纤维蛋白在软骨组织工程中的应用

纤维蛋白是纤维蛋白原经激活后形成的聚合物凝胶，作为软骨组织工程研究的载体材料，在相关的生物学和物理特性上具有其一定的优势。对纤维蛋白在软骨组织工程中应用的现状和发展方向进行综述。     

纤维蛋白在软骨组织工程中的应用

纤维蛋白是纤维蛋白原经激活后形成的聚合物凝胶,作为软骨组织工程研究的载体材料,在相关的生物学和物理特性上具有其一定的优势。对纤维蛋白在软骨组织工程中应用的现状和发展方向进行综述。     

Karyotyping of human chondrocytes in scaffold-assisted cartilage tissue engineering

Scaffold-assisted autologous chondrocyte implantation (ACI) is an effective clinical procedure for cartilage repair. The aim of our study was to evaluate the chromosomal stability of human chondrocytes subjected to typical cell culture procedures needed for regenerative approaches in polymer-scaffold-assisted cartilage repair. Chondrocytes derived from post mortem donors and from donors scheduled for ACI were expanded, cryopreserved and re-arranged in polyglycolic acid (PGA)-fibrin scaffolds for tissue culture. Chondrocyte redifferentiation was analyzed by electron microscopy, histology and gene expression analysis. Karyotyping was performed using GTG banding and fluorescence in situ hybridization on a single cell basis. Chondrocytes showed de- and redifferentiation accompanied by the formation of extracellular matrix and induction of typical chondrocyte marker genes like type II collagen in PGA-fibrin scaffolds. Post mortem chondrocytes showed up to 1.7% structural and high numbers of numerical (up to 26.7%) chromosomal aberrations, while chondrocytes from living donors scheduled for ACI showed up to 1.8% structural and up to 1.3% numerical alterations. Cytogenetically, cell culture procedures and PGA-fibrin scaffolds did not significantly alter chromosomal integrity of the chondrocyte genome. Human chondrocytes derived from living donors subjected to regenerative medicine cell culture procedures like cell expansion, cryopreservation and culture in resorbable polymer-based scaffolds show normal chromosomal integrity and normal karyotypes.     

以聚羟基烷酸酯为支架同种异体工程化软骨构建

目的 探讨以聚羟基烷酸酯(polyhydroxyalkanotes,PHBV)为支架材料的同种异体软骨细胞构建组织工程化软骨的能力.方法 分离、培养、扩增、传代培养兔软骨细胞,接种在PHBV支架材料上,体外培养7 d后,将细胞-材料复合物种植在成年兔皮下,6周取材,对获得的同种异体工程化软骨进行组织学评价及扫描电镜观察.结果 组织学观察示PHBV有成熟软骨组织形成,软骨细胞形态正常,胶原形成较多;电镜观察示软骨基质内弹性纤维较丰富,在弹性纤维包绕中有散在的卵圆形软骨细胞.单纯PHBV体内培养无软骨组织形成.结论 以PHBV为支架材料同种异体软骨细胞在有免疫力的动物体内可形成工程化软骨.     

Long-term stable fibrin gels for cartilage engineering

It is essential that hydrogel scaffold systems maintain long-term shape stability and mechanical integrity for applications in cartilage tissue engineering. Within this study, we aimed at the improvement of a commercially available fibrin gel in order to develop a long-term stable fibrin gel and, subsequently, investigated the suitability of the optimized gel for in vitro cartilage engineering. Only fibrin gels with a final fibrinogen concentration of 25mg/ml or higher, a Ca(2+) concentration of 20mm and a pH between 6.8 and 9 were transparent and stable for three weeks, the duration of the experiment. In contrast, when preparing fibrin gels with concentrations out of these ranges, turbid gels were obtained that shrank and completely dissolved within a few weeks. In rheological characterization experiments, the optimized gels showed a broad linear viscoelastic region and withstood mechanical loadings of up to 10,000 Pa. Bovine chondrocytes suspended in the optimized fibrin gels proliferated well and produced the extracellular matrix (ECM) components glycosaminoglycans and collagen type II. When initially seeding 3 million cells or more per construct (5mm diameter, 2mm thick), after 5 weeks of culture, a coherent cartilaginous ECM was obtained that was homogenously distributed throughout the whole construct. The developed fibrin gels are suggested also for other tissue engineering applications in which long-term stable hydrogels appear desirable.     

