Influence of radiation crosslinked carboxymethyl-chitosan/gelatin hydrogel on cutaneous wound healing

A series of carboxymethyl chitosan (CM-chitosan) and gelatin hydrogels were prepared by radiation crosslinking. A pre-clinical study was performed by implantation model and full-thickness cutaneous wound model in Sprague–Dawley rats to preliminarily evaluate the biocompatibility, biodegradability and effects on healing. In the implantation test, as a component of the hydrogels, CM-chitosan showed a positive effect on promoting cell proliferation and neovascularization, while gelatin was efficient to stabilize the structure and prolong the degradation time. To evaluate the function on wound healing, the hydrogels were applied to the relatively large full-thickness cutaneous wounds (Φ3.0 cm). Compared with the control groups, the hydrogel group showed significantly higher percentage of wound closure on days 9, 12 and 15 postoperatively, which was consistent with the significantly thicker granulation tissue on days 3 and 6. All results apparently revealed that the radiation crosslinked CM-chitosan/Gelatin hydrogels could induce granulation tissue formation and accelerate the wound healing.     

Evaluation of proanthocyanidin-crosslinked electrospun gelatin nanofibers for drug delivering system

Electrospun nanofibers are excellent candidates for various biomedical applications. We successfully fabricated proanthocyanidin‐crosslinked gelatin electrospun nanofibers. Proanthocyanidin, a low cytotoxic collagen crosslinking reagent, increased the gelatin crosslinking percentage in the nanofibers from 53% to 64%. The addition of proanthocyanidin kept the nanofibers from swelling, and, thus, made the fibers more stable in the aqueous state. The compatibility and the release behavior of the drug in the nanofibers were examined using magnesium ascorbyl phosphate as the model drug. Proanthocyanidin also promoted drug loading and kept the drug release rate constant. These properties make the proanthocyanidin‐crosslinked gelatin nanofibers an excellent material for drug delivery. In the cell culture study, L929 fibroblast cells had a significantly higher proliferation rate when cultured with the gelatin/proanthocyanidin blended nanofibers. This characteristic showed that proanthocyanidin‐crosslinked gelatin electrospun nanofibers could potentially be employed as a wound healing material by increasing cell spreading and proliferation.     

Cell-laden microengineered pullulan methacrylate hydrogels promote cell proliferation and 3D cluster formation

The ability to encapsulate cells in three-dimensional (3D) environments is potentially of benefit for tissue engineering and regenerative medicine. In this paper, we introduce pullulan methacrylate (PulMA) as a promising hydrogel platform for creating cell-laden microscale tissues. The hydration and mechanical properties of PulMA were demonstrated to be tunable through modulation of the degree of methacrylation and gel concentration. Cells encapsulated in PulMA exhibited excellent viability. Interestingly, while cells did not elongate in PulMA hydrogels, cells proliferated and organized into clusters, the size of which could be controlled by the hydrogel composition. By mixing with gelatin methacrylate (GelMA), the biological properties of PulMA could be enhanced as demonstrated by cells readily attaching to, proliferating, and elongating within the PulMA/GelMA composite hydrogels. These data suggest that PulMA hydrogels could be useful for creating complex, cell-responsive microtissues, especially for applications that require controlled cell clustering and proliferation.     

Influence of viscosity on chondrogenic differentiation of mesenchymal stem cells during 3D culture in viscous gelatin solution-embedded hydrogels

Differentiation of human bone marrow-derived mesenchymal stem cells(hMSCs)is regulated by a variety of cues of their surrounding microenvironments.In particular,mechanical properties of cell culture matrices have been recently disclosed to play a pivotal role in stem cell differentiation.However,it remains elusive how viscosity affects the chondrogenic differentiation of hMSCs during three-dimensional(3 D)culture.In this study,a 3 D culture system that was established by embedding viscous gelatin solution in chemically cross-linked gelatin hydrogels was used for 3 D culture of hMSCs in gelatin solutions with different viscosities.The influence of solution viscosity on chondrogenic differentiation of hMSCs was investigated.Viscous gelatin solutions promoted cell proliferation in the order of low,middle and high viscosity while elastic hydrogels restricted cell proliferation.High viscosity gelatin solution led to increased production of the cartilaginous matrix.Under the synergistic stimulation of chondrogenic induction factors,high viscosity was beneficial for the chondrogenic differentiation of hMSCs.The results suggested the role of viscosity should be considered as one of the dominant mechanical cues affecting stem cell differentiation.     

