Type â…¡ Collagen Hydrogel-Cell Complexes (Injectable Tissue-engineered Cartilage) In The Regeneration Of Full-thickness Articular Cartilage Defects Of Mini Pigs

IntroductionWithout blood circulation, lymphatic drainage or innervation, mature articular cartilageshows poor healing abilities. The articular cartilage defect whose diameter exceeds3mm isrepaired by fibrous tissue rather than hyaline cartilage. The lack of the biochemical andbiomechanical characteristics of the later always leads to degeneration of the repaired tissueunder physiological load, which may develop into joint dysfunction or even the loss of workability. In clinical practice, articular cartilage lesion is very common. According to thestatistics of the United States, each year there are250,000to500,000patients with cartilagelesions waiting for medical intervention. However, the existing technologies are far fromsatisfaction. Joint debridement can only relieve symptoms temporarily, but can not stop thecourse of worsening. Joint fusion or joint resection angioplasty makes joint dysfunction. Jointreplacement, the most popular surgery today, is no more than a substitute implantation. Theproblems of loosening of prosthesis, osteolysis and high cost prevent joint replacement frombeing widely applied.In1984, Swedish physician Peterson first reported the technology of autologouschondrocyte transplantation (ACT) to repair the articular cartilage defect of rabbit.16weeksafter the surgery, the articular cartilage defects were repaired with hyaline cartilage. In1987,Brittberg et al. from Gothenburg Medical Center of Sweden reported the successful treatmentof articular cartilage defects with the help of ACT. All these successful attempts built a solidbase for further development of cartilage engineering. However, nowadays researchers arestill searching for ideal seed cells. Harvesting the autologous chondrocytes may cause extradamage to the joint, besides the small amount of cells, the risk of a second operation andanaesthesia, the long-term treatment cycles and the chondrocytes' instability in monolayerculture. Allogenetic chondrocyte transplantation may result in immune rejection and thepossible disease transmission. Embryonic stem cells have the abilities of pluripotency and unlimited proliferation, but the tumorigenicity and ethics problems confine its application.Adult stem cells, especially bone marrow-derived mesenchymal stem cells (MSCs), havebeen considered as an attractive source of cells for cartilage engineering owing to their ease ofavailability and their high capacity of expansion in vitro. But the separating and purifyingprocesses of MSCs remain complex and uncertain.The choice of scaffold plagues researchers as well. Natural materials, synthetic materials,composite materials or gene activated materials all have their limitations. With the progressgained in cartilage engineering, naturally derived hydrogels have received better attention.Silverman et al. combined fibrin gel with porcine cartilage cells. Then they injected thecomposition into subcutaeous of nude mice.6or12weeks later, the composition formed solidcartilage. Comparing with those in the control group or the fibrin gel group, the componentsin the composition group were more similar to natural cartilage through histologicalobservation, glycosaminoglycan and type Ⅱ collagen content analysis. Recent work ofChung et al. showed that hexadecenoic acid (HA) hydrogel can not only maintain the vitalityand phenotype of chondrocytes cultured in vitro or in vivo, but also induce MSCs todifferentiate into chondrocytes. As the carrier of seed cells, hydrogel has many advantages,including ease of handling where cells are simply mixed in a solution prior to gelationenabling a highly uniform cell seeding, a highly hydrated tissue-like environment, and theability to form in vivo. In addition, natural polymers have good biocompatibility. Activegroups in the surface can promote cells proliferation and differentiation, and easily degradeinto non-toxic products in the body. In vitro experiments have confirmed that type Ⅱcollagen, the major component of articular cartilage, could induce bovine MSCs tochondrogenic differentiation, which also showed better cell morphology and chondrogenicgene expression than the type Ⅰ collagen. However, currently no unified standard have beenmade about preparation of type Ⅱ collagen hydrogel. In vivo experimental results of it differapparently. The large animal experiments have been rarely reported.Chinese researchers have achieved remarkable outcomes in cartilage engineering. Theengineered cartilage shaped like a human ear was successfully cultured in the back of a nudemouse by Cao Yilin in1997. Many basic researches keep up with international advancedlevel in recent years. Yet the transformation of achievements and clinical applications arequite insufficient. A large number of foreign patients with cartilage damages have benefited from the ACT or MACI technology. But in China very few domestically engineered cartilageproducts have moved from bench to bedside and industrial application standards remainslacking. Joint Surgery Center of Southwest Hospital of Third Military Medical Universitydevotes into the research and development of engineered cartilage and the related fields since1997. We raised the concept of biomimetic engineered cartilage construction and accumulateda wealth of experience in the separation, purification, culture and identification of bonemarrow-derived MSCs, as well as quantitative studies about articular cartilage structure andthe interface of chondro-osseous junctional. As for this