Comparison of different chondrocytes for use in tissue engineering of cartilage model structures

This study compares bovine chondrocytes harvested from four different animal locations--nasoseptal, articular, costal, and auricular--for tissue-engineered cartilage modeling. While the work serves as a preliminary investigation for fabricating a human ear model, the results are important to tissue- engineered cartilage in general. Chondrocytes were cultured and examined to determine relative cell proliferation rates, type II collagen and aggrecan gene expression, and extracellular matrix production. Respective chondrocytes were then seeded onto biodegradable poly(L-lactide-epsilon-caprolactone) disc-shaped scaffolds. Cell-copolymer constructs were cultured and subsequently implanted in the subcutaneous space of athymic mice for up to 20 weeks. Neocartilage development in harvested constructs was assessed by molecular and histological means. Cell culture followed over periods of up to 4 weeks showed chondrocyte proliferation from the tissue sources varied, as did levels of type II collagen and aggrecan gene expression. For both genes, highest expression was found for costal chondrocytes, followed by nasoseptal, articular, and auricular cells. Retrieval of 20-week discs from mice revealed changes in construct dimensions with different chondrocytes. Greatest disc diameter was found for scaffolds seeded with auricular chondrocytes, followed by those with costal, nasoseptal, and articular cells. Greatest disc thickness was measured for scaffolds containing costal chondrocytes, followed by those with nasoseptal, auricular, and articular cells. Retrieved copolymer alone was smallest in diameter and thickness. Only auricular scaffolds developed elastic fibers after 20 weeks of implantation. Type II collagen and aggrecan were detected with differing expression levels on quantitative RT-PCR of discs implanted for 20 weeks. These data demonstrate that bovine chondrocytes obtained from different cartilaginous sites in an animal may elicit distinct responses during their respective development of a tissue-engineered neocartilage. Thus, each chondrocyte type establishes or maintains its particular developmental characteristics, and this observation is critical in the design and elaboration of any tissue-engineered cartilage model.     

Rabbit articular chondrocytes seeded on collagen-chitosan-GAG scaffold for cartilage tissue engineering in vivo

In this study, we prepared a tri-copolymer porous matrices by natural polymer, collagen (Col), Chitosan (Chi) and Chondroitin (CS). Rabbit articular chondrocytes were isolated from the shoulder articular joints of a rabbit, seeded in Col-Chi-CS scaffold, and implanted subcutaneously in the dorsum of athymic nude mice to tissue engineer articular cartilage in vivo. In vitro studies show that Chondrocytes adhered to the scaffold, where they proliferated and secreted extracellular matrices with time, filling the space within the scaffold. The results of hematoxylin and eosin staining scanning electron microscopy revealed that most of the chondrocytes maintained their typically rounded morphology. After 28 days of culture within Col-Chi-CS scaffold in vitro, the results of histological staining showed forming of cartilage-specific morphological appearance and structural characteristics such as lacunae. Subcutaneous implantation studies in nude mice demonstrated that a homogeneous cartilaginous tissue, which was similar to those of natural cartilage, formed when chondrocytes were seeded in Col-Chi-CS matrix after implant 12 weeks. The tri-copolymer matrix could therefore have potential applications as a three-dimensional scaffold for cartilage tissue engineering.     

Incorporation of exudates of human platelet-rich fibrin gel in biodegradable fibrin scaffolds for tissue engineering of cartilage

The goal of this study was to assess the incorporation of exudates of human platelet-rich fibrin (hPRF) that is abundant in platelet cytokines and growth factors into biodegradable fibrin (FB) scaffolds as a regeneration matrix for promoting chondrocyte proliferation and re-differentiation. hPRF was obtained from human blood by centrifugation without an anticoagulant, and the exudate of hPRF was collected and mixed with bovine fibrinogen, and then thrombin was added to form the FB scaffold. Proliferation and differentiation of human primary chondrocytes and a human chondrosarcoma cell line, the SW-1353, embedded in the three-dimensional (3D) scaffolds and on the two-dimensional (2D) surface of the FB scaffolds so produced were evaluated in comparison with an agarose (AG) scaffold serving as the control. Results demonstrated that the amounts of these cytokines and growth factors in hPRF exudates were higher than those in the blood-derived products except for TGF-β1. Chondrocytes and SW1353 cells on the 2D and 3D FB scaffolds with the addition of the exudates of PRF exhibited more-available proliferation and differentiation than cells on 2D and 3D FB and AG scaffolds. It was concluded that FB scaffolds can provide an appropriate environment for chondrocyte proliferation and re-differentiation, and it could be improved by adding exudates of hPRF. These 3D scaffolds have great promise for cartilage tissue engineering.     

