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Growth factors like BMP2 have been tested for osteochondral repair, but transfer methods used until now were insufficient. Therefore, the aim of this study was to analyse if stable BMP2 expression after retroviral vector (Bullet) transduction is able to regenerate osteochondral defects in rabbits. Fibrin clots colonized by control or BMP2-transduced chondrocytes were generated for in vitro experiments and implantation into standardized corresponding osteochondral defects (n=32) in the rabbit trochlea. After 4 and 12 weeks repair tissue was analysed by histology (HE, alcian-blue, toluidine-blue), immunohistochemistry (Col1, Col2, aggrecan, aggrecan-link protein), ELISA (BMP2), and quantitative RT-PCR (BMP2, Col1, Col2, Col10, Cbfa1, Sox9). In vitro clots were also analysed by BMP2-ELISA, histology (alcian-blue), quantitative RT-PCR and in addition by electron microscopy. BMP2 increased Col2 expression, proteoglycan production and cell size in vitro. BMP2 transduction by Bullet was efficient and gene expression was stable in vivo over at least 12 weeks. Proteoglycan content and ICRS-score of repair tissue were improved by BMP2 after 4 and 12 weeks and Col2 expression after 4 weeks compared to controls. However, in spite of stable BMP2 expression, a complete repair of osteochondral defects could not be demonstrated. Therefore, BMP2 is not suitable to regenerate osteochondral lesions completely.

In the field of tissue engineering, techniques have been described to generate cartilage tissue with isolated chondrocytes and bioresorbable or nonbioresorbable biomaterials serving as three-dimensional cell carriers. In spite of successful cartilage engineering, problems of uneven degradation of biomaterial, and unforeseeable cell-biomaterial interactions remain. This study represents a novel technique to engineer cartilage by an in vitro macroaggregate culture system without the use of biomaterials. Human nasoseptal or auricular chondrocytes were enzymatically isolated and amplified in conventional monolayer culture before the cells were seeded into a cell culture insert with a track-etched membrane and cultured in vitro for 3 weeks. The new cartilage formed within the in vitro macroaggregates was analyzed by histology (toluidine blue, von Kossa-safranin O staining), and immunohistochemistry (collagen types I, II, V, VI, and X and elastin). The total glycosaminoglycan (GAG) content of native and engineered auricular as well as nasal cartilage was assayed colorimetrically in a safranin O assay. The biomechanical properties of engineered cartilage were determined by biphasic indentation assay. After 3 weeks of in vitro culture, nasoseptal and auricular chondrocytes synthesized new cartilage with the typical appearance of hyaline nasal cartilage and elastic auricular cartilage. Immunohistochemical staining of cartilage samples showed a characteristic pattern of staining for collagen antibodies that varied in location and intensity. In all samples, intense staining for cartilage-specific collagen types I, II, and X was observed. By the use of von Kossa-safranin O staining a few positive patches-a possible sign of beginning mineralization within the engineered cartilages-were detected. The unique pattern for nasoseptal cartilage is intense staining for type V collagen, whereas auricular cartilage is only weakly positive for collagen types V and VI. Engineered nasal and auricular macroaggregates were negative for anti-elastin antibody (interterritorially). The measurement of total GAG content demonstrated higher GAG content for reformed nasoseptal cartilage compared with elastic auricular cartilage. However, the total GAG content of engineered macroaggregates was lower than that of native cartilage. In spite of the mechanical stability of the auricular macroaggregates, there was no equilibrium of indentation. The histomorphological and immunohistochemical results demonstrate successful cartilage engineering without the use of biomaterials, and identify characteristics unique to hyaline as well as elastic cartilage. The GAG content of engineered cartilage was lower than in native cartilage and the biomechanical properties were not determinable by indentation assay. This study illustrates a novel in vitro macroaggregate culture system as a promising technique for tissue engineering of cartilage grafts. Further long-term in vitro and in vivo studies must be done before this method can be applied to reconstructive surgery of the nose or auricle.

