant failure. To overcome this, strategies that induce angiogenic processes should be combined with potent bone graft substitutes in new bone regeneration approaches. To this end, we describe a unique approach to immobilize the pro-angiogenic growth factor VEGF165 in its native state on the surface of nanosized bioactive glass particles (nBGs) via a binding peptide (PR1P). We demonstrate that covalent coupling of the peptide to amine functional groups grafted on the nBG surface allows immobilization of VEGF with high efficiency and specificity. The amount of coupled peptide could be controlled by varying amine density, which eventually allows tailoring the amount of bound VEGF within a physiologically effective range. In vitro analysis of endothelial cell tube formation in response to VEGF-carrying nBG confirmed that the biological activity of VEGF is not compromised by the immobilization. Instead, comparable angiogenic stimulation was found for lower doses of immobilized VEGF compared to exogenously added VEGF. The described system, for the first time, employs a binding peptide for growth factor immobilization on bioactive glass nanoparticles and represents a promising strategy to overcome the problem of insufficient neovascularization in large bone defect regeneration.
Nanostructured compounds already validated as performant reinforcements for biomedical applications together with different fabrication strategies have been often used to channel the biophysical and biochemical features of hydrogel networks. Ergo, a wide array of nanostructured compounds has been employed as additive materials integrated with hydrophilic networks based on naturally-derived polymers to produce promising scaffolding materials for specific fields of regenerative medicine. To date, nanoengineered hydrogels are extensively explored in (bio)printing formulations, representing the most advanced designs of hydrogel (bio)inks able to fabricate structures with improved mechanical properties and high print fidelity along with a cell-interactive environment. The development of printing inks comprising organic–inorganic hybrid nanocomposites is in full ascent as the impact of a small amount of nanoscale additive does not translate only in improved physicochemical and biomechanical properties of bioink. The biopolymeric nanocomposites may even exhibit additional particular properties engendered by nano-scale reinforcement such as electrical conductivity, magnetic responsiveness, antibacterial or antioxidation properties. The present review focus on hydrogels nanoengineered for 3D printing of biomimetic constructs, with particular emphasis on the impact of the spatial distribution of reinforcing agents (0D, 1D, 2D). Here, a systematic analysis of the naturally-derived nanostructured inks is presented highlighting the relationship between relevant length scales and size effects that influence the final properties of the hydrogels designed for regenerative medicine.
This article is protected by copyright. All rights reserved.
In order to better recapitulate both anatomic and physiological aspects of bone, it is important to build constructs that are hierarchically organized from the nano to the macro-scale. For this reason, many strategies rely on nanomaterials as base units for the development of new biomaterials, from nanofibers (1D) to nano-coatings (2D) or nanocomposite scaffolds (3D). Nanoparticles have been widely used because of their versatility in composition, size and shape, easily fitting different applications. Besides their structural roles, nanobiomaterials incorporated in devices for bone repair can affect the cellular behavior towards bone regrowth or their integration in the defect site. In particular for bone regeneration, bioactive nanomaterials are integrated in different biomaterials in order to induce biomineralization.
This chapter overviews recent advances in the nanoscale design of new biomaterials for biomineralization in bone tissue engineering. The focus is on bioactive silica nanoparticles and their combination with different types of biomaterials.
Hydrogels have attracted much attention for biomedical and pharmaceutical applications due to the similarity of their biomimetic structure to the extracellular matrix of natural living tissues, tunable soft porous microarchitecture, superb biomechanical properties, proper biocompatibility, etc. Injectable hydrogels are an exciting type of hydrogels that can be easily injected into the target sites using needles or catheters in a minimally invasive manner. The more comfortable use, less pain, faster recovery period, lower costs, and fewer side effects make injectable hydrogels more attractive to both patients and clinicians in comparison to non-injectable hydrogels. However, it is difficult to achieve an ideal injectable hydrogel using just a single material (i.e., polymer). This challenge can be overcome by incorporating nanofillers into the polymeric matrix to engineer injectable nanocomposite hydrogels with combined or synergistic properties gained from the constituents. This work aims to critically review injectable nanocomposite hydrogels, their preparation methods, properties, functionalities, and versatile biomedical and pharmaceutical applications such as tissue engineering, drug delivery, and cancer labeling and therapy. The most common natural and synthetic polymers as matrices together with the most popular nanomaterials as reinforcements, including nanoceramics, carbon-based nanostructures, metallic nanomaterials, and various nanosized polymeric materials, are highlighted in this review.