Effect of gelatin on the drug release behaviors for the organic hybrid gels based on <Emphasis Type="Italic">N</Emphasis>-isopropylacrylamide and gelatin

A series of organic hybrid gels were prepared based on poly(N-isopropylacrylamide), poly(NIPAAm), and gelatin. The hybrid gels were crosslinked through a two-step process with genipin or glutaraldehyde. The swelling behavior and physical properties of the gels were investigated in the previous report. In this study, we loaded sulfanilamide, caffeine, vitamin B12, phenol red, and neutral red in the hybrid gels. The effects of gelatin on the drug release profile were demonstrated. The ionicity of hybrid gels strongly influenced the release of phenol red (anionic) and neutral red (cationic). However, the releases of sulfanilamide, caffeine and vitamin B12 were not influenced by the ionicity of hybrid gel. The drug released from the gels crosslinked with genipin was significantly smaller than that released from the gels crosslinked with glutaraldehyde.     

Biocompatibility of chemically cross-linked gelatin hydrogels for ophthalmic use

Biocompatibility is a major requirement for the development of functional biomaterials for ophthalmic applications. In this study, we investigated the effect of cross-linker functionality on ocular biocompatibility of chemically modified gelatin hydrogels. The test materials were cross-linked with glutaraldehyde (GTA) or 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC), and were analyzed using in vitro and in vivo assays. Primary rat iris pigment epithelial cultures were incubated with various gelatin discs for 2 days, and the cellular responses were monitored by cell proliferation, viability, and pro-inflammatory gene and cytokine expression. The results demonstrated that the cells exposed to EDC cross-linked gelatins had relatively lower lactate dehydrogenase activity, cytotoxicity, and interleukin-1β and tumor necrosis factor-α levels than did those to GTA treated samples. In addition, the gelatin implants were inserted in the anterior chamber of rabbit eyes for 12 weeks and characterized by clinical observations and scanning electron microscopy studies. The EDC cross-linked gelatin hydrogels exhibited good biocompatibility and were well tolerated without causing toxicity and adverse effects. However, a significant inflammatory reaction was elicited by the presence of GTA treated materials. It was noted that, despite its biocompatibility, the potential application of non-cross-linked gelatin for local delivery of cell and drug therapeutics would be limited due to rapid dissolution in aqueous environments. In conclusion, these findings suggest ocular cell/tissue response to changes in cross-linker properties. In comparison to GTA treatment, the EDC cross-linking is more suitable for preparation of chemically modified gelatin hydrogels for ophthalmic use.     

Differential physical, rheological, and biological properties of rapid in situ gelable hydrogels composed of oxidized alginate and gelatin derived from marine or porcine sources

Marine derived gelatin is not known to associate with any communicable diseases to mammals and could be a reasonable substitute for gelatin derived from either bovine or porcine sources. The low melting point of marine gelatin (8°C) also offers greater formulation flexibility than mammalian derived gelatins. However, the sub-optimal physical properties of marine gelatin generally limit the interest to further develop it for biomedical applications. This study aimed at investigating the feasibility of using oxidized alginate (Oalg) as a high activity macromolecular crosslinker of marine gelatin to formulate in situ gelable hydrogels with the goal of enhancing the latter’s physical properties. The performance of Oalg/marine gelatin hydrogel was compared to Oalg/porcine gelatin hydrogel; in general, the physicomechanical properties of both hydrogels were comparable, with the hydrogels containing porcine gelatin exhibiting moderately higher mechanical strengths with shorter gelation times, smaller size pores, and higher swelling ratios. On the contrary, the biological performances of the two hydrogels were significantly difference. Cells cultured in the marine gelatin derived hydrogel grew significantly faster, with greater than 60% more cells by 7 days and they exhibited more spread-out conformations as compared those cultured in the porcine derived hydrogel. Production of ECM by cells cultured in the Oalg/marine gelatin hydrogel was up to 2.4 times greater than that of in the Oalg/porcine gelatin hydrogel. The biodegradation rate of the hydrogel formulated from marine gelatin was greater than its counterpart prepared from porcine gelatin. These differences have important implications in the biomedical applications of the two hydrogels. Huijuan Liao and Hanwei Zhang are the first authors.     

