Gelatin Methacrylamide Research Articles

Pre-Conditioning Stem Cells in a Biomimetic Environment for Enhanced Cardiac Tissue Repair: In Vitro and In Vivo Analysis.

Stem cell-based therapies represent a valid approach to restore cardiac function due to their beneficial effect in reducing scar area formation and promoting angiogenesis. However, their translation into the clinic is limited by the poor differentiation and inability to secrete sufficient therapeutic factors. To address this issue, several strategies such as genetic modification and biophysical preconditioning have been used to enhance the efficacy of stem cells for cardiac tissue repair. In this study, a biomimetic approach was used to mimic the natural mechanical stimulation of the myocardium tissue. Specifically, human adipose-derived stem cells (hASCs) were cultured on a thin gelatin methacrylamide (GelMA) hydrogel disc and placed on top of a beating cardiomyocyte layer. qPCR studies and metatranscriptomic analysis of hASCs gene expression were investigated to confirm the correlation between mechanical stimuli and cardiomyogenic differentiation. In vivo intramyocardial delivery of pre-conditioned hASCs was carried out to evaluate their efficacy to restore cardiac function in mice hearts post-myocardial infarction. The cyclic strain generated by cardiomyocytes significantly upregulated the expression of both mechanotransduction and cardiomyogenic genes in hASCs as compared to the static control group. The inherent angiogenic secretion profile of hASCs was not hindered by the mechanical stimulation provided by the designed biomimetic system. Finally, in vivo analysis confirmed the regenerative potential of the pre-conditioned hASCs by displaying a significant improvement in cardiac function and enhanced angiogenesis in the peri-infarct region. Overall, these findings indicate that cyclic strain provided by the designed biomimetic system is an essential stimulant for hASCs cardiomyogenic differentiation, and therefore can be a potential solution to improve stem-cell based efficacy for cardiovascular repair.

Development of Organic/Inorganic Compatible and Sustainably Bioactive Composites for Effective Bone Regeneration.

In this paper, we demonstrate a strategy of covalently bonding bioactive molecules onto inorganic hydroxyapatite (HAp) to improve the compatibility between organic and inorganic components and endow the bone composites with sustainable bioactivity. Bone morphogenetic protein-2 (BMP-2) peptide covalently immobilized nano-hydroxyapatite (nHAp-BMP-2) is developed to preserve the bioactivity and slow the release of the BMP-2 peptide. Then nHAp-BMP-2 was further incorporated into an ultraviolet-curable mixture of gelatin methacrylamide (GelMA) and four-armed PEG methacrylamide (four-armed PEGMA) to form a Gel/(nHAp-BMP-2) composite. The hydrogen bonding between gelatin and BMP-2 on nHAp-BMP-2 enhanced the compatibility between inorganic and organic components. The Gel/(nHAp-BMP-2) composite exhibited superior biocompatibility caused by gelatin and nHAp-BMP-2, except in a two-dimensional cell culture, the hydrogel was also capable of a three-dimensional cell culture. In addition, the introduction of nHAp-BMP-2 had a positive influence on bone marrow mesenchymal stem cell proliferation, differentiation, and the subsequent calcification on the composite. After treatment of a rat calvarial defect model for 12 weeks, the Gel/(nHAp-BMP-2) group showed the largest new bone volume and the highest ratio of new bone (50.54 ± 13.51 mm3 and 64.38 ± 17.22%, respectively) compared to those of the other groups. These results demonstrate that this way of controlling BMP-2 release is effective and the Gel/(nHAp-BMP-2) composite has great potential in bone regeneration therapy.

Fabrication of a Double-Cross-Linked Interpenetrating Polymeric Network (IPN) Hydrogel Surface Modified with Polydopamine to Modulate the Osteogenic Differentiation of Adipose-Derived Stem Cells.

