| Articular cartilage defects are common outcomes of trauma and joint disease, alwaysleading to osteoarthritis (OA) with severe pain. The articular cartilage is lack of bloodsupplied and chondrocytes belong to stable cells with nature of low division, proliferationand metabolism, so the articular cartilage has limited self-repair capacity. Moreover,subchondral bone often undergoes degeneration, sclerosis with overlying chondral defectat the same time. The neocartilage is hard to integrate with the degenerated and sclerotizedsubchondral bone. Therefore, lesions of subchondral bone should be treated together withthe chondral defects. Till now, many surgerical strategies have resulted in poor results. Theimproved tissue-engineering technique is the most promising method for repair of articularosteochondral defects. Recent years, some people have employed osteochondral composite scaffolds to repair the articular osteochondral defects. Although some initialsuccesses have been achieved, two major factors still limit the widespread implementationof these techniques. Firstly, the weak integration between chondral scaffold and bonyscaffold always result in mechanical failure in vivo. Secondly, the regeneration andproliferation capacity of the seed cells in the biphasic scaffold is very low, leading tounsatisfied regeneration of osteochondral tissue.To resolve the aboved problems, the micro structure of osteochondral tissue and thesurvivable environment of chondrocytes and osteoblasts should be adequately understood.The normal articular surface is composed by cartilage and subchondral bone. Articularcartilage has four-layered structure composed of superficial, middle, deep and calcifiedlayers. The calcified layer has a compact structure. Virtually, it is a depletion layerbetween cartilage and subchondral bone, serving the function of insulating and integrating.Articular cartilage derives nutrients primarily from the synovial fluid, where oxygenpartial pressure is much lower than in vascularized subchondral bone. The chondrocytesare well adapted to a hypoxic environment and the hypoxic tension environment may bebeneficial to speed differentiation and proliferation. Once blood infiltrates into thearticular cavity, the antigenic products of leukocytes may be released into the synovialfluid, provoking the inflammation that causes necrosis in the newborn chondrocytes. Atthe same place, if the synovial fluid permeates into subchondral bone, the osteogenicactivity will be detrimented. Therefore, especially for the chondrocytes with limitedregeneration capacity, the calcified layer is a prerequisite to keep in good state. The idealosteochondral scaffold should mimic the native physiological structure of osteochondraltissue. So, only a thing works like calcified layer is developed in the osteochondralcomposite scaffold, the regeneration of osteochondral tissue can be promoted.In previous experiment, the extracellular matrix (ECM) was obtained after bovinearticular cartilage was physically shattered and decellularized sequentially. An orientedmicrotubule-structure scaffold was fabricated using a temperature gradient-guidedthermal-induced phase separation (TIPS) technique. The bony scaffold, possessing a3-Dcore-sheath structure, was composed of a PLGA/β-TCP skeleton wrapped with Type I collagen. The core-sheath structure composite scaffold was fabricated using rapidprototyping (RP) technique and low-temperature deposition manufacturing (LDM)technique. Previous in vitro and in vivo experiments have demonstraetd that both the bonyand chondral phases exhibit super characteristics of fabrication and molding, high porosity,significant hydrophilia and biocompatibility, and suitable mechanical strength anddegradation velocity. Moreover, they could meet the standard of ideal tissue-engineeringosteochondral scaffold.In this study, the objective of adding the compact layer between the chondral andbony scaffolds was to integrate them more closely and enhance the mechanical property ofthe osteochondral composite scaffold. Furthermore, it was expected that provided withtwo independent living environments by the compact layer, the proliferation anddifferentiation of new-born chondrocytes and osteocytes, and the repair of osteochondraltissue would be promoted. The detail is as follows:Part I Fabrication of osteochondral composite scaffold with thecompact layer and research on the characterists of the compact layerIn this study, the compact layer with about0.5mm thickness was formed by extrudingslurry of PLGA/β-TCP onto the top of bony scaffold with RP technique. The compactlayer and chondral scaffolds were connected with dissolving-conglutination method. Thenthe osteochondral composite scaffold with compact layer was formed. The osteochondralcomposite scaffolds without compact layers were also made. The bony and chondralscaffolds were bonded directly using the same process. The osteochondral compositescaffolds with compact layers were assigned into the experimental group, and the oneswithout the compact layers were assigned into the control group.The compact layer was observed with scanning electron microscope, and the poriness,absorption rate and permeability for fluid and cells were detected. The mechanicalproperties of the osteochondral composite scaffolds in the two groups were also evaluated.It was demonstrated that the poriness and absorption rate were very small, and thehydrophobicity was strong. Moreover, the