In the treatment of bone non-unions an alternative to bone autografts is the use of bone morphogenetic proteins (BMP-2, BMP-7) with powerful osteoinductive and osteogenic properties. In clinical settings, BMPs are applied using absorbable collagen sponges. Supraphysiological doses are needed and major side effects may occur as induce ectopic bone formation, chronic inflammation and excessive bone resorption. In order to increase the efficiency of the delivered for BMPs we designed cryostructured collagen scaffolds functionalized with hydroxyapatite, mimicking the structure of cortical bone (aligned porosity, anisotropic, ANI) or trabecular bone (random distributed porosity, isotropic, ISO). We hypothesize that anisotropic structure would enhance osteoconductive properties of the scaffolds increasing rhBMP-2 regenerative properties.
In the treatment of nonunions, and other complications of bone repair, an attractive alternative to bone autografts would be the use of a combination of autologous mesenchymal progenitors cells (MSCs), biomaterials and growth factors. Our goal was to determine the therapeutic potential and contribution to the repair process of different sources of mesenchymal stem cells for the treatment of nonunions. The right femur of Sprague-Dawley (SD) rats was stabilized with an aluminum plate (20 mm long, 4 mm wide, 2 mm thick) and four screws (1.5 mm diameter, 8 mm long). A diaphyseal critical size defect was performed (5 mm). Six groups (n=6–8 animals each) were created. A nonunion group (Control group, empty defect); LBA group, live bone allograft; BMP2 group, rhBMP-2 (2 μg) in collagen sponge; PCL group, polycaprolactone scaffold; PMSCs group, PCL scaffold loaded with 5×106 periosteum-derived MSCs; and BMSCs group, PCL scaffold loaded with 5×106 bone marrow-derived MSCs. For cell tracking purposes, LBA and MSCs were derived from SD-GFP transgenic rats. The repair process was followed up by x-rays up to sacrifice, week 10. After sacrifice, femurs were analyzed by micro computed tomography (μCT), histology and immunohistochemistry. For multiple comparisons one-way ANOVA followed by Dunnett”s test for single comparisons was used. Statistical significance was established for p<0.05.INTRODUCTION
METHODS
Hyaline cartilage is a support tissue with a poor capacity to self repair. In the last years, tissue engineering and cell therapy have focused its efforts in the development of scaffolds that may support the differentiation and the implantation of mesnechymal stem cells (MSC) in the site of lesions performed in femoral cartilage. Among synthetic materials used for the construction of these scaffolds, poly(L-lactic acid) (PLLA) is a suitable option, since some studies have offered promising results. The use of PLLA, nevertheles has an important handicap, as cell seeding easily results in a non uniform distribution and a poor density of cells, wich have been proposed as key steps for the differentiation of MSCs to chondrocytes. In our work we have cultured sheep MSCs, and proved its potentiallity by differentiation to chondrocytes in micromass culture. PLLA scaffolds 1 mm thick and 6 mm in diameter were characterized by determining their porosity and their mechanical properties, and subsequently were used to assay the seeding of MSCs. We measured efficiency and retention by quantification of DNA, and density and distribution by light microscopy of paraffin sections. Our results describe a simple technique of cell seeding by aspirating cells with a syringe that achieves a uniform distribution and a high density of cells. Finally 3D seeded MSCs were cultured with condrogenic medium containing TGF-β3 for 21 days and results analyzed by massons trichrome staining in paraffin embedded sections.
