Feasibility of constructing artificial cartilage with rabbit mesenchymal stem cells and polyglycolic acid scaffold / 中国组织工程研究

ABSTRACT

BACKGROUND: Whether choosing a suitable biological scaffold compounding with mesenchymal stem cells (MSCs) can construct an ideal tissue-engineering cartilage or not should be researched further.OBJ ECTIVE To investigate the feasibility of constructing artificial cartilage by using amplified rabbit MSCs which were inoculated on poly-glycolic acid (PGA).DESIGN: Single sample observation.SETTING: Department of Otolaryngology-Head & Neck Surgery, Affiliated Hospital of Sichuan Luzhou Medical College.MATERIALS The experiment was carried out in the Luzhou Medical College from October 2004 to October 2005. A total of 8 Japanese large ear rabbits, of both genders, clean grade, aged from 2 to 3 months, weighing 1.5-2.0 kg, were fed in normal temperature and humidity. Poly-glycolic acid was provided by Albany Company, USA.METHODS: Rabbit MSCs were separated, obtained and amplified. In addition, poly-glycolic acid was sheared into pieces with the size of 1 cm × 1 cm × 1 cm and embedded with poly-L-lysine. Amplified MSCs were inoculated on the surface of poly-glycolic acid, and then, they were averagely grown on pre-wet scaffold of poly-glycolic acid according to 4 mL/cm3multi-points spreading style and cultured in vitro for 3 weeks. After the operations mentioned above, samples were regarded as the experimental group. Scaffolds of poly-glycolic acid without MSCs were considered as the control group.Samples in both experimental group and control group were transplanted into abdominal cavity of 4 rabbits, respectively,cultured in vivo for 6-12 weeks, taken out and observed generally. Meanwhile, the samples were fixed with 100 g/L neutrality formaldehyde, cut into sections with the thickness of 5 μm, stained with haematine-eosin (HE) and observed their histomorphological characteristics. Moreover, the samples were stained with alcian blue for observation of glycosaminoglycans formation, with toluidine blue for observation of metachromasia matrix formation, and with immunohistochemical staining for detection of type- Ⅱ collagen expression.MAIN OUTCOME MEASURES: Generally observational results of two kinds ofscaffolds at 6 and 12 weeks after transplanting into experimental rabbits, various staining results and expression of type- Ⅱ collagen.RESULTS: A total of 8 experimental rabbits were involved in the final analysis. ① Generally observational results of two kinds of scaffolds at 6 and 12 weeks after transplanting into experimental rabbits At 6 weeks after transplanting scaffold into abdominal cavity of rabbits, samples in the experimental group were still coated with greater omentum of abdominal cavity. After clearing greater omentum, three samples were light yellow, smooth and moderate quality; meanwhile, their appearances were coincidence with those before transplantation. However, one sample was gray black and soft quality;meanwhile, it was not able to take shape. Twelve weeks later, appearances of samples in the experimental group were still coincidence with those before transplantation. They were gray white, smooth but hard quality. However, samples in the control group were mostly absorbed at 6 and 12 weeks after transplantation, especially, samples were remarkably absorbed at 12 weeks after transplantation. Any tissue like cartilage did not form in both two durations. ② Various staining results and expression of type- Ⅱ collagen at 6 and 12 weeks after transplanting scaffolds into experimental rabbits Results of HE staining showed that, at 6 weeks after transplantation, structure of cartilage lacuna-like was started form, and PGA scaffold began to be degraded. In contrast, at 12 weeks postoperatively, some cartilage-like tissue were observed in compound, cartilage lacuna-like structure formed obviously; cells arrayed regularly and geminately existed In cartilage lacuna-like atructure; the smaller cell sizes observed at borders and the bigger ones observed at the center;PGA degraded completely. Results of alcian blue staining showed that, at 6 weeks after transplantation, a partial of regions in tissue were light blue; meanwhile, at 12 weeks after transplantation, matrixes in tissue were mostly blue. This suggested that a lot of glycosaminoglycans were formed. Results of toluidine blue staining suggested that, at 6 weeks after transplantation, blue metachromasia matrixes were observed in tissue; meanwhile, at 12 weeks after transplantation, blue metachromasia matrixes were stronger and stronger in tissue. Results of immunohistochemical staining indicated that, at 6 weeks after transplantation, buffy positive granules were observed in plasma and a few of type-Ⅱ collagen expressed in matrix; meanwhile, at 12 weeks after transplantation, powerfully positive expressions were observed in both plasma and matrix. Expression of type- Ⅱ collage showed that, at 6 weeks after transplantation, dark buffy positive granules were observed in plasma and a few of type- Ⅱ collagen mRNA expressed in matrix; meanwhile, at 12 weeks after transplantation, powerfully positive expressions were observed in both plasma and matrix. Samples in the control group were completely absorbed and any tissue-engineering samples did not form.CONCLUSION: Affecting by osteogenic inducer, rabbit MSCs can generate tissue-engineering cartilage after culture in vitro and in vivo.