compact layer could not be permeated through by fluid and cells. The maximal anti-tensile strength and the maximal anti-shear strenghtin the experimental group were remarkably higher than those in the control group(P<0.05). It colud be concluded that after the osteochondral composite scaffolds wereimplanated into osteochondral defects, being separated by the compact layers, thechondrocytes and osteoblasts would be provided with different enivronments. Furthermore,the compact layer might significantly enhance the biomechanical properties of theosteochondral composite scaffold to bear stress in the articular cavities.Part II The research on the effect of the compact layer on fabricatingtissue-engineering osteochondral composite in vitroIn this study, after the bone marrow stem cells (BMSCs) were obtained from adultrabbits’ iliac bones, they were purified, and amplified. Then the BMSCs were induced tochondrocytes and osteoblasts with different revulsive, respectively. The osteochondralcomposite scaffold was placed into the osteochondral culture chamber invented by ourself.The chondrocytes and osteoblasts induced from BMSCs were dropped onto the chondraland bony scaffolds of osteochondral composite scaffold, respectively. The cells seededwere kept on to induce to chondrocytes and osteoblasts. Due to the compact layers, thecells in the chondral and bony scaffolds were separated into different environments. SEMwas used to observe the morphous of cells seeded onto the osteochondral compositescaffolds, and MTT method was used to detect the cells proliferation. Many techniqueswere employed to evaluate the differentiation of chondrocytes and osteoblasts.SEM micrographs showed that provided with two independent living environmentsby the compact layers, the cells stretched out pseudopodiums on the scaffolds, becamethin and flat, got in touch with each other and confluensed, proliferated in deep sites of thescaffolds, and covered the whole surface of the micro pores. The results of MTT revealedthat with prolongation of culture, the number of cells in both chondral and bony scaffoldsincreased remarkably.Having being induced and cultured in the osteochondral scaffolds for20days, the chondrogenic and osteogenic induced BMSCs was digested off the scaffolds. The cellswere cultured and induced in chondrogenic or osteogenic induction medium, respectively.All chondrogenic induced BMSCs were stained positive for safranin O, toluidine blue, andtype Ⅱ collagen, but negative for collagen type I. The osteogenic induced BMSCs werestained positive by BCIP/NBT for alkaline phosphatase (ALP) and alizarin bordeaux forcalcium nodus.The aboved results demonstrated that provided with two independent livingenvironments by the compact layers, the cells on chondral and bony scaffolds couldadhere and proliferate well, exhibit typical differentiated phenotype of chondrocytes andosteoblasts, and form tissue-engineering osteochondral composite in vivo. Moreover, nonedegeneration was found.Part III The research on the impact of the compact layer in osteochondralcomposite scaffold on repair of rabbits’ articular osteochondral defect in vivoIn this study, the cells-scaffolds composites were implanted into articularosteochondral defects of autogeneic rabbits’ knees. At3and6months after surgicalimplantation, the regenerated cartilage was examined macroscopically. Furthermore, thecompressive modulus and property of molecular biology of neocartilage, bone mineraldensity of subchondral bone, and3-D reconstruction of Micro-CT and histologicalstaining of regenerated osteochondral tissue were also evaluated. The results were used toanalyze the impact of the compact layers in osteochondral composite scaffolds on repair ofarticular osteochondral defects in vivo.The gross morphology of the regenerated neocartilage exhibited that theosteochondral defects in the experimental group were repaired more satisfactorily thanthose in the control group. The scores of the gross morphology, the compressive modulus,the expression level of the type II Collagen and its mRNA of neocartilages in theexperimental group were significantly higher than those in the control group (P <0.05). At6months after surgery, there were no significant deviation between the results of theneocartilage in the experimental group and those of native cartilage. The histological appearance showed that the regenerated osteochondral tissue of theexperimental group was much better than that of the control group. Especially at6monthsafter surgery, it was very similar to the native osteochondral tissue. The histological scoresof the regenerated osteochondral tissue in experimental group were statistically superior tothat in the control group (P<0.05). The3-D images of Micro-CT were similar to thehistological images. The BMD of regenerated subchondral bone of experimental groupwere remarkably higher than that of the control group (P<0.05).The aboved results showed that compared with the newborn osteochondral tissues ofcontrol group, those of experimental group got better outcomes at the aspects ofmorphology, biomechanics and molecular biology. It was demonstrated that provided withdifferent environment by the compact layers, the osteoblasts and chondrocytes proliferatedand differentiated well.Therefor, the compact layers play an important role in repair of articularosteochondral defects by osteochondral composite scaffolds. |
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