The osteogenic differentiation of dog bone marrow mesenchymal stem cells in a thermo-sensitive injectable chitosan/collagen/β-glycerophosphate hydrogel: in vitro and in vivo

Type I collagen was added to the composite chitosan solution in a ratio of 1:2 to build a physical cross-linked self-forming chitosan/collagen/β-GP hydrogel. Osteogenic properties of this novel injectable hydrogel were evaluated. Gelation time was about 8 min which offered enough time for handling a mixture containing cells and the subsequent injection. Scanning electronic microscopy (SEM) observations indicated good spreading of bone marrow mesenchymal stem cells (BMSCs) in this hydrogel scaffold. Mineral nodules were found in the dog-BMSCs inoculated hydrogel by SEM after 28 days. After subcutaneous injection into nude mouse dorsum for 4 weeks, partial bone formation was observed in the chitosan/collagen/β-GP hydrogel loaded with pre-osteodifferentiated dog-BMSCs, which indicated that chitosan/collagen/β-GP hydrogel composite could induce osteodifferentiation in BMSCs without exposure to a continual supply of external osteogenic factors. In conclusion, the novel chitosan/collagen/β-GP hydrogel composite should prove useful as a bone regeneration scaffold.     

Fabrication of chitosan/poly(ε-caprolactone) composite hydrogels for tissue engineering applications

The aim of this study was to fabricate three-dimensional (3D) porous chitosan/poly(ε-caprolactone) (PCL) hydrogels with improved mechanical properties for tissue engineering applications. A modified emulsion lyophilisation technique was developed to produce 3D chitosan/PCL hydrogels. The addition of 25 and 50 wt% of PCL into chitosan substantially enhanced the compressive strength of composite hydrogel 160 and 290%, respectively, compared to pure chitosan hydrogel. The result of ATR–FTIR imaging corroborated that PCL and chitosan were well mixed and physically co-existed in the composite structures. The composite hydrogels were constructed of homogenous structure with average pore size of 59.7 ± 14 μm and finer pores with average size of 4.4 ± 2 μm on the wall of these larger pores. The SEM and confocal laser scanning microscopy images confirmed that fibroblast cells were attached and proliferated on the 3D structure of these composite hydrogels. The composite hydrogels acquired in this study possessed homogeneous porous structure with improved mechanical strength and integrity. They may have a high potential for the production of 3D hydrogels for tissue engineering applications.     

Preparation and characterization of electrospun PCL/PLGA membranes and chitosan/gelatin hydrogels for skin bioengineering applications

In this study, two distinct systems of biomaterials were fabricated and their potential use as a bilayer scaffold (BS) for skin bioengineering applications was assessed. The initial biomaterial was a polycaprolactone/poly(lacto-co-glycolic acid) (PCL/PLGA) membrane fabricated using the electrospinning method. The PCL/PLGA membrane M-12 (12% PCL/10% PLGA, 80:20) displayed strong mechanical properties (stress/strain values of 3.01 ± 0.23 MPa/225.39 ± 7.63%) and good biocompatibility as demonstrated by adhesion of keratinocyte cells on the surface and ability to support cell proliferation. The second biomaterial was a hydrogel composed of 2% chitosan and 15% gelatin (50:50) crosslinked with 5% glutaraldehyde. The CG-3.5 hydrogel (with 3.5% glutaraldehyde (v/v)) displayed a high porosity, ≥97%, good compressive strength (2.23 ± 0.25 MPa), ability to swell more than 500% of its dry weight and was able to support fibroblast cell proliferation. A BS was fabricated by underlaying the membrane and hydrogel casting method to combine these two materials. The physical properties and biocompatibility were preliminarily investigated and the properties of the two biomaterials were shown to be complementary when combined. The upper layer membrane provided mechanical support in the scaffold and reduced the degradation rate of the hydrogel layer. Cell viability was similar to that in the hydrogel layer which suggests that addition of the membrane layer did not affect the biocompatibility.     

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.     

Preparation of collagen modified photopolymers: a new type of biodegradable gel for cell growth

In this study a new branched methacrylated poly(propylene glycol-co-lactic acid) (PPG–PLA–IEM) and methacrylated cellulose acetate butyrate resin (CAB–IEM) were synthesized. Hydrogels with various amounts of PPG–PLA–IEM and CAB–IEM (25, 50 and 75 wt% IEM modified) were prepared by photopolymerization. Collagen tethered PEG–monoacrylate (PEGMA–collagen) was prepared and introduced as a bioactive moiety to modify the hydrogel in order to enhance cell affinity. In vitro attachment and growth of 3T3 mouse fibroblasts and human umbilical vein endothelial cells (HUVEC) on the hydrogels with and without collagen were also investigated. It was observed that, the collagen improves the cell adhesion onto the hydrogel surface. With the increasing amount of collagen, cell viability increased by 28% for ECV304 (P < 0.05) and 30% for 3T3 (P < 0.05).     