3D printing enables a better control over the microstructure of bone restoring constructs, addresses the challenges seen in the preparation of patient-specific bone scaffolds, and overcomes the bottlenecks that can appear in delivering drugs/growth factors promoting bone regeneration. Here, 3D printing is employed for the fabrication of an osteogenic construct made of hydrogel nanocomposites. Alginate dialdehyde-gelatin (ADA-GEL) hydrogel is reinforced by the incorporation of bioactive glass nanoparticles, i.e. mesoporous silica-calcia nanoparticles (MSNs), in two types of drug (icariin) loading. The composites hydrogel is printed as superhydrated composite constructs in a grid structure. The MSNs not only improve the mechanical stiffness of the constructs but also induce formation of an apatite layer when the construct is immersed in simulated body fluid (SBF), thereby promoting cell adhesion and proliferation. The nanocomposite constructs can hold and deliver icariin efficiently, regardless of its incorporation mode, either as loaded into the MSNs or freely distributed within the hydrogel. Biocompatibility tests showed that the hydrogel nanocomposites assure enhanced osteoblast proliferation, adhesion, and differentiation. Such optimum biological properties stem from superior biocompatibility of ADA-GEL, bioactivity of the MSNs, and the supportive effect of icariin in relation to cell proliferation and differentiation. Taken together, given the achieved structural and biological properties and effective drug delivery capability, the hydrogel nanocomposites show promising potential for bone tissue engineering.
The emergence of nanotechnology provides a new method for the development and application of nanomaterials in the medical field. Nanoparticles could promote the expression of vascular endothelial growth factor (VEGF), dilate the coronary artery, improve the cardiac mechanism and reduce the recurrence rate of angina pectoris. Nanoparticles have the advantages of large specific surface area, high surface reactivity, high catalytic efficiency and strong adsorption capacity, which provide a new method for biomedical research. This paper mainly studies the application of nanoparticles in the diagnosis and treatment of coronary artery disease under the intervention of exercise rehabilitation. Then the prepared nanoparticles were added into the medium for co culture. The effect of nanoparticles on coronary cells was observed. It was found that the detection of zinc in the extracellular medium and in the cell increased with the increase of the concentration of ZnO NPs, and the differences were statistically significant (F = 13012.34, P < 0.001; F = 7033.54, P < 0.001). The results showed that the nanoparticles could promote the expression of vascular endothelial growth factor (VEGF), dilate the coronary artery, improve the cardiac mechanism and reduce the recurrence rate of angina pectoris.
Alginate is a natural polysaccharide that is easily chemically modified or compounded with other components for various types of functionalities. The alginate derivatives are appealing not only because they are biocompatible so that they can be used in biomedicine or tissue engineering but also because of the prospering bioelectronics that require various biomaterials to interface between human tissues and electronics or to serve as electronic components themselves. The study of alginate-based materials, especially hydrogels, have repeatedly found new frontiers over recent years. In this Review, we document the basic properties of alginate, their chemical modification strategies, and the recent development of alginate-based functional composite materials. The newly thrived functions such as ionically conductive hydrogel or 3D or 4D cell culturing matrix are emphasized among other appealing potential applications. We expect that the documentation of relevant information will stimulate scientific efforts to further develop biocompatible electronics or smart materials and to help the research domain better address the medicine, energy, and environmental challenges faced by human societies.
Because of its ideal degradation rate and features, oxidized alginate (OA) is selected as one of the appropriate substitutes and has been introduced into hydrogels, microspheres, 3D–printed/composite scaffolds, membranes, and electrospinning and coating materials. By taking advantage of OA, the OA-based materials can be easily functionalized and deliver drugs or growth factors to promote tissue regeneration. In 1928, it was first found that alginate could be oxidized using periodate, yielding OA. Since then, considerable progress has been made in the research on the modification and application of alginate after oxidation. In this article, we summarize the key properties and existing applications of OA and various OA-based materials and discuss their prospects in regenerative medicine.
