Makoto Tamura

Recombinant human basic fibroblast growth factor accelerates fracture healing by enhancing callus remodeling in experimental dog tibial fracture

Effect of recombinant human basic fibroblast growth factor (bFGF) on fracture healing was investigated using a tibial fracture in beagle dogs. Transverse fractures in the middle of the diaphyses were created in the right tibiae and bFGF was injected into the fracture sites at a single dose of 200 μg. The time course of changes in callus volume and morphology of the fracture sites were evaluated at weeks 2, 4, 8, 16, and 32 after treatment, and the fracture strength was analyzed at weeks 16 and 32. At week 2, a radiogram of the fracture site showed obvious membranous ossification in the group injected with bFGF. Basic FGF extended the callus area at week 4 and increased the bone mineral content (BMC) in the callus at week 8. bFGF also increased the osteoclast number in the periosteal callus at weeks 2 and 4. In the bFGF group, a maximal increase in the osteoclast index was found at week 4, and an identical increase was recognized in the control group at weeks 8 and 16. These findings strongly suggested that bFGF stimulated not only callus formation but osteoclastic callus resorption. BMC in the bFGF group was followed by a rapid decrease from week 8, while that in the control group was identical from week 4. Fracture strength of the bFGF group showed significant recovery by week 16, and recovery was still evident by week 32. We conclude that bFGF promotes the fracture healing in dogs by the stimulation of bone remodeling.

Bone regeneration by basic fibroblast growth factor complexed with biodegradable hydrogels

The objective of this study is to enhance the bone induction activity of basic fibroblast growth factor (bFGF) for reconstruction of skull bone defects which has been clinically recognized as almost impossible. For this purpose, we prepared biodegradable hydrogels from gelatin with an isoelectric point of 4.9 which is capable of polyionic complexing with basic bFGF. When implanted in rabbit skull defects of 6 mm in diameter (6 defects per experimental group), the gelatin hydrogels incorporating 100 microg of bFGF promoted bone regeneration at the defect in marked contrast to free bFGF of the same dose, finally closing the bone defects after 12 weeks of implantation as is apparent from histological examination. In dual energy X-ray absorptometry analysis, the bone mineral density at the skull defects enhanced by the hydrogels was significantly higher than that by free bFGF at doses ranging from 2 to 200 microg/defect (P < 0.05). The extent of bone regeneration induced by gelatin hydrogels incorporating 100 microg of bFGF increased with a decrease in their water content. Histological examination indicated that more slowly degrading hydrogels of lower water content prolonged the retention period of osteoblasts in the bone defects. This led to enhanced bone regeneration compared with faster degrading hydrogels of higher water content. It was concluded that this biodegradable hydrogel system was a promising surgical tool to assist self-reconstruction of the skull bone.

Effects of FGF-2 on metaphyseal fracture repair in rabbit tibiae

Fibroblast growth factor-2 (FGF-2) has been found to have stimulatory effects on fracture repair at diaphysis, while its effect on metaphyseal fracture repair, where spongiosal bone is dominant, has not been studied. This study was conducted to investigate the effect of FGF-2 on metaphyseal fracture healing in a rabbit proximal tibial metaphyseal model. The proximal tibial metaphysis of 6-month-old Japanese white rabbits was osteotomized bilaterally. Then 400 µg of FGF-2, mixed with gelatin hydrogel, and gelatin hydrogel alone (the control) were injected to each osteotomy site of the rabbit proximal tibiae, and the osteotomies were fixed with staples. One and 2 weeks after surgery, the osteoid area in the repairing spongiosal bone at the fracture site was significantly larger in the FGF-2 group than in the control group (P < 0.05). On immunohistochemistry, proliferating-cell nuclear antigen-positive cells had a tendency to show greater numbers in the FGF-2 group. After 4 and 8 weeks, values for bone mineral density and the cancellous bone area in the healing region of the fracture site were significantly larger in the FGF-2 group (P < 0.05). These data suggest that local application of FGF-2 may have an accelerating effect on the repair of metaphyseal fractures. Exogenous recombinant human rhFGF-2 may have potential clinical applications in metaphyseal fracture treatment.

Promoted Bone Healing at a Rabbit Skull Gap Between Autologous Bone Fragment and the Surrounding Intact Bone with Biodegradable Microspheres Containing Transforming Growth Factor-β1

This study is a trial to promote repairing of the rabbit skull bone gap between an autologous bone flap and the intact bone with biodegradable gelatin microspheres containing transforming growth factor-beta1 (TGF-beta1). A 10-mm diameter bone defect was prepared in rabbit skulls by drilling out a bone flap of 6 mm in diameter. After a surrounding gap defect of 2 mm was created and treated with 0.5 microg of free TGF-beta1 and gelatin microspheres containing 0.5 microg of free TGF-beta1, the circular autologous bone flap was placed in the center. Significant bone healing at the gap defect was observed 3 weeks after implantation of the TGF-beta1-containing gelatin microspheres. The bone mineral density (BMD) was significantly higher than that of other experimental groups. On the contrary, when applied with free TGF-beta1, a fibrous tissue initially infiltrated into the gap defect, resulting in impairing bone healing. The tissue response was similar to that at the defect implanted with empty gelatin microspheres and TGF-beta1-free phosphate-buffered saline solution alone. There was more space in the gap-filling bone in the 16-week view than the 3-week view. It is possible that this was an intermediate step along the way toward normal healing and formation of cancellous bone. We conclude that gelatin microspheres containing TGF-beta1 show promise as an agent to promote bone regeneration of subcritical size defects between surgically positioned autologous bone flaps and surrounding host bone.

Transient exposure of fibroblast growth factor-2 induced proliferative but not destructive changes in mouse knee joints.

Fibroblast growth factor-2 (FGF-2) has the potential to regenerate damaged articular cartilage tissue due to its exerting anabolic effects on chondrocytes. However, FGF-2 is involved in pathogenesis of rheumatoid arthritis, where the joint is destructed. The study aims at clarifying the effects of FGF-2 on joints. When radiolabeled FGF-2 was injected into knee joints of C57Bl/10 mice, a transient binding was observed in the superficial and intermediate zones of the articular cartilage as well as in the synovium and perichondrium. An FGF-2 injection (5 μg) caused synovial hyperplasia adjacent to the articular cartilage on day 7, cartilage formation adjacent to the articular cartilage on day 14, and osteophyte on day 21. The intensity of safranin-O staining of the articular cartilage increased on day 14. These changes were dose-dependent. No destructive changes in the joints were observed. In a joint, transient exposure of FGF-2 caused proliferative changes, but not destructive changes.