Hydrogel surface properties can be modified to form bioactive interfaces to modulate the osteogenic differentiation of stem cells. In this work, a hydrogel made of gelatin methacrylamide (GelMA) and alginate was designed and tested as a scaffold to control stem-cell osteogenic differentiation. The hydrogel's surface was treated with polydopamine (pDA) to create an adhesive layer for the adsorption of the osteoinductive drug dexamethasone (Dex). The presence of the pDA coating enhanced Dex adsorption and retention over 21 days. This effect resulted in a delay in the osteogenic differentiation of hASCs cultured on the hydrogel treated with a pDA layer.

Regulation Effects of Biomimetic Hybrid Scaffolds on Vascular Endothelium Remodeling.

The formation of complete and well-functioning endothelium is critical for the success of tissue-engineered vascular grafts yet remaining a fundamental challenge. Endothelium remodeling onto the lumen of tissue-engineered vascular grafts is affected by their topographical, mechanical, and biochemical characteristics. For meeting multiple requirements, composite strategies have recently emerged for fabricating hybrid scaffolds, where the integrated properties are tuned by varying their compositions. However, the underlying principle how the integrated properties of hybrid scaffolds regulate vascular endothelium remodeling remains unclear. To uncover the regulation effects of hybrid scaffolds on vascular endothelium remodeling, we prepared different biomimetic hybrid scaffolds using gelatin methacrylamide (GelMA) and poly-ε-caprolactone (PCL) and then investigated vascular endothelial cell responses on them. GelMA and PCL, respectively, conferred the resulting scaffolds with biomimetic bioactivity and mechanical properties, which were tuned by varying GelMA/PCL mass ratios (3:1, 1:1, or 1:3). On different GelMA/PCL hybrid scaffolds, distinct vascular endothelial cell responses were observed. Firm cell-scaffold/cell-cell interactions were rapidly established on the hybrid scaffolds with the highest mass ratio of bioactive GelMA. However, they were mechanically insufficient as vascular grafts. On the contrary, the scaffolds with the highest mass ratio of PCL showed significantly reinforced mechanical properties but poor biological performance. Between the two extremes, the scaffolds with the same GelMA/PCL mass ratio balanced the pros and cons of two materials. Therefore, they could meet the mechanical requirements of vascular grafts and support the early-stage vascular endothelial cell remodeling by appropriate biological signaling and mechanotransduction. This investigation experimentally proves that scaffold bioactivity is the dominant factor affecting vascular endothelial cell adhesion and remodeling, whereas mechanical properties are crucial factors for the integrity of endothelium. This work offers a universal design strategy for desirable vascular grafts for improved endothelium remodeling.

Tyrosinase-doped bioink for 3D bioprinting of living skin constructs

Three-dimensional bioprinting is an emerging technology for fabricating living 3D constructs, and it has shown 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 the 3D bioprinting of living skin tissues. Ty has the dual function of being an essential bioactive compound in the skin regeneration process and also as an enzyme to facilitate the crosslink of Col and GelMA. Further, 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 shows that three major cell lines: human melanocytes (HEM), human keratinocytes (HaCat) and human dermal fibroblasts (HDF) exhibit high cell viabilities. The viability of these three cell lines is above 90%. The proliferation and scratching test show that Ty can enhance the proliferation of HEM, inhibit the growth and migration of HDF and not affect HaCat significantly. Animal tests show that the doped bioinks for 3D bioprinting can help form an epidermis and dermis, and thus have high potential as a skin bioink.

Controlling Adult Stem Cell Behavior Using Nanodiamond-Reinforced Hydrogel: Implication in Bone Regeneration Therapy

Nanodiamonds (NDs) have attracted considerable attention as drug delivery nanocarriers due to their low cytotoxicity and facile surface functionalization. Given these features, NDs have been recently investigated for the fabrication of nanocomposite hydrogels for tissue engineering. Here we report the synthesis of a hydrogel using photocrosslinkable gelatin methacrylamide (GelMA) and NDs as a three-dimensional scaffold for drug delivery and stem cell-guided bone regeneration. We investigated the effect of different concentration of NDs on the physical and mechanical properties of the GelMA hydrogel network. The inclusion of NDs increased the network stiffness, which in turn augmented the traction forces generated by human adipose stem cells (hASCs). We also tested the ability of NDs to adsorb and modulate the release of a model drug dexamethasone (Dex) to promote the osteogenic differentiation of hASCs. The ND-Dex complexes modulated gene expression, cell area, and focal adhesion number in hASCs. Moreover, the integration of the ND-Dex complex within GelMA hydrogels allowed a higher retention of Dex over time, resulting in significantly increased alkaline phosphatase activity and calcium deposition of encapsulated hASCs. These results suggest that conventional GelMA hydrogels can be coupled with conjugated NDs to develop a novel platform for bone tissue engineering.