Leveraging 3D bioprinting for processing stem cell-laden biomaterials has unlocked a tremendous potential for fabricating living 3D constructs for bone tissue engineering. Even though several bioinks developed to date display suitable physicochemical properties for stem cell seeding and proliferation, they generally lack the nanosized minerals present in native bone bioarchitecture. To enable the bottom-up fabrication of biomimetic 3D constructs for bioinstructing stem cells pro-osteogenic differentiation, herein we developed multi-bioactive nanocomposite bioinks that combine the organic and inorganic building blocks of bone. For the organic component gelatin methacrylate (GelMA), a photocrosslinkable denaturated collagen derivative used for 3D bioprinting was selected due to its rheological properties display of cell adhesion moities to which bone tissue precursors such as human bone marrow derived mesenchymal stem cells (hBM-MSCs) can attach to. The inorganic building block was formulated by incorporating mesoporous silica nanoparticles functionalized with calcium, phosphate and dexamethasone (MSNCaPDex), which previously proven to induce osteogenic differentiation. The newly formulated photocrosslinkable nanocomposite GelMA bioink incorporating MSNCaPDex nanoparticles and laden with hBM-MSCs was sucessfully processed into a 3D bioprintable construct with structural fidelity and well dispersed nanoparticles throughout the hydrogel matrix. These nanocomposite constructs could induce the deposition of apatite in vitro, thus showing attractive bioactivity properties. Viability and differentiation studies showed that hBM-MSCs remained viable and exhibited osteogenic differentiation biomarkers when incorporated in GelMA/MSNCaPDex constructs and without requiring further biochemical nor mechanical stimuli. Overall, our nanocomposite bioink has demonstrated excellent processability via extrusion bioprinting into osteogenic constructs with potential application in bone tissue repair and regeneration.
Escalating bone graft scarcity and donor site morbidity worldwide are alarming reminders that highlight the need for alternatives to gold standard tissue rejuvenation methods. Over the last few decades, many efforts have been made in bone tissue engineering (BTE) to fabricate artificial bone transplants. Conventional BTE techniques do not render pertinent spatial organization of cells, and they fail in mimicking the extracellular matrix of native bone tissue. This setback can be overcome by using the emerging technology of three-dimensional bioprinting (3DBP). 3DBP is a state-of-the-art technology that provides accurate hierarchal biomaterial structures that accommodate live-cell patterning to mimic their native counterparts. Herein, we provide an overview on the recent progress of cell-laden 3DBP technologies and also discuss the various biomaterials utilized (natural polymers such as chitosan, collagen, gelatin, hyaluronic acid, and silk fibroin and synthetic polymers such as PCL, PVP, and ceramics) to engineer scaffolds with requisite structural, mechanical, and biological complexity. We also highlight some of the persisting challenges and the solutions to surmount them, paving the way for progress in the field. Finally, we discuss how the combination of novel modalities with 3DBP can pave the way for new frontiers, like four-dimensional bioprinting (4DBP), to bring customized, stimuli-responsive, and highly effective regenerative scaffolds to bone tissue engineering.
3D printing is a pioneering technology which has shown immense potential in different area of product design, manufacturing, engineering, and biology. This technology has evolved over the time which has led to the development of high-end 3D printing machines to obtain the product with high resolution. In all cases, the object to be printed is created using computer aided design (CAD) file which is the exported as a file to be printed. Different types of 3D printing techniques have shown enormous application in the area of medicine and surgery. 3D printing equipment's and technology is supposed to revolutionize the pharmaceutical industry by providing personalized and patient oriented products. Advances the in area of high-resolution 3D printing will complement the patient specific diagnostics and personalized medicine. Overall, this technology holds many promises for the future and is expected to advance the healthcare.
Native human tissues are supported by a viscoelastic extracellular matrix (ECM) that can adapt its intricate network to dynamic mechanical stimuli. To recapitulate the unique ECM biofunctionality, hydrogel design is shifting from typical covalent crosslinks toward covalently adaptable networks. To pursue such properties, herein hybrid polysaccharide‐polypeptide networks are designed based on dynamic covalent assembly inspired by natural ECM crosslinking processes. This is achieved through the synthesis of an amine‐reactive oxidized‐laminarin biopolymer that can readily crosslink with gelatin (oxLAM‐Gelatin) and simultaneously allow cell encapsulation. Interestingly, the rational design of oxLAM‐Gelatin hydrogels with varying aldehyde‐to‐amine ratios enables a refined control over crosslinking kinetics, viscoelastic properties, and degradability profile. The mechanochemical features of these hydrogels post‐crosslinking offer an alternative route for imprinting any intended nano‐ or microtopography in ECM‐mimetic matrices bearing inherent cell‐adhesive motifs. Different patterns are easily paved in oxLAM‐Gelatin under physiological conditions and complex topographical configurations are retained along time. Human adipose‐derived mesenchymal stem cells contacting mechanically sculpted oxLAM‐Gelatin hydrogels sense the underlying surface nanotopography and align parallel to the anisotropic nanoridge/nanogroove intercalating array. These findings demonstrate that covalently adaptable features in ECM‐mimetic networks can be leveraged to combine surface topography and cell‐adhesive motifs as they appear in natural matrices.