3D Bioplotting of Gelatin/Alginate Scaffolds for Tissue Engineering:Influence of Crosslinking Degree and Pore Architecture on Physicochemical Properties

Gelatin/Alginate hydrogels were engineered for bioplotting in tissue engineering. One major drawback of hydrogel scaffolds is the lack of adequate mechanical properties. In this study, using a bioplotter, we constructed the scaffolds with different pore architectures by deposition of gelatin/alginate hydrogels layerby-layer. The scaffolds with different crosslinking degree were obtained by post-crosslinking methods.Their physicochemical properties, as well as cell viability, were assessed. Different crosslinking methods had little influence on scaffold architecture, porosity, pore size and distribution. By contrast, the water absorption ability, degradation rate and mechanical properties of the scaffolds were dramatically affected by treatment with various concentrations of crosslinking agent(glutaraldehyde). The crosslinking process using glutaraldehyde markedly improved the stability and mechanical strength of the hydrogel scaffolds. Besides the post-processing methods, the pore architecture can also evidently affect the mechanical properties of the scaffolds. The crosslinked gelatin/alginate scaffolds showed a good potential to encapsulate cells or drugs.     

In vitro evaluation of crosslinked electrospun fish gelatin scaffolds

Gelatin from cold water fish skin was electrospun, crosslinked and investigated as a substrate for the adhesion and proliferation of cells. Gelatin was first dissolved in either water or concentrated acetic acid and both solutions were successfully electrospun. Cross-linking was achieved via three different routes: glutaraldehyde vapor, genipin and dehydrothermal treatment. Solution's properties (surface tension, electrical conductivity and viscosity) and scaffold's properties (chemical bonds, weight loss and fiber diameters) were measured. Cellular viability was analyzed culturing 3T3 fibroblasts plated on the scaffolds and grown up to 7 days. The cells were fixed and observed with SEM or stained for DNA and F-actin and observed with confocal microscopy. In all scaffolds, the cells attached and spread with varying degrees. The evaluation of cell viability showed proliferation of cells until confluence in scaffolds crosslinked by glutaraldehyde and genipin; however the rate of growth in genipin crosslinked scaffolds was slow, recovering only by day five. The results using the dehydrothermal treatment were the less satisfactory. Our results show that glutaraldehyde treated fish gelatin is the most suitable substrate, of the three studied, for fibroblast adhesion and proliferation.     

Bio-nanocomposites by assembling of gelatin and layered perovskite mixed oxides

A new class of bio-nanocomposites based on hybrid gelatin-perovskite hydrogels was prepared by mixing exfoliated perovskite-tetraalkylammonium species in aqueous suspensions with gelatin solutions. Colloidal nanosheets derived from the CsCa2Nb3O10 layered perovskite re-stacked in the gelatin solutions give bio-nanocomposite materials with different content in the inorganic moiety. These films can be easily processed as homogeneous self-supported films. The partial exfoliation of the pristine mixed oxide is produced from alkylammonium exchanged phases, being the tetraalkylammonium ions (tetraethylammonium, TEA+) an efficient intermediate to give the colloid phase constituted by well exfoliated materials useful to generate homogeneous films. The nanosheets are highly oriented in the bio-nanocomposite films in agreement with the XRD patterns and the FTIR dichroism. This orientation could be considered as a characteristic of this type of hybrid materials leading to new potential applications. In this way, we have observed that the assembling of perovskite to gelatin produces a greater increase of the dielectric permittivity than the dielectric loss in the studied samples.     

