Matsaba, Thato Nelly2014-05-222014-05-222014-05-22http://hdl.handle.net10539/14701Several members of the bone morphogenetic protein/osteogenic protein (BMP/OP) and transforming growth factor beta (TGF-B) families are molecular regulators o f cartilage and bone regeneration. However, their precise mode o f signal transduction and combined interactions are poorly understood. The presence of several molecular forms o f these growth factors suggests multiple functions in vivo as well as synergistic interactions during both embryonic bone development and regeneration o f cartilage and bone in postnatal life. A functional bioassay for identification of osteogenic proteins within the bone matrix has been established. The heterotopic bioassay in rats, together with the improved purification methods, has led to the purification o f native BMPs. In rats, heterotopic and orthotopic implantation o f TGF- 13 singly fails to initiate new bone formation whereas implantation o f BMPs/OPs elicit the local differentiation o f new bone, at both sites. This study presents data which shows that co-administration of TGF-B1 with OP-1 delivered by a collagenous carrier, and implanted in the subcutaneous site o f rats results in synergistic induction o f bone formation. Changes in hist-logical, biochemical and molecular response o f the effects o f the morphogens were vu bed over 7, 12 and 21 days post implantation. Induced cartilage and bone were analyzed by alkaline phosphatase activity, calcium content, Northern blots and histological examination of subcutaneous implants in the rats. Single applications o f TGF-B1 (0.01pg, 0.03pg and O.lpg) on days 7, 12 and 21 gave rise to negligible alkaline phosphatase activity (0.03-0.09 U/ mg protein) and calcium content (0.05-0.6 pg/mg tissue). Histological examination showed that all TGF-B1 implants did not exhibit any sign o f bone formation. On day 7, OP-1 implants (O .lpg and 0.3 pg) elicited negligible alkaline phosphatase activity and calcium content. However, higher doses o f OP-1 (1 pg and 3 pg) elicited alkaline phosphatase activity o f 0.1 U/mg protein and 0.2 U/mg protein. Combination TGF-Bl (O.Olpg, 0.0.3pg and O.lpg) with OP-1 (O.lpg, 0.3pg, Ip g and 3pg) slightly increased the activity o f alkaline phosphatase activity (0.2 U/mg protein-0.4 U/mg protein). OP-1 singly gave rise to very low calcium levels o f 0.4 pg/mg tissue to 0.5 pg/mg tissue. Addition o f TGF-Bl to OP-1 resulted in calcium content rising from 0.2 to 1 pg/mg tissue. Histologically, the specimens o f single OP-1 applications did not show any sign o f bone formation. On addition o f TGF-Bl to OP-1 the specimens showed signs o f the beginning o f chondroblastic differentiation. On day 12 alkaline phosphatase activity elicited by single applications o f OP-1 ranged from 0.1 U/mg protein to 1 U/mg protein. Addition of l i , : 7- Bl increased the alkaline phosphatase from 0.8 U/mg protein to 7 U/mg protein. Calcium levels resulting from single applications of OP-1 ranged from 0.1 to 15 pg/mg tissue Auer addition of TGF-Bl to OP-1 calcium levels rose from 5 to 20 pg/mg tissue. Histological analysis showed formation o f cartilage in specimens both of OP-1 solo and OP-1 in combination with TGF-B1. On day 21 alkaline phosphatase activity was reduced to a range o f 0.1-0.5 U/mg protein upon single applications o f OP-1. Addition TGF-131 resulted in a further decrease in alkaline phosphatase activity. Calcium levels were 10-68 jag/mg tissue on single applications o f OP-1. Addition o f TGF-131 to OP-1 increased the calcium levels in the range o f 2-70 gg/mg tissue. Histological examination o f the 3 jig OP-1 solo specimens showed complete chondrolysis whereas the OP-1 (3|ig)/ TGF-B1 (0.03 and 0.1 gg) specimens showed the differentiation o f bone marrow. Tissues generated in the rat subcutaneous space at 7, 12 and 21 days post implantation elicited mKNA expression o f OP-1, BMP-3 and TGF-B1. These results indicate that at least in part, the matrix-induced endochondral bone formation involves the expression o f some members o f the TGF-B superfamily. Type II collagen (chondrogenesis marker) and type IV collagen (angiogenesis marker) mRNAs were also detected on days 12 and 21, respectively. The present data suggests that TGF-B up regulates the temporal activity o f OP-1 to induce bone formation. Co-administiation of TGF-B 1 to OP-1 caused an increase in the alkaline phosphatase activity and calcium content, markers o f bone formation, which implies that when TGF-B is mixed with OP-1, the cascade o f bone formation is accelerated. These results may have important therapeutic implications. The rapidity o f tissue morphogenesis with bone marrow formation is important for regeneration o f bone in older patients where repair phenomena are temporally delayed and the healing process is slower than in younger patients. The expression of multiple members o f the TGF-13 superfamily indicates that the cascade of bone formation incorporates some members o f the TGF-B superfamily and this may form the basis for synergistic molecular therapeutics for cartilage and bone regeneration in clinical contexts.enOsteogenesisBone developmentThesis