Due to the relatively poor cell-material interaction of alginate hydrogel, alginate-gelatin crosslinked (ADA-GEL) hydrogel was synthesized through covalent crosslinking of alginate di-aldehyde (ADA) with gelatin that supported cell attachment, spreading and proliferation. This study highlights the evaluation of the physico-chemical properties of synthesized ADA-GEL hydrogels of different compositions compared to alginate in the form of films. Moreover, in vitro cell-material interaction on ADA-GEL hydrogels of different compositions compared to alginate was investigated by using normal human dermal fibroblasts. Viability, attachment, spreading and proliferation of fibroblasts were significantly increased on ADA-GEL hydrogels compared to alginate. Moreover, in vitro cytocompatibility of ADA-GEL hydrogels was found to be increased with increasing gelatin content. These findings indicate that ADA-GEL hydrogel is a promising material for the biomedical applications in tissue-engineering and regeneration.
The encapsulation of living mammalian cells within a semi-permeable hydrogel matrix is an attractive procedure for many biomedical and biotechnological applications, such as xenotransplantation, maintenance of stem cell phenotype and bioprinting of three-dimensional scaffolds for tissue engineering and regenerative medicine. In this review, we focus on naturally derived polymers that can form hydrogels under mild conditions and that are thus capable of entrapping cells within controlled volumes. Our emphasis will be on polysaccharides and proteins, including agarose, alginate, carrageenan, chitosan, gellan gum, hyaluronic acid, collagen, elastin, gelatin, fibrin and silk fibroin. We also discuss the technologies commonly employed to encapsulate cells in these hydrogels, with particular attention on microencapsulation.
Current tissue engineering techniques have various drawbacks: they often incorporate uncontrolled and imprecise scaffold geometries, whereas the current conventional cell seeding techniques result mostly in random cell placement rather than uniform cell distribution. For the successful reconstruction of deficient tissue, new material engineering approaches have to be considered to overcome current limitations. An emerging method to produce complex biological products including cells or extracellular matrices in a controlled manner is a process called bioprinting or biofabrication, which effectively uses principles of rapid prototyping combined with cell-loaded biomaterials, typically hydrogels. 3D tissue printing is an approach to manufacture functional tissue layer-by-layer that could be transplanted in vivo after production. This method is especially advantageous for stem cells since a controlled environment can be created to influence cell growth and differentiation. Using printed tissue for biotechnological and pharmacological needs like in vitro drug-testing may lead to a revolution in the pharmaceutical industry since animal models could be partially replaced by biofabricated tissues mimicking human physiology and pathology. This would not only be a major advancement concerning rising ethical issues but would also have a measureable impact on economical aspects in this industry of today, where animal studies are very labor-intensive and therefore costly. In this review, current controlled material and cell positioning techniques are introduced highlighting approaches towards 3D tissue printing.
Achieving cell spreading and proliferation inside hydrogels that are compatible with microencapsulation technology represents a major challenge for tissue engineering scaffolding and for the development of three-dimensional cell culture models. In this study, microcapsules of 650-900 μm in diameter were fabricated from oxidized alginate covalently cross-linked with gelatine (AlGel). Schiff's base bond formed in AlGel, detected by Fourier transform infrared spectroscopy, which confirmed the cross-linking of oxidized alginate with gelatine. Biological properties of alginate based hydrogels were studied by comparing the viability and morphology of MG-63 osteosarcoma cells encapsulated in gelatine and RGD-modified alginate. We hypothesized that the presence of gelatine and RGD will support cell adhesion and spreading inside the microcapsules and finally, also vascular endothelial growth factor (VEGF) secretion. After 4 days of incubation, cells formed extensive cortical protrusions and after 2 weeks they proliferated, migrated, and formed cellular networks through the AlGel material. In contrast, cells encapsulated in pure alginate and in RGD-modified alginate formed spherical aggregates with limited cell mobility and VEGF secretion. Metabolic activity was doubled after 5 days of incubation, making AlGel a promising material for cell encapsulation.
Microencapsulation of cells by using biodegradable hydrogels offers numerous attractive features for a variety of biomedical applications including tissue engineering. This study highlights the fabrication of microcapsules from alginate-gelatin cross-linked hydrogel (ADA-GEL) and presents the evaluation of the physico-chemical properties of the new microcapsules which are relevant for designing suitable microcapsules for tissue engineering. Alginate di-aldehyde (ADA) was synthesized by periodate oxidation of alginate which facilitates crosslinking with gelatin through Schiff’s base formation between the free amino groups of gelatin and the available aldehyde groups of ADA. Formation of Schiff’s base in ADA-GEL and aldehyde groups in ADA was confirmed by FTIR and NMR spectroscopy, respectively. Thermal degradation behavior of films and microcapsules fabricated from alginate, ADA and ADA-GEL was dependent on the hydrogel composition. The gelation time of ADA-GEL was found to decrease with increasing gelatin content. Swelling ratio of ADA-GEL microcapsules of all compositions was significantly decreased, whereas the degradability was found to increase with increasing of gelatin ratio. Surface morphology of the ADA-GEL microcapsules was totally different to that of alginate and ADA microcapsules, observed by SEM. Two different buffer solutions (with and without calcium salt) have influence on the stability of microcapsules that drew a significant difference on gelatin release profile from ADA-GEL microcapsules in these two buffer solutions.
