Double. Bfgf / Collagen Matrix Composites Repair Articular Cartilage Defects Related Research

Wound healing is a spatiotemporal controlled process coordinated by a variety of growth factors. Although exogenous growth factor has potential application in the field of tissue repair, there is still great challenge to construct a spatiotemporal controlled release system for delivery of growth factors, and then achieve the regeneration of wound tissue. The development of tissue engineering provides a new way for the repair of tissue defect. Construction a tissue engineering scaffold with spatiotemporal controlled release of growth factors is becoming a new research direction. Spatiotemporal controlled release means (i) the released growth factors must target the desired site and desired cells with spatial gradient, and reduce the undesired releasing amount to around injury site as far as possible; and (ii) the growth factors should be released in sustainable mode in tissues for a relatively long period to obtain the proposed effect.The release of growth factors is closely related to scaffolds structure. Therefore, it should be feasible to change the release kinetics of growth factors by adjusting the structure of biomaterials or scaffolds. In this study, in order to realize spatial controlled release of loaded growth factors by adjusting biomaterials structure, a kind of double layered materials was designed and prepared with different density, one dense layer and one loose layer, respectively. The materials prepared were utilized to repair cartilage defects which can not achieve effective repair by traditional therapies such as subchondral drilling operation, micro fracture, autologous osteochondral enchase molding, autologous chondrocyte transplantation and etc.1. Collagen is used as the main ingredient to construct tissue engineering scaffolds. Basic Fibroblast Growth Factor (bFGF) was selected as model growth factors for the reasons that bFGF is one of the most effective mitogens in the body which ont only promotes mitosis of cartilage cells and mesenchymal stem cells significantly, but also accelerates differentiation of undifferentiated mesenchymal cells to cartilage cells as well as promotes vascular proliferation. Composite scaffolds were obtained by sandwiching bFGF loaded nanoparticles between the double layers with different density of collagen membrane. The scaffolds were investigated by in vitro release assay, in vitro cell culture and articular cartilage repair research. The detailed contents were as follows. A kind of double layered collagen membranes incorporated with bFGF loaded chitosan-heparin (CS-Hep) nanoparticles was prepared by freeze-drying and air-drying, named as CFM and CAM respectively and characterized by transmission electron microscopy (TEM) as well as Fourier transform infrared (FTIR). Continuous flow apparatus method was employed to investigate the effect of this composite material on the release behavior of model protein HSA in PBS and collagenase solution mediums respectively. The release kinetics of fluorescein isothiocyanate (FITC) labeled HSA in PBS was observed by confocal laser scanning microscope (CLSM).2. Fibroblasts L929were cultured in vitro and were seeded on the surfaces of double layered collagen matrix composite with and without bFGF respectively as well as crosslinking freeze-dried collagen membrane (CFM). MTT assay and CLSM are utilized to evaluate the conglutination and proliferation of the cultured cells on the third and fifth day.3. bFGF/double layered collagen membrane extract was prepared. In addition, its bio-safety was evaluated by acute systemic toxicity test, hemolytic test, pyrogen test and cytotoxicity test.4. Full-thickness defects were conducted on60Japanese white rabbits. Before operation, rabbits were divided into five groups randomly. They are follows:bDM with CFM layer faced the subchondral bone termed as bDM-CFM group, bDM with CAM layer faced the subchondral bone termed as bDM-CAM group, DM with CFM layer faced the subchondral bone termed as DM-CFM group, DM with CAM layer faced the subchondral bone termed as DM-CAM group, and defects without implants treatment as the control defect group. The full-thickness defects models were constructed at both right and left patellas of rabbits. Material was implanted in the defect with CFM and CAM layer toward subchondral bone surface. The speciments were collected in1,2,4,8,12and16weeks post-operation respectively. General observation, histological grading, immunohistochemistry for type Ⅱ collagen were applied to analyse the cartilage repair of defects in different groups. At the same time, synovial fluid was extracted at3d,1w,4w,8w,12w respectively to detect the expression of20species of growth factors, cytokines and chemokines with antibody array system.The results of all experiments show that bFGF/double layered collagen membrane have different density structure. In vitro release study, the release of HSA flowing from CAM to CFM was faster than that from CFM to CAM, when PBS was used as release media, while the reverse result was obtained in the medium of collagenase solution. The release study on FITC labeled HSA was conducted to verify double materials can uneven release the protein. These results clearly show the constructed inhomogeneous layered collagen membrane incorporating with growth factors loaded nanoparticles provides a spatiotemporal control release profile. The results from in vitro cell culture assay demonstrate that all materials could promote normal cells adhesion and growth. Both membranes loaded with bFGF displayed higher cells growth rate than that of membranes without bFGF (P<0.05), and the number of cells on the CFM surfaces of bDM (bDM-CFM) was much more than that on the CAM surfaces of bDM (bDM-CAM)(P<0.05). It is probable that the spatiotemporal control release of growth factors did induce different biological functions. The3D image by CLSM also confirmed this view.The results of biological evaluation showed that pH of the leaching liquor from bFGF/double-layered collagen composite was6.55±0.13, which was close to neutral. The materials were demomstrated to be biological safety resulting from no systemic acute toxicity, no cytotoxicity and pyrogen reactions as well as no hemolytic effect. The materials had good cell compatibility and promoted cell growth, the released growth factor can maintain biological activity.In the articular cartilage repair process, these released growth factors, cytokines and chemokines in synovial fluid could be detected using antibody array system. The results showed that the changing process of these growth factors may indirectly reflect the articular cartilage repairing process and the effects of implanted materials and administrated growth factors. In articular cartilage defects of rabbits'knees, bDG-CFM group was better than other groups. At16weeks, the reparative tissue formed new tissues in the defect without obvious margins. The reparative tissue surface was nearly similar to normal cartilage in the bDM-CFM group. The results of above showed that the structure and released characteristic of bDM-CFM was considered as positive to cartilage repair.