Fibroblast Growth Factor Enhances Bone Formation Induced by Bone Morphogenetic Protein in Mice
Li Yafei, Song Haiyan, Lu Rong, Hu Yunyu.
Institute of Orthopaedics ,Xijing Hospital.,710032,Xian,PRC.

Abstract Implantation of bone morphogenetic protein( BMP) in rodents elicits a series of cellular events leading to the formation of new bone inside and adjacent to the implants. It has been suggested that local growth factors may further regulate the process once it has been initiated.The experiment was designed to study the effect of adding a growth factor to the inductive implant.100ng of recombinant human basic fibroblast growth fator(bFGF) was added to the suspension containing 4 mg of partially purified bBMP( group A) which was injected into muscle of mice.The other implants served as controls. They contained either 4mg of bBMP without bFGF(group B) or 100 of bFGF alone(group C). Bone formation was induced in group A and B after 3 weeks.The amount of mineralized tissue in the implants of group A was 3 times greater than in the group B (P<0.01). 
Key Words: Fibroblast Growth Factor; Bone Morphogenetic Protein;
Bone Induction 
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The implantation of BMP in rodents elicits a series of cellullar events leading to the formation of new bone inside and adjacent to the implants. Mesenchymal cells at the recipient site migrate into the implant and are induced to multiply and differentiate into chondroblasts .Bone induction by this mechanism has similarities both with bone formation in the growth plate and with fracture healing. It has been suggested that