TISSUE INHIBITOR OF METALLOPROTEINASES II (TIMP-2) IS AN OSTEOANABOLIC FACTOR IN VITRO AND IN VIVO

OBJECTIVE: Critical size defects (CSDs) of bone are defined as defects that do not heal spontaneously to new bone during the lifetime of an adult individual. In contrast, immature animals are capable to heal defects of identical size. It was our hypothesis that age-related paracrine effects are relevant for this difference in regeneration. METHODS: The pooled supernatant of primary rat calvarial osteoblast-like cell cultures (POBC) derived from prenatal or postnatal donors was concentrated and applied into CSDs of adult recipient organisms (n = 10). In addition, the supernatant of POBC derived from prenatal donors was pooled and purified by reverse-phase chromatography. Each pre-purified fraction was tested in a proliferation indicating bioassay. Peptide fractions containing proliferative activities were re-chromatographed and re-tested in a bioassay. Finally, a proliferative activity was purified, identified by sequence analysis and applied into CSDs of adult recipients. RESULTS: The application of POBC derived from prenatal donors resulted in osseous regeneration of a CSD in adult recipients, while the supernatant of postnatal donors had much smaller effects. The morphologic features resembled the spontaneous osseous healing of calvarial defects of the same size in immature organisms. The polypeptide "tissue inhibitor of metalloproteinases type II"(TIMP-2) was isolated from the supernatant of cultures of POBC derived from prenatal donors by measuring the induction of their proliferation. Additionally, the application of human TIMP-2 injected into calvarial CSDs of adult organisms resulted in osseous healing. CONCLUSION: We conclude that one component responsible for the healing effect of CSDs of POBC supernatants derived from prenatal donors is TIMP-2.