Lie Ma

Collagen/chitosan porous scaffolds with improved biostability for skin tissue engineering

Porous scaffolds for skin tissue engineering were fabricated by freeze-drying the mixture of collagen and chitosan solutions. Glutaraldehyde (GA) was used to treat the scaffolds to improve their biostability. Confocal laser scanning microscopy observation confirmed the even distribution of these two constituent materials in the scaffold. The GA concentrations have a slight effect on the cross-section morphology and the swelling ratios of the cross-linked scaffolds. The collagenase digestion test proved that the presence of chitosan can obviously improve the biostability of the collagen/chitosan scaffold under the GA treatment, where chitosan might function as a cross-linking bridge. A detail investigation found that a steady increase of the biostability of the collagen/chitosan scaffold was achieved when GA concentration was lower than 0.1%, then was less influenced at a still higher GA concentration up to 0.25%. In vitro culture of human dermal fibroblasts proved that the GA-treated scaffold could retain the original good cytocompatibility of collagen to effectively accelerate cell infiltration and proliferation. In vivo animal tests further revealed that the scaffold could sufficiently support and accelerate the fibroblasts infiltration from the surrounding tissue. Immunohistochemistry analysis of the scaffold embedded for 28 days indicated that the biodegradation of the 0.25% GA-treated scaffold is a long-term process. All these results suggest that collagen/chitosan scaffold cross-linked by GA is a potential candidate for dermal equivalent with enhanced biostability and good biocompatibility.

Layer by layer chitosan/alginate coatings on poly(lactide-co-glycolide) nanoparticles for antifouling protection and Folic acid binding to achieve selective cell targeting.

Polyelectrolyte multilayers (PEMs) composed of two natural polysaccharides-chitosan (Chi) and alginate (Alg) were deposited by Layer by layer (LbL) assembly on top of biocompatible poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs). Folic acid (FA) or FA grafted poly(ethylene glycol) (PEG-FA) were covalently bounded to the PEMs via carbodiimide chemistry. The assembly of biocompatible PEMs was monitored on planar surfaces by means of the quartz crystal microbalance with dissipation (QCM-D) technique and on top of PLGA NPs by means of ζ-potential measurements. BSA was used as model protein to characterize protein adsorption on PEMs. QCM-D showed protein deposition could not be observed on the Chi/Alg multilayer, for both Chitosan and Alginate as top layers. Finally, cellular uptake experiments were carried out by co-culture of HepG2 cells in presence of NPs. Flow Cytometry and confocal laser scanning microscopy (CLSM) were used to investigate the influence of the surface chemistry of the NPs on uptake. For the HepG2 cell line significantly less uptake of PLGA NPs coated with Chi/Alg than the bare NPs was observed but the uptake increased after attachment of FA molecules.

Chitosan-Based Biomaterials for Tissue Repair and Regeneration

Tissue repair and regeneration is an interdisciplinary field focusing on development of biological and bioactive substitutes. Chitosan is a natural polysaccharide exhibiting excellent biocompatibility, biodegradability, affinity to biomolecules, and wound-healing activity. It can also be easily modified via chemical and physical reactions to obtain derivatives of various structures, properties, functions, and applications. This paper focuses on chitosan and its derivatives as biomaterials for tissue repair and regeneration. Tuning the structure and properties such as biodegradability, mechanical strength, gelation property, and cell affinity can be achieved through chemical reaction, immobilization of specific ligands such as peptide and sugar molecules, combination with other biomaterials, and chemical or physical crosslinking. To obtain applicable three-dimensional scaffolding materials such as porous sponges, hydrogels, and rods, the formulation and stimuli-responsiveness of this material can also be modified. Moreover, chitosan and its derivatives can function as vectors for delivery of cell growth factors and particularly of functional genes encoding cell growth factors, which are easier to integrate with the formulated materials to obtain scaffolds of higher activity. Recent studies have shown that such scaffolds are of particular importance in mediating the proliferation, migration, and differentiation of stem cells. Finally, integration of chitosan with cell growth factors and associated genes and/or with cells (stem cells) produces chitosan-based biomaterials with applications in repair or regeneration of skin, cartilage, bone, and other tissue.

The healing of full-thickness burns treated by using plasmid DNA encoding VEGF-165 activated collagen-chitosan dermal equivalents.