The present study investigates a new family of bioactive glass nanoparticles with and without Sr-doping focusing on the influence of the nanoparticles on human bone marrow stromal cells (hBMSCs) in vitro. The bioactive glass nanoparticles were fabricated by flame spray synthesis and a particle diameter of 30–35 nm was achieved. Glass nanoparticles were undoped (BG 13-93-0Sr) or doped with 5 wt% strontium (Sr) (BG 13-93-5Sr) and used at concentrations of 10 and 100 μg/cm² (particles per culture plate area), respectively. Cells were cultured for 14 days after which the samples were analysed regarding metabolic activity and expression of various bone-specific genes. Cell growth and morphology indicated the high cytocompatibility of the nanoparticulate bioactive glass. The presence of the nanoparticles enhanced cell growth compared to the plain polystyrene control group. At a concentration of 100 μg/cm², Sr-doped particles led to significantly enhanced gene expression of osteocalcin, collagen type 1 and vascular endothelial growth factor. Thus, Sr-doped nanoparticles showing a dose-dependent increase of osteogenic differentiation in hBMSCs are a promising biomaterial for bone regeneration purposes.
Inspired by the rolling of water drops over the lotus leaf, we propose a new biomimetic fabrication process for hydrogel and polymeric spheres over a superhydrophobic substrate. This method may be potentially applied in tissue engineering or as support for cell expansion, cell encapsulation and as spherical structures for controlled release of molecule.
New manufacturing technologies under the banner of rapid prototyping enable the fabrication of structures close in architecture
to biological tissue. In their simplest form, these technologies allow the manufacture of scaffolds upon which cells can grow
for later implantation into the body. A more exciting prospect is the printing and patterning in three dimensions of all the
components that make up a tissue (cells and matrix materials) to generate structures analogous to tissues; this has been termed
bioprinting. Such techniques have opened new areas of research in tissue engineering and regenerative medicine.
Organ printing can be defined as layer-by-layer additive robotic biofabrication of three-dimensional functional living macrotissues and organ constructs using tissue spheroids as building blocks. The microtissues and tissue spheroids are living materials with certain measurable, evolving and potentially controllable composition, material and biological properties. Closely placed tissue spheroids undergo tissue fusion - a process that represents a fundamental biological and biophysical principle of developmental biology-inspired directed tissue self-assembly. It is possible to engineer small segments of an intraorgan branched vascular tree by using solid and lumenized vascular tissue spheroids. Organ printing could dramatically enhance and transform the field of tissue engineering by enabling large-scale industrial robotic biofabrication of living human organ constructs with "built-in" perfusable intraorgan branched vascular tree. Thus, organ printing is a new emerging enabling technology paradigm which represents a developmental biology-inspired alternative to classic biodegradable solid scaffold-based approaches in tissue engineering.
There is significant interest in the development of injectable carriers for cell transplantation to engineer bony tissues. In this study, we hypothesized that adhesion ligands covalently coupled to hydrogel carriers would allow one to control pre-osteoblast cell attachment, proliferation, and differentiation. Modification of alginate with an RGD-containing peptide promoted osteoblast adhesion and spreading, whereas minimal cell adhesion was observed on unmodified hydrogels. Raising the adhesion ligand density increased osteoblast proliferation, and a minimum ligand density (1.5-15 femtomoles/cm2) was needed to elicit this effect. MC3T3-E1 cells demonstrated increased osteoblast differentiation with the peptide-modified hydrogels, as confirmed by the up-regulation of bone-specific differentiation markers. Further, transplantation of primary rat calvarial osteoblasts revealed statistically significant increases of in vivo bone formation at 16 and 24 weeks with G4RGDY-modified alginate compared with unmodified alginate. These findings demonstrate that biomaterials may be designed to control bone development from transplanted cells.
