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Three-Dimensional Printable Gelatin Hydrogels Incorporating Graphene Oxide to Enable Spontaneous Myogenic Differentiation
Abstract
Three-dimensional (3D) bioprinting has attracted considerable attention for producing 3D engineered cellular microenvironments that replicate complex and sophisticated native extracellular matrices (ECM) as well as the spatiotemporal gradients of numerous physicochemical and biological cues. Although various hydrogel-based bioinks have been reported, the development of advanced bioink materials that can reproduce the complexity of ECM accurately and mimic the intrinsic property of laden cells is still a challenge. This paper reports 3D printable bioinks composed of phenol-rich gelatin (GHPA) and graphene oxide (GO) as a component for a myogenesis-inducing material, which can form a hydrogel network in situ by a dual enzyme-mediated cross-linking reaction. The in situ curable GO/GHPA hydrogel can be utilized successfully as 3D-printable bioinks to provide suitable cellular microenvironments with facilitated myogenic differentiation of C2C12 skeletal myoblasts. Overall, we suggest that functional bioinks may be useful in muscle tissue engineering and regenerative medicine.
... According to Bhattacharyya et al., the bioink formulation must meet the following criteria: (1) the cells to be printed should be selected based on their viability during in vitro and in vivo measurements; (2) the polymer matrix or the additives incorporated into the polymeric composite should be biocompatible, and the additives should be bioactive to enhance the physicochemical and mechanical properties as well as the biofunctionalities of the printed gel; (3) the cross-linkage conditions should be amicable without stressing the printed cells, and they should not affect the cell survival rate after crosslinking; (4) the bioink layers should be able to maintain structural stability in the cell culture medium for a long time [93] . In the subsequent sections, we discuss the various CFNscontaining biomaterial inks and CFNs-containing bioinks, along with the CFNs' dimensions utilized, the specification of bioprinters, and the biological outcomes in various TE applications, as shown in Tables 1 [94][95][96][97][98][99][100][101][102][103] and 2 [97,[104][105][106][107][108] , respectively. ...
... Muscle tissue engineering [106] GO HCAECs showed cellular proliferation and lumen-like formation over 10 days of treatment. Myocardial tissue engineering [108] MWCNTs All the scaffolds demonstrated biocompatibility in MC3T3 and NIH3T3 cells up to 21 days of investigation. ...
... Additionally, we have fabricated a 3D-printable bioink that is combined with phenol-rich gelatin (GHPA), GO, and C2C12 myoblasts via a dual enzyme-mediated crosslinking reaction (glucose oxidase and horseradish peroxidase) for skeletal muscle TE [106] . The 3D-printed construct obtained via EBB retained its shape fidelity immediately after printing. ...
Biofabrication approaches, such as three-dimensional (3D) bioprinting of hydrogels, have recently garnered increasing attention, especially in the construction of 3D structures that mimic the complexity of tissues and organs with the capacity for cytocompatibility and post-printing cellular development. However, some printed gels show poor stability and maintain less shape fidelity if parameters such as polymer nature, viscosity, shear-thinning behavior, and crosslinking are affected. Therefore, researchers have incorporated various nanomaterials as bioactive fillers into polymeric hydrogels to address these limitations. Carbon-family nanomaterials (CFNs), hydroxyapatites, nanosilicates, and strontium carbonates have been incorporated into printed gels for application in various biomedical fields. In this review, following the compilation of research publications on CFNs-containing printable gels in various tissue engineering applications, we discuss the types of bioprinters, the prerequisites of bioink and biomaterial ink, as well as the progress and challenges of CFNs-containing printable gels in this field.
... Graphene oxide is known to trigger myogenesis [113]. Park KD et al. used a graphene oxide hydroxyphenyl propionic acid euhedral gelatin hydrogel (GO/GHPA) as a bioink to print structures with an optimal microenvironment for the growth and differentiation of myoblasts [114]. GO can enhance the adsorption of fibronectin and albumin, upregulate intercellular signals, and induce spontaneous myogenic differentiation of C2C12 cells. ...
As an emerging 3D printing technology, 3D bioprinting has shown great potential in tissue engineering and regenerative medicine. Decellularized extracellular matrices (dECM) have recently made significant research strides and have been used to create unique tissue-specific bioink that can mimic biomimetic microenvironments. Combining dECMs with 3D bioprinting may provide a new strategy to prepare biomimetic hydrogels for bioinks and hold the potential to construct tissue analogs in vitro, similar to native tissues. Currently, the dECM has been proven to be one of the fastest growing bioactive printing materials and plays an essential role in cell-based 3D bioprinting. This review introduces the methods of preparing and identifying dECMs and the characteristic requirements of bioink for use in 3D bioprinting. The most recent advances in dECM-derived bioactive printing materials are then thoroughly reviewed by examining their application in the bioprinting of different tissues, such as bone, cartilage, muscle, the heart, the nervous system, and other tissues. Finally, the potential of bioactive printing materials generated from dECM is discussed.
... G is one of the two-dimensional (2D) nanomaterials that is composed of monolayered sp 2 -bonded hexagonal carbon atoms [87]. G is obtained by kinds of methods such as mechanical exfoliation, liquid phase exfoliation, chemical vapor deposition, electric arc production, and thermal decomposition [81]. ...
Recent advances in inorganic nanomaterial-based theranostics enabled imaging-guided molecular targeting and drug delivery, and various combinations of theranostic systems. The term “theranostics” is defined as diagnosis processed with therapy simultaneously with a specific connection between therapy and diagnosis. The inorganic nanomaterials, representatively carbon, metal, ceramic, and semiconductor-based nanomaterials, exhibit their unique characteristics to be used in theranostic applications. However, the unveiled human biosafety of nanomaterials for clinical use has become a major concern. Therefore, in this review, we compiled recent research on in vitro and in vivo biosafety of inorganic nanomaterials in various theranostic applications, along with a discussion of how the particle formulation, size, surface functionalization, test species, and test condition affect biocompatibility. Furthermore, the progress and challenges of the development of biocompatible inorganic nanomaterials for theranostic applications were discussed. In conclusion, with appropriate precautions on the biosafe condition to be administered, inorganic nanomaterials can be proposed to have excellent potential in the future theranostic application.
... For example, cues for differentiation could be recreated, by including specific catalysts in the matrix material, such as graphene. In recently published studies graphene was shown to induce spontaneous myogenic differentiation without additional chemical cues by enhancing the adsorption of fibronectin and albumin and upregulating intercellular signaling [67][68][69] . ...
Recent advances in tissue engineering and biofabrication technology have yielded a plethora of biological tissues. Among these, engineering of bioartificial muscle stands out for its exceptional versatility and its wide range of applications. From the food industry to the technology sector and medicine, the development of this tissue has the potential to affect many different industries at once. However, to date, the biofabrication of cultured meat, biorobotic systems, and bioartificial muscle implants are still considered in isolation by individual peer groups. To establish common ground and share advances, this review outlines application-specific requirements for muscle tissue generation and provides a comprehensive overview of commonly used biofabrication strategies and current application trends. By solving the individual challenges and merging various expertise, synergetic leaps of innovation that inspire each other can be expected in all three industries in the future. This review discusses the recent advances in the engineering of bioartificial muscle. The applications highlighted include cultured meat, biorobotics, and muscle implants.
... This was in line with what was observed elsewhere 15,42 and can be explained by the enhanced cell-cell contact that promotes an enhanced inter-cellular communication, as previously reported by others. 43 Thus, the potential of this construct to maturate into a functional contractile muscle surrogate will be further evaluated in the future. ...
The treatment of skeletal muscle defects is still a topic of noteworthy concern since surgical intervention is not capable of recovering muscle function. Herein, we propose myoblasts laden in laminin-inspired biofunctionalized gellan gum hydrogels as promising tissue-engineered skeletal muscle surrogates. Gellan gum-based hydrogels were developed by combining native gellan gum (GG) and GG tethered with laminin-derived peptides (CIKVAVS (V), KNRLTIELEVRTC (T) or RKRLQVQLSIRTC (Q)), using different polymer content (0.75%-1.875%). Hydrogels were characterized in terms of compressive modulus, molecules trafficking, and C2C12 adhesion. Hydrogels with higher polymeric content (1.125%-1.875%) showed higher stiffness whereas hydrogels with lower polymer content (0.75%-1.125%) showed higher fluorescein isothiocyanate-dextran molecules diffusion. Cell spreading was achieved regardless of the laminin-derived peptide but preferred in hydrogels with higher polymer content (1.125%-1.875%). Taken together, hydrogels with 1.125% of polymer content were selected for printability analysis. GG-based inks showed a non-newtonian, shear-thinning, and thixotropic behavior suitable for printing. Accordingly, all inks were printable, but inks tethered with T and Q peptides presented some signs of clogging. Cell viability was affected after printing but increased after 7 days of culture. After 7 days, cells were spreading but not showing significant signs of cell-cell communications. Therefore, cell density was increased, thus, myocytes loaded in V-tethered GG-based inks showed higher cell-cell communication, spreading morphology, and alignment 7, 14 days post-printing. Overall, myoblasts laden in laminin-inspired biofunctionalized GG-based hydrogels are a promising skeletal muscle surrogate with the potential to be used as in vitro model or explored for further in vivo applications.
... Three-dimensional bioprinted phenol-rich gelatin/ graphene oxide hydrogel as a component for a myogenesis-inducing material is considered as potential materials in muscle tissue engineering and regenerative medicine application [116]. Bioderived aliphatic hyperbranched polyurethane/3-aminopropyltriethoxysilane-modified graphene oxide nanocomposites produce smart materials with considerable enhanced mechanical and selfhealing properties under microwave and sunlight. ...
