Isolation of growth factors from adult bovine bone

Fetal rat calvariae synthesize transforming growth factor beta (TGF beta), beta 2 microglobulin (beta 2 m), and insulinlike growth factor I (IGF I), but, except for TGF beta, it is not known if these polypeptides are also present in adult bone tissue. Pulverized bovine bone, extracted with 0.5 N HCl and 4 M guanidine HCl and fractionated by gel filtration, was found to contain several biologically active components when tested for its effects on DNA synthesis in osteoblast-rich cell cultures. TGF beta, beta 2 m, and IGF I were identified and further purified using high performance liquid chromatography (HPLC). TGF beta, identified by a standard TGF beta bioassay or by immunoreactivity, was purified by muBondapak C18 and muBondapak CN reversed phase HPLC. beta 2 m, identified by immunoreactivity, required an additional fractionation step on a DEAE-HPLC column for complete purification. IGF I, identified by immunoreactivity, was purified by HPLC using a muBondapak C18 and a DEAE-HPLC column. Purified TGF beta, beta 2 m, and IGF I migrated as single bands on polyacrylamide gel electrophoresis with respective molecular masses of 24,000, 10,000, and 7,500. In conclusion, adult bone matrix, like fetal bone cultures, contains TGF beta, beta 2 m, and IGF I and these factors may play a role in adult skeletal remodeling.