study, we aimed to develop apreparation method of type Ⅱ collagen hydrogel, in which seed cells were encapsulated toconstruct a injectable engineered cartilage. After the detection of its biological safety, thisengineered cartilage was used to repair the articular cartilage full-thickness defects of largeanimals (mini pigs), and verify the effect of the tactic.In this study, we compared bone marrow nucleated cells (BNCs) with bonemarrow-derived mesenchymal stem cells (MSCs) as seed cells in the treatment of cartilagedefects through animal experiments to explore a more secure, economic and convenienttechnology for repairing articular cartilage defects in clinic.Research Methods1. The fresh porcine articular cartilages were selected as raw materials. Afterwithdrawing the proteoglycans with guanidine hydrochloride, digesting of pepsin, salting ofsodium chloride, dialyzing of ultrapure water and centrifuge enrichment, we had extractedcollagen type Ⅱ, which were used to make temperature sensitive hydrogel. The identificationof the extract was performed through amino acid analysis. The characteristic of gelation, thegross and histological structures as well as preliminary biocompatibility tests of the hydrogelscaffold were observed.2. New Zealand white rabbits were selected as experimental animals. Bonemarrow-derived MSCs were separated and cultured to be encapsulated in hydrogel scaffold,which were wrapped in a diffusion chamber made of semi-permeable membrane. Thediffusion chamber was placed in the rabbit knee joint cavity through sterile surgery.8weekslater, the initial transmutation of injectable engineered cartilage in vivo was observed.3. Twenty skeletally mature Guizhou minipigs were used to create full-thicknesschondral defects of6.0mm in diameter in the knee joints and divided between two time points (four and eight weeks) for final assessment. At every time point, animals wereseparated into four groups: the CON group which underwent no implantation (A group); thecollagen type Ⅱ hydrogel group (B group); the collagen type Ⅱ hydrogel+bonemarrow-derived MSCs group (C group); and the collagen type Ⅱ hydrogel+BNCs group(D group). The samples were grossly examined, observed through MRI, a stereo microscope,histologically analysed and evaluated with the O'Driscoll scoring system, respectively.Results1. The extraction and preparation techniques for collagen type Ⅱ hydrogels weredeveloped successfully. The hydrogels had good biocompatibility and temperature sensitivitygelation property, which means that the gelation were accomplished after5to10minutes at37℃.2. The diffusion chambers used to wrap the hydrogel-MSCs composite were placed inthe rabbit knee joint cavities through sterile surgery.4weeks after the surgery, MRI showedno rupture of the diffusion chambers or significant articular dropsy.8weeks after the surgery,the wounds healed well without deformity. When the knee joints were exposed, no significanthyperaemia and hyperplasia of synovial membranes and synovial fluid were observed. Thegray and translucent chondrogenic tissues could be found clearly in the diffusion chambers.Histological observation showed a large number of GAGs expression, the cell morphologysimilar to the chondrocyte and the existence of some cartilage lacunae.3. Knee joint full-thickness chondral defect models of Guizhou mini pigs and theimplanting technique of injectable engineered cartilage were established successfully.8weeksafter the surgery, gross observation showed that the defects of the cell-treated groups(experimental groups) were repaired with cartilage-like tissues. Yet those of the control groupand the collagen group were repaired incompletely. The surfaces of the regenerating tissueswere smooth. The interfaces of regenerating tissues with adjacent tissues were integratedsatisfactorily in the experimental groups through a stereo microscope observation.Histological observation confirmed that the repairing tissues were hyaline cartilage-liketissues in the experimental groups. The O'Driscoll histological scores for repairing tissuesshowed that the experimental groups were significantly better than the control group and thecollagen group. However, there were no significant differences between the BNCs group andthe BMSCs group. Conclusions1. The extraction and preparation techniques for collagen type Ⅱ hydrogels weredeveloped successfully. The hydrogels had good biocompatibility and temperature sensitivitygelation property.2. The separation, purification and culture techniques of seed cells (BMSCs and BNCs)were standardized. Seed cells were encapsulated in collagen type Ⅱ hydrogels to constructinjectable engineered cartilages, whose cells density were105-6/ml.3. The hydrogel-MSCs composites wrapped in the diffusion chambers were placed inthe rabbit knee joint cavities. After8weeks of culturing, it was confirmed initially that theinjectable engineered cartilages had good chondrogenic ability, which provided an evidencefor the follow-up experiment in big animals.4. Anesthesia, surgery and recovery technologies for mini pigs were standardized. Kneejoint full-thickness chondral defect models of mini pigs were established successfully toprovide a good evaluation carrier for animal study of engineered cartilage.5. With collagen type Ⅱ hydrogels as scaffolds, the transplantation of BMSCs orBNCs contributes to articular cartilage repair of Guizhou mini pigs, in which the regeneratedcartilaginous tissues integrated closely with the circumjacent cartilages. There was nosignificant difference in the repair results between the BNCs group and the BMSCs group8weeks after the surgery.