Repair of deep burn by use of the dermal equivalent relies strongly on the angiogenesis and thereby the regeneration of dermis. To enhance the dermal regeneration, in this study plasmid DNA encoding vascular endothelial growth factor-165 (VEGF-165)/N,N,N-trimethyl chitosan chloride (TMC) complexes were loaded into a bilayer porous collagen-chitosan/silicone membrane dermal equivalents (BDEs), which were applied for treatment of full-thickness burn wounds. The DNA released from the collagen-chitosan scaffold could remain its supercoiled structure but its degree was decayed along with the prolongation of incubation time. The released DNA could transfect HEK293 cells in vitro with decayed efficiency too. Human umbilical vein endothelial cells (HUVECs) in vitro cultured in the scaffold loaded with TMC/pDNA-VEGF complexes expressed a significantly higher level of VEGF and showed higher viability than those cultured in the controls, i.e. blank scaffold, and scaffolds loaded with naked pDNA-VEGF and TMC/pDNA-eGFP, respectively. The four different BDEs were then transplanted in porcine full-thickness burn wounds. Results showed that the TMC/pDNA-VEGF group had a significantly higher number of newly-formed and mature blood vessels, and fastest regeneration of the dermis. RT-qPCR and western blotting found that the experimental group also had the highest expression of VEGF, CD31 and α-SMA in both mRNA and protein levels. Furthermore, ultra-thin skin grafting was performed on the regenerated dermis 14 days later, leading to complete repair of the burn wounds with normal histology. Moreover, the tensile strength of the repaired tissue increased along with the time prolongation of post grafting, resulting in a value of approximately 70% of the normal skin at 105 days.

Gelatin Hydrogel Prepared by Photo‐initiated Polymerization and Loaded with TGF‐β1 for Cartilage Tissue Engineering

Gelatin is a nature-derived protein having good cytocompatibility, and widely used in tissue engineering particularly in a form of a hydrogel. To obtain the hydrogel with good enough mechanical properties, however, measures are still need to be taken. In this work, the gelatin molecule was modified with methacrylic acid (MA) to obtain crosslinkable gelatin (GM), which formed a chemically crosslinked hydrogel by photoinitiating polymerization. The gelation time could be easily tuned and showed an inverse relationship with the GM concentration. After photo-irradiation for 20 min there was no detectable double carbon bond in the hydrogen spectrum of high resolution magic angle spinning nuclear magnetic resonance spectroscopy ((1)H HR-MAS NMR). With the increase of the GM concentration, storage modulus and loss modulus of the hydrogels increased, but their swelling ratio and mesh size decreased. Weight loss of the hydrogels was also affected by the polymer concentration. Transform growth factor-beta1 (TGF-beta1) was incorporated into the GM hydrogel to improve its bioactivity. In vitro chondrocyte culture showed that the GM hydrogel had indeed good performance to support chondrocyte growth and maintain chondrocytic phenotype. Incorporation of TGF-beta1 could further improve the biological activity in terms of cell proliferation and extracellular matrix secretion.

Fabrication and physical and biological properties of fibrin gel derived from human plasma

The fast development of tissue engineering and regenerative medicine drives the old biomaterials, for example, fibrin glue, to find new applications in these areas. Aiming at developing a commercially available hydrogel for cell entrapment and delivery, in this study we optimized the fabrication and gelation conditions of fibrin gel. Fibrinogen was isolated from human plasma by a freeze-thaw circle. Gelation of the fibrinogen was accomplished by mixing with thrombin. Absorbance of the fibrinogen/thrombin mixture at 550 nm as a function of reaction time was monitored by UV-VIS spectroscopy. It was found that the clotting time is significantly influenced by the thrombin concentration and the temperature, while less influenced by the fibrinogen concentration. After freeze-drying, the fibrin gel was characterized by scanning electron microscopy (SEM), revealing fibrous microstructure. Thermal gravimetric analysis found that the degradation temperature of the crosslinked fibrin gel starts from 288 degrees C, which is about 30 degrees C higher than that of the fibrinogen. The hydrogel has an initial water-uptake ratio of approximately 50, decreased to 30-40 after incubation in water for 11 h depending on the thrombin concentration. The fibrin gels lost their weights in PBS very rapidly, while slowly in DMEM/fetal bovine serum and DMEM. In vitro cell culture found that human fibroblasts could normally proliferate in the fibrin gel with spreading morphology. In conclusion, the fibrin gel containing higher concentration of fibrinogen (20 mg ml(-1)) and thrombin (5 U ml(-1)) has suitable gelation time and handling properties, and thus is applicable as a delivery vehicle for cells such as fibroblasts.

Thermal dehydration treatment and glutaraldehyde cross-linking to increase the biostability of collagen–chitosan porous scaffolds used as dermal equivalent

A biodegradable scaffold for skin-tissue engineering was designed using collagen and chitosan, which are common materials for biomedical application. The scaffolds containing different amounts of chitosan were prepared by mixing the collagen and chitosan solutions followed by removal of the solvent using a freeze-drying method. The cross-linking treatment of these scaffolds was performed using the dehydrothermal treatment (DHT) method or glutaraldehyde (GA) to increase their biostability. The effect of the chitosan concentration and the cross-linking methods on the morphology of these scaffolds was studied by SEM. The water retention and the biodegradability in vitro of various collagen-chitosan scaffolds were investigated. Finally the biocompatibility of the collagen-chitosan (10 wt% chitosan) scaffold treated with different cross-linking methods was evaluated using a in vivo animal test. A mild inflammatory reaction could be detected in the early stages, and GA treatment can decrease the inflammatory reaction in a long-term implantation. After implantation for four weeks, all kinds of scaffolds, especially the GA-treated scaffolds (Col-GA) were filled with a large number of fibroblasts and were vascularized to a certain extent. These results suggest that the GA-treated scaffold has an increased biostability and excellent biocompatibility. It can be a potential candidate for skin-tissue engineering.

