Matrix metalloproteinases are important in the physiological and pathological degradation of extracellular matrix including that of bone and cartilage. The process of bone defect healing is associated with formation of cartilage callus and cancelous bone. With maturation and aging, the response of skeletal tissues to injury is limited. The ability of growth factors to enhance bone defect healing in aged rats was studied. Partial bone defects were induced in femurs of aged rats. A single dose of IGF-1, TGF-beta+IGF-1 or saline was inserted in the defect and bones were examined after 2 and 4 weeks. Morphology revealed that after 2 weeks of treatment with TGF-beta the defects were filled with mesenchyme-like tissue and delicate bone trabeculae. Positive staining for metalloproteinase-2 (MMP-2) was shown at the sites of new bone formation. In defects treated with IGF-1 or TGF-beta+IGF-1 nodules of cartilage and fine bone trabeculae along with positive staining for both MMP-2 and MMP-3 were demonstrated in the healing defects. After 4 weeks radiology revealed mineralization in defects treated with TGF-beta and less pronounced mineralization after treatment with IGF-1, or with TGF-beta+IGF-1, whereas only partial healing of the defects was observed in control specimens. MMP-2 and MMP-3 were detected at sites of new bone formation after treatment with TGF-beta, IGF-1, and TGF-beta+IGF-1. It is concluded that TGF-beta and IGF-1 induced bone defect healing in aged rats. TGF-beta induced bone formation while IGF-1 induced cartilage and than bone formation via endochondral ossification. The localization of MMP-2 and MMP-3 in the healing defects reflected the synthesis of bone or cartilage matrices in the defect, reflecting the involvement of MMPs in the process of bone formation and endochondral ossification. The ability to induce bone defect healing in aging is of great clinical importance and understanding the involvement of MMPs in this process can contribute to future treatment with growth factors to enhance bone defect healing in 20-month-old female rats.