Structural analysis of photocrosslinkable methacryloyl-modified protein derivatives.

Biochemically modified proteins have attracted significant attention due to their widespread applications as biomaterials. For instance, chemically modified gelatin derivatives have been widely explored to develop hydrogels for tissue engineering and regenerative medicine applications. Among the reported methods, modification of gelatin with methacrylic anhydride (MA) stands out as a convenient and efficient strategy to introduce functional groups and form hydrogels via photopolymerization. Combining light-activation of modified gelatin with soft lithography has enabled the materialization of microfabricated hydrogels. So far, this gelatin derivative has been referred to in the literature as gelatin methacrylate, gelatin methacrylamide, or gelatin methacryloyl, with the same abbreviation of GelMA. Considering the complex composition of gelatin and the presence of different functional groups on the amino acid residues, both hydroxyl groups and amine groups can possibly react with methacrylic anhydride during functionalization of the protein. This can also apply to the modification of other proteins, such as recombinant human tropoelastin to form MA-modified tropoelastin (MeTro). Here, we employed analytical methods to quantitatively determine the amounts of methacrylate and methacrylamide groups in MA-modified gelatin and tropoelastin to better understand the reaction mechanism. By combining two chemical assays with instrumental techniques, such as proton nuclear magnetic resonance (1H NMR) and liquid chromatography tandem-mass spectrometry (LC-MS/MS), our results indicated that while amine groups had higher reactivity than hydroxyl groups and resulted in a majority of methacrylamide groups, modification of proteins by MA could lead to the formation of both methacrylamide and methacrylate groups. It is therefore suggested that the standard terms for GelMA and MeTro should be defined as gelatin methacryloyl and methacryloyl-substituted tropoelastin, respectively, to remain consistent with the widespread abbreviations used in literature.

Flexible oligomer spacers as the key to solid-state photopolymerization of hydrogel precursors

Efficient crosslinking of conventional photoreactive hydrogel precursors relies on the mobility of the reactive groups and is typically addressed from the liquid state. However, this represents a major limitation for many processing techniques of hydrogel materials. Herein, a model precursor is introduced that overcomes this challenge using a prepolymer designed to enable successful crosslinking in the solid state. The precursor is synthesized by connecting a flexible, mono-acrylated spacer to a semi-crystalline hydrophilic backbone polymer using a di-isocyanate linker. The resulting prepolymers exhibit excellent solid-state photoreactivity, even in the absence of a photo-initiator. As a proof of concept, the precursor has successfully been processed by: (1) solution electrospinning with subsequent solid-state photopolymerization (SSPP), (2) melt-based additive manufacturing with subsequent SSPP and (3) two-photon polymerization in the solid state. No cell adhesion takes place on bare crosslinked 3D-printed scaffolds whereas excellent cell adhesion is recovered after application of a gelatin methacrylamide coating. With this novel class of UV-reactive precursors unprecedented hydrogel processing avenues are opened.

Cellulose Nanofibers for the Enhancement of Printability of Low Viscosity Gelatin Derivatives

Inadequate rheological properties of gelatin methacrylamide (GelMA) were successfully improved by incorporating cellulose nanofibers (CNFs), such that the printed scaffolds could maintain their structural fidelity during the three-dimensional (3D) bio-printing process. The CNFs provided an outstanding shear thinning property, and the GelMA/CNF inks exhibited high zero shear viscosity and structural fidelity under a low dispensing pressure. After evaluating the printability, composite inks containing 2% w/v CNF were observed to have an optimal concentration of CNF to prepare 3D print stable constructs. Therefore, these inks were used to manufacture human nose and ear structures, producing highly porous structures in the printed composite hydrogels. Furthermore, the mechanical stability of the GelMA/CNF composite hydrogel was increased when CNFs were incorporated, which indicated that CNFs played an important role in enhancing the structural properties of the composite hydrogels. Additionally, the biocompatibility of CNF-reinforced hydrogels was evaluated using a fibroblast cell line.

