Development of a novel collagen–GAG nanofibrous scaffold via electrospinning

Collagen and glycosaminoglycan (GAG) are native constituents of human tissues and are widely utilized to fabricate scaffolds serving as an analog of native extracellular matrix (ECM).The development of blended collagen and GAG scaffolds may potentially be used in many soft tissue engineering applications since the scaffolds mimic the structure and biological function of native ECM. In this study, we were able to obtain a novel nanofibrous collagen–GAG scaffold by electrospinning with collagen and chondroitin sulfate (CS), a widely used GAG. The electrospun collagen–GAG scaffold exhibited a uniform fiber structure in nano-scale diameter. By crosslinking with glutaraldehyde vapor, the collagen–GAG scaffolds could resist from collagenase degradation and enhance the biostability of the scaffolds. This led to the increased proliferation of rabbit conjunctiva fibroblast on the scaffolds. Incorporation of CS into collagen nanofibers without crosslinking did not increase the biostability but still promoted cell growth. In conclusion, the electrospun collagen–GAG scaffolds, with high surface-to-volume ratio, may potentially provide a better environment for tissue formation/biosynthesis compared with the traditional scaffolds.     

Genipin-crosslinked gelatin scaffolds for articular cartilage tissue engineering with a novel crosslinking method

A novel crosslinking method with directly crosslinking the gelatin gel, being cut to a disc of chosen size beforehand, for the fabrication of porous gelatin scaffold was proposed. This novel method of gel-crosslinking was compared with the traditional methods of mixing-crosslinking and scaffold-crosslinking. The structure of the scaffold fabricated by the gel-crosslinking method shows uniformly distributed and interconnected pores which can be much smaller than those made by the other two methods. All three methods have the last step as freeze-drying; nevertheless, freeze-drying once more will increase the uniformity of the structure and the interconnecting pores. Crosslinking of gelatin was carried out at room temperature with glutaraldehyde (GTA) or genipin (GP). In vitro cell culture of Wistar rat's joint chondrocytes demonstrates that the GTA-crosslinked scaffold is much worse than the GP-crosslinked one; a tissue containing collagen and glycosaminoglycan was produced in the GP-crosslinked scaffold in just 9 days after cell seeding, and a tissue with a cell distribution resembling that of the native cartilage was developed after 30 day cell culture. It was concluded that the novel method is feasible for application in articular cartilage tissue engineering.     

Construction of collagen II/hyaluronate/chondroitin-6-sulfate tri-copolymer scaffold for nucleus pulposus tissue engineering and preliminary analysis of its physico-chemical properties and biocompatibility

To construct a novel scaffold for nucleus pulposus (NP) tissue engineering, The porous type II collagen (CII)/hyaluronate (HyA)–chondroitin-6-sulfate (6-CS) scaffold was prepared using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) cross-linking system. The physico-chemical properties and biocompatibility of CII/HyA–CS scaffolds were evaluated. The results suggested CII/HyA–CS scaffolds have a highly porous structure (porosity: 94.8 ± 1.5%), high water-binding capacity (79.2 ± 2.8%) and significantly improved mechanical stability by EDC/NHS crosslinking (denaturation temperature: 74.6 ± 1.8 and 58.1 ± 2.6°C, respectively, for the crosslinked scaffolds and the non-crosslinked; collagenase degradation rate: 39.5 ± 3.4 and 63.5 ± 2.0%, respectively, for the crosslinked scaffolds and the non-crosslinked). The CII/HyA–CS scaffolds also showed satisfactory cytocompatibility and histocompatibility as well as low immunogenicity. These results indicate CII/HyA–CS scaffolds may be an alternative material for NP tissue engineering due to the similarity of its composition and physico-chemical properties to those of the extracellular matrices (ECM) of native NP.     

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.     

Preparation and characterization of genipin-crosslinked rat acellular spinal cord scaffolds

