Accumulation, Localization, and Compartmentation of Transforming Growth Factor 13 During Endochondral Bone Development

Endochondral bone formation was induced in postnatal rats by implantation of demineralized rat bone matrix. Corresponding control tissue was generated by implanting inactive extracted bone matrix, which did not induce bone formation. At various times, implants were removed and sequentially extracted with guanidine hydrochloride, and then EDTA and guanidine hydrochloride. Transforming growth factor 13 (TGF13) in the extracts was quantitated by a radioreceptor assay. TGF13 was present in demineralized bone matrix before implantation, and the concentration had decreased by 1 d after implantation. Thereafter, TGF13 was undetectable by radioreceptor assay until day 9. From day 9-21 the TGF13 was extracted only after EDTA demineralization, indicating tight association with the mineralized matrix. During this time, the content of TGF13 per milligram soluble protein rose steadily and remained high through day 21. This increased concentration correlated with the onset of vascularization and calcification of cartilage. TGF13 was detected only between days 3-9 in the controls; i.e., non-bone-forming implants. Immunolocalization of TGF13 in bone-forming implants revealed staining of inflammatory cells at early times, followed later by staining of chondrocytes in calcifying cartilage and staining of osteoblasts. The most intense staining of TGFI3 was found in calcified cartilage and mineralized bone matrix, again indicating preferential compartmentalization of TGF13 in the mineral phase. In contrast to the delayed expression of TGF13 protein, northern blot analysis showed TGF13 mRNA in implants throughout the sequence of bone formation. The timedependent accumulation of TGF13 when cartilage is being replaced by bone in this in vivo model of bone formation suggests that TGF13 may play a role in the regulation of ossification during endochondral bone