Indirect Solid Freeform Fabrication of an Initiator-Free Photocrosslinkable Hydrogel Precursor for the Creation of Porous Scaffolds.

In the present work, a photopolymerized urethane-based poly(ethylene glycol) hydrogel is applied as a porous scaffold material using indirect solid freeform fabrication (SFF). This approach combines the benefits of SFF with a large freedom in material selection and applicable concentration ranges. A sacrificial 3D poly(ε-caprolactone) structure is generated using fused deposition modeling and used as template to produce hydrogel scaffolds. By changing the template plotting parameters, the scaffold channel sizes vary from 280 to 360 μm, and the strut diameters from 340 to 400 μm. This enables the production of scaffolds with tunable mechanical properties, characterized by an average hardness ranging from 9 to 43 N and from 1 to 6 N for dry and hydrated scaffolds, respectively. Experiments using mouse calvaria preosteoblasts indicate that a gelatin methacrylamide coating of the scaffolds results in an increased cell adhesion and proliferation with improved cell morphology.

Gelatin methacrylamide hydrogel with graphene nanoplatelets for neural cell-laden 3D bioprinting.

Nervous system is extremely complex which leads to rare regrowth of nerves once injury or disease occurs. Advanced 3D bioprinting strategy, which could simultaneously deposit biocompatible materials, cells and supporting components in a layer-by-layer manner, may be a promising solution to address neural damages. Here we presented a printable nano-bioink composed of gelatin methacrylamide (GelMA), neural stem cells, and bioactive graphene nanoplatelets to target nerve tissue regeneration in the assist of stereolithography based 3D bioprinting technique. We found the resultant GelMA hydrogel has a higher compressive modulus with an increase of GelMA concentration. The porous GelMA hydrogel can provide a biocompatible microenvironment for the survival and growth of neural stem cells. The cells encapsulated in the hydrogel presented good cell viability at the low GelMA concentration. Printed neural construct exhibited well-defined architecture and homogenous cell distribution. In addition, neural stem cells showed neuron differentiation and neurites elongation within the printed construct after two weeks of culture. These findings indicate the 3D bioprinted neural construct has great potential for neural tissue regeneration.

3D bioprinting of BM-MSCs-loaded ECM biomimetic hydrogels for in vitro neocartilage formation

In this work we demonstrate how to print 3D biomimetic hydrogel scaffolds for cartilage tissue engineering with high cell density (>107 cells ml−1), high cell viability (85 ÷ 90%) and high printing resolution (≈100 μm) through a two coaxial-needles system. The scaffolds were composed of modified biopolymers present in the extracellular matrix (ECM) of cartilage, namely gelatin methacrylamide (GelMA), chondroitin sulfate amino ethyl methacrylate (CS-AEMA) and hyaluronic acid methacrylate (HAMA). The polymers were used to prepare three photocurable bioinks with increasing degree of biomimicry: (i) GelMA, (ii) GelMA + CS-AEMA and (iii) GelMA + CS-AEMA + HAMA. Alginate was added to the bioinks as templating agent to form stable fibers during 3D printing. In all cases, bioink solutions were loaded with bone marrow-derived human mesenchymal stem cells (BM-MSCs). After printing, the samples were cultured in expansion (negative control) and chondrogenic media to evaluate the possible differentiating effect exerted by the biomimetic matrix or the synergistic effect of the matrix and chondrogenic supplements. After 7, 14, and 21 days, gene expression of the chondrogenic markers (COL2A1 and aggrecan), marker of osteogenesis (COL1A1) and marker of hypertrophy (COL10A1) were evaluated qualitatively by means of fluorescence immunocytochemistry and quantitatively by means of RT-qPCR. The observed enhanced viability and chondrogenic differentiation of BM-MSCs, as well as high robustness and accuracy of the employed deposition method, make the presented approach a valid candidate for advanced engineering of cartilage tissue.