Graphene-based materials are widely applied due to their interesting physical and chemical properties, but their hydrophobic surface and toxicity to living creatures limit their application in some fields. Biopolymers are incorporated with graphene-based materials to overcome these issues and improve their biodegradability, biocompatibility, and ecological friendliness, and the synergetic effect enhances other properties as well. These properties make graphene-based materials a novel subject of interest in science and industry. In this study, the various applications of developed biopolymer/graphene-based composites are broadly addressed, and recent progress in the field is emphasized. Modification, stability, and compatibility are among the key merits for developing highly advanced composites with desirable properties. The major challenges and some recommendations in various applications based on reviewed studies are covered. However, the development of environmentally friendly, low-cost, high-quality, and large-scale biopolymer/graphene-based composites for specified applications is challenging. Studies based on application and trend are conducted. Opportunities and limitations can guide researchers in the field to solve challenges, provide directions for future studies, and optimize sustainable biopolymer/graphene-based composites for specified industrial applications.
... The family of G related materials includes graphene oxide (GO), and reduced graphene oxide (rGO), obtained after GO reduction [16]. Graphene-based composites have been suggested as promising novel biomaterials in the field of regenerative medicine [19], particularly in skeletal muscle regeneration [20][21][22][23]. Composites based on natural polymers, such as gelatin with graphene oxide (GO) nanosheets [24] and aligned polysaccharides with graphene fibers [25], have been shown to promote spontaneous differentiation of C2C12 myoblasts. ...
Graphene derivatives such as reduced graphene oxide (rGO) are used as components of novel biomaterials for their unique electrical properties. Electrical conductivity is a crucial factor for muscle cells, which are electrically active. This study reports the development of a new type of semi-interpenetrated polymer network based on two biodegradable FDA-approved biomaterials, sodium alginate (SA) and polycaprolactone (PCL), with Ca²⁺ ions as SA crosslinker. Several drawbacks such as the low cell adhesion of SA and weak structural stability can be improved with the incorporation of PCL. Furthermore, this study demonstrates how this semi-IPN can be engineered with rGO nanosheets (0.5 and 2% wt/wt rGO nanosheets) to produce electroactive nanohybrid composite biomaterials. The study focuses on the microstructure and the enhancement of physical and biological properties of these advanced materials, including water sorption, surface wettability, thermal behaviour and thermal degradation, mechanical properties, electrical conductivity, cell adhesion and myogenic differentiation. The results suggest the formation of a complex nano-network with different interactions between the components: bonds between SA chains induced by Ca²⁺ ions (egg-box model), links between rGO nanosheets and SA chains as well as between rGO nanosheets themselves through Ca²⁺ ions, and strong hydrogen bonding between rGO nanosheets and SA chains. The incorporation of rGO significantly increases the electrical conductivity of the nanohybrid hydrogels, with values in the range of muscle tissue. In vitro cultures with C2C12 murine myoblasts revealed that the conductive nanohybrid hydrogels are not cytotoxic and can greatly enhance myoblast adhesion and myogenic differentiation. These results indicate that these novel electroactive nanohybrid hydrogels have great potential for biomedical applications related to the regeneration of electroactive tissues, particularly in skeletal muscle tissue engineering.
... Kang et al. [111] GO/GelMA/PCL Nanofibers Peripheral nerve regeneration rGO improved the mechanical and electrical properties of the formulation and, at lower concentration of about 0.25-0.5 wt%, enhanced Schwann cell (RSC96) proliferation. ...
The field of tissue engineering is constantly evolving as it aims to develop bioengineered and functional tissues and organs for repair or replacement. Due to their large surface area and ability to interact with proteins and peptides, graphene oxides offer valuable physiochemical and biological features for biomedical applications and have been successfully employed for optimizing scaffold architectures for a wide range of organs, from the skin to cardiac tissue. This review critically focuses on opportunities to employ protein–graphene oxide structures either as nanocomposites or as biocomplexes and highlights the effects of carbonaceous nanostructures on protein conformation and structural stability for applications in tissue engineering and regenerative medicine. Herein, recent applications and the biological activity of nanocomposite bioconjugates are analyzed with respect to cell viability and proliferation, along with the ability of these constructs to sustain the formation of new and functional tissue. Novel strategies and approaches based on stem cell therapy, as well as the involvement of the extracellular matrix in the design of smart nanoplatforms, are discussed.
... In particular, polymeric, carbon-based, and metallic nanomaterials are of interest to develop novel antibacterial agents and often functionalized to have enhanced biocompatibility and bioactivity (Han and Atabaev 2020;Han and Chrzanowski 2018;. Recent advances in two-dimensional (2D) nanomaterials proposed their clinical potential including bioimaging, drug delivery, antimicrobial and antibacterial effect, and regenerative therapy (SelestinRaja et al. 2020;Lin et al. 2018;Lingamdinne et al. 2021;Kang et al. 2021). Especially, graphene and their derivatives are the most highlighted 2D nanomaterial in terms of their exceptional antibacterial property. ...
Graphene is sp2-hybridized carbon structure-based two-dimensional (2D) sheet. Graphene-based nanomaterials possess several features such as unique mechanical, electronic, thermal, and optical properties, high specific surface area, versatile surface functionalization, and biocompatibility, which attracted researcher’s interests in various fields including biomedicine. In this chapter, we particularly focused on the biomedical imaging applications of graphene-based nanomaterials like graphene oxide (GO), reduced graphene oxide (rGO), graphene quantum dots (GQDs), graphene oxide quantum dots (GOQDs), and other derivatives, which utilize their outstanding optical properties. There are some biomedical imaging modalities using Graphene-based Nanomaterials, among which we will highlight fluorescence imaging, Raman imaging, magnetic resonance imaging, and photoacoustic imaging. We also discussed the brief perspectives and future application related to them.
... The positive charge on CS can inhibit the growth and reproduction of some bacteria and viruses, which is beneficial to reduce the infection (Abid et al., 2019;Hao et al., 2021;Yin et al., 2021). The three-dimensional network structure of chitosan temperature-sensitive hydrogels can provide a good microenvironment for cell proliferation, migration, and differentiation and can guide the long entry of host cells Kang et al., 2021;Sordi et al., 2021;Yang et al., 2021). Sodium alginate (SA) is a natural polysaccharide that is a byproduct of iodine and mannitol extraction from the brown algae kelp or sargassum, which has promising biocompatible and biodegradable (Mujtaba and Alotaibi, 2020). ...
This study aims to explore the feasibility of the novel temperature-sensitive hydrogel-based dual sustained-release system (Van/SBA-15/CS-GP-SA) in the repair and treatment of infectious jaw defects. Van/SBA-15 was prepared using the mesoporous silica (SBA-15) as a carrier for vancomycin hydrochloride (Van), and Van/SBA-15 was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersive spectrometry (EDS), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FTIR), Brunauer–Emmett–Teller (BET), and Barrett–Joyner–Halenda (BJH). The characterization results confirm that Van is loaded in SBA-15 successfully. Van/SBA-15/CS-GP-SA is constructed by encapsulating Van/SBA-15 in chitosan–sodium glycerophosphate–sodium alginate hydrogel (CS-GP-SA). The microstructures, sustained-release ability, biocompatibility, and antibacterial properties of Van/SBA-15/CS-GP-SA were systematically studied. Van/SBA-15/CS-GP-SA is found to have promising sustained-release ability, outstanding biocompatibility, and excellent antibacterial properties. This study provides new ideas for the management of infectious jaw defects.
... This makes GO hydrophilic, readily dispersible in water and solvents, and easily modified to make composite materials [111]. Moreover, the functional moieties facilitate cell-matrix interaction between the implant surface and surrounding cells, enhancing cellular behaviors including adhesion, proliferation, migration, and differentiation into specific lineages [29,[111][112][113]. GO was incorporated on Y-Zr ceramics for dental implants application [79]. ...
While conventional dental implants focus on mechanical properties, recent advances in functional carbon nanomaterials (CNMs) accelerated the facilitation of functionalities including osteoinduction, osteoconduction, and osseointegration. The surface functionalization with CNMs in dental implants has emerged as a novel strategy for reinforcement and as a bioactive cue due to their potential for mechanical reinforcing, osseointegration, and antimicrobial properties. Numerous developments in the fabrication and biological studies of CNMs have provided various opportunities to expand their application to dental regeneration and restoration. In this review, we discuss the advances in novel dental implants with CNMs in terms of tissue engineering, including material combination, coating strategies, and biofunctionalities. We present a brief overview of recent findings and progression in the research to show the promising aspect of CNMs for dental implant application. In conclusion, it is shown that further development of surface functionalization with CNMs may provide innovative results with clinical potential for improved osseointegration after implantation.
Contemporary advances in three-dimensional (3D) bioprinting technologies have enabled the fabrication of tailored live 3D tissue mimetics. Furthermore, the development of advanced bioink materials has been highlighted to accurately reproduce the composition of a native extracellular matrix and mimic the intrinsic properties of laden cells. Recent research has shown that MXene is one of promising nanobiomaterials with osteogenic activity for bone grafts and scaffolds due to its unique atomic structure of three titanium layers between two carbon layers. In this study, the MXene-incorporated gelatin methacryloyl (GelMA) and hyaluronic acid methacryloyl (HAMA) (i.e., GelMA/HAMA-MXene) bioinks were prepared to explore if they have the potential to enable the spontaneous osteodifferentiation of human mesenchymal stem cells (hMSCs) when the hMSCs-laden GelMA/HAMA-MXene bioinks were 3D printed. The physicochemical and rheological characteristics of the GelMA/HAMA-MXene hydrogels were proven to be unprecedentedly favorable supportive matrices suited for the growth and survival of hMSCs. Furthermore, hMSCs were shown to spontaneously differentiate into osteoblasts within GelMA-HAMA/MXene composites to provide favorable microenvironments for osteogenesis. Therefore, our results suggest that the remarkable biofunctional advantages of the MXene-incorporated GelMA/HAMA bioink can be utilized in a wide range of strategies for the development of effective scaffolds in bone tissue regeneration.
