| In the natural joint, articular cartilage and subchondral bone form theload-bearing system, which was regulated by the biological signals. After theaccomplishment of development, relative invariable anatomical configuration andstable function was maintained, while regenerative ability was lost even thoughexiguity physiological tissue rebuilding. Furthermore, articular cartilage andsubchondral bone would undergo agenesis and undevelopment as the result ofexotic obstacle. It suggested that joint deformity and loss of joint could notrecover by itself, thus requiring surgical procedures for treatment ofosteochondral defects. However, present medical treatments have not yetsucceeded in this field.The aim of this study is to show potential of using a tissue engineeringapproach for regeneration of osteochondral defects. In vitro bone and cartilageengineering via combining a novel composite, biphasic scaffold technology withcells has been shown a high potential in the defect regeneration in the animal models. The fitable seeding cells and scaffolds were chosen to construct theanatomical osteochondral composites and relative studies of scaffolds systemloaded with seeding cells in the repair of large osteochondral defects was alsoprovided as follows:I Selection of seeding cells of osteochondral composites1. Obtainment and culture of bone mesenchymal stem cells (BMSCs) andchondrocytes in vitroTwo-week-old New Zealand White rabbits were selected for BMSCs andchondrocytes isolation and culture. Trypsin-collagenase digested method,adhesive culture method, high or low-density culture method were adopted andbiological characters such as proliferative capability, cell phenotype and cellexcretion of primitive cells and third-generation cells were inveatigated. Theresults showed that high-density culture chondrocytes, especially purifiedprimitive cells, have well proliferative capability and cell excretion. Moreover, alarge quantity of primitive cells obtained from different parts could be guaranteed,because the biological characters of BMSCs from ears or joints were almost same.Adhesive culture method was proved to be an effective method to provide a largenumber of cells. All above was the favorable base of future study.2. Inducement and differentiation of BMSCsCelluar non-contact co-culture induced method and conditioned mediuminduced method were taken to futher choose the seeding cells in this study. Theresults suggested that BMSCs induced by different methods could bedifferentiated into desired cells and the induced cells had the well proliferativecapability evaluated by MTT, mitotic cycle, GAG secretion, ALP, cell-stainingmethods. The results also displayed that the biological characters of co-cultureinduced cells was seemed to be better than conditioned medium induced cells,which was related with the growth factors and matrices from chondrocytes, eventhough poorer than primitive cells. Osteoinductive ability and osteogenetictendency of BMSC was definite. Therefore, primitive chondrocytes andthird-generation osteoinductive BMSC were comfirmed as the ultimate seedingcells in this study.II Selection of scaffolds of osteochondral composites1. Morphology and physical characters of scaffolds of osteochondral compositesFour kinds of scaffolds were made by different biomaterials, and the surfacemorphology, mechanical propertyand in vitro degradation of them wasinvestigated. Gelatin–chondortin–hyaluronan hybrid scaffold and Gelatin–CBBscaffold were the ultimate scaffolds.2. Construction and biocompatibility of osteochondral compositesTo investigate the cytotoxicity and the effect on cell proliferation of thescaffolds, the cells were seeded on them. And the novel three-dimensional specialscaffold was developed into a biphasic scaffolds with a controlled release ofbFGF, which provided structural support and stimulated repair. The in vitroformation of osteochondral composites was observed under the condition ofosteoinduction or un-osteoinduction. The results testified the formation ofosteochondral composite tissue on the biphasic scaffolds by morphological andmatrix secretion assay.Construction of osteochondral composites and in vivo study1. Heterotopic transplantation of osteochondral composites in vivoThree-dimensional biphasic scaffolds seeded with cultured fluorescence mouse costal chondrocytes and BMSCs were transplanted subcutaneously intonude mice after 1day, 7day and 14day, and analyzed histologically during 8weeks after the operation. Our findings showed that chondral and boneregenerations of the subchondral phases were observed via fluorescent analysisand the results demonstrated the newel tissue was derived from transplantedosteochondral composites. On the other hand, the earlier transplantedosteochondral composites were contributed to tissue maturation, which impliedthe nutrient content and blood vessel ingrowth in the early stage.2. Repair and regeneration of osteochondral defects in rabbit articular jointsThe aim of this study was to discuss the regenerative capability ofanatomical osteochondral composites in in repairing large articular joint defects.The scaffolds were fabricated via Perfusion Rapid Prototyping technology andcontrolled release system of growth factors ,and the resulted anatomicalosteochondral composites were orthotopic transplantated. In the study, hyalinecartilage-like regeneration tissue was observed at the border of the normalcartilage with histologically 24 weeks postoperatively, for physiologicalstimulation,such as growth factors and biomechanics, were existed in jointmicroenvironment. Both morphology and motor function were recovered byfunctional rebuilding.In conclusion, osteochondral composites were constructed successfully invitro after selection of seeding cells and scaffolds. The in vivo bone and cartilageengineering via combining a novel composite, biphasic scaffold technology withtwo kinds of seed cells has been shown a high potential in the large defectregeneration in the animal models. The present study implied the great potentialof the novel strategy as a new way to promote clinical relative diseases and it might serve as a desirable approach to contruct other tissue engineering organs. |