Gelatin methacrylamide as coating material in cell culture

Unmodified gelatin (uG) is widely used as a coating material in cell culture for improving surface properties. In this study, the authors investigated if gelatin methacrylamide (GM) with a medium degree of methacrylamide modification (GM1.5) and a high degree of methacrylamide modification (GM4) are equally suitable for this purpose. Therefore, gold surfaces were coated with uG, GM1.5, and GM4 by adsorption of the polymers on the surfaces. Coating success was confirmed by spectroscopic ellipsometry, contact angle measurements, surface plasmon resonance spectroscopy (SPRS), and atomic force microscopy (AFM). The authors found that upon adsorption of uG, GM1.5, a nd GM4 on gold, thin films with thicknesses of 2.95 nm, 2.50 nm, and 2.26 nm were formed. The coated surfaces showed advancing contact angles of 46° (uG and GM1.5) and 52° (GM4) without alteration of the surface roughness determined by AFM. Protein adsorption taking place on the coated surfaces was measured during contact of the surfaces with fetal calf serum by SPRS. Protein adsorption on the coated surfaces was reduced by the factor of 6.4 (uG), 5.4 (GM1.5), and 4.6 (GM4) compared to gold surfaces. Human fibroblasts cultured on the surfaces showed excellent viability shown by water soluble tetrazolium salt assay as well as live/dead staining with propidium iodide and fluorescein diacetate. No cytotoxic effects of the GM coated surfaces were observed, giving rise to the conclusion that GMs are suitable materials as coatings in cell culture.

A Hydrogel Model Incorporating 3D-Plotted Hydroxyapatite for Osteochondral Tissue Engineering.

The concept of biphasic or multi-layered compound scaffolds has been explored within numerous studies in the context of cartilage and osteochondral regeneration. To date, no system has been identified that stands out in terms of superior chondrogenesis, osteogenesis or the formation of a zone of calcified cartilage (ZCC). Herein we present a 3D plotted scaffold, comprising an alginate and hydroxyapatite paste, cast within a photocrosslinkable hydrogel made of gelatin methacrylamide (GelMA), or GelMA with hyaluronic acid methacrylate (HAMA). We hypothesized that this combination of 3D plotting and hydrogel crosslinking would form a high fidelity, cell supporting structure that would allow localization of hydroxyapatite to the deepest regions of the structure whilst taking advantage of hydrogel photocrosslinking. We assessed this preliminary design in terms of chondrogenesis in culture with human articular chondrocytes, and verified whether the inclusion of hydroxyapatite in the form presented had any influence on the formation of the ZCC. Whilst the inclusion of HAMA resulted in a better chondrogenic outcome, the effect of HAP was limited. We overall demonstrated that formation of such compound structures is possible, providing a foundation for future work. The development of cohesive biphasic systems is highly relevant for current and future cartilage tissue engineering.

Quickly promoting angiogenesis by using a DFO-loaded photo-crosslinked gelatin hydrogel for diabetic skin regeneration.

Changes in blood vessel formation, especially microvasculature formation, are one of the most important factors contributing to the poor wound healing capabilities of diabetic patients. Furthermore, recovery of the vascular network in the early stages after injury is a key factor in the prevention of wound expansion and ulcer formation. A hydrogel is a popular scaffold type and has many biological advantages, however, it is incapable of rapidly recruiting angiogenesis-related cells and cytokines to the wound area under the disturbed microcirculatory conditions of diabetics. For the above reasons, we devised a desferrioxamine (DFO)-loaded photo-crosslinked hydrogel (gelatin methacrylamide (Gelma)) for quickly developing the vascular network and accelerating skin reconstruction. The controlled release of DFO peaking at 16 h followed by a steady release after 48 h through the swelling of the Gelma hydrogel led to a significant increase of neovascularization. The in vitro results showed that DFO-Gelma provided an excellent microenvironment for cell viability, adhesion and proliferation, and up-regulated the expression of HIF-1α, which was critical for blood vessel formation. The in vivo studies showed new blood vessels, high quality granulation tissues, and early epithelialization in wound beds by treating them with DFO-loaded hydrogels. Through this investigation, the mechanism associated with wound healing was further investigated. This study demonstrated that DFO-Gelma was safe, reliable, and highly effective for the diabetic wound healing process.