In situ bioprinting provides a reliable solution to the problem of in vitro tissue culture and vascularization by printing tissue directly at the site of injury or defect and maturing the printed tissue using the natural cell microenvironment in vivo. As an emerging field, in situ bioprinting is based on computer-assisted scanning results of the defect site and is able to print cells directly at this site with biomaterials, bioactive factors, and other materials without the need to transfer prefabricated grafts as with traditional in vitro 3D bioprinting methods, and the resulting grafts can accurately adapt to the target defect site. However, one of the important reasons hindering the development of in situ bioprinting is the absence of suitable bioinks. In this review, we will summarize bioinks developed in recent years that can adapt to in situ printing scenarios at the defect site, considering three aspects: the in situ design strategy of bioink, the selection of commonly used biomaterials, and the application of bioprinting to different treatment scenarios.
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.
Gelatin's versatile functionalization offers prospects of facile and effective crosslinking as well as combining with other materials (e.g., metal nanoparticles, carbonaceous, minerals, and polymeric materials exhibiting desired functional properties) to form hybrid materials of improved thermo-mechanical, physio-chemical and biological characteristics. Gelatin-based hydrogels (GHs) and (nano)composite hydrogels possess unique functional features that make them appropriate for a wide range of environmental, technical, and biomedical applications. The properties of GHs could be balanced by optimizing the hydrogel design. The current review explores the various crosslinking techniques of GHs, their properties, composite types, and ultimately their end-use applications. GH's ability to absorb a large volume of water within the gel network via hydrogen bonding is frequently used for water retention (e.g., agricultural additives), and absorbency towards targeted chemicals from the environment (e.g., as wound dressings for absorbing exudates and in water treatment for absorbing pollutants). GH's controllable porosity makes its way to be used to restrict access to chemicals entrapped within the gel phase (e.g., cell encapsulation), regulate the release of encapsulated cargoes within the GH (e.g., drug delivery, agrochemicals release). GH's soft mechanics closely resembling biological tissues, make its use in tissue engineering to deliver suitable mechanical signals to neighboring cells. This review discussed the GHs as potential materials for the creation of biosensors, drug delivery systems, antimicrobials, modified electrodes, water adsorbents, fertilizers and packaging systems, among many others. The future research outlooks are also highlighted.
Recent advances in three‐dimensional (3D) bioprinting technologies enabled the fabrication of sophisticated live 3D tissue analogs. Although various hydrogel‐based bioink has been reported, the development of advanced bioink materials that can reproduce the composition of native extracellular matrix (ECM) accurately and mimic the intrinsic property of laden cells is still challenging. In this work, 3D printed skin equivalents incorporating hair follicle structures and epidermal‐papillary‐dermal layers are fabricated with gelatin methacryloyl (GelMA)/hyaluronic acid (HA) MA (HAMA) hydrogel (GelMA/HAMA) bioink. The composition of collagen and glycosaminoglycan (GAG) of native skin was recapitulated by adjusting the combination of GelMA and HAMA. The GelMA/HAMA bioink was proven to have excellent viscoelastic and physicochemical properties, 3D printability, cytocompatibility, and functionality to maintain the hair inductive potency and facilitated spontaneous hair pore development. Overall, we suggest that the GelMA/HAMA hydrogels can be promising candidates as bioinks for the 3D printing of skin equivalents with epidermal‐papillary‐dermal multi‐layers and hair follicle structures, and they might serve as a useful model in skin tissue engineering and regeneration.
Recently, several three-dimensional (3D) bioprinting techniques have emerged for the synthesis of 3D tissue analogs. Accordingly, many researchers have focused on the development of novel bioinks that can mimic the natural extracellular matrix with cytocompatibility and biofunctionality. Hyaluronic acid and collagen are the most abundant proteins in the extracellular matrix of the skin and are known to support several cellular behaviors. Herein, we developed tyramine-conjugated hyaluronic acid and collagen (HA-Tyr/Col-Tyr) hydrogel bioinks, which are photocrosslinkable in the presence of riboflavin and ammonium peroxydisulfate, to fabricate dermis-mimetic constructs. The physicochemical properties and 3D printability of the HA-Tyr/Col-Tyr hydrogel were examined. 3D printing of the lattice structure with the HA-Tyr/Col-Tyr hydrogel enabled a sophisticated micron-sized fine structure without any clogging or coagulation. Approximately 80% of normal human dermal fibroblasts (NHDFs) in the printed constructs were alive after 24 h of culture. Moreover, the 3D printed constructs supported 4.57-fold cell proliferation and 4.23-fold f-actin expansion over four days in culture, indicating that HA-Tyr/Col-Tyr hydrogels provide cytocompatible microenvironments. The findings of this study suggest that HA-Tyr/Col-Tyr hydrogels are promising candidates as bioinks for the 3D printing of dermis-mimetic constructs.
Recent research has shown that graphene as a novel “green” antibacterial material possess excellent antibacterial properties with no risk of bacterial resistance for daily life due to its physical damage-based bactericidal mechanism. Therefore, an increasing amount of research has been focused towards evaluating the antibacterial effects of graphene and graphene-based hybrid materials. In this chapter, we reviewed the antibacterial activity and mechanism of graphene-based nanomaterials and highlighted the importance of size, morphology, and composites in the application of antibacterial materials development. Finally, we made a summary and outlook on this research field.
Electric field stimulation has supported biophysical and biological cues for tissue regeneration approaches to affect cell morphology, alignment, and even cellular phenotypes types. Here, an innovative bioprinting approach supported by in situ electrical stiumlation (E‐printing) is used to fabricate a bioengineered skeletal muscle construct composed of human adipose stem cells and methacrylated decellularized extracellular matrix (dECM‐Ma) derived from porcine muscle. To obtain highly ordered myofiber‐like structures, various parameters of the printing process are optimized. The E‐printed structure exhibits higher cell viability and fully aligned cytoskeleton than the conventionally printed cell‐bearing structures, due to activation of voltage‐gated ion channels that affect various signaling pathways. When using the E‐printed structure, expression of myogenesis‐related genes is upregulated by 1.9–2.5‐fold higher than when using a dECM‐Ma structure produced without electrical stimulation. Furthermore, when implanted into a rat model of volumetric muscle loss, the structure yields outstanding myogenesis relative to the conventionally bioprinted structure. Using an innovative bioprinting approach supported by in situ electrical stimulation, a bioengineered skeletal muscle construct composed of human adipose stem cells and a methacrylated decellularized extracellular matrix derived from porcine muscle is fabricated. When implanted into a rat model of volumetric muscle loss, the structure yields outstanding myogenesis relative to the conventionally bioprinted structure.
The physiological process of muscle regeneration is quite limited due to low satellite cell quantity and also the inability to regenerate and reconstruct niche tissue. The purpose of the study was to examine whether a graphene oxide scaffold is able to stimulate myogenic progenitor cell proliferation and the endocrine functions of differentiating cells, and therefore, their active participation in the construction of muscle tissue. Studies were carried out using mesenchymal cells taken from 6-day-old chicken embryos and human umbilical vein endothelial cells (HUVEC) were used to assess angiogenesis. The graphene scaffold was readily colonized by myogenic progenitor cells and the cells dissected from heart, brain, eye, and blood vessels did not avoid the scaffold. The scaffold strongly induced myogenic progenitor cell signaling pathways and simultaneously activated proangiogenic signaling pathways via exocrine vascular endothelial growth factor (VEGF) secretion. The present study revealed that the graphene oxide (GO) scaffold initiates the processes of muscle cell differentiation due to mechanical interaction with myogenic progenitor cell.
Biofabrication can be a tool to three-dimensionally (3D) print muscle cells embedded inside hydrogel biomaterials, ultimately aiming to mimic the complexity of the native muscle tissue and to create in-vitro muscle analogues for advanced repair therapies and drug testing. However, to 3D print muscle analogues of high cell alignment and synchronous contraction, the effect of biofabrication process parameters on myoblast growth has to be understood. A suitable biomaterial matrix is required to provide 3D printability as well as matrix degradation to create space for cell proliferation, matrix remodelling capacity, and cell differentiation. We demonstrate that by the proper selection of nozzle size and extrusion pressure, the shear stress during extrusion-bioprinting of mouse myoblast cells (C2C12) can achieve cell orientation when using oxidized alginate-gelatin (ADA-GEL) hydrogel bionk. The cells grow in the direction of printing, migrate to the hydrogel surface over time, and differentiate into ordered myotube segments in areas of high cell density. Together, our results show that oxidized alginate gelatin hydrogel can be a simple and cost-efficient biodegradable bioink that allows the successful 3D bioprinting and cultivation of C2C12 cells in-vitro to study muscle engineering.
