Duneas N, Crooks J, Ripamonti U
Bone Research Laboratory, Medical Research Council/University of the Witwatersrand, Medical School, Johannesburg, South Africa.
Growth Factors. 1998;15(4):259-77. doi: 10.3109/08977199809017482.
Certain members of the bone morphogenetic protein (BMP) and transforming growth factor-beta (TGF-beta) families are inducers of endochondral bone formation in vivo. TGF-betas, however, do not initiate bone formation when implanted in heterotopic (extraskeletal) sites of rodents. Here we show that platelet-derived porcine TGF-beta 1 (pTGF-beta 1) induces endochondral bone in heterotopic sites of the baboon (Papio ursinus) at doses of 5 microgram per 100 mg of guanidinium-inactivated collagenous bone matrix as carrier, with an inductive efficiency comparable to 5 and 25 micrograms of recombinant osteogenic protein-1 (hOP-1, BMP-7), a well characterized inducer of bone formation. We further demonstrate that pTGF-beta 1 and hOP-1 interact synergistically to induce large ossicles in the rectus abdominis of the primate as evaluated by key parameters of bone formation on day 14 and 30. Tissue generated on day 30 by 5 microgram pTGF-beta 1 or 25 micrograms hOP-1 induced comparable expression levels of OP-1, BMP-3 and type IV collagen mRNA transcripts, whereas TGF-beta 1 and type II collagen expression was 2 to 3 fold higher in pTGF-beta 1-treated implants, as determined by Northern analysis. In ossicles generated by 25 micrograms hOP-1 in combination with relatively low doses of pTGF-beta 1 (0.5, 1.5 and 5 micrograms), type II collagen expression increased in a pTGF-beta 1 dose-dependent manner, whilst type IV collagen was synergistically upregulated with a 3 to 4 fold increase compared to ossicles generated by a single application of 5 micrograms pTGF-beta 1 or 25 micrograms hOP-1. Morphogen combinations (5 micrograms pTGF-beta 1 with 20 micrograms hOP-1, and 5 and 15 micrograms pTGF-beta 1 with 100 micrograms hOP-1 per g of collagenous matrix as carrier) induced exuberant tissue formation and greater amounts of osteoid than hOP-1 alone when implanted in calvarial defects of the baboon as evaluated. on day 30 and 90, with displacement of the temporalis muscle above the defects. Since a single application of TGF-beta 1 in the primate did not induce bone formation in calvarial defects, whilst it induces endochondral bone differentiation in heterotopic sites, our data indicate that the bone inductive activity of TGF-beta 1 is site and tissue specific. mRNA expression of multiple members of the TGF-beta superfamily suggests complex autocrine and paracrine activities of the ligands and different signalling pathways on responding cells during the cascade of endochondral bone formation in the primate. The present findings may provide the basis for synergistic molecular therapeutics for cartilage and bone regeneration in clinical contexts.
骨形态发生蛋白（BMP）家族和转化生长因子-β（TGF-β）家族的某些成员是体内软骨内骨形成的诱导剂。然而，当将TGF-β植入啮齿动物的异位（骨骼外）部位时，并不会引发骨形成。在此我们表明，以每100mg胍灭活的胶原骨基质作为载体，剂量为5微克的血小板衍生猪TGF-β1（pTGF-β1）可在狒狒（山魈）的异位部位诱导软骨内骨形成，其诱导效率与5微克和25微克的重组骨生成蛋白-1（hOP-1，BMP-7，一种已充分表征的骨形成诱导剂）相当。我们进一步证明，通过第14天和第30天骨形成的关键参数评估，pTGF-β1和hOP-1在灵长类动物的腹直肌中协同作用以诱导大的骨块。第30天由5微克pTGF-β1或25微克hOP-1诱导产生的组织中OP-1、BMP-3和IV型胶原mRNA转录本的表达水平相当，而通过Northern分析确定，在pTGF-β1处理的植入物中TGF-β1和II型胶原的表达高2至3倍。在由25微克hOP-1与相对低剂量的pTGF-β1（0.5、1.5和5微克）联合产生的骨块中，II型胶原的表达以pTGF-β1剂量依赖性方式增加，而IV型胶原与单次应用5微克pTGF-β1或单次应用25微克hOP-1产生的骨块相比协同上调3至4倍。形态发生素组合（每克胶原基质作为载体，5微克pTGF-β1与20微克hOP-1，以及5微克和15微克pTGF-β1与100微克hOP-1）在植入狒狒颅骨缺损时，与单独使用hOP-1相比，诱导了旺盛的组织形成和更多的类骨质，在第30天和第90天评估时，缺损上方的颞肌发生移位。由于在灵长类动物的颅骨缺损中单次应用TGF-β1不会诱导骨形成，而它在异位部位可诱导软骨内骨分化，我们的数据表明TGF-β1的骨诱导活性具有部位和组织特异性。TGF-β超家族多个成员的mRNA表达表明，在灵长类动物软骨内骨形成级联反应过程中，配体具有复杂的自分泌和旁分泌活性以及对反应细胞的不同信号通路。本研究结果可能为临床环境中软骨和骨再生的协同分子治疗提供基础。