Biomimetic gelatin methacrylamide hydrogel scaffolds for bone tissue engineering.

Bone tissue engineering is an exciting research area that develops functional strategies for the most challenging bone related clinical issues. Among the numerous materials used in this area, biomimetic materials have been developed amazingly over the past decades. In this study, biomimetic gelatin methacrylamide (Bio-GelMA) hydrogel scaffolds have been fabricated to mimic both the physical architecture and chemical composition of the natural bone extracellular matrix (ECM) by using the thermally induced phase separation (TIPS) technique, to provide three-dimensional templates and extracellular matrix microenvironments. Adipose derived stem cells (ADSCs) also play a pivotal role in osteogenesis when co-cultured with Bio-GelMA at days 7, 14, and 21. The effects of cell-biomaterial interactions such as adhesion, proliferation, and osteogenic differentiation were systematically investigated. The results showed that Bio-GelMA significantly enhanced cell attachment and viability, alkaline phosphatase (ALP) activity, and mineral deposition, as well as mRNA expression levels of osteogenic genes of ADSCs. In a subcutaneous model, H&E staining and dual immunofluorescent staining differed significantly. More importantly, in a critical-size rat calvarial bone defect model, the results of Micro-CT, H&E staining and Masson trichrome staining confirmed that the combination of the Bio-GelMA and ADSCs could enhance osteogenesis significantly. Altogether, the observations prove that the Bio-GelMA scaffolds can act as cell carriers for ADSCs, promote greater osteogenic differentiation of ADSCs and may have great potential in future clinical applications.

Tailoring Hydrogel Viscoelasticity with Physical and Chemical Crosslinking

Biological tissues are viscoelastic, demonstrating a mixture of fluid and solid responses to mechanical strain. Whilst viscoelasticity is critical for native tissue function, it is rarely used as a design criterion in biomaterials science or tissue engineering. We propose that viscoelasticity may be tailored to specific levels through manipulation of the hydrogel type, or more specifically the proportion of physical and chemical crosslinks present in a construct. This theory was assessed by comparing the mechanical properties of various hydrogel blends, comprising elastic, equilibrium, storage and loss moduli, as well as the loss tangent. These properties were also assessed in human articular cartilage explants. It was found that whilst very low in elastic modulus, the physical crosslinks found in gellan gum-only provided the closest approximation of loss tangent levels found in cartilage. Blends of physical and chemical crosslinks (gelatin methacrylamide (GelMA) combined with gellan gum) gave highest values for elastic response. However, a greater proportion of gellan gum to GelMA than investigated may be required to achieve native cartilage viscoelasticity in this case. Human articular chondrocytes encapsulated in hydrogels remained viable over one week of culture. Overall, it was shown that viscoelasticity may be tailored similarly to other mechanical properties and may prove a new criterion to be included in the design of biomaterial structures for tissue engineering.

Indirect additive manufacturing as an elegant tool for the production of self-supporting low density gelatin scaffolds.

The present work describes for the first time the production of self-supporting low gelatin density (<10w/v%) porous scaffolds using methacrylamide-modified gelatin as an extracellular matrix mimicking component. As porous scaffolds starting from low gelatin concentrations cannot be realized with the conventional additive manufacturing techniquesin the abscence of additives, we applied an indirect fused deposition modelling approach. To realize this, we have printed a sacrificial polyester scaffold which supported the hydrogel material during UV crosslinking, thereby preventing hydrogel structure collapse. After complete curing, the polyester scaffold was selectively dissolved leaving behind a porous, interconnective low density gelatin scaffold. Scaffold structural analysis indicated the success of the selected indirect additive manufacturing approach. Physico-chemical testing revealed scaffold properties (mechanical, degradation, swelling) to depend on the applied gelatin concentration and methacrylamide content. Preliminary biocompatibility studies revealed the cell-interactive and biocompatible properties of the materials developed.