The feasibility of rat acellular spinal cord scaffolds for tissue engineering applications was investigated. Fresh rat spinal cords were decellularized and crosslinked with genipin (GP) to improve their structural stability and mechanical properties. The GP-crosslinked spinal cord scaffolds possessed a porous structure with an average pore diameter of 31.1 μm and a porosity of 81.5%. The resultant scaffolds exhibited a water uptake ratio of 229%, and moderate in vitro degradation rates of less than 5% in phosphate-buffered saline (PBS) and slightly more than 20% in trypsin-containing buffer, within 14 days. The ultimate tensile strength and elastic modulus of GP-crosslinked spinal cord scaffolds were determined to be 0.193 ± 0.064 MPa and 1.541 ± 0.082 MPa, respectively. Compared with glutaraldehyde (GA)-crosslinked acellular spinal cord scaffolds, GP-crosslinked scaffolds demonstrated similar microstructure and mechanical properties but superior biocompatibility as indicated by cytotoxicity evaluation and rat mesenchymal stem cell (MSC) adhesion behavior. Cells were able to penetrate throughout the crosslinked scaffold due to the presence of an interconnected porous structure. The low cytotoxicity of GP facilitated cell proliferation and extracellular matrix (ECM) secretion in vitro on the crosslinked scaffolds over 7 days. Thus, these GP-crosslinked spinal cord scaffolds show great promise for tissue engineering applications.     

Glutaraldehyde and oxidised dextran as crosslinker reagents for chitosan-based scaffolds for cartilage tissue engineering

Chitosan crosslinked with glutaraldehyde or oxidised dextran was studied as a potential scaffold material in tissue engineering for cartilage regeneration. By mixing two solutions of both components it became a gel, which was frozen. After lyophilisation a scaffold was generated with interconnected pores with diameters ranging between 120–350 μm. The mechanical properties (yielding point, elastic and viscous moduli), absolute porosity, pore morphology were determined depending on the ratio of chitosan to crosslinker. ATDC5 (murine cell line) and bovine articular chondrocytes (primary cells) were cultured for 14 days on the scaffolds. Cultivation with ATDC5 cells and bovine chondrocytes showed no negative influence of glutaraldehyde on cell vitality and growth.     

Properties of biocomposites based on titanium scaffolds with a different porosity

Open-porous titanium scaffolds have been widely investigated for orthopaedic and dental applications because of their ability to form composites via bone ingrowth into pores and promote implant fixation with mother bone. In this work, porous titanium scaffolds coated with a diamond-like carbon were produced, and their ability to form biocomposites was evaluated through in vivo experiments. Three types of the open-porous scaffolds made of spongy titanium granules (porosity 0.3, 0.4 and 0.5, Young’s modulus 4.4, 3.5 and 0.6 GPa) were implanted into a bone defect of sheep. Time dependences of the Young’s modulus of titanium scaffold–bone tissue biocomposites were determined through the measurement of Young’s modulus of the extracted scaffolds after 4, 8, 24 and 52 weeks of surgery. The Young’s modulus of biocomposite is dependent not only on the time of composite formation but also on the porosity of scaffold.     

Fabrication and in vitro evaluations with osteoblast-like MG-63 cells of porous hyaluronic acid-gelatin blend scaffold for bone tissue engineering applications

In this study, hyaluronic acid–gelatin (HyA–Gel) scaffolds were prepared with HyA:Gel ratios of 15:85, 50:50, and 85:15 with the goal of obtaining a porous biocompatible scaffold for bone tissue engineering applications. Scanning electron microscopy and Fourier-transform infrared spectroscopy were done to characterize the morphological orientations of the scaffolds. The biocomposite structure was highly porous and the pores in the scaffolds were interconnected. The compressive strength of the scaffold was 7.39 ± 0.2 MPa for the HyA–Gel when fabricated at a ratio of 15:85. To assess the biocompatibility and cell behavior on the HyA–Gel biocomposite, the proliferation of MG-63 osteoblast cell on the scaffolds was examined using the MTT assay, optical microscopy, and confocal microscopy. Collagen type I and osteopontin expression of cells cultured on the scaffolds were examined using immunoblotting. The scaffolds fabricated with a 15:85—HyA:Gel ratio showed excellent biocompatibility, good mechanical properties, and high porosity, which suggest that the highly porous scaffold holds great promise for use in bone tissue engineering applications.     

Fibro/chondrogenic differentiation of dental stem cells into chitosan/alginate scaffolds towards temporomandibular joint disc regeneration