A novel injectable chlorhexidine thermosensitive hydrogel for periodontal application: preparation, antibacterial activity and toxicity evaluation

The aim of this paper was to evaluate the application potential of CS–HTCC/GP–0.1%Chx thermosensitive hydrogel which was synthesized using chitosan (CS), quaternized CS, and α,β-glycerophosphate (α,β-GP) loading with 0.1% chlorhexidine (Chx) (w/v) for periodontal treatment. An aqueous solution of CS–HTCC/GP–0.1%Chx was transformed into hydrogel at 6 min when the temperature was increased to 37°C. The scan electron microscopy (SEM) image of the gel was a porous, loose and crosslinked network. In vitro, Chx released over 18 h from the CS–HTCC/GP thermosensitive hydrogel in artificial saliva pH 6.8. Release rate could be controlled through adjustment of α,β-GP or Chx concentration. CS–HTCC/GP–0.1%Chx thermosensitive hydrogel exhibited excellent inhibitory activity against primary periodontal pathogens. CS–HTCC/GP–0.1%Chx thermosensitive hydrogel had no acute toxicity; the maximum tolerated dose in rats was 400 mg/ml. All results indicated that CS–HTCC/GP–0.1%Chx thermosensitive hydrogel is a strong candidate as a local drug delivery system for periodontal treatment.     

Influence of some factors affecting antibacterial activity of PVA/Chitosan based hydrogels synthesized by gamma irradiation

Poly (vinyl alcohol) hydrogels containing different concentrations of chitosan with molecular weight of 471 and 101 kDa were crosslinked by gamma irradiation at a dose of 25 kGy. The swelling behavior, gel content and morphological structure of the blend were investigated. The antibacterial effect, as a function of chitosan content and molecular weight in the hydrogel, was investigated against Escherichia coli and Bacillus subtilis. With increasing chitosan content the equilibrium degree of swelling of the blend increased and the gel fraction decreased. Results of antibacterial activity of chitosan revealed that chitosan was more effective in inhibiting growth of gram positive bacteria than that of gram negative ones. It was observed that, the chitosan content as well as its molecular weight has a direct influence on bacteria growth inhibition. The higher the chitosan content in the blend and the higher its initial molecular weight, the larger was the inhibition zone diameter. The bacteria growth inhibition was attributed to the diffusion of entrapped chitosan from the hydrogel blend to the culture medium.     

In-situ forming biodegradable glycol chitosan-based hydrogels: Synthesis,characterization, and chondrocyte culture

In-situ forming hydrogels from thiolated glycol chitosan (GCH-SH) and vinyl sulfone-modified PEG (PL-VS) were designed, prepared and successfully applied as biodegradable, non-toxic bio-scaffolds for chondrocyte culture. The hydrogels could be formed in situ under physiological conditions via Michael-type addition between the GCH-SH and PL-VS at a low polymer concentration of 1–3% (w/v). Gelation times varied from 0.75 to 50 min, depending on the polymer concentration and the arm number of PEG-VS. Moreover, a high arm number and a high polymer concentration may lead to efficient network formation of GCH-SH/PEG-VS hydrogels. These hydrogels were found biodegradable in the presence of lysozyme, a cationic protein in the body, for a long period of time. Rheological studies indicated that these hydrogels generally displayed highly elastic property and had higher mechanical strength than those from thiolated hyaluronic acid/PEG-VS reported previously. SEM observation revealed that these hydrogels possessed well-interconnected microporous morphology. Besides these, the chondrocytes could be incorporated and homogeneously distributed in the hydrogel based on GCH-SH and 4-arm PL-VS. Importantly, after cell culture of 14 days, the chondrocytes in the hydrogel remained viable, as determined by a live–dead assay, and the cells kept their round chondrocytic phenotype. These results suggest that Michael-type addition is an effective method in the preparation of in-situ forming, biodegradable GCH-based hydrogels serving as bio-scaffolds for chondrocyte culture.     