Thomas Distler et al 2020 Biofabrication 12 045005 | https://doi.org/10.1088/1758-5090/ab98e4 | The article has been published open-access under the CC BY 4.0 creative commons license | https://creativecommons.org/licenses/by/4.0/
3D printing technology has been widely explored for the rapid design and fabrication of hydrogels, as required by complicated soft structures and devices. Here, a new 3D printing method is presented based on the rheology modifier of Carbomer for direct ink writing of various functional hydrogels. Carbomer is shown to be highly efficient in providing ideal rheological behaviors for multifunctional hydrogel inks, including double network hydrogels, magnetic hydrogels, temperature‐sensitive hydrogels, and biogels, with a low dosage (at least 0.5% w/v) recorded. Besides the excellent printing performance, mechanical behaviors, and biocompatibility, the 3D printed multifunctional hydrogels enable various soft devices, including loadable webs, soft robots, 4D printed leaves, and hydrogel Petri dishes. Moreover, with its unprecedented capability, the Carbomer‐based 3D printing method opens new avenues for bioprinting manufacturing and integrated hydrogel devices. This paper describes a highly efficient rheology modifier—Carbomer (minimum content 0.5% w/v)—enabled approach for printing multifunctional hydrogels, including double network hydrogels, magnetic hydrogels, temperature‐sensitive hydrogels, and biocompatible hydrogels. Besides the excellent printing performance, mechanical behaviors, and biocompatibility, the printed multifunctional hydrogels enable various soft devices, including loadable webs, soft robots, four‐dimensional printed leaves, and hydrogel Petri dishes.
Abstract Inkjet printing is widely considered a promising strategy to pattern hydrogels and living cells into three-dimensional (3D) constructs that structurally resemble tissues in our body. However, this approach is currently constrained by the limited control over multi-component deposition: the variable droplet ejection characteristics of different bioinks and dispensing units make synchronized printing very challenging. This invariably results in artificial tissues that lack the complexity and function of their native counterparts. By careful optimization of the printing parameters for two different bioink formulations, here we report the inkjet-based 3D-patterning of hydrogels according to relatively complex blueprints. 3D printing of bioinks containing living cells resulted in high-resolution, multi-component living constructs. Finally, we describe a sacrificial material approach to inkjet print perfuseable channels for improved long-term cultures of larger samples. We believe that this work provides a foundation for the generation of more complex 3D tissue models by inkjet printing.
Black phosphorus (BP) is a monolayer/multilayer two-dimensional (2D) nanomaterial, which has recently emerged as one of the most attractive 2D nanomaterials due to its fascinating physicochemical and optoelectronical properties. Layered BP may have promising applications in biomedical fields, such as drug delivery, photodynamic/photothermal therapy and bioimaging, although its intrinsic toxicity has not been fully elucidated yet. In the present study, the cytotoxicological effects of layered BP on both cell metabolic activity and membrane integrity were investigated. Layered BPs were prepared using a modified ultrasonication-assisted solution method, and their physicochemical properties were characterized. The dose- and time-dependent cytotoxicity of layered BP was assessed against L-929 fibroblasts. Our findings indicate that the cytotoxicity of BPs is proportionally dependent on their concentration and exposure time, which is affected by the oxidative stress-mediated enzyme activity reduction and membrane disruption. On the other hand, layered BPs did not exhibit significant cytotoxicity at concentrations lower than 4 μg/mL. Therefore, it is suggested that layered BPs can be effectively utilized as therapeutic delivery carriers and imaging agents.
3D printing, an additive manufacturing based technology for precise 3D construction, is currently widely employed to enhance applicability and function of cell laden scaffolds. Research on novel compatible biomaterials for bioprinting exhibiting fast crosslinking properties is an essential prerequisite toward advancing 3D printing applications in tissue engineering. Printability to improve fabrication process and cell encapsulation are two of the main factors to be considered in development of 3D bioprinting. Other important factors include but are not limited to printing fidelity, stability, crosslinking time, biocompatibility, cell encapsulation and proliferation, shear-thinning properties, and mechanical properties such as mechanical strength and elasticity. In this review, we recite recent promising advances in bioink development as well as bioprinting methods. Also, an effort has been made to include studies with diverse types of crosslinking methods such as photo, chemical and ultraviolet (UV). We also propose the challenges and future outlook of 3D bioprinting application in medical sciences and discuss the high performance bioinks.
3D Bioprinting is an emerging technology to fabricate 3D living constructs and it has shown a great promise in tissue engineering. Bioinks are scaffold materials mixed with cells used by 3D bioprinting to form a required cell-laden structure. In this paper, a novel bioink made of Gelatin Methacrylamide (GelMA) and Collagen (Col) doped with Tyrosinase (Ty) is presented for 3D bioprinting of living skin tissues. Ty has dual functions: one to serve as an essential bioactive compound in the skin regeneration process and the other to serve as enzyme to facilitate the crosslink of Col and GelMA. Further, the enzyme crosslinking together with photocrosslinking can enhance the mechanical strength of the bioink. The experimental results show that the bioink is able to form stable 3D living constructs using the 3D bioprinting process. The cell culture has shown that three major cell lines: human melanocytes (HEM), human keratinocytes (HaCat) and human dermal fibroblasts (HDF) exhibit high cell viabilities. The viability of these 3 cell lines is above 90%. The proliferation and scratching test show that the Ty could enhance the proliferation of HEM, inhibit the growth and migration of HDF and did not affect HaCat significantly. The animal tests show that the doped bioinks using 3D bioprinting could help form epidermis and dermis and thus are with a high potential as a skin bioink.
Different concentrations of gold nanoparticles (Au NPs) have been doped to polyacrylamide/chitosan (PAM/CS) blend by casting method to demonstrate the effect of nano particles as a doped on the structural, morphological and thermal behavior for PAM/CS blend. Nanocomposite films were characterized before and after addition of the gold. The FT-IR measurement shows some changes in the chemical structure with increase of the gold nanoparticles. The X-ray diffraction indicated that degree of crystallinity for PAM/CS blend was decreased after adding the nano-gold. Transmission electron microscope (TEM) images of the samples contain the gold nanoparticles in PAM/CS matrices showed that the spherical particle has a size range nearly from 5 to 30 nm. The parameters of kinetic thermodynamic like activation energies, enthalpy, Gibbs free energy and entropy were calculated from the thermogravimetric (TG) curves. The thermal data irregularly decrease with increasing of gold nanoparticles. TG analysis showed the thermal stability of nanocomposites films after addition of Au NPs was enhanced.
We present a new strategy for the fabrication of artificial skeletal muscle tissue with functional morphologies based on an innovative 3D bioprinting approach. The methodology is based on a microfluidic printing head coupled to a co-axial needle extruder for high-resolution 3D bioprinting of hydrogel fibers laden with muscle precursor cells (C2C12). To promote myogenic differentiation, we formulated a tailored bioink with a photocurable semi-synthetic biopolymer (PEG-Fibrinogen) encapsulating cells into 3D constructs composed of aligned hydrogel fibers. After 3–5 days of culture, the encapsulated myoblasts started migrating and fusing, forming multinucleated myotubes within the 3D bioprinted fibers. The obtained myotubes showed high degree of alignment along the direction of hydrogel fiber deposition, further revealing maturation, sarcomerogenesis, and functionality. Following subcutaneous implantation in the back of immunocompromised mice, bioprinted constructs generated organized artificial muscle tissue in vivo. Finally, we demonstrate that our microfluidic printing head allows to design three dimensional multi-cellular assemblies with an exquisite compartmentalization of the encapsulated cells. Our results demonstrate an enhanced myogenic differentiation with the formation of parallel aligned long-range myotubes. The approach that we report here represents a robust and valid candidate for the fabrication of macroscopic artificial muscle to scale up skeletal muscle tissue engineering for human clinical application.
In skeletal muscles, levels and activity of Matrix MetalloProteinases (MMPs) and Tissue Inhibitors of MetalloProteinases (TIMPs) have been involved in myoblast migration, fusion and various physiological and pathological remodeling situations including neuromuscular diseases. This has opened perspectives for the use of MMPs' overexpression to improve the efficiency of cell therapy in muscular dystrophies and resolve fibrosis. Alternatively, inhibition of individual MMPs in animal models of muscular dystrophies has provided evidence of beneficial, dual or adverse effects on muscle morphology or function. We review here the role played by MMPs/TIMPs in skeletal muscle inflammation and fibrosis, two major hurdles that limit the success of cell and gene therapy. We report and analyze the consequences of genetic or pharmacological modulation of MMP levels on the inflammation of skeletal muscles and their repair in light of experimental findings. We further discuss how the interplay between MMPs/TIMPs levels, cytokines/chemokines, growth factors and permanent low-grade inflammation favor cellular and molecular modifications resulting in fibrosis.
Bioprinting is a process based on additive manufacturing from materials containing living cells. These materials, often referred to as bioink, are based on cytocompatible hydrogel precursor formulations, which gel in a manner compatible with different bioprinting approaches. The bioink properties before, during and after gelation are essential for its printability, comprising such features as achievable structural resolution, shape fidelity and cell survival. However, it is the final properties of the matured bioprinted tissue construct that are crucial for the end application. During tissue formation these properties are influenced by the amount of cells present in the construct, their proliferation, migration and interaction with the material. A calibrated computational framework is able to predict the tissue development and maturation and to optimize the bioprinting input parameters such as the starting material, the initial cell loading and the construct geometry. In this contribution relevant bioink properties are reviewed and discussed on the example of most popular bioprinting approaches. The effect of cells on hydrogel processing and vice versa is highlighted. Furthermore, numerical approaches were reviewed and implemented for depicting the cellular mechanics within the hydrogel as well as for prediction of mechanical properties to achieve the desired hydrogel construct considering cell density, distribution and material-cell interaction.
Recently, graphene and other graphene-based materials have become an essential part of composite science and technology. Their unique properties are not only restricted to graphene but also shared with derivative compounds like graphene oxide, reduced graphene oxide, functionalized graphene, and so forth. One of the most structurally important materials, graphene oxide (GO), is prepared by the oxidation of graphite. Though removal of the oxide groups can create vacancies and structural defects, reduced graphene oxide (rGO) is used in composites as effective filler similar to GO. Authors developed a new polyurethane nanocomposite using a derivative of grapheme, thermally reduced graphene oxide (rGO), to modify the matrix of polyurethane elastomers, by rGO.