Tissue engineering (TE) may provide effective alternative treatment for challenging temporomandibular joint (TMJ) pathologies associated with disc malpositioning or degeneration and leading to severe masticatory dysfunction. Aim of this study was to evaluate the potential of chitosan/alginate (Ch/Alg) scaffolds to promote fibro/chondrogenic differentiation of dental pulp stem cells (DPSCs) and production of fibrocartilage tissue, serving as a replacement of the natural TMJ disc. Ch/Alg scaffolds were fabricated by crosslinking with CaCl2 combined or not with glutaraldehyde, resulting in two scaffold types that were physicochemically characterized, seeded with DPSCs or human nucleus pulposus cells (hNPCs) used as control and evaluated for cell attachment, viability, and proliferation. The DPSCs/scaffold constructs were incubated for up to 8 weeks and assessed for extracellular matrix production by means of histology, immunofluorescence, and thermomechanical analysis. Both Ch/Alg scaffold types with a mass ratio of 1:1 presented a gel-like structure with interconnected pores. Scaffolds supported cell adhesion and long-term viability/proliferation of DPSCs and hNPCs. DPSCs cultured into Ch/Alg scaffolds demonstrated a significant increase of gene expression of fibrocartilaginous markers (COLI, COL X, SOX9, COM, ACAN) after up to 3 weeks in culture. Dynamic thermomechanical analysis revealed that scaffolds loaded with DPSCs significantly increased storage modulus and elastic response compared to cell-free scaffolds, obtaining values similar to those of native TMJ disc. Histological data and immunochemical staining for aggrecan after 4 to 8 weeks indicated that the scaffolds support abundant fibrocartilaginous tissue formation, thus providing a promising strategy for TMJ disc TE-based replacement.

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Tenocyte proliferation on collagen scaffolds protects against degradation and improves scaffold properties

Tissue engineering scaffolds encourage cell proliferation whilst degrading to facilitate tissue regeneration. Their mechanical properties therefore change, decreasing due to scaffold degradation and increasing due to extracellular matrix deposition. This work compares the changing properties of collagen scaffolds incubated in culture medium, with and without human tenocytes, in order to investigate the relationship between degradation and tenocyte proliferation. The material properties of scaffolds are compared over 26 days using mechanical testing, differential scanning calorimetry, infra-red spectroscopy, and histology and biochemical assays. For medium-only scaffolds, the mechanical properties decrease rapidly, while culture medium sulfhydryl content increases significantly, with no significant changes in the denaturation temperature of scaffold collagen content. Conversely, the mechanical properties and collagen content of tenocyte-seeded scaffolds increase significantly while culture medium sulfhydryl content decreases and denaturation temperature remains the same. These results indicate that tenocytes proliferation both reduces the degradation of collagen scaffolds incubated in culture medium and produces scaffolds with improved properties.     

EDC/NHS-crosslinked type II collagen-chondroitin sulfate scaffold: characterization and in vitro evaluation

Three-dimensional biodegradable porous type II collagen scaffolds are interesting materials for cartilage tissue engineering. This study reports the preparation of porous type II collagen-chondroitin sulfate (CS) scaffold using variable concentrations of 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The physico-chemical properties and ultrastructural morphology of the collagen scaffolds were determined. Then, isolated chondrocytes were cultured in porous type II collagen scaffolds either in the presence and/or absence of covalently attached CS up to 14 days. Cell proliferation, the total amount of proteoglycans and type II collagen retained in the scaffold and chondrocytes morphology were evaluated. The results suggest that EDC-crosslinking improves the mechanical stability of collagen-CS scaffolds with increasing EDC concentration. Cell proliferation and the total amount of proteoglycans and type II collagen retained in the scaffolds were higher in type II collagen-CS scaffolds. Histological analysis showed the formation of a denser cartilaginous layer at the scaffold periphery. Scanning electron microscopy (SEM) revealed chondrocytes distributed the porous surface of both scaffolds maintained their spherical morphology. The results of the present study also indicate that type II collagen-CS scaffolds have potential for use in tissue engineering.     

明胶/硫酸钙复合生物材料

将具有传骨性的生物可吸收无机材料硫酸钙与具有诱骨性的明胶组合起来,构制新型可吸收复合生物材料。该材料可避免硫酸钙颗粒的脱落,可以作为骨填充物或支架材料。明胶的加入减缓了半水硫酸钙向二水硫酸钙的转化,形成了CSH/CSD/CLG e l复合材料。在该复合材料表面培养了人成骨细胞,细胞在支架表面粘附良好,可见该支架和细胞有较好的生物相容性。硫酸钙吸收后形成一可供细胞生长的多孔支架,所以明胶/硫酸钙(CLG e l/CS)复合材料实为原位成孔支架材料,解决了多孔支架材料初期强度低的致命弱点。     

Attachment of stem cells on porous chitosan scaffold crosslinked by Na5P3O10

In this study, porous chitosan scaffolds were prepared by freeze-dried method using Na₅P₃O₁₀ as a crosslinking agent. The three-dimensional pore structure of the scaffold was interconnected with a mean pore size about 40 to 100 μm. The remained weight of crosslinked scaffold was about 76% after being exposed to PBS for 30 days. Mouse embryonic stem (E. S.) cells could grow on these crosslinked scaffolds. E. S. cells differentiated to other cells after 21 days of culturing on the scaffolds. The growth rate of E. S. cells was improved by post surface treatment of the scaffolds with collagen. However, there was no significant increase in growth rate of E. S. cells when scaffolds were surface treated with argon plasma. These porous chitosan scaffolds present a promising approach for tissue engineering applications.     