Preparation of fluoride-containing gelatin nanofiber scaffold

A novel material, fluoride-containing gelatin nanofiber scaffold, was prepared by electrospinning process successfully. Scanning electron microscopy (SEM) image showed that the morphology of nanofibers was uniform and smooth, and the average diameter was about 200 nm. An even distribution in the fiber matrix of some nanoparticles which is about 20 nm was also observed in transmission electron microscopy (TEM) images. X-ray diffraction (XRD) demonstrated that the nanoparticles dispersed in the gelatin fiber matrix were CaF₂ crystals. This scaffold was crosslinked with glutaraldehyde (GTA) vapor at room temperature for 4 days in order to improve the water-resistant ability. After soaking in Dulbecco's Modified Eagle's Medium (DMEM) solution (37 °C) for 4 weeks the crosslinked scaffold still maintained a good appearance and morphology. All the properties of this novel material show a good potential to be a bone tissue engineering scaffold.     

Hydrogels of collagen/chondroitin sulfate/hyaluronan interpenetrating polymer network for cartilage tissue engineering

The network structure of a three-dimensional hydrogel scaffold dominates its performance such as mechanical strength, mass transport capacity, degradation rate and subsequent cellular behavior. The hydrogels scaffolds with interpenetrating polymeric network (IPN) structure have an advantage over the individual component gels and could simulate partly the structure of native extracellular matrix of cartilage tissue. In this study, to develop perfect cartilage tissue engineering scaffolds, IPN hydrogels of collagen/chondroitin sulfate/hyaluronan were prepared via two simultaneous processes of collagen self-assembly and cross linking polymerization of chondroitin sulfate-methacrylate (CSMA) and hyaluronic acid-methacrylate. The degradation rate, swelling performance and compressive modulus of IPN hydrogels could be adjusted by varying the degree of methacrylation of CSMA. The results of proliferation and fluorescence staining of rabbit articular chondrocytes in vitro culture demonstrated that the IPN hydrogels possessed good cytocompatibility. Furthermore, the IPN hydrogels could upregulate cartilage-specific gene expression and promote the chondrocytes secreting glycosaminoglycan and collagen II. These results suggested that IPN hydrogels might serve as promising hydrogel scaffolds for cartilage tissue engineering.     

Mechanical properties and in vitro behavior of nanofiber-hydrogel composites for tissue engineering applications

Hydrogel-based biomaterial systems have great potential for tissue reconstruction by serving as temporary scaffolds and cell delivery vehicles for tissue engineering (TE). Hydrogels have poor mechanical properties and their rapid degradation limits the development and application of hydrogels in TE. In this study, nanofiber reinforced composite hydrogels were fabricated by incorporating electrospun poly(ε-caprolactone) (PCL)/gelatin 'blend' or 'coaxial' nanofibers into gelatin hydrogels. The morphological, mechanical, swelling and biodegradation properties of the nanocomposite hydrogels were evaluated and the results indicated that the moduli and compressive strengths of the nanofiber reinforced hydrogels were remarkably higher than those of pure gelatin hydrogels. By increasing the amount of incorporated nanofibers into the hydrogel, the Young's modulus of the composite hydrogels increased from 3.29 ± 1.02 kPa to 20.30 ± 1.79 kPa, while the strain at break decreased from 66.0 ± 1.1% to 52.0 ± 3.0%. Compared to composite hydrogels with coaxial nanofibers, those with blend nanofibers showed higher compressive strength and strain at break, but with lower modulus and energy dissipation properties. Biocompatibility evaluations of the nanofiber reinforced hydrogels were carried out using bone marrow mesenchymal stem cells (BM-MSCs) by cell proliferation assay and immunostaining analysis. The nanocomposite hydrogel with 25 mg ml(-1) PCL/gelatin 'blend' nanofibers (PGB25) was found to enhance cell proliferation, indicating that the 'nanocomposite hydrogels' might provide the necessary mechanical support and could be promising cell delivery systems for tissue regeneration.     