The injectable polymer scaffolds which are biocompatible and biodegradable are important biomaterials for tissue engineering and drug delivery. Hydrogels derived from natural proteins and polysaccharides are ideal scaffolds for tissue engineering since they resemble the extracellular matrices of the tissue comprised of various amino acids and sugar-based macromolecules. Here, we report a new class of hydrogels derived from oxidized alginate and gelatin. We show that periodate-oxidized sodium alginate having appropriate molecular weight and degree of oxidation rapidly cross-links proteins such as gelatin in the presence of small concentrations of sodium tetraborate (borax) to give injectable systems for tissue engineering, drug delivery and other medical applications. The rapid gelation in the presence of borax is attributed to the slightly alkaline pH of the medium as well as the ability of borax to complex with hydroxyl groups of polysaccharides. The effect of degree of oxidation and concentration of alginate dialdehyde, gelatin and borax on the speed of gelation was examined. As a general rule, the gelling time decreased with increase in concentration of oxidized alginate, gelatin and borax and increase in the degree of oxidation of alginate. Cross-linking parameters of the gel matrix were studied by swelling measurements and trinitrobenzene sulphonic acid (TNBS) assay. In general, the degree of cross-linking was found to increase with increase in the degree of oxidation of alginate, whereas the swelling ratio and the degree of swelling decreased. The gel was found to be biocompatible and biodegradable. The potential of the system as an injectable drug delivery vehicle and as a tissue-engineering scaffold is demonstrated by using primaquine as a model drug and by encapsulation of hepatocytes inside the gel matrix, respectively.
Developments in mesoporous ceramics in drug delivery, nanomedicine, and bone tissue regeneration have opened promising developments in biomedical research, many applicable in the clinic in the near future. Due to the ability to fine-tune the physicochemical properties of these materials, the field has experienced an impressive burst in the number of publications. As controlled drug delivery systems are one of the most promising applications for human health care, it is now necessary to set the milestones of this technology, from the very basic to the most advanced. Biomedical Applications of Mesoporous Ceramics: Drug Delivery, Smart Materials and Bone Tissue Engineering is a comprehensive overview of silica-based mesoporous materials with special attention given to their use in drug delivery systems, sophisticated stimuli-responsive materials, and bone tissue engineering. The book provides a comprehensive overview of the basic aspects of the properties of mesoporous materials, with a focus on textural properties such as surface and porosity. Starting from this consolidated knowledge, it then addresses various aspects of more sophisticated stimuli-responsive materials and bone tissue engineering, detailing the research and development of these biomedical applications.
There is a growing demand for three-dimensional scaffolds for expanding applications in regenerative medicine, tissue engineering, and cell culture techniques. The material requirements for such three-dimensional structures are as diverse as the applications themselves. A wide range of materials have been investigated in the recent decades in order to tackle these requirements and to stimulate the anticipated biological response. Among the most promising class of materials are inorganic/organic hydrogel composites for regenerative medicine. The generation of synergetic effects by hydrogel composite systems enables the design of materials with superior properties including biological performance, stiffness, and degradation behavior in vitro and in vivo. Here, we review the most important organic and inorganic materials used to fabricate hydrogel composites. We highlight the advantages of combining different materials with respect to their use for biofabrication and cell encapsulation as well as their application as injectable materials for tissue enhancement and regeneration. [GRAPHICS] .
Using additive manufacturing to create hydrogel scaffolds which incorporate homogeneously distributed, immobilized cells in the context of biofabrication approaches represents an emerging and expanding field in tissue engineering. Applying hydrogels for additive manufacturing must consider the material processing properties as well as their influence on the immobilized cells. In this work alginate-dialdehyde (ADA), a partially oxidized alginate, was used as a basic material to improve the physico-chemical properties of the hydrogel for cell immobilization. At first, the processing ability of the gel using a bioplotter and the compatibility of the process with MG-63 osteoblast like cells were investigated. The metabolic and mitochondrial activities increased at the beginning of the incubation period and they balanced at a relatively high level after 14-28 days of incubation. During this incubation period the release of vascular endothelial growth factor-A also increased. After 28 days of incubation the cell morphology showed a spreading morphology and cells were seen to move out of the scaffold struts covering the whole scaffold structure. The reproducible processing capability of alginate-gelatine (ADA-GEL) and the compatibility with MG-63 cells were proven, thus the ADA-GEL material is highlighted as a promising matrix for applications in biofabrication.
Hydrogel-based biomaterials are ideal scaffolding matrices for microencapsulation, but they need to be modified to resemble the mechanical, structural and chemical properties of the native extracellular matrix. Here, we compare the mechanical properties and the degradation behavior of unmodified and modified alginate hydrogels in which cell adhesive functionality is conferred either by blending or covalently cross-linking with gelatin. Furthermore, we measure the spreading and proliferation of encapsulated osteoblast-like MG-63 cells. Alginate hydrogels covalently crosslinked with gelatin show the highest degree of cell adhesion, spreading, migration, and proliferation, as well as a faster degradation rate, and are therefore a particularly suitable material for microencapsulation.