A novel surface modification on calcium polyphosphate scaffold for articular cartilage tissue engineering

The surface of porous three-dimensional (3D) calcium polyphosphate (CPP) scaffold was modified by treatment of quenching-after-sintering in the fabrication process. Scanning electron microscopic examination and degradation tests confirmed a new type of surface modification. A rotary-shaking culture was compared to that of a stationary culture and the results showed that rotary shaking led to enhanced extracellular matrices (ECM) secretion of both proteoglycans and collagen. Rotary-shaking cultured results showed that the quenching-treated CPP scaffold produced a better cartilage tissue, with both proteoglycans and collagen secretions enhanced, than the air-cooled-after-sintering scaffolds. Moreover, β-CPP scaffolds were better for the ECM secretion of both proteoglycans and collagen than the β-CPP + γ-CPP multiphase scaffold. However, the multiphase scaffold led to higher growth rate than that of β-CPP scaffold; the quenching-after-sintering treatment reversed this. In addition, the ECM secretions of both proteoglycans and collagen in the quenching-treated β-CPP scaffold were higher than those in the air-cooled one. Thus, the novel treatment of quenching-after-sintering has shown merits to the porous 3D CPP scaffolds for articular cartilage tissue engineering.     

Preparation, characterization and in vitro biological study of biomimetic three-dimensional gelatin–montmorillonite/cellulose scaffold for tissue engineering

This work focused on studying the effect of blending gelatin (Gel) with Cellulose (Cel), in the presence of montmorillonite (MMT), on the swelling behavior, in vitro degradation and surface morphology. Additionally, the effect of the prepared biocomposites on the characteristics of the human osteosarcoma cells (Saos-2), including proliferation, scaffold/cells interactions, apoptosis and their potential of the cells to induce osteogenesis and differentiation was evaluated. The crosslinked biocomposites with glutaraldehyde (GA) or N,N-methylene-bisacrylamide (MBA) was prepared via an intercalation process and freeze-drying technique. Properties including SEM morphology, X-ray diffraction characterization and in vitro biodegradation were investigated. The successful generation of 3-D biomimetic porous scaffolds incorporating Saos-2 cells indicated their potential for de novo bone formation that exploits cell–matrix interactions. In vitro studies revealed that the scaffolds containing 12 and 6% MMT crosslinked by 5 and 0.5% GA seem to be the two most efficient and effective biodegradable scaffolds, which promoted Saos-2 cells proliferation, migration, expansion, adhesion, penetration, spreading, and differentiation, respectively. MMT improved cytocompatibility between the osteoblasts and the biocomposite. In vitro analysis indicated good biocompatibility of the scaffold and presents the scaffold as a new potential candidate as suitable biohybrid material for tissue engineering.     

Development of poly (lactic-co-glycolic acid)-collagen scaffolds for tissue engineering

Collagen as an important extra-cellular matrix (ECM) in many tissues is weakly antigenic and the structure of collagen sponges is highly porous with interconnected pores effective for cell infiltration and mass transfer of oxygen and nutrients. Its application as a scaffold is limited by poor mechanical strength and rapid biodegradation. In this paper, we attempt to graft hydrolyzed PLGA fiber surfaces with collagen by N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) in combination with N-hydroxysuccinimide (NHS), and then embed these collagen-grafted PLGA fibers in collagen sponge to form a hybrid PLGA-collagen scaffold. For further stability, we cross-linked the collagen in the scaffold and used it in rat liver cell cultivation. The scaffold was characterized by mechanical micro-tester, scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Results showed that (1) the scaffolds exhibited isotropic and interconnected porous structure; (2) the compression modulus of this scaffold was enhanced 50 fold compared to the collagen scaffolds. The cell attachment and cytotoxicity of this scaffold were studied. Cell attachment was improved remarkably and the cytotoxicity of the hybrid PLGA-collagen scaffold was lower than that of the un-grafted PLGA-collagen scaffolds using alamarBlue™ assay normalized to the DNA content in each scaffold. This new hybrid scaffold has potential applications for tissue engineering.     