物理交联聚乙烯醇/明胶复合水凝胶透明性的溶剂敏感性

以二甲基亚砜(DMSO)水溶液为分散介质,采用冷冻-解冻法制备了聚乙烯醇/明胶复合水凝胶,并研究了其透明性的溶剂敏感性。研究发现,将水凝胶交替置于去离子水和不同浓度的DMSO水溶液中,其透光率呈周期性变化;随DM-SO浓度的升高,其透光率的响应速率加快。通过X射线衍射(XRD)研究了聚乙烯醇/明胶复合水凝胶和纯聚乙烯醇水凝胶的聚集态结构。结果表明,两种水凝胶在不同溶剂中结构差异很大,并具有可逆转变性。随复合水凝胶中水含量的增加,其结晶度减小。该复合水凝胶的溶剂敏感性可能与其内部的结晶形态在不同溶剂中的转变或溶剂诱导相分离有关。     

A novel osteochondral scaffold of ceramic–gelatin assembly for articular cartilage repair

A novel ceramic–gelatin assembly (CGA) has been designed as an osteochondral scaffold for articular cartilage repair. The CGA scaffold consists of four layers, that is, a porous ceramic layer as osseous component and also as anchor, a dense ceramic layer to prevent blood vessel penetration and also to stand shear stress, a porous ceramic layer for fixation of bone to cartilage, i.e. for joining the ceramic part to the porous gelatin layer, the latter being used as cartilaginous component. The joining was done by the infiltration of gelatin solution into the porous ceramic layer, gelling and crosslinking. This CGA scaffold can offer solutions to the so-far not satisfactorily resolved issues of the osteochondral scaffold, i.e. anchoring, blood vessel penetration, shear stress distribution during articular joint motion, and enough strength to join the cartilaginous component to the osseous component to prevent delamination. This novel scaffold was tested by in vitro cell culture with Wistar rat's joint chondrocytes. DNA assay, GAGs assay, RT-PCR, and histological evaluations with hematoxylin–eosin and Safranin-O staining were carried out to show that cartilage tissue can be developed in four weeks.     

复合明胶涂层对钙磷陶瓷多孔支架的增强作用

采用有机泡沫浸渍工艺制备了高孔隙率的钙磷多孔陶瓷支架, 将多孔陶瓷样品浸于明胶溶液中渗涂得到陶瓷/明胶复合支架; 采用复合明胶涂层的方法对钙磷多孔陶瓷支架进行增强处理, 在不破坏多孔支架孔隙特征的情况下, 成功地在样品的孔壁上复合了明胶涂层。复合明胶涂层提高了样品的压缩强度和压缩模量, 与未涂覆样品相比, 涂敷样品受压时的应变特性发生了明显变化。尤其是渗涂5%明胶溶液的多孔样品, 在保持高孔隙率(82.8%)的条件下其压缩强度和压缩模量分别由原来的1.04MPa 和 0.105GPa增加到5.17MPa和0.325GPa。研究结果表明, 孔壁上复合明胶涂层可以有效地增强多孔陶瓷支架。      

Reduced Graphene Oxide‐GelMA Hybrid Hydrogels as Scaffolds for Cardiac Tissue Engineering

Biomaterials currently used in cardiac tissue engineering have certain limitations, such as lack of electrical conductivity and appropriate mechanical properties, which are two parameters playing a key role in regulating cardiac cell behavior. Here, the myocardial tissue constructs are engineered based on reduced graphene oxide (rGO)‐incorporated gelatin methacryloyl (GelMA) hybrid hydrogels. The incorporation of rGO into the GelMA matrix significantly enhances the electrical conductivity and mechanical properties of the material. Moreover, cells cultured on composite rGO‐GelMA scaffolds exhibit better biological activities such as cell viability, proliferation, and maturation compared to ones cultured on GelMA hydrogels. Cardiomyocytes show stronger contractility and faster spontaneous beating rate on rGO‐GelMA hydrogel sheets compared to those on pristine GelMA hydrogels, as well as GO‐GelMA hydrogel sheets with similar mechanical property and particle concentration. Our strategy of integrating rGO within a biocompatible hydrogel is expected to be broadly applicable for future biomaterial designs to improve tissue engineering outcomes. The engineered cardiac tissue constructs using rGO incorporated hybrid hydrogels can potentially provide high‐fidelity tissue models for drug studies and the investigations of cardiac tissue development and/or disease processes in vitro.     