Background:
Hydrogels can serve as three-dimensional (3D) scaffolds for cell culture and be readily injected into the body. Recent advances in the image technology for 3D scaffolds like hydrogels have attracted considerable attention to overcome the drawbacks of ordinary imaging technologies such as optical and fluorescence microscopy. Multiphoton microscopy (MPM) is an effective method based on the excitation of two-photons. In the present study, C2C12 myoblasts differentiated in 3D gelatin hydroxyphenylpropionic acid (GHPA) hydrogels were imaged by using a custom-built multiphoton excitation fluorescence microscopy to compare the difference in the imaging capacity between conventional microscopy and MPM.
Results:
The physicochemical properties of GHPA hydrogels were characterized by using scanning electron microscopy and Fourier-transform infrared spectroscopy. In addition, the cell viability and proliferation of C2C12 myoblasts cultured in the GHPA hydrogels were analyzed by using Live/Dead Cell and CCK-8 assays, respectively. It was found that C2C12 cells were well grown and normally proliferated in the hydrogels. Furthermore, the hydrogels were shown to be suitable to facilitate the myogenic differentiation of C2C12 cells incubated in differentiation media, which had been corroborated by MPM. It was very hard to get clear images from a fluorescence microscope.
Conclusions:
Our findings suggest that the gelatin-based hydrogels can be beneficially utilized as 3D scaffolds for skeletal muscle engineering and that MPM can be effectively applied to imaging technology for tissue regeneration.
A microvalve-based bioprinting system for the manufacturing of high-resolution, multimaterial 3D-structures is reported. Applying a straightforward fluid-dynamics model, the shear stress at the nozzle site can precisely be controlled. Using this system, a broad study on how cell viability and proliferation potential are affected by different levels of shear stress is conducted. Complex, multimaterial 3D structures are printed with high resolution. This work pioneers the investigation of shear stress-induced cell damage in 3D bioprinting and might help to comprehend and improve the outcome of cell-printing studies in the future.
For the effective bone regeneration with appropriate pathological/physiological properties, a variety of bone fillers have been adapted as a therapeutic treatment. However, the development of ideal bone fillers is still remained as a big challenge in clinical practice. The main aims of this study are i) fabrication of a highly porous PCL beads; and ii) the estimation of the potential use of the porous PCL beads as a bone filler through preliminary animal study.
The porous PCL beads with size range of 53 ~ 600 μm (425 ~ 500 μm dominantly) are fabricated by a spray/precipitation method using a double nozzle spray and PCL solution (in tetraglycol). The PCL beads show highly porous inner pore structure and the pores are interconnected with outer surface pores. For the preliminary animal study, we recognize that the porous PCL bead can induce the new bone formation from the outer surface of bone defect toward the bone marrow cavity through the bead matrix.
From the preliminary results, we can suggest that the highly porous PCL beads may be a promising candidate as a bone filler (scaffolding matrix) for the effective bone regeneration.
In recent days Zinc oxide (ZnO) attracted much interest due to its versatibility and size dependent opto-electronic properties. ZnO flake structures have been hydrothermally synthesized in the reaction of aqueous solution of cetyl trimethyl ammonium bromide (CTAB) and NaOH at 373.15 K. It was found that, the volume of added ammonia, surfactant (CTAB), and mixed solvent play crucial roles in morphological control of ZnO nanostructures. Based on the structural information of XRD and SEM, a growth mechanism was proposed for the formation of ZnO flake structures.
Additive manufacturing, otherwise known as three-dimensional (3D) printing, is driving major innovations in many areas, such as engineering, manufacturing, art, education and medicine. Recent advances have enabled 3D printing of biocompatible materials, cells and supporting components into complex 3D functional living tissues. 3D bioprinting is being applied to regenerative medicine to address the need for tissues and organs suitable for transplantation. Compared with non-biological printing, 3D bioprinting involves additional complexities, such as the choice of materials, cell types, growth and differentiation factors, and technical challenges related to the sensitivities of living cells and the construction of tissues. Addressing these complexities requires the integration of technologies from the fields of engineering, biomaterials science, cell biology, physics and medicine. 3D bioprinting has already been used for the generation and transplantation of several tissues, including multilayered skin, bone, vascular grafts, tracheal splints, heart tissue and cartilaginous structures. Other applications include developing high-throughput 3D-bioprinted tissue models for research, drug discovery and toxicology.
The aim of this present study was to provide a scaffold as a tool for the investigation of the effect of mechanical stimulation on three-dimensionally cultured cells. For this purpose, we developed an artificial self-assembling peptide (SPG-178) hydrogel scaffold. The structural properties of the SPG-178 peptide were confirmed by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and transmission electron microscopy (TEM). The mechanical properties of the SPG-178 hydrogel were studied using rheology measurements. The SPG-178 peptide was able to form a stable, transparent hydrogel in a neutral pH environment. In the SPG-178 hydrogel, mouse skeletal muscle cells proliferated successfully (increased by 12.4 ± 1.5 times during 8 days of incubation; mean ± SEM). When the scaffold was statically stretched, a rapid phosphorylation of ERK was observed (increased by 2.8 ± 0.2 times; mean ± SEM). These results demonstrated that the developed self-assembling peptide gel is non-cytotoxic and is a suitable tool for the investigation of the effect of mechanical stimulation on three-dimensional cell culture.
Myotonic dystrophy type 1 (DM1) is a dominant neuromuscular disorder caused by a trinucleotide (CTG) repeat expansion. Mutant DMPK 3'-untranslated region (3'-UTR) transcripts aggregate in nuclear foci and are thought to impose dominant-negative effects by interacting with RNA binding proteins. We demonstrated previously that the mutant 3'-UTR RNA disrupted C2C12 myoblast differentiation, and that the CUG expansion was necessary for this effect. Several proteins are known to interact with the CUG tract or the region 3' (distal) to it. Here, using a library of transfected C2C12 clones, we show that although transcripts containing a CUG expansion alone or a CUG expansion plus the distal region of the DMPK 3'-UTR accumulate into RNA foci, neither of these RNAs affect C2C12 myogenesis. Thus, RNA foci formation, and perturbation of any RNA binding factors involved in this process, are not sufficient to block myoblast differentiation. Interestingly, we found that transcripts containing expanded CUG tracts can form both nuclear and cytoplasmic RNA foci, demonstrating that factors involved in foci formation are present in the nucleus and cytoplasm. RNA analysis of myogenic markers revealed that the mutant DMPK 3'-UTR mRNA does not affect myoblast determination factors MyoD or Myf5, but significantly impedes upregulation of the differentiation factors myogenin and p21. C2C12 provide a good model to study adult muscle regeneration. Our observations in this system may be relevant to the lack of a regenerative response to continued muscle wasting in DM, and point to defects in early events in the myogenic response to muscle damage.
Tumor necrosis factor alpha (TNFalpha) has been implicated as a mediator of muscle wasting through nuclear factor kappa B (NF-kappaB) -dependent inhibition of myogenic differentiation. The aim of the present study was to identify the regulatory molecule(s) of myogenesis targeted by TNFalpha/NF-kappaB signaling. TNFalpha interfered with cell cycle exit and repressed the accumulation of transcripts encoding muscle-specific genes in differentiating C2C12 myoblasts. Overexpression of a p65 (RelA) mutant lacking the transcriptional activation domain attenuated the TNFalpha-mediated inhibition of muscle-specific gene transcription. The ability of muscle regulatory factor MyoD to induce muscle-specific transcription in 10T1/2 fibroblasts was also disrupted by wild-type p65, demonstrating that NF-kappaB transcriptional activity interferes with the function of MyoD. Inhibition of muscle-specific gene expression by TNFalpha was restored by overexpression of MyoD, whereas endogenous MyoD protein abundance and stability were reduced by TNFalpha through increased proteolysis of MyoD by the ubiquitin proteasome pathway. Last, the inhibitory effects of TNFalpha on myogenic differentiation were demonstrated in a mouse model of skeletal muscle regeneration, in which TNFalpha caused a delay in myoblast cell cycle exit. These results implicate that TNFalpha inhibits myogenic differentiation through destabilizing MyoD protein in a NF-kappaB-dependent manner, which interferes with skeletal muscle regeneration and may contribute to muscle wasting.
Cells respond directly to the chemical and topographical cues of the engineered substrate. To date, recent extensive studies have been witnessed on the wide development of biomimetic substrates that can regulate the cellular behaviors by establishing the specific cues of the substrate. It is well known that the topographical features with nanoscale and microscale strongly modulate the behaviors of cells, including adhesion, migration, proliferation, and differentiation. Herein, we present a simple and robust strategy to generate the patterned arrays of reduced graphene oxide (rGO) on a substrate to be used for the cellular interfaces. The rGO patterned arrays were prepared by an evaporative self-assembly process, which is a highly efficient technique for the controlled deposition of rGO sheets on a flat substrate. Such periodic patterned arrays of rGO could be utilized as a micron topographic substratum for living cell culture to observe the growth and alignment behaviors of C2C12 skeletal and vascular smooth muscle cells (VSMCs). The exquisite evaluations showed that both cells were regularly grown along the rGO patterned arrays leading to the well-defined contact guidance, but the only C2C12 myoblasts exhibited slightly higher level in the morphological alignment features to the rGO patterned arrays, compared to the VSMCs. Our findings suggest that the nanotextured thin films and patterned arrays of rGO can serve as promising biomimetic substrates for skeletal muscle cells and provide subtle effects on cellular morphology discriminating in their responses.
