| BackgroundCartilage injury caused by trauma or osteopathy is very common in clinics, and all thecurrent treatments for cartilage repair have limitations. It is believed that tissue engineeringand regeneration medicine would provide a new strategy for repairing articularosteochondral lesions. The key factors of tissue engineering include cells, growth factors,and scaffolds, with scaffolds being the most essential one of the three for tissue engineering.There are two main categories of osteochondral scaffold. One category uses hierarchicalstructure that construct bone and cartilage tissue separately, and absorption suture materialsor fibrin glue is used to bond the constructed bone and cartilage together. Then it is culturedin vitro or implanted in vivo to repair defects. The other category uses integration structurethat construct complex osteochondral scaffold directly. Then cultured with osteoblasts orchondrocytes in vitro and implated into the defects. Studies show that the engineered tissuefrom these scaffolds faces problems such as poor mechanical properties, tissue fibrosis, andpoor integration, thus making the repaired tissue difficult to reach the long termeffectiveness.According to the basic principle of tissue engineering and the new development ofosteochondral tissue engineering home and abroad, we believed that the reasons for theaforementioned problems are:①Lack of thorough understanding in the biochemistry,biomechanics, and structure of osteochondral tissue;②Currentstrategies for osteochondraltissue engineering have obvious drawbacks, due to the lack of subchondral bone-hyalinecartilage interface–the calcified cartilage zone. Among them, lack of thoroughunderstanding of the concept for articular cartilage connecting interfaceand bionic structure conception is the most important reason and bottleneck of thetechnology in clinical application.Based on morphological structure and physical function of articular calcified cartilagezone and the new engineering conception of integration construction: hyaline cartilage-calcified cartilage zone-subchondral bone, here we used decellularizedosteochondral tissue as scaffold, which completely preserves the calcified cartilage zone.With excellent biocompatible properties, these scaffolds preserve the extracellular matrixcomponents of natural bone and cartilage. Extracellular matrix scaffold from cartilage andbone respectively fit for the different requirements of two tissues. This study preserved thatnatural structure of calcified cartilage zone, so it is more in line with the physiologicalcharacteristics of subchondral bone and cartilage. Hence, it may be an ideal scaffold forosteochondral tissue engineering. This study illustrated boundary layer structure inaccordance with necessity and feasibility of construction of tissue in bone and cartilage, andprovided theoretical basis and technical support for the new insight on application ofregeneration repair of articular cartilage defects.Methods1. Preparation and identification of decellularized bone with natural calcified cartilagelayer:8mm diameter bone blocks with calcified layer was drilled from the bone surface,the cartilage layer was then carefully removed without damaging the calcified cartilagestructure. After decellularization, any residual cell and the integrity of calcified cartilagelayer was examined by histological analysis.2. Preparation and identification of collagen type Ⅱ: Cartilage slices from freshporcine knee joints were lyophilized, shattered and digesting process was performed tofabricate collagen type Ⅱ. The extraction rate, purity, and property of gel were measured.3. Preparation and identification of osteochondral scaffold: decellularized bone blockwas fit into a cylindrical mode, followed by2mm thickness collagen type Ⅱ coating.Lyophilization and cross-linking were applied on the osteochondral complex. Porosity andbore diameter of the osteochondral scaffold were evaluated by anhydrous ethanolreplacement method and scanning electron microscope. The osteochondral scaffold waslater seeded with BMSCs, the biocompatibility was examined in osteochondral scaffoldwith/without calcified cartilage layer. At last, the cell survival of BMSCs in each group wasevaluated by green fluorescent protein transfection (lentivirus vector).4. SigmaPlot12.0was used to summarize the data，SPSS18.0was used to analysisdiameter of pores and porosity of the osteochondral scaffold，the data are expressed as mean±SD. Results:1. Preparation and identification of decellularized bone with natural calcified cartilagelayer: The result of decellularizing process was excellent, no residual cell was found byhistological analysis. Combined with micro-CT result, our data suggested the calcifiedcartilage zone was intact.2. Preparation and identification of collagen type Ⅱ: The fabricated type II collagendisplayed the color of ivory-white. The extraction rate of collagen was3.9%. SDS-PAGEresult showed its molecular weight was slightly more than130KD. HPLC result showedthe purity of collagen type II was similar to that of collagen type II made by Sigma.3. Preparation and identification of osteochondral scaffold: The parameters of scaffoldin each group were measured. The porosity of collagen type Ⅱ scaffold was (91.1±3.8)%, and the diameter of pores was (79.7±17.1) μm; the porosity of decellularized bonescaffold was (73.5±2.6)%, and the diameter of pores was (470.2±158.8) μm. In addition,BMSCs seeded in both group showed good cell compatibility, suggesting that thesescaffolds met the requirements of tissue growth.Conclusion：The osteochondral scaffold with triple layers was fabricated with bonedecellularization combined with collagen type Ⅱ lyophilization and cross-linking method.Our fabricated scaffold reserved the natural calcified cartilage layer. Furthermore, thecartilage and subchondral bone scaffolds had proper bore diameter and porosity, whichinsured the cellular growth. Our data form SD rat BMSCs growth and attachment suggestedthe scaffold had good cell compatibility. However, the bio-function of the scaffold needsfurther in vivo test. |
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