A sandwich model for engineering cartilage with acellular cartilage sheets and chondrocytes

Acellular cartilage can provide a native extracellular matrix for cartilage engineering. However, it is difficult for cells to migrate into acellular cartilage because of its non-porous structure. The aim of this study is to establish a sandwich model for engineering cartilage with acellular cartilage sheets and chondrocytes. Cartilage from adult pig ear was cut into a circular cylinder with a diameter of approximately 6?mm and freeze-sectioned at thicknesses of 10?μm and 30?μm. The sheets were then decellularized and lyophilized. Chondrocytes isolated from newborn pig ear were expanded for 2 passages. The acellular sheets and chondrocytes were then stacked layer-by-layer, in a sandwich model, and cultured in dishes. After 4 weeks of cultivation, the constructs were then either maintained in culture for another 12 weeks or implanted subcutaneously in nude mouse. Histological analysis showed that cells were completely removed from cartilage sheets after decellularization. By re-seeding cells and stacking 20 layers of sheets together, a cylinder-shaped cell sheet was achieved. Cartilage-like tissues formed after 4 weeks of culture. Histological analyses showed the formation of cartilage with a typical lacunar structure. Cartilage formation was more efficient with 10?μm-thick sheets than with 30?μm sheets. Mature cartilage was achieved after 12 weeks of implantation, which was demonstrated by histology and confirmed by Safranin O, Toluidine blue and anti-type II collagen antibody staining. Furthermore, we achieved cartilage with a designed shape by pre-shaping the sheets prior to implantation. These results indicate that the sandwich model could be a useful model for engineering cartilage in vitro and in vivo.     

软骨组织工程种子细胞的来源、培养和评价

可靠而安全的软骨种子细胞来源是保证软骨组织工程持续深入研究的前提。本文介绍了目前软骨组织工程种子细胞来源、培养和评价等方面的最新研究进展     

Delivery of TGF-beta1 and chondrocytes via injectable, biodegradable hydrogels for cartilage tissue engineering applications

In this work, novel hydrogel composites, based on the biodegradable polymer, oligo(poly(ethylene glycol) fumarate) (OPF) and gelatin microparticles (MPs) were utilized as injectable cell and growth factor carriers for cartilage tissue engineering applications. Specifically, bovine chondrocytes were embedded in composite hydrogels co-encapsulating gelatin MPs loaded with transforming growth factor-beta1 (TGF-beta1). Hydrogels with embedded cells co-encapsulating unloaded MPs and those with no MPs served as controls in order to assess the effects of MPs and TGF-beta1 on chondrocyte function. Samples were cultured up to 28 days in vitro. By 14 days, cell attachment to embedded gelatin MPs within the constructs was observed via light microscopy. Bioassay results showed that, over the 21 day period, there was a statistically significant increase in cellular proliferation for samples containing gelatin MPs, but no increase was exhibited in samples without MPs over the culture period. The release of TGF-beta1 further increased cell construct cellularity. Over the same time period, glycosaminoglycan content per cell remained constant for all formulations, suggesting that the dramatic increase in cell number for samples with TGF-beta1-loaded MPs was accompanied by maintenance of the cell phenotype. Overall, these data indicate the potential of OPF hydrogel composites containing embedded chondrocytes and TGF-beta1-loaded gelatin MPs as a novel strategy for cartilage tissue engineering.     

Three-dimensional tissue engineering of hyaline cartilage: comparison of adult nasal and articular chondrocytes

Adult chondrocytes are less chondrogenic than immature cells, yet it is likely that autologous cells from adult patients will be used clinically for cartilage engineering. The aim of this study was to compare the postexpansion chondrogenic potential of adult nasal and articular chondrocytes. Bovine or human chondrocytes were expanded in monolayer culture, seeded onto polyglycolic acid (PGA) scaffolds, and cultured for 40 days. Engineered cartilage constructs were processed for histological and quantitative analysis of the extracellular matrix and mRNA. Some engineered constructs were implanted in athymic mice for up to six additional weeks before analysis. Using adult bovine tissues as a cell source, nasal chondrocytes generated a matrix with significantly higher fractions of collagen type II and glycosaminoglycans as compared with articular chondrocytes. Human adult nasal chondrocytes proliferated approximately four times faster than human articular chondrocytes in monolayer culture, and had a markedly higher chondrogenic capacity, as assessed by the mRNA and protein analysis of in vitro-engineered constructs. Cartilage engineered from human nasal cells survived and grew during 6 weeks of implantation in vivo whereas articular cartilage constructs failed to survive. In conclusion, for adult patients nasal septum chondrocytes are a better cell source than articular chondrocytes for the in vitro engineering of autologous cartilage grafts. It remains to be established whether cartilage engineered from nasal cells can function effectively when implanted at an articular site.     