Protective effects of reactive functional groups on chondrocytes in photocrosslinkable hydrogel systems

Photocrosslinkable hydrogels are frequently used in cartilage tissue engineering, with crosslinking systems relying on cytotoxic photoinitiators and ultraviolet (UV) light to form permanent hydrogels. These systems are rarely assessed in terms of optimization of photoinitiator or UV dosage, with non-cytotoxic concentrations from literature deemed sufficient. We hypothesized that the number of reactive functional groups present within a hydrogel polymer is highly relevant when crosslinking, affording cytoprotection to chondrocytes by preferentially interacting with the highly reactive radicals that are formed during UV-mediated activation of a photoinitiator. This was tested using two photocrosslinkable hydrogel systems: gelatin methacrylamide (GelMA) and gellan gum methacrylate (GGMA). We further assessed the effects of two different UV dosages on chondrocyte differentiation while subject to a single photoinitiator dosage in the GGMA system. Most notably, we found that a higher ratio of reactive groups to photoinitiator molecules offers cytoprotective effects, and future developments in photocrosslinkable hydrogel technology may involve assessment of such ratios. In contrast, we found there to be no effect of UV on chondrocyte differentiation at the two chosen dosages. Overall the optimization of photocrosslinkable systems is of great value in cartilage tissue engineering and these data provide a groundwork for such concepts to be developed further. Statement of SignificancePhotocrosslinkable hydrogels, which use photoinitiators and predominantly ultraviolet light to form stable matrices for cell encapsulation and tissue development, are promising for cartilage tissue engineering. While both photoinitiators and ultraviolet light can damage cells, these systems have generally not been optimized. We propose that the ratio of reactive functional groups within a polymer to photoinitiator molecules is a critical parameter for optimization of photocrosslinkable hydrogels. Using photocrosslinkable gelatin and gellan gum, we found that a higher ratio of reactive groups to photoinitiator molecules protected chondrocytes, but did not affect chondrocyte differentiation. The principle of cytoprotection by functional groups developed in this work will be of great value in optimizing photocrosslinkable hydrogel systems for cartilage and other tissue engineering applications.

Enhancing structural integrity of hydrogels by using highly organised melt electrospun fibre constructs

Applying additive manufacturing technology to the principles of fibre reinforcement of hydrogels, we have fashioned weak hydrogels into mechanically enhanced composites. We combined the extracellular matrix-like structure of gelatin-methacrylamide (GelMA) and GelMA/hyaluronic acid-methacrylamide (HAMA) hydrogels with highly oriented poly(ε-caprolactone) (PCL) fibres fabricated by Melt Electrospinning Writing (MEW) to achieve fibre-reinforced GelMA/HAMA composites with improved compressive properties. Stacked fibres with lay-down patterns of 0°–90° and 0°–60°–120°, and spacing of 400 and 800μm were prepared by MEW. These defined fibrous structures were infiltrated with hydrogels, namely GelMA (10%) and GelMA/HAMA (0.125%, 0.25% and 0.5%) in custom-made moulds and crosslinked by a reduction–oxidation initiating system (ammonium persulphate/tetramethylethylenediamine). Mechanical properties and deformation characteristics of the constructs were evaluated under uniaxial compression loading conducted at 37°C in culture media with an integrated camera. Reinforced constructs showed more than a 35-fold-increase of the compressive Young’s modulus. However, the compressive Young’s moduli were highly strain-rate dependent. The fibre reinforcement has a particular impact on the Poisson’s ratio of the composite constructs, decreasing from values of approximately 0.4 to 0.01. The high interfacial surface area between the fibre structure and the hydrogel matrix is believed to be one of the main factors responsible for the significant increase in the mechanical properties of the constructs. In summary, we have found that reinforcement of hydrogels with defined MEW fibre architectures achieves an outstanding increase in the mechanical properties at high strain rates.