Hydrogel-elastomer composite biomaterials: 2. Effects of aging methacrylated gelatin solutions on the preparation and physical properties of interpenetrating polymer networks

This study was conducted to understand the effects of aging methacrylated gelatin solutions on the properties of gelatin-HydroThane^TM Interpenetrating Polymer Network (IPN) films. The latter were prepared from methacrylated gelatin solutions that were either freshly made or stored at different concentrations and temperatures for various periods. The morphology, swelling stability and mechanical properties of the IPNs were then accordingly characterized. The IPNs prepared with aged solutions showed a reduced phase separation; changed from a network-like structure to a continuous phase structure; and demonstrated higher swelling stabilities and higher elasticity under optimal aging conditions, compared to the IPN prepared with a fresh methacrylated gelatin solution. An increase in viscosity and a change in phase transition of aged methacrylated gelatin solutions were also observed, presumably due to the physical structuring of methacrylated gelatin chains (e.g., by the formation of a helix structure), thus altering the resulting IPN characteristics. A better understanding of the effects of aging methacrylated gelatin solution on the formation and properties of gelatin-HydroThane^TM IPNs should enable us to further develop our composite biomaterials for different dressing applications.     

An open-pored gelatin/hydroxyapatite composite as a potential bone substitute

Gelatin matrix composites reinforced with fine hydroxyapatite (HA) dispersants were investigated in an exploratory study for their suitability as surgical implants. The criteria for the candidate implant material were that it: (1) be benign, (2) have useful mechanical properties under quasi-in vivo environmental conditions, (3) be dimensionally stable, (4) be sterilizable, (5) be completely assimilable, and (6) exhibit contiguous porosity to encourage invasion by the live host tissue. The synthesis of a composite comprised of a HA particulate reinforcement of a cross-linked gelatin matrix was undertaken to provide preliminary data on its swelling behavior and compressive stiffness that is retained after extended immersion in normal saline solution. A new approach leading to a tailorable, open pore microstructure is described. At a sufficiently high ratio of HA to gelatin the attainable compressive stiffness and the resistance to swelling suggests that this composite system offers potential as a versatile surgical implant material. Suggestions for further studies are offered.     

Implantable biodegradable sponges: effect of interpolymer complex formation of chitosan with gelatin on the release behavior of tramadol hydrochloride

The effect of interpolymer complex formation between positively charged chitosan and negatively charged gelatin (Type B) on the release behavior of tramadol hydrochloride from biodegradable chitosan-gelatin sponges was studied. Mixed sponges were prepared by freeze-drying the cross-linked homogenous stable foams produced from chitosan and gelatin solutions where gelatin acts as a foam builder. Generation of stable foams was optimized where concentration, pH of gelatin solution, temperature, speed and duration of whipping process, and, chitosan-gelatin ratio drastically affect the properties and the stability of the produced foams. The prepared sponges were evaluated for their morphology, drug content, and microstructure using scanning electron microscopy, mechanical properties, uptake capacity, drug release profile, and their pharmacodynamic activity in terms of the analgesic effect after implantation in Wistar rats.

It was revealed that whipping 7% (w/w) gelatin solution, of pH 5.5, for 15 min at 25°C with a stirring speed of 1000 rpm was the optimum conditions for stable gelatin foam generation. Moreover, homogenous, uniform chitosan-gelatin foam with small air bubbles were produced by mixing 2.5% w/w chitosan solution with 7% w/w gelatinsolution in 1:5 ratio. Indeed, polyionic complexation between chitosan and gelatin overcame the drawbacks of chitosan sponge mechanical properties where, pliable, soft, and compressible sponge with high fluid uptake capacity was produced at 25°Cand 65% relative humidity without any added plasticizer. Drugreleasestudies showed a successful retardation of the incorporated drug where the t_50% values of the dissolution profiles were 0.55, 3.03, and 4.73 hr for cross-linked gelatin, un-cross-linked chitosan-gelatin, and cross-linked chitosan-gelatin sponges, respectively. All the release experiments followed Higuchi's diffusion mechanism over 12 hr. The achieved drug prolongation was a result of a combined effect of both cross-linking and polyelectrolyte complexation between chitosan and gelatin. The analgesic activity of the implanted tramadol hydrochloride mixed chitosan-gelatin sponge showed reasonable analgesic effect that was maintained for more than 8 hr. Therefore, the use of chitosan and gelatin together appears to allow the formulator to manipulate both the drug release profiles and the mechanical properties of the sponge that could be effectively implanted.