Enhanced angiogenesis of gene-activated dermal equivalent for treatment of full thickness incisional wounds in a porcine model

Angiogenesis of dermal equivalent is one of the key issues for treatment of full thickness skin defects. To develop a gene-activated bilayer dermal equivalent (BDE), N,N,N-trimethyl chitosan chloride (TMC), a cationic gene delivery vector, was used to form complexes with the plasmid DNA encoding vascular endothelial growth factor-165 (VEGF-165), which was then incorporated into a collagen-chitosan/silicone membrane scaffold. To evaluate the angiogenesis property in vivo, full thickness skin defects were made on the back of pigs, into which the TMC/pDNA-VEGF complexes loaded BDE and other three control BDEs, i.e. the blank BDE, and the BDEs loaded with pDNA-VEGF and TMC/pDNA-eGFP complexes, respectively, were transplanted. Biopsy specimens were harvested at day 7, 10 and 14 after surgery for histology, immunohistochemistry, immunofluorescence, real-time quantitative PCR (RT-qPCR) and western blotting analyses. The results showed that the TMC/pDNA-VEGF group had the strongest VEGF expression in mRNA and protein levels, resulting in the highest densities of newly-formed and mature vessels. The ultra-thin skin graft was further transplanted onto the dermis regenerated by the TMC/pDNA-VEGF complexes loaded BDE at day 10 and well survived. At 112 days grafting, the healing skin had a similar structure and approximately 80% tensile strength of the normal skin.

Enhanced angiogenesis of porous collagen scaffolds by incorporation of TMC/DNA complexes encoding vascular endothelial growth factor

Angiogenesis of an implanted construct is one of the most important issues in tissue engineering and regenerative medicine, and can often take as long as several weeks. The vascular endothelial growth factor (VEGF) shows a positive effect on enhancing angiogenesis in vivo. But the incorporation of growth factors has many limitations, since they typically have half-lives only on the order of minutes. Therefore, in this work the DNA encoding VEGF was applied to enhance the angiogenesis of a collagen scaffold. A cationic gene delivery vector, N,N,N-trimethyl chitosan chloride (TMC), was used to form complexes with the plasmid DNA encoding VEGF. The complexes were then incorporated into the collagen scaffold, the loading being mediated by the feeding concentration and release in a sustained manner. In vitro cell culture demonstrated a significant improvement in the VEGF expression level from the TMC/DNA complexes containing scaffolds, in particular with a large amount of DNA. The scaffolds containing the TMC/DNA complexes were subcutaneously implanted into Sprague-Dawley mice to study their angiogenesis via macroscopic observation, hematoxylin-eosin staining and immunohistochemical staining. The results demonstrated that the incorporation of TMC/DNA complexes could effectively enhance the in vivo VEGF expression and thereby the angiogenesis of implanted scaffolds.

Fabrication and characterization of poly(L-lactide-co-glycolide) knitted mesh-reinforced collagen-chitosan hybrid scaffolds for dermal tissue engineering.

Mechanical properties are essential considerations for the design of porous scaffolds in the field of tissue engineering. To develop a well-supported hybrid dermal substitute, poly(L-lactide-co-glycolide) (PLGA) yarns were knitted into a mesh with relative fixed loops, followed by incorporation into collagen-chitosan scaffolds (CCS) to obtain PLGA knitted mesh-reinforced CCS (PLGAm/CCS). The morphology and tensile strength in both the dry and wet state of PLGAm/CCS were investigated in vitro. To characterize the tissue response, specifically angiogenesis and tissue regeneration, PLGAm/CCS was embedded subcutaneously in Sprague-Dawley rats and compared with two control implants, i.e., PLGA mesh (PLGAm) and CCS. At weeks 1, 2, and 4 post surgery, tissue specimens were harvested for histology, immunohistochemistry, real-time quantitative PCR and Western blot analysis. These results demonstrated that the incorporation of PLGA knitted mesh into CCS can improve the mechanical strength with little influence on its mean pore size and porosity. After implantation, PLGAm/CCS can resist contraction and promote cell infiltration, neotissue formation, and blood vessel ingrowth, effectively. In conclusion, the mechanical strength of scaffolds can play a synergetic role in tissue regeneration and vascularization by maintaining its 3D microstructure. The ability of PLGAm/CCS to promote angiogenesis and induce in situ tissue formation demonstrates its strong potential in the field of skin tissue engineering.