Introduction to tissue engineering and application for cartilage engineering

Tissue engineering is a multidisciplinary field that applies the principles of engineering, life sciences, cell and molecular biology toward the development of biological substitutes that restore, maintain, and improve tissue function. In Western Countries, tissues or cells management for clinical uses is a medical activity governed by different laws. Three general components are involved in tissue engineering: (1) reparative cells that can form a functional matrix; (2) an appropriate scaffold for transplantation and support; and (3) bioreactive molecules, such as cytokines and growth factors that will support and choreograph formation of the desired tissue. These three components may be used individually or in combination to regenerate organs or tissues. Thus the growing development of tissue engineering needs to solve four main problems: cells, engineering development, grafting and safety studies.     

Attachment of periosteal grafts to articular cartilage with fibrin sealant.

Favorable results using fibrin sealants in vascular surgery and soft tissue reconstruction have prompted investigation of these biologic adhesives for orthopedic applications. One important recent application was as a sealant for periosteal grafts applied to defects in articular surfaces, a procedure that contained injected chondrocytes cultured in vitro. The low and variable adhesive strength of autologous fibrin substances prompted our investigation of allogeneic fibrin. An in vitro test method was developed to investigate the use of a fibrin sealant for attaching periosteal (bovine) patches to articular cartilage (bovine). Dermis-dermis (porcine) adhesion also was evaluated. In tests of the periosteum-to-cartilage bond performed in a physiological environment, we determined the effects of the following variables on the adhesive shear strength: set time, source of fibrinogen (bovine versus human), and fibrinogen concentration. A specially designed test rig was developed to avoid nonshear force components. Adhesive shear strength increased with fibrin set time for both fibrinogen concentrations and sources (p <.03). The 30-min set time yielded data with less variance than the 5-min set time in all cases except with the higher human fibrinogen concentration (50-80 mg/mL). While there was a trend at each set time towards greater shear strength with increased protein concentration (50-80 mg/mL versus 25-40 mg/mL), only the 5-min trial of the bovine product provided a significant advantage (p <.006). There was no significant difference in adhesive strength between the fibrin products produced with human and bovine fibrinogen. The periosteum-cartilage adhesive strengths obtained in our model were comparable to values recorded for the dermis-dermis bonding. The greater strength at the 30-min set time suggests that a certain time period of joint immobilization might be beneficial in procedures in which grafts are glued to articular cartilage. This study has shown that adhesive strengths achieved with fibrin glues in treating skin wounds also can be achieved in the attachment of periosteal grafts to articular cartilage.     

Scaffolds for tissue engineering of cartilage

Articular cartilage lesions resulting from trauma or degenerative diseases are commonly encountered clinical problems. It is well-established that adult articular cartilage has limited regenerative capacity, and, although numerous treatment protocols are currently employed clinically, few approaches exist that are capable of consistently restoring long-term function to damaged articular cartilage. Tissue engineering strategies that focus on the use of three-dimensional scaffolds for repairing articular cartilage lesions offer many advantages over current treatment strategies. Appropriate design of biodegradable scaffold conduits (either preformed or injectable) allow for the delivery of reparative cells bioactive factors, or gene factors to the defect site in an organized manner. This review seeks to highlight pertinent design considerations and limitations related to the development, material selection, and processing of scaffolds for articular cartilage tissue engineering, evidenced over the last decade. In particular, considerations for novel repair strategies that use scaffolds in combination with controlled release of bioactive factors or gene therapy are discussed, as are scaffold criteria related to mechanical stimulation of cell-seeded constructs. Furthermore, the subsequent impact of current and future aspects of these multidisciplinary scaffold-based approaches related to in vitro and in vivo cartilage tissue engineering are reported herein.     