Copyright © 2015. Published by Elsevier B.V.
Hierarchical polymeric carriers with high encapsulation efficiencies are fabricated via a biocompatible strategy developed using superhydrophobic (SH) surfaces. The carries are obtained by the incorporation of cell/BSA-loaded dextran-methacrylate (DEXT-MA) microparticles into alginate (ALG) macroscopic beads. Engineered devices like these are expected to boost the development of innovative and customizable systems for biomedical and biotechnological purposes.
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Biopolymeric hydrogels that mimic the properties of extracellular matrix have great potential in promoting cellular migration and proliferation for tissue regeneration. We have reported earlier that rapidly gelling, biodegradable, injectable hydrogels can be prepared by self-cross-linking of periodate oxidized alginate and gelatin in the presence of borax without using any toxic cross-linking agents. The present paper investigates the suitability of this hydrogel as a minimally invasive injectable, cell attractive and adhesive scaffold for cartilage tissue engineering for the treatment of osteoarthritis. Time and frequency sweep rheology analysis confirmed gel formation within 20 s. Hydrogel integrated well with the cartilage tissuehaving aburst pressure of 70 ± 3 mm of Hg indicating its adhesive nature. Hydrogel induced negligible inflammatory and oxidative stress responses, a prerequisite for the management and treatment of osteoarthritis. SEM images of primary murine chondrocytes encapsulated within the matrix revealed attachment of cells onto the hydrogel matrix. Chondrocytes demonstrated viability, proliferation and migration within the matrix, while maintaining their phenotype, as seen by expression of collagen type II and aggrecan and functionality, as seen by enhanced glycosoaminoglycan (GAG) deposition with time. DNA content and GAG deposition of chondrocytes within the matrix can be tuned by incorporation of bioactive signaling molecules such as dexamethasone, chondroitin sulphate, platelet derived growth factor (PDGF-BB) and combination of these three agents. The results suggest that self-cross-linked oxidized alginate/gelatin hydrogel may be a promising injectable, cell attracting adhesive matrix for neo-cartilage formation in the management and treatment of osteoarthritis.
There are a variety of both natural and synthetic polymeric systems that have been investigated for the controlled release of proteins. Many of the procedures employed to incorporate proteins into a polymeric matrix can be harsh and often cause denaturation of the active agent. Alginate, a naturally occurring biopolymer extracted from brown algae (kelp), has several unique properties that have enabled it to be used as a matrix for the entrapment and/or delivery of a variety of biological agents. Alginate polymers are a family of linear unbranched polysaccharides which contain varying amounts of 1,4'-linked beta-D-mannuronic acid and alpha-L-guluronic acid residues. The residues may vary widely in composition and sequence and are arranged in a pattern of blocks along the chain. Alginate can be ionically crosslinked by the addition of divalent cations in aqueous solution. The relatively mild gelation process has enabled not only proteins, but cells and DNA to be incorporated into alginate matrices with retention of full biological activity. Furthermore, by selection of the type of alginate and coating agent, the pore size, degradation rate, and ultimately release kinetics can be controlled. Gels of different morphologies can be prepared including large block matrices, large beads (>1 mm in diameter) and microbeads (<0.2 mm in diameter). In situ gelling systems have also been made by the application of alginate to the cornea, or on the surfaces of wounds. Alginate is a bioadhesive polymer which can be advantageous for the site specific delivery to mucosal tissues. All of these properties, in addition to the nonimmunogenicity of alginate, have led to an increased use of this polymer as a protein delivery system. This review will discuss the chemistry of alginate, its gelation mechanisms, and the physical properties of alginate gels. Emphasis will be placed on applications in which biomolecules have been incorporated into and released from alginate systems.
Nanosized bioactive glass (nBG) particles show high in vitro reactivity as a result of their high specific surface making them promising materials for bone tissue engineering. In this study, we investigate the in vitro reactivity of Sr-containing nanosized bioactive glass particles (type 1393) in simulated body fluid (SBF). Hydroxyapatite (HAp) formation was monitored by Fourier Transform Infrared (FT-IR) spectroscopy and X-ray diffraction (XRD) showing that HAp crystallization was delayed with increasing Sr-content due to the inhibitory effect of Sr on HAp mineralization. The HAp forming ability of bioactive glasses gives insight into their surface reactivity which is relevant for application of nanoscaled bioactive glass particles in bone regeneration.