Introduction: Mesenchymal stem cells (MSC) have the capacity to differentiate into chondrocytes with the synthesis of cartilage. This report presents the use of human adult bone marrow derived mesenchymal stem cells for tissue engineering of autologous cartilage grafts., Methods: Human bone marrow aspirates were obtained from the iliac crest and fractionated on a Percoll gradient. The isolated hMSC were plated at 20 x 10 (6) cells per 100 mm (2) culture dish. After 21 days in culture at 37 degrees C with 5 % CO 2, the adherent multiplied MSC were trypsinized, counted, and tested for viability by trypan blue assay. The hMSCs were loaded into a sterile 15 ml polypropylene tube (0.5 Mio cells/ml) and centrifuged on the bottom of the tube at 500 g for 5 minutes. The MSC were cultivated for 3 weeks in vitro in a specific chondrogenetic medium composed of Dulbecco's Modified Eagles Medium-High Glucose supplemented with 10 ng/ml transforming growth factor-beta 1, 1 % ITS-Premix medium, 80 micro M ascorbic acid, and 100 nM dexamethasone., Results: Histological and immunohistochemical studies performed after 3 weeks in three dimensional culture demonstrated the expression of cartilage specific collagen type II and X as well as proteoglycans., Conclusion: Human adult mesenchymal stem cells derived from bone marrow aspirates have the ability to differentiate into chondrocytes under specific culture conditions by growth factors. The use of adult mesenchymal stem cells may be a promising tool for tissue engineering of autologous cartilage grafts in reconstructive surgery in the future.

For tissue engineering of cartilage, chondrocytes can be seeded in a scaffold and stimulated to produce a cartilage-like matrix. In the present study, we investigated the effect of alginate as a chondrocyte-delivery substance for the construction of cartilage grafts. E210 (a non-woven fleece of polyglactin) was used as a scaffold. When bare' E210 (without alginate and without chondrocytes) was implanted subcutaneously in nude mice for 8 weeks. the explanted tissue consisted of fat and fibrous tissue only. When E210 with alginate but without chondrocytes was implanted in nude mice, small areas of newly formed cartilage were found. Alginate seems to stimulate chondrogenesis of ingrowing cells. When chondrocytes were seeded in E210, large amounts of cartilage were found, independent of the use of alginate. This was expressed by a high concentration of glycosaminoglycans (30 microg/mg w.w.) and the presence of collagen type II (1.5 microg/mg w.w.). Macroscopically the grafts of E210 without alginate were shrunk and warped, whereas the grafts with alginate had kept their original shape during the 8 weeks of implantation. The use of alginate did not lead to inflammatory reactions nor increased capsule formation. In conclusion, the use of alginate to seed chondrocytes in E210 does not influence the amount of cartilage matrix proteins produced per tissue wet weight. However, it provides retention of the graft shape.

In vitro multiplication of isolated autologous chondrocytes is required to obtain an adequate number of cells to generate neo-cartilage, but is known to induce cell-dedifferentiation. The aim of this study was to investigate whether multiplied chondrocytes can be used to generate neo-cartilage in vivo. Adult bovine articular chondrocytes, of various differentiation stages, were suspended in alginate at densities of 10 or 50 million/ml, either directly after isolation (P0) or after multiplication in monolayer for one (P1) or three passages (P3). Alginate with cells was seeded in demineralized bovine bone matrix (DBM) or a fleece of polylactic/polyglycolic acid (E210) and implanted in nude mice for 8 weeks. The newly formed tissue was evaluated by Alcian Blue and immunohistochemical staining for collagen type-II and type-I. Structural homogeneity of the tissue, composed of freshly isolated as well as serially passaged cells, was found to be enhanced by high-density seeding (50 million/ml) and the use of E210 as a carrier. The percentage of collagen type-II positive staining P3-cells was generally higher when E210 was used as a carrier. Furthermore, seeding P3-chondrocytes at the highest density (50 million/ml) enhanced collagen type-II expression. This study shows promising possibilities to generate structurally regular neo-cartilage using multiplied chondrocytes in alginate in combination with a fleece of polylactic/polyglycolic acid.