Electrospinning collagen and hyaluronic acid nanofiber meshes

Collagen and hyaluronic acid (HA) are main components of the extracellular matrix and have been utilized in electrospinning; a technique that creates nanosized fibers for tissue scaffolds. A collagen/HA polymer solution was electrospun into a scaffold material for osteoporosis patients who have reduced bone strength. To synthesize nanofibers, a high voltage was applied to the polymer solution to draw out nanofibers that were collected on a ground plate as a uniform mesh. The meshes were then crosslinked to render them insoluble and conjugated with gold nanoparticles to promote biocompatibility. Characterization of the mesh was performed using scanning electron microscope, electron dispersive spectroscopy and fourier transform infrared spectroscopy. A WST-1 assay determined the potential biocompatibility. The results show that collagen/HA scaffolds were developed that were insoluble in aqueous solutions and promoted cellular attachment that could be used as a tissue engineered scaffold to promote cell growth.     

Bone scaffolds with homogeneous and discrete gradient mechanical properties

Bone TE uses a scaffold either to induce bone formation from surrounding tissue or to act as a carrier or template for implanted bone cells or other agents. We prepared different bone tissue constructs based on collagen, gelatin and hydroxyapatite using genipin as cross-linking agent. The fabricated construct did not present a release neither of collagen neither of genipin over its toxic level in the surrounding aqueous environment. Each scaffold has been mechanically characterized with compression, swelling and creep tests, and their respective viscoelastic mechanical models were derived. Mechanical characterization showed a practically elastic behavior of all samples and that compressive elastic modulus basically increases as content of HA increases, and it is strongly dependent on porosity and water content.Moreover, by considering that gradients in cellular and extracellular architecture as well as in mechanical properties are readily apparent in native tissues, we developed discrete functionally graded scaffolds (discrete FGSs) in order to mimic the graded structure of bone tissue.These new structures were mechanically characterized showing a marked anisotropy as the native bone tissue. Results obtained have shown FGSs could represent valid bone substitutes.     

PLLA–collagen and PLLA–gelatin hybrid scaffolds with funnel-like porous structure for skin tissue engineering

In skin tissue engineering, a three-dimensional porous scaffold is necessary to support cell adhesion and proliferation and to guide cells moving into the repair area in the wound healing process. Structurally, the porous scaffold should have an open and interconnected porous architecture to facilitate homogenous cell distribution. Moreover, the scaffolds should be mechanically strong to protect deformation during the formation of new skin. In this study, the hybrid scaffolds were prepared by forming funnel-like collagen or gelatin sponge on a woven poly(l-lactic acid) (PLLA) mesh. The hybrid scaffolds combined the advantages of both collagen or gelatin (good cell-interactions) and PLLA mesh (high mechanical strength). The hybrid scaffolds were used to culture dermal fibroblasts for dermal tissue engineering. The funnel-like porous structure promoted homogeneous cell distribution and extracellular matrix production. The PLLA mesh reinforced the scaffold to avoid deformation. Subcutaneous implantation showed that the PLLA–collagen and PLLA–gelatin scaffolds promoted the regeneration of dermal tissue and epidermis and reduced contraction during the formation of new tissue. These results indicate that funnel-like hybrid scaffolds can be used for skin tissue regeneration.     

PLLA–collagen and PLLA–gelatin hybrid scaffolds with funnel-like porous structure for skin tissue engineering

Abstract

In skin tissue engineering, a three-dimensional porous scaffold is necessary to support cell adhesion and proliferation and to guide cells moving into the repair area in the wound healing process. Structurally, the porous scaffold should have an open and interconnected porous architecture to facilitate homogenous cell distribution. Moreover, the scaffolds should be mechanically strong to protect deformation during the formation of new skin. In this study, the hybrid scaffolds were prepared by forming funnel-like collagen or gelatin sponge on a woven poly(l-lactic acid) (PLLA) mesh. The hybrid scaffolds combined the advantages of both collagen or gelatin (good cell-interactions) and PLLA mesh (high mechanical strength). The hybrid scaffolds were used to culture dermal fibroblasts for dermal tissue engineering. The funnel-like porous structure promoted homogeneous cell distribution and extracellular matrix production. The PLLA mesh reinforced the scaffold to avoid deformation. Subcutaneous implantation showed that the PLLA–collagen and PLLA–gelatin scaffolds promoted the regeneration of dermal tissue and epidermis and reduced contraction during the formation of new tissue. These results indicate that funnel-like hybrid scaffolds can be used for skin tissue regeneration.