Native and DPPA cross-linked collagen sponges seeded with fetal bovine epiphyseal chondrocytes used for cartilage tissue engineering

Collagen-based biomaterials in the form of sponges (bovine type I collagen, both native and cross-linked by treatment with diphenylphosphorylazide, noted control and DPPA sponges respectively) were tested as three-dimensional scaffolds to support chondrocyte proliferation with maintenance of the phenotype in order to form neocartilage. Control and DPPA sponges were initially seeded with 10(6) or 10(7) foetal bovine epiphyseal chondrocytes and maintained for 4 weeks in culture under static conditions in RPMI/NCTC medium with 10% FCS and without addition of fresh ascorbic acid. Both supports were always present during the study and a partial decrease in size and weight was detected only with control sponges, both seeded and unseeded. Cell proliferation was only noted in the 10(6) cells-seeded sponges (4-fold increase after 4 weeks of culture). Specific cartilage collagens (types II and XI) were deposited in the matrix throughout the culture and traces of type I collagen were noticed only in the culture medium after 2-3 weeks and 4 weeks in the case of 10(6) and 10(7) cells-seeded sponges, respectively. Glycosaminoglycans accumulated in the matrix, up to 1.8 and 9.8% of total dry weight after one month with both seeding conditions, which was much lower than in the natural tissue. In the 10(7) cells-seeded sponges, mineral deposition, observed with unseeded sponges, was significantly decreased (2- to 3-fold). These in vitro results indicate that both collagen matrices can support the development of tissue engineered cartilage.     

Potential of 3-D tissue constructs engineered from bovine chondrocytes/silk fibroin-chitosan for in vitro cartilage tissue engineering

The use of cell-scaffold constructs is a promising tissue engineering approach to repair cartilage defects and to study cartilaginous tissue formation. In this study, silk fibroin/chitosan blended scaffolds were fabricated and studied for cartilage tissue engineering. Silk fibroin served as a substrate for cell adhesion and proliferation while chitosan has a structure similar to that of glycosaminoglycans, and shows promise for cartilage repair. We compared the formation of cartilaginous tissue in silk fibroin/chitosan blended scaffolds seeded with bovine chondrocytes and cultured in vitro for 2 weeks. The constructs were analyzed for cell viability, histology, extracellular matrix components glycosaminoglycan and collagen types I and II, and biomechanical properties. Silk fibroin/chitosan scaffolds supported cell attachment and growth, and chondrogenic phenotype as indicated by Alcian Blue histochemistry and relative expression of type II versus type I collagen. Glycosaminoglycan and collagen accumulated in all the scaffolds and was highest in the silk fibroin/chitosan (1:1) blended scaffolds. Static and dynamic stiffness at high frequencies was higher in cell-seeded constructs than non-seeded controls. The results suggest that silk/chitosan scaffolds may be a useful alternative to synthetic cell scaffolds for cartilage tissue engineering.     

Cellulose-based scaffold materials for cartilage tissue engineering

Non-woven cellulose II fabrics were used as scaffolds for in vitro cartilage tissue engineering. The scaffolds were activated in a saturated Ca(OH)(2) solution and subsequently coated with a calcium phosphate layer precipitated from a supersaturated physiological solution. Chondrocyte cell response and cartilage development were investigated. The cell adherence was significantly improved compared to untreated cellulose fabrics, and the proliferation and vitality of the adhered chondrocytes were excellent, indicating the biocompatibility of these materials. A homogeneous distribution of the seeded cells was possible and the development of cartilageous tissue could be proved. In contact with a physiological chondrocyte solution, calcium is expected to be leached out from the precipitated layer, which might lead to a microenvironment that triggers the development of cartilage in a way similar to cartilage repair in the vicinity of subchondral bone.     

RGD-peptides for tissue engineering of articular cartilage

One keypoint in the development of a biohybrid implant for articular cartilage defects is the specific binding of cartilage cells to a supporting structure. Mimicking the physiological adhesion process of chondrocytes to the extracellular matrix is expected to improve cell adhesion of in vitro cultured chondrocytes. Our approach involves coating of synthetic scaffolds with tailor-made, cyclic RGD-peptides, which bind to specific integrin receptors on the cell surface. In this study we investigated the expression pattern of integrins on the cell surface of chondrocytes and their capability to specifically bind to RGD-peptide coated materials in the course of monolayer cultivation. Human chondrocytes expressed integrins during a cultivation period of 20 weeks. Receptors proved to be functionally active as human and pig chondrocytes attached to RGD-coated surfaces. A competition assay with soluble RGD-peptide revealed binding specificity to the RGD-entity. Chondrocyte morphology changed with increasing amounts of cyclic RGD-peptides on the surface.