Hydrogels that mimic the composition and properties of the extracellular matrix have attracted great attention as potential polymeric scaffolds for tissue engineering applications. In this study, an injectable hydrogel composed of hyaluronic acid and gelatin was developed via the oxidative coupling reaction using horseradish peroxidase (HRP). The hydrogel was prepared by mixing two phenol-conjugated polymer solutions, hyaluronic acid-tyramine (HA-TA) and gelatin-hydroxyphenyl propionic acid (GH), in the presence of HRP and hydrogen peroxide (H202). The gelation rate and mechanical properties of composite hydrogel were controlled by adjusting the HRP and H202 concentrations, respectively Compared to the pure HA-TA hydrogels, the stability and cellular behaviors of composite hydrogel improved significantly In addition, the injectable hydrogel system showed good performance in 3D printing with high cell viability after one day of printing. The results suggest that enzymatically crosslinked HA-TA and GH hybrid (HA-TA/GH) composite of HA-TA and GH hydrogel has the potential as a material for tissue engineering and 3D printing biofabrication.
Three-dimensional (3D) printing enables the production of personalized tissue-engineered products with high tunability and complexity. It is thus an attractive and promising technology in the pharmaceutical and medical fields. Printable and biocompatible hydrogels are attractive materials for 3D printing applications because they offer favorable biomimetic environments for live cells, such as high water content, porous structure, bioactive molecule incorporation, and tunable mechanical properties and degradation rates. However, most conventional hydrogel materials are brittle and mechanically weak and hence cannot meet the mechanical needs for handling and soft and elastic tissue use. Thus, the development of printable, high-strength, and elastic hydrogel materials for 3D printing in tissue repair and regeneration is critical and interesting. In this review, we summarized the recent reports on high-strength and elastic hydrogels for printing use and categorized them into three groups, namely double-network hydrogels, nanocomposite hydrogels, and single-network hydrogels. The reinforcing mechanisms of these high-strength hydrogels and the strategies to improve their printability and biocompatibility were further discussed. These high-strength and elastic hydrogels may offer opportunities to accelerate the development of 3D printing technology and provide new insights for 3D-printed product design in biomedicine.
Statement of Significance
Biocompatible and biodegradable hydrogels are highly attractive in 3D printing because of their desirable printability and friendly environment for loading bioactive molecules and living cells. The development of high-strength and elastic hydrogels changes the conventional impression of weak and brittle hydrogels and provides new opportunities and inspirations for 3D printing and biomedical applications. In this review, we analyzed the hydrogel reinforcement mechanisms, summarized recent progresses in developing high-strength and elastic hydrogels for 3D printing, and discussed the strategies to improve the printability and biocompatibility of the hydrogel inks.
Graphene, two-dimensional (2D) carbon nanomaterial has received great attention owing to their advantageous characteristics, including superior electrical, chemical and physical properties. Especially, the exfoliated graphite with strong oxidants has been explored to prepare a suspension of individual 2D graphene oxide (GO) nanosheets dispersed in various types of solvents. This colloidal suspension can readily be employed in a wide range of promising potential applications. However, one major challenge is to assemble such individual nanosheets on defined areas of surfaces forming specific structures. Here, we developed a simple and robust one-step strategy to create highly regular ordered nanostructures composed of chemically reduced graphene oxide (rGO) nanosheet building blocks on SiO2/Si or glass substrate over large areas. An aqueous suspension of rGO nanosheets dispersed in a volatile solvent was subjected to a confined geometry to induce a spontaneous formation of rGO patterned arrays with unprecedented regularity by utilizing both a controlled coffee ring effect and consecutive stick-slip motions of rGO solutions during the drying process. As a result, the generation of ultrathin nanotextured films in the form of micropatterns (i.e., concentric gradient rGO coffee rings) was effectively tailored. As a biological approach, to fully utilize a patterned rGO associated with the nanoscale surface topography, we used these patterned rGO arrays as a biomimetic in vitro architecture to study interfacial interactions of living cells such as fibroblasts, myoblasts, and neuronal cells. The observed results show that the patterned rGO arrays can be used as manipulated cellular responsive templates to form living cell assemblies and related patterns that are useful for regenerative tissue engineering.
A bio-inspired hydrogel for 3D bioprinting of soft free-standing neural tissues is presented. The novel filler-free bioinks were designed by combining natural polymers for extracellular matrix biomimicry with synthetic polymers to endow desirable rheological properties for 3D bioprinting. Crosslinking of thiolated Pluronic F-127 with dopamine-conjugated (DC) gelatin and DC hyaluronic acid through a thiol-catechol reaction resulted in thermally gelling bioinks with Herschel-Bulkley fluid rheological behavior. Microextrusion 3D bioprinting was used to fabricate free-standing cell-laden tissue constructs. The bioinks exhibited flattened parabolic velocity profiles with tunable low shear regions. Two pathways were investigated for curing the bioink: chelation and photocuring. The storage modulus of the cured bioinks ranged from 6.7 to 11.7 kPa. The iron (III) chelation chemistry produced crosslinked neural tissues of relatively lower storage modulus than the photocuring approach. In vitro cell viability studies using the 3D bioprinted neural tissues showed that the cured bioink was biocompatible based on minimal cytotoxic response observed over seven days in culture relative to control studies using alginate hydrogels. Rodent Schwann cell-, rodent neuronal cell-, and human glioma cell-laden tissue constructs were printed and cultured over seven days and exhibited comparable viability relative to alginate bioink controls. The ability to fabricate soft, free-standing 3D neural tissues with low modulus has implications in the biofabrication of microphysiological neural systems for disease modeling as well as neural tissues and innervated tissues for regenerative medicine.
Three-dimensional (3D) bioprinting of biomaterials shows great potential for producing cell-encapsulated scaffolds to repair nerves after injury or disease. For this, preparation of biomaterials and bioprinting itself are critical to create scaffolds with both biological and mechanical properties appropriate for nerve regeneration, yet remain unachievable. This paper presents our study on bioprinting Schwann cell-encapsulated scaffolds using composite hydrogels of alginate, fibrin, hyaluronic acid, and/or RGD peptide, for nerve tissue engineering applications. For the preparation of composite hydrogels, suitable hydrogel combinations were identified and prepared by adjusting the concentration of fibrin based on the morphological spreading of Schwann cells. In bioprinting, the effects of various printing process parameters (including the air pressure for dispensing, dispensing head movement speed, and crosslinking conditions) on printed structures were investigated and, by regulating these parameters, mechanically-stable scaffolds with fully interconnected pores were printed. The performance of Schwann cells within the printed scaffolds were examined in terms of viability, proliferation, orientation, and ability to produce laminin. Our results show that the printed scaffolds can promote the alignment of Schwann cells inside scaffolds and thus provide haptotactic cues to direct the extension of dorsal root ganglion neurites along the printed strands, demonstrating their great potential for applications in the field of nerve tissue engineering.
In tissue engineering and regenerative medicine, a biomaterial provides mechanical support and biochemical signals to encourage cell attachment and modulate cell behaviour. Nature’s template for a biomaterial is the extracellular matrix (ECM). The ECM contains intrinsic biochemical and mechanical cues that regulate cell phenotype and function in development, in homeostasis and in response to injury. The use of ECM-based materials in biomedical research has advanced from coating cell culture plates with purified ECM components to the design of ECM-mimicking biomaterials and the engineering of decellularized tissues aimed at recapitulating the dynamics, composition and structure of the ECM. In this Review, we highlight important matrix properties and functions in the context of tissue engineering and regenerative medicine, consider techniques such as proteomics for the investigation of matrix structure and composition and discuss different engineering strategies for the design of matrix-mimicking biomaterials. Tissue, whole organ and cell culture decellularization approaches are examined for their potential to preserve the tissue-specific biochemical composition and ultrastructure of the ECM and for the development of biomaterials that promote the formation of functional tissues in clinical applications. Finally, we investigate challenges and opportunities of ECM biomaterials for the design of organotypic models to study disease progression, for the ex vivo creation of engineered tissue and for the clinical translation of functional tissue reconstruction strategies in vivo.
Hydrogels currently used in electroactive tissues (cardiac tissues, skeletal muscles, and nerves) have certain shortcomings such as a lack of electrical conductivity and adhesiveness, both of which play a key role in the success of hydrogels in biomedical applications. In this study, chitosan (CS)/graphene oxide (GO) composite hydrogels with self-adhesive and self-healing properties, as well as electrical conductivity, were prepared by the incorporation of the mussel-inspired protein polydopamine (PDA). During the oxidizing process of dopamine (DA), graphene oxide was reduced by PDA and dispersed into the hydrogel network to form electric pathways. The covalent bonds, supramolecular interactions, hydrogen bonding, and π-π stacking gave the CS/GO composite hydrogels high stability, strong mechanical behaviors, good adhesiveness, self-healing properties, and a fast recovery ability. The electrical conductivity of the CS/GO reached 1.22 mS/cm and the adhesive strength of the composite hydrogel increased by 300% compared to CS-DA hydrogels. Cell culture results demonstrated that the conductive CS/GO hydrogels enhanced the cell viability and proliferation of human embryonic stem cell-derived fibroblasts (HEF1) and cardiomyocytes (CMs) compared to CS-DA hydrogels. Moreover, CMs showed a faster spontaneous beating rate than those in the control groups. Our work demonstrates a simple approach to fabricating polydopamine-based, adhesive, conductive, self-healing, and fast-recovering hydrogels that have great potential in electroactive tissue engineering applications.