Novel chitosan hydrogel formed by ethylene glycol chitosan, 1,6-diisocyanatohexan and polyethylene glycol-400 for tissue engineering scaffold: in vitro and in vivo evaluation

Traditional chitosan hydrogels were prepared by chemical or physical crosslinker, and both of the two kinds of hydrogels have their merits and demerits. In this study, researchers attempted to prepare one kind of chitosan hydrogel by slightly crosslinker, which could combine the advantages of the two kinds of hydrogels. In this experiment, the crosslinker was formed by a reaction between the isocyanate group of 1,6-diisocyanatohexan and the hydroxyl group of polyethylene glycol-400 (PEG-400), then the crosslinker reacted with the amidine and the hydroxyl group of ethylene glycol chitosan to form the network structure. Physical properties of the hydrogel were tested by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and biodegradation. Biocompatibility was assessed by cell implantation in vitro and the scaffold was used as a cartilage tissue engineering scaffold to repair a defect in rabbit knee joints in vivo. FTIR results show the formation of a covalent bond during thickening of the ethylene glycol chitosan. SEM and degradation experiments showed that the ethylene glycol chitosan hydrogel is a 3-D, porous, and degradable scaffold. The hydrogel contained 2 % ethylene glycol chitosan and 10 μl crosslinker was selected for the biocompatibility experiment in vitro and in vivo. After chondrocytes were cultured in the ethylene glycol chitosan hydrogel scaffold for 1 week cells exhibited clustered growth and had generated extracellular matrix on the scaffold in vitro. The results in vivo showed that hydrogel-chondrocytes promoted the repair of defect in rabbits. Based on these results, it could be concluded that ethylene glycol chitosan hydrogel is a scaffold with excellent physicochemical properties and it is a promising tissue engineering scaffold.     

Bioactive injectable triple acting thermosensitive hydrogel enriched with nano-hydroxyapatite for bone regeneration: in-vitro characterization,Saos-2 cell line cell viability and osteogenic markers evaluation

Hydrogels forming in-situ have gained great attention in the area of bone tissue engineering recently, they were also showed to be a good and less invasive alternative to surgically applied ones. The primal focus of this study was to prepare chitosan-glycerol phosphate thermosensitive hydrogel formed in-situ and loaded with risedronate (bone resorption inhibitor) in an easy way with no requirement of complicated processes or large number of equipment. Then we investigated its effectiveness for bone regeneration. In-situ forming hydrogels were prepared using chitosan cross-linked with glycerol phosphate and loaded with risedronate and nano-hydroxyapatite as bone cement. The prepared hydrogels were characterized by analyzing their gelation time at 37?°C, % porosity, swelling index, in-vitro degradation, rheological properties, and in-vitro drug release. Results showed that the in-situ hydrogels prepared using 2.5% (w/v) chitosan cross-linked with 50% (w/v) glycerol phosphate in the ratio (9:1, v/v) reinforced with 20?mg/mL and nano-hydroxyapatite possessed the most sustained drug release profile. This optimized formulation was further evaluated using DSC and FTIR studies, in addition to their morphological properties using scanning electron microscopy. The effect on Saos-2 cell line viability was evaluated also using MTT assay on the optimized hydrogel formulation in addition to their action on cell proliferation using fluorescence microscope. Moreover, calcium deposition on the hydrogel and alkaline phosphatase activity were evaluated. Risedronate-nano-hydroxyapatite loaded hydrogels significantly enhanced the Saos-2 cell proliferation in addition to enhanced alkaline phosphatase activity and calcium deposition. Such results suggest that risedronate-nano-hydroxyapatite loaded hydrogels present great biocompatibility for bone regeneration. Proliferation of cells, as well as deposition of mineral on the hydrogel, was an evidence of the biocompatible nature of the hydrogel. This hydrogel formed in-situ present a good less invasive alternative for bone tissue engineering.     

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.     

Injectable thermosensitive hydrogel based on chitosan and quaternized chitosan and the biomedical properties

A novel injectable thermosensitive hydrogel (CS–HTCC/α β-GP) was successfully designed and prepared using chitosan (CS), quaternized chitosan (HTCC) and α,β-glycerophosphate (α,β-GP) without any additional chemical stimulus. The gelation point of CS–HTCC/α β-GP can be set at a temperature close to normal body temperature or other temperature above 25°C. The transition process can be controlled by adjusting the weight ratio of CS to HTCC, or different final concentration of α,β-GP. The optimum formulation is (CS + HTCC) (2% w/v), CS/HTCC (5/1 w/w) and α,β-GP 8.33% or 9.09% (w/v), where the sol–gel transition time was 3 min at 37°C. The drug released over 3 h from the CS–HTCC/α,β-GP thermosensitive hydrogel in artificial saliva pH 6.8. In addition, CS–HTCC/α,β-GP thermosensitive hydrogel exhibited stronger antibacterial activity towards two periodontal pathogens (Porphyromonas gingivalis, P.g and Prevotella intermedia, P.i). CS–HTCC/α, β-GP thermosensitive hydrogel was a considerable candidate as a local drug delivery system for periodontal treatment.     