Engineered cell instructive microenvironments with the ability to stimulate specific cellular responses are a topic of high interest in the fabrication and development of biomaterials for application in tissue engineering. Cells are inherently sensitive to the in vivo microenvironment that is often designed as the cell "niche." The cell "niche" comprising the extracellular matrix and adjacent cells, influences not only cell architecture and mechanics, but also cell polarity and function. Extensive research has been performed to establish new tools to fabricate biomimetic advanced materials for tissue engineering that incorporate structural, mechanical, and biochemical signals that interact with cells in a controlled manner and to recapitulate the in vivo dynamic microenvironment. Bioactive tunable microenvironments using micro and nanofabrication have been successfully developed and proven to be extremely powerful to control intracellular signaling and cell function. This Review is focused in the assortment of biochemical signals that have been explored to fabricate bioactive cell microenvironments and the main technologies and chemical strategies to encode them in engineered biomaterials with biological information.
The interaction between collagen and a natural derived cross-linker alginate dialdehyde (ADA) was investigated. Fourier transform infrared (FTIR) spectroscopy and the circular dichroism (CD) measurements indicate that the structure integrity of collagen is still maintained after the ADA treatment, while the differential scanning calorimetry (DSC) study suggests that ADA could promote collagen–ADA membrane's thermostability compared to pure collagen. And the atomic force microscopy (AFM) of cross-linked collagen reveals a denser network structure. Besides, the water contact angle test indicates that the hydrophilic property of collagen–ADA membrane is promoted, which is favorable for cell's attachment and proliferation. Meanwhile, the cytocompatibility results imply that not only no extra cytotoxicity is introduced into the collagen–ADA membrane after ADA treatment, but also collagen–ADA membrane facilitates cell's proliferation when the content of ADA is less than 20%. In conclusion, our study reveals that ADA stabilizes collagen as a cross-linker and preserves its triple helical structure.
Fifteen years ago, the field of cell and organ printing began with a few research groups looking to take newly developed direct-write tools and apply them to living cells. Initial experiments demonstrated cell viability and functionality post-deposition. Recently, research has begun in earnest to create three-dimensional cellular constructs that mimic both the heterogeneous structure and function of natural tissue. Several companies are now marketing cell printers, expanding access to a wider group of scientists and accelerating the pace of development. This article describes the past decade and a half of research by showing examples of some of the most sophisticated work, comparing the approaches and tools used in the field, and predicting the products that will arrive in the not too distant future.
Herein, we present a new type of biphasic organic/inorganic scaffold, which can be fabricated by multi-channel 3D plotting under mild conditions based on a highly concentrated alginate paste and a ready-to-use calcium phosphate cement (CPC) for bone and osteochondral tissue engineering. The structures of scaffolds were characterised by light and scanning electron microscopy (SEM). Results indicated that the concentrated alginate and CPC pastes had comparable plotting consistency, and therefore could be combined in one (biphasic) scaffold applying predesigned plotting parameters. After crosslinking of alginate and setting of CPC, the biphasic scaffold obtained mechanical and structural stability. Mechanical test data revealed that biphasic CPC/alginate scaffolds had significantly increased compressive strength and modulus compared to pure alginate as well as mixed calcium phosphate (CaP)/alginate scaffolds in wet state, and improved strength and toughness compared to pure CPC scaffolds in both dry and wet conditions. Culture of human mesenchymal stem cells (hMSC) on these scaffolds over 3 weeks demonstrated the good cytocompatibility of the selected materials. Because of the mild preparation conditions, bovine serum albumin (BSA) as a model protein was loaded in alginate and CPC pastes prior to plotting with high loading efficiency. Release studies in vitro showed that BSA released much faster from alginate strands than from CPC strands, which might allow amount-controlled protein release from biphasic CPC/alginate scaffolds. Furthermore, an upgraded bipartite osteochondral scaffold consisting of an alginate part for chondral and a biphasic CPC/alginate part for bony repair was fabricated based on this technique. This scaffold showed a strong organic/inorganic interface binding due to interlocking and crosslinking of the alginate strands.
Among the large number of applications of mesoporous MCM-41 materials, we have recently developed their new use as drug delivery system. Since this kind of materials consist on a disordered network of siloxane bridges and free silanol groups, these latter could be the reacting sites against appropriate guest chemical species. In this work, this application of mesoporous MCM-41 as drug delivery system has been studied from the host-guest interaction point of view. Two factors could affect that interaction: the structure of pore wall surface, and the functional groups present in the organic molecule. Hence, two approaches have been performed: functionalising pore wall groups and changing the drug.