Processing of hydrogels represents a main challenge for the prospective application of additive manufacturing (AM) to soft tissue engineering. Furthermore, direct manufacturing of tissue precursors with a cell density similar to native tissues has the potential to overcome the extensive in vitro culture required for conventional cell-seeded scaffolds seeking to fabricate constructs with tailored structural and functional properties. In this work, we present a simple AM methodology that exploits the thermoresponsive behavior of a block copolymer (Pluronic®) as a means to obtain good shape retention at physiological conditions and to induce cellular alignment. Pluronic/alginate blends have been investigated as a model system for the processing of C2C12 murine myoblast cell line. Interestingly, C2C12 cell model demonstrated cell alignment along the deposition direction, potentially representing a new avenue to tailor the resulting cell histoarchitecture during additive manufacturing process. Furthermore, the fabricated constructs exhibited high cell viability, as well as a significantly improved expression of myogenic genes vs. conventional 2D cultures. This article is protected by copyright. All rights reserved.
The purpose of 3D bioprinting technology is to design and create functional 3D tissues or organs in situ for in vivo applications. 3D cell-printing, or additive biomanufacturing, allows the selection of biomaterials and cells (bioink), and the fabrication of cell-laden structures in high resolution. 3D cell-printed structures have also been used for applications such as research models, drug delivery and discovery, and toxicology. Recently, numerous attempts have been made to fabricate tissues and organs by using various 3D printing techniques. However, challenges such as vascularization are yet to be solved. This article reviews the most commonly used 3D cell-printing techniques with their advantages and drawbacks. Furthermore, up-to-date achievements of 3D bioprinting in in vivo applications are introduced, and prospects for the future of 3D cell-printing technology are discussed. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2017.
3D cell printing is an emerging technology for fabricating complex cell-laden constructs with precise and pre-designed geometry, structure and composition to overcome the limitations of 2D cell culture and conventional tissue engineering scaffold technology. This technology enables spatial manipulation of cells and biomaterials, also referred to as 'bioink', and thus allows study of cellular interactions in a 3D microenvironment and/or in the formation of functional tissues and organs. Recently, many efforts have been made to develop new bioinks and to apply more cell sources for better biocompatibility and biofunctionality. However, the influences of printing parameters on the shape fidelity of 3D constructs as well as on cell viability after the cell printing process have been poorly characterized. Furthermore, parameter optimization based on a specific cell type might not be suitable for other types of cells, especially cells with high sensibility. In this study, we systematically studied the influence of bioink properties and printing parameters on bioink printability and embryonic stem cell (ESC) viability in the process of extrusion-based cell printing, also known as bioplotting. A novel method was established to determine suitable conditions for bioplotting ESCs to achieve both good printability and high cell viability. The rheological properties of gelatin/alginate bioinks were evaluated to determine the gelation properties under different bioink compositions, printing temperatures and holding times. The bioink printability was characterized by a newly developed semi-quantitative method. The results demonstrated that bioinks with longer gelation times would result in poorer printability. The live/dead assay showed that ESC viability increased with higher printing temperatures and lower gelatin concentrations. Furthermore, an exponential relationship was obtained between ESC viability and induced shear stress. By defining the proper printability and acceptable viability ranges, a combined parameters region was obtained. This study provides guidance for parameter optimization and the fine-tuning of 3D cell printing processes regarding both bioink printability and cell viability after bioplotting, especially for easily damaged cells, like ESCs.
Horseradish peroxidase (HRP) and hydrogen peroxide (H2O2)-mediated crosslinking reaction has become an attractive method to create in situ forming hydrogels. While the crosslinking system has been widely utilized, there are certain issues require improvement to extend their biomedical applications, including creation of stiff hydrogels without compromising cytocompatibility due to initially high concentrations of H2O2. A gelatin-based hydrogels formed through a dual enzyme-mediated crosslinking reaction using HRP and glucose oxidase (GOx) as an H2O2-generating enzyme to gradually supply a radical source in HRP-mediated crosslinking reaction is reported. The physicochemical properties can be controlled by varying enzyme concentrations. Furthermore the hydrogel matrices provide 3D microenvironments for supporting the growth and spreading of human dermal fibroblasts with minimized cytotoxicity, despite the cells being encapsulated within stiff hydrogels. These hydrogels formed with HRP/GOx have great potential as artificial microenvironments for a wide range of biomedical applications.
Recently, numerous studies have focused on the development of scaffolds for skeletal tissue engineering and regeneration with various structures. Among various structures, in situ forming hydrogels have attracted considerable attention because they can provide three-dimensional microenvironments for cells, and their stiffness and elasticity can be easily controlled by physical or chemical means. Over the last decade, graphene oxide (GO) has been widely explored as a potential candidate for biomaterials because of its excellent physicochemical properties and outstanding biocompatibility. In this study, horseradish peroxide-reactive gelatin polymer (GH) hydrogels incorporated with GO were prepared and their physicochemical and biomechanical properties were characterized by scanning electron microscopy, Raman and Fourier transform-infrared spectroscopy, thermogravimetric analysis and rheological study. The cellular behaviors of the C2C12 myoblasts within the GO-incorporated GH (GH/GO) hydrogels were examined by a cell counting kit-8 assay and immunocytochemistry. GO was uniformly distributed inside the GH hydrogels without affecting their physicochemical and biomechanical properties. GH/GO hydrogels facilitated the myogenic differentiation of C2C12 cells without hindering their proliferation. These results suggest that GH/GO hydrogels can be exploited to craft a range of strategies for the development of promising scaffolds to accelerate skeletal tissue regeneration because of their potential to stimulate myogenesis.
This study explored the influence of polyethylene glycol-linked multi-walled carbon nanotube (PEG-CNT) films on skeletal myogenic differentiation of human mesenchymal stem cells (hMSCs). PEG-CNT films were prepared with nanoscale surface roughness, orderly arrangement of PEG-CNTs, high hydrophilicity and high mechanical strength. Notably, PEG-CNT films alone could direct the skeletal myogenic differentiation of hMSCs in the absence of myogenic induction factors. The quantitative real-time polymerase chain reaction (RT-PCR) showed that the non-induced hMSCs plated on the PEG-CNT films, compared to the negative control, presented significant up-regulation of general myogenic markers including early commitment markers of myoblast differentiation protein-1 (MyoD) and desmin, as well as a late phase marker of myosin heavy chain-2 (MHC). Corresponding protein analysis by immunoblot assays corroborated these results. Skeletal muscle-specific markers, fast skeletal troponin-C (TnC) and ryanodine receptor-1 (Ryr) were also significantly increased in the non-induced hMSCs on PEG-CNT films by RT-PCR. For these cells, the commitment to specific skeletal myoblasts was further proved by the absence of enhanced adipogenic, chondrogenic and osteogenic markers. This study elucidated that PEG-CNT films supported a dedicated differentiation of hMSCs into a skeletal myogenic lineage and can work as a promising material towards skeletal muscle injury repair.
The regeneration of functional skin remains elusive, due to poor engraftment, deficient vascularization, and excessive scar formation. Aiming to overcome these issues, the present study proposed the combination of a three-dimensional graphene foam (GF) scaffold loaded with bone marrow derived mesenchymal stem cells (MSCs) to improve skin wound healing. The GFs demonstrated good biocompatibility and promoted the growth and proliferation of MSCs. Meanwhile, the GFs loaded with MSCs obviously facilitated wound closure in animal model. The dermis formed in the presence of the GF structure loaded with MSCs was thicker and possessed a more complex structure at day 14 post-surgery. The transplanted MSCs correlated with upregulation of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), which may lead to neo-vascularization. Additionally, an anti-scarring effect was observed in the presence of the 3D-GF scaffold and MSCs, as evidenced by a downregulation of transforming growth factor-beta 1 (TGF-β1) and alpha-smooth muscle actin (α-SMA) together with an increase of TGF-β3. Altogether, the GF scaffold could guide the wound healing process with reduced scarring, and the MSCs were crucial to enhance vascularization and provided a better quality neo-skin. The GF scaffold loaded with MSCs possesses necessary bioactive cues to improve wound healing with reduced scarring, which may be of great clinical significance for skin wound healing.
Native surfaces of Al, Cu and Au are exposed to diluted solutions of 4,4′-methylene diphenyl diisocyanate (4,4′-MDI). The resulting 4,4′-MDI adsorption state is characterised by in situ infrared external reflection absorption spectroscopy. On Au, the adsorbate state differs from bulk but the interactions are only weak. On Al and Cu, rich spectral features and resistance against desorption indicate strong adhesion which is most likely chemical by nature. However, further quantum mechanical modelling is needed for final conclusions on the adhesion mechanism.
This study aims at generating highly aligned functional myotubes using graphene as the underlying scaffold. Graphene not only supports the growth of C2C12 muscle cells but also enhances its differentiation and leads to spontaneous patterning of myotubes.
Here we present Fourier transform infrared (FTIR) and Raman images of Steinernema kraussei nematode worms and, in conjunction with chemometric analysis. We also distinguished the biochemical differences associated with different regions of these images. The nematodes are complex, multicellular organisms that have a simple, but defined body plan with distinct digestive, muscular and reproductive systems. As such, nematodes have often been used as model organisms for the study of biological processes in higher organisms. We show that FTIR spectra, collected in transmission mode, contain information from the entire thickness of the nematode, but still exhibit biochemical differences reflecting different tissues or anatomical structures, such as the digestive tract. Raman spectroscopy, in contrast, can be used to investigate the distribution of biological molecules on one plane within a constrained depth of the nematode, allowing imaging of fine details within the body of the worm, including the distribution of lipid, protein and collagen. Together these vibrational spectroscopic techniques provide complementary, non-invasive and label-free information on the spatial distribution of biomolecules within multicellular organisms and, therefore, have potential future applications in developmental studies of such model organisms.
Graphene-based nanomaterials have received much attention in biomedical applications for drug/gene delivery, cancer therapy, imaging, and tissue engineering. Despite the capacity of 2D carbon materials as a nontoxic and implantable platform, their effect on myogenic differentiation has been rarely studied. We investigated the myotube formation on graphene-based nanomaterials, particularly graphene oxide (GO) and reduced graphene oxide (rGO). GO sheets were immobilized on amine-modified glass to prepare GO-modified glass, which was further reduced by hydrazine treatment for the synthesis of rGO-modified substrate. We studied the behavior, including adhesion, proliferation, and differentiation, of mouse myoblast C2C12 on unmodified, GO-, and rGO-modified glass substrates. According to our analyses of myogenic protein expression, multinucleate myotube formation, and expression of differentiation-specific genes (MyoD, myogenin, Troponin T, and MHC), myogenic differentiation was remarkably enhanced on GO, which resulted from serum protein adsorption and nanotopographical cues. Our results demonstrate the ability of GO to stimulate myogenic differentiation, showing a potential for skeletal tissue engineering applications.
In our postgenomic era, understanding of protein-protein interactions by characterizing the structure of the corresponding protein complex is becoming increasingly important. An important problem is that many protein complexes are only stable for a few minutes. Dissociation will occur when using the typical, time-consuming purification methods such as tandem affinity purification and multiple chromatographic separations. Therefore, there is an urgent need for a quick and efficient protein-complex purification method for 3D structure characterization. The graphene oxide (GO)·streptavidin complex is prepared via a GO·biotin·streptavidin strategy and used for affinity purification. The complex shows a strong biotin recognition capability and an excellent loading capacity. Capturing biotinylated DNA, fluorophores and Au nanoparticles on the GO·streptavidin complexes demonstrates the usefulness of the GO·streptavidin complex as a docking matrix for affinity purification. GO shows a high transparency towards electron beams, making it specifically well suited for direct imaging by electron microscopy. The captured protein complex can be separated via a filtration process or even via on-grid purification and used directly for single-particle analysis via cryo-electron microscopy. Therefore, the purification, sample preparation, and characterization are rolled into one single step.
The skeletal muscle extracellular matrix (ECM) plays an important role in muscle fiber force transmission, maintenance, and repair. In both injured and diseased states, ECM adapts dramatically, a property that has clinical manifestations and alters muscle function. Here we review the structure, composition, and mechanical properties of skeletal muscle ECM; describe the cells that contribute to the maintenance of the ECM; and, finally, overview changes that occur with pathology. New scanning electron micrographs of ECM structure are also presented with hypotheses about ECM structure–function relationships. Detailed structure–function relationships of the ECM have yet to be defined and, as a result, we propose areas for future study.
Significant enhancement in mechanical stiffness (10-200%) and fracture strength (approximately 50%) of graphene oxide paper, a novel paperlike material made from individual graphene oxide sheets, can be achieved upon modification with a small amount (less than 1 wt %) of Mg(2+) and Ca(2+). These results can be readily rationalized in terms of the chemical interactions between the functional groups of the graphene oxide sheets and the divalent metals ions. While oxygen functional groups on the basal planes of the sheets and the carboxylate groups on the edges can both bond to Mg(2+) and Ca(2+), the main contribution to mechanical enhancement of the paper comes from the latter.
Skeletal muscle differentiation requires a cascade of transcriptional events to control the spatial and temporal expression of muscle-specific genes. Until recently, muscle-specific transcription was primarily attributed to prototypic enhancer-binding factors, while the role of core promoter recognition complexes in directing myogenesis remained unknown. Here, we report the development of a purified reconstituted system to analyze the properties of a TAF3/TRF3 complex in directing transcription initiation at the Myogenin promoter. Importantly, this new complex is required to replace the canonical TFIID to recapitulate MyoD-dependent activation of Myogenin. In vitro and cell-based assays identify a domain of TAF3 that mediates coactivator functions targeted by MyoD. Our findings also suggest changes to CRSP/Mediator in terminally differentiated myotubes. This switching of the core promoter recognition complex during myogenesis allows a more balanced division of labor between activators and TAF coactivators, thus providing another strategy to accommodate cell-specific regulation during metazoan development.
Cell cycle progression in mammalian cells is strictly regulated by both integrin-mediated adhesion to the extracellular matrix and by binding of growth factors to their receptors. This regulation is mediated by G1 phase cyclin-dependent kinases (CDKs), which are downstream of signaling pathways under the integrated control of both integrins and growth factor receptors. Recent advances demonstrate a surprisingly diverse array of integrin-dependent signals that are channeled into the regulation of the G1 phase CDKs. Regulation of cyclin D1 by the ERK pathway may provide a paradigm for understanding how cell adhesion can determine cell cycle progression.
Surface properties of polymeric devices that are used to regenerate nervous damage are a point to be considered for axon regeneration in nerve system. In our previous studies, we prepared a wettability gradient on polyethylene (PE) surfaces using a corona discharge treatment from a knife-type electrode whose power increases gradually along the sample length. The PE surfaces were oxidized gradually with increasing power. The effect of surface wettability on the different types of cells has an important role for cell adhesion and proliferation. The purpose of this study is to investigate neurite formation on polymer surfaces with different wettability. Induction and growth of neurites from the rat pheochromocytoma (PC-12) cells attached on the polymer surfaces with different hydrophilicity were investigated using the wettability gradient PE surfaces prepared by a corona discharge treatment. Neurites were investigated for number and length of neurites in terms of surface wettability. It was observed that neurite formation of PC-12 cells was increased more onto the positions with moderate hydrophilicity of the wettability gradient surface than onto the more hydrophobic or hydrophilic positions. From those results, it could be assumed that initial adhesion of PC-12 cells was caused by more calf serum (CS) protein than nerve growth factor (NGF), whereas the neurite formation of PC-12 cells was caused by more NGF than CS protein. It follows from what has been said thus far that PC-12 cells are a differentiated neuronal phenotype with a long neurite at around the position 2.5 cm (water contact angle of about 55 deg). In conclusion, surface wettability plays an important role for neurite formation on the polymer surfaces for axon regeneration.
A biofunctional scaffold was constructed with human mesenchymal stem cells (hMSCs) encapsulated in polyelectrolyte complexation (PEC) fibers. Human MSCs were either encapsulated in PEC fibers and constructed into a fibrous scaffold or seeded on PEC fibrous scaffolds. The proliferation, chondrogenic and osteogenic differentiation of the encapsulated and seeded hMSCs were compared for a culture period of 5.5 weeks. Gene expression and extracellular matrix production showed evidences of chondrogenesis and osteogenesis in the cell-encapsulated scaffolds and cell-seeded scaffolds when the samples were cultured in the chondrogenic and osteogenic differentiation media, respectively. However, better cell proliferation and differentiation were observed on the hMSC-encapsulated scaffolds compared to the hMSC-seeded scaffolds. The study demonstrated that the cell-encapsulated PEC fibers could support proliferation and chondrogenic and osteogenic differentiation of the encapsulated-hMSCs. Together with our previous works, which demonstrated the feasibility of PEC fiber in controlled release of drug, protein and gene delivery, the reported PEC fibrous scaffold system will have the potential in composing a multi-component system for various tissue-engineering applications.
Our long-term goal is to develop an artificial implant as a conduit for axonal regeneration after peripheral nerve injury. In this study, biodegradable, aligned poly-epsilon-caprolactone (PCL) and collagen/PCL (C/PCL) nanofibers designed as guidance structures were produced by electrospinning and tested in cell culture assays. We compared fibers of 100% PCL with fibers consisting of a 25:75% C/PCL blend. To test their biocompatibility, assays of cell adhesion, survival, migration, effects on cell morphology, axonal growth and axonal guidance were performed. Both types of eletrospun fibers supported oriented neurite outgrowth and glial migration from dorsal root ganglia (DRG) explants. Schwann cell migration, neurite orientation, and process formation of Schwann cells, fibroblasts and olfactory ensheathing cells were improved on C/PCL fibers, when compared to pure PCL fibers. While the velocity of neurite elongation from DRG explants was higher on PCL fibers, analysis of isolated sensory neurons showed significantly better axonal guidance by the C/PCL material. The data demonstrate that electrospun fibers composed of a collagen and PCL blend represent a suitable substrate for supporting cell proliferation, process outgrowth and migration and as such would be a good material for artificial nerve implants.
The basic helix-loop-helix myogenic regulatory factors play critical roles in skeletal myogenesis. Among the myogenic regulatory factors (MRFs), MRF4 shows a biphasic expression pattern during the formation of myotomes, although its function remains unclear. In this study, we used BEF (spontaneously immortalized bovine embryonic fibroblast that shows myogenic differentiation by overexpression of MyoD) and C2C12 cells to investigate the function of MRF4. Ectopic expressions of MRF4 did not stimulate myogenic differentiation in the BEF and C2C12 cells, but did show a marked increase of cell proliferation, upregulation of cyclin E, and downregulation of p21WAF1. Furthermore, MRF4 was found to induce degradation of the MyoD protein, which acts as a transcriptional activator for p21WAF1, and thus indicates that MRF4 accelerates cell proliferation by suppressing MyoD-dependent p21WAF1 expression. However, forced expression of MyoD in the MRF4-overexpressing cells inhibited cell proliferation and partially induced myogenic differentiation, which suggests that MyoD is a potential negative intercessor of MRF4 in the regulation of the cell cycle. Taken together, these results indicate that MRF4 and MyoD play competitive roles in myogenesis by stimulating cell proliferation and differentiation, respectively.
- Schwartz M. A.