Thermoresponsive hyperbranched copolymer with multi acrylate functionality for in situ cross-linkable hyaluronic acid composite semi-IPN hydrogel

Thermoresponsive polymers have been widely used for in situ formed hydrogels in drug delivery and tissue engineering as they are easy to handle and their shape can easily conform to tissue defects. However, non-covalent bonding and mechanical weakness of these hydrogels limit their applications. In this study, a physically and chemically in situ cross-linkable hydrogel system was developed from a novel thermoresponsive hyperbranched PEG based copolymer with multi acrylate functionality, which was synthesized via an ‘one pot and one step’ in situ deactivation enhanced atom transfer radical co-polymerization of poly(ethylene glycol) diacrylate (PEGDA, M_n = 258 g mol⁻¹), poly(ethylene glycol) methyl ether methacrylate (PEGMEMA, M_n = 475 g mol⁻¹) and (2-methoxyethoxy) ethyl methacrylate (MEO₂MA). This hyperbranched copolymer was tailored to have the lower critical solution temperature to form physical gelation around 37°C. Meanwhile, with high level of acrylate functionalities, a chemically cross-linked gel was formed from this copolymer using thiol functional cross-linker of pentaerythritol tetrakis (3-mercaptopropionate) (QT) via thiol-ene Michael addition reaction. Furthermore, a semi-interpenetrated polymer networks (semi-IPN) structure was developed by combining this polymer with hyaluronic acid (HA), leading to an in situ cross-linkable hydrogel with significantly increased porosity, enhanced swelling behavior and improved cell adhesion and viability both in 2D and 3D cell culture models.     

Cell delivery with genipin crosslinked gelatin microspheres in hydrogel/microcarrier composite

Tissue engineering using injectable cellular constructs such as hydrogel is an emerging field in regenerative medicine. Common non-adhesive hydrogels have the advantages of biocompatibility and injectability but modest cell settlement and viability due to its hydrophilic nature which makes it a challenge for effective and sustainable applications in cell-based therapies. Recently, a hydrogel/microcarrier (GC) model has been developed to provide cells with ‘anchors’ to support cell adhesion within the hydrogel bulk. However, complex fabrications steps and minimal bio-degradability of the microcarrier have limited the spatial growth of the cells. This study aims to overcome challenges of the current GC model through the use of bio-degradable and bio-adhesive gelatin microspheres stabilized with natural crosslinking agent, genipin. This genipin crosslinked microcarrier is capable of maintaining high viability and the stretched morphology of the cells when encapsulated in the hydrogel bulk. This suggests that the microcarrier developed is suitable for GC composite model and has potential applications in biomedical applications.     

Preparation and HL-7702 cell functionality of titania/chitosan composite scaffolds

Titania/chitosan composite scaffolds were prepared through a freeze-drying technique. The composite scaffolds were highly porous with the average pore size of 120–300 μm, and the titania (TiO₂) powders were uniformly dispersed on the surface of the pore walls. The compressive strength of the composite scaffolds was significantly improved compared to that of pure chitosan scaffolds. Composite scaffold with 0.3 of TiO₂/chitosan weight ratio showed the maximum compressive strength of 159.7 ± 21 kPa. Hepatic immortal cell line HL-7702 was used as seeding cells on the scaffolds, and after different culture periods, cell attachment and function was analyzed. HL-7702 cells attached on the pore walls of the scaffolds with the spheroid shape after 1 day of culture, but more cell aggregations formed within the TiO₂/chitosan composite scaffolds as compared to pure chitosan scaffolds. Liver-specific functions, albumin secretion and urea synthesis were detected using a spectrometric method. The results showed that albumin secretion and urea synthesis rate of HL-7702 cells slightly decreased with the culture time, and there was no significant difference between composite scaffolds and pure chitosan scaffolds. In conclusion, the TiO₂/chitosan composite scaffolds possessed an improved mechanical strength compared to pure chitosan scaffolds and supported the attachment and functional expression of hepatocyte, implying their potential application in liver tissue engineering.     

Corneal epithelialisation on surface-modified hydrogel implants: artificial cornea

The objective was to investigate corneal re-epithelialisation of surface-modified polymethacrylate hydrogel implants in order to evaluate them as potential materials for an artificial cornea. Polymethacrylate hydrogels were modified with amines and then coated with different extracellular matrix proteins (collagen I, IV, laminin and fibronectin). The modified hydrogels were surgically implanted into bovine corneas maintained in a 3-D culture system for 5 days. The epithelial growth across the implant surface was evaluated using fluorescent, light and electron microscopy. Full epithelialisation was achieved on 1,4-diaminobutane-modified hydrogels after coating with collagen IV. Hydrogels modified with 1,4-diaminobutane but without further coating only showed partial re-epithelialisation. Hydrogels modified with other amines (1,2-diaminoethane or 1,3-diaminopropane) showed only partial re-epithelialisation; further coating with extracellular matrix proteins improved epithelialisation of these surfaces but did not result in complete re-epithelialisation. Evaluation of the corneas implanted with the 1,4-diaminobutane-modified hydrogels coated with collagen IV showed that the artificial corneas remain clear, integrate well and become covered by a healthy stratified epithelium. In conclusion the 1,4-diaminobutane surface-modified hydrogel coated with collagen IV supported the growth of a stable stratified epithelium. With further refinement this hydrogel has the potential to be used clinically for an artificial cornea.     

Composite poly-l-lactic acid/poly-(α,β)-dl-aspartic acid/collagen nanofibrous scaffolds for dermal tissue regeneration

Tissue engineering scaffolds for skin tissue regeneration is an ever expounding area of research, as the products that meet the necessary requirements are far and elite. The nanofibrous poly-l-lactic acid/poly-(α,β)-dl-aspartic acid/Collagen (PLLA/PAA/Col I&III) scaffolds were fabricated by electrospinning and characterized by SEM, contact angle and FTIR analysis for skin tissue regeneration. The cell-scaffold interactions were analyzed by cell proliferation and their morphology observed in SEM. The results showed that the cell proliferation was significantly increased (p ≤ 0.05) in PLLA/PAA/Col I&III scaffolds compared to PLLA and PLLA/PAA nanofibrous scaffolds. The abundance and accessibility of adipose derived stem cells (ADSCs) may prove to be novel cell therapeutics for dermal tissue regeneration. The differentiation of ADSCs was confirmed using collagen expression and their morphology by CMFDA dye extrusion technique. The current study focuses on the application of PLLA/PAA/Col I&III nanofibrous scaffolds for skin tissue engineering and their potential use as substrate for the culture and differentiation of ADSCs. The objective for inclusion of a novel cell binding moiety like PAA was to replace damaged extracellular matrix and to guide new cells directly into the wound bed with enhanced proliferation and overall organization. This combinatorial epitome of PLLA/PAA/Col I&III nanofibrous scaffold with stem cell therapy to induce the necessary paracrine signalling effect would favour faster regeneration of the damaged skin tissues.     

Enhanced mechanical properties of thermosensitive chitosan hydrogel by silk fibers for cartilage tissue engineering

Articular cartilage has limited repair capability following traumatic injuries and current methods of treatment remain inefficient. Reconstructing cartilage provides a new way for cartilage repair and natural polymers are often used as scaffold because of their biocompatibility and biofunctionality. In this study, we added degummed chopped silk fibers and electrospun silk fibers to the thermosensitive chitosan/glycerophosphate hydrogels to reinforce two hydrogel constructs which were used as scaffold for hyaline cartilage regeneration. The gelation temperature and gelation time of hydrogel were analyzed by the rheometer and vial tilting method. Mechanical characterization was measured by uniaxial compression, indentation and dynamic mechanical analysis assay. Chondrocytes were then harvested from the knee joint of the New Zealand white rabbits and cultured in constructs. The cell proliferation, viability, production of glycosaminoglycans and collagen type II were assessed. The results showed that mechanical properties of the hydrogel were significantly enhanced when a hybrid with two layers of electrospun silk fibers was made. The results of GAG and collagen type II in cell-seeded scaffolds indicate support of the chondrogenic phenotype for chondrocytes with a significant increase in degummed silk fiber–hydrogel composite for GAG content and in two-layer electrospun fiber–hydrogel composite for Col II. It was concluded that these two modified scaffolds could be employed for cartilage tissue engineering.