Accumulation, Localization, and Compartmentation of Transforming Growth Factor β during Endochondral Bone Development

• Published in: The Journal of cell biology Vol. 107; no. 5; pp. 1969 - 1975
• Main Authors: Carrington, Jill L., Roberts, Anita B., Flanders, Kathleen C., Roche, Nanette S., Reddi, A. Hari
• Format: Journal Article
• Language: English
• Published: New York, NY Rockefeller University Press 01-11-1988; The Rockefeller University Press
• Subjects: Animals; Antibodies; Biological and medical sciences; Blotting, Northern; Bone Development; Bone formation; Bones; Cartilage; Cell growth; Chondrocytes; Collodion; DNA - analysis; Fundamental and applied biological sciences. Psychology; Guanidine; Guanidines - pharmacology; Human growth; Immunohistochemistry; Male; Molecular and cellular biology; Osteoblasts; Rats; RNA; RNA - analysis; RNA, Messenger - analysis; Skeletal development; Skeleton and joints; Transforming Growth Factors - metabolism; Vertebrates: osteoarticular system, musculoskeletal system; Human; Demineralization; Rat; Induction; Transforming growth factor; Time metabolism relation; Rodentia; Implantation; Accumulation; Immunological method; Characterization; Vertebrata; Mammalia; Newborn; Articular cartilage; Isolation; Bone; Localization; Bone matrix; Formation mechanism
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.107.5.1969

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 β (TGFβ) in the extracts was quantitated by a radioreceptor assay. TGFβ was present in demineralized bone matrix before implantation, and the concentration had decreased by 1 d after implantation. Thereafter, TGFβ was undetectable by radioreceptor assay until day 9. From day 9-21 the TGFβ was extracted only after EDTA demineralization, indicating tight association with the mineralized matrix. During this time, the content of TGFβ 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. TGFβ was detected only between days 3-9 in the controls; i.e., non-bone-forming implants. Immunolocalization of TGFβ 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 TGFβ was found in calcified cartilage and mineralized bone matrix, again indicating preferential compartmentalization of TGFβ in the mineral phase. In contrast to the delayed expression of TGFβ protein, northern blot analysis showed TGFβ mRNA in implants throughout the sequence of bone formation. The time-dependent accumulation of TGFβ when cartilage is being replaced by bone in this in vivo model of bone formation suggests that TGFβ may play a role in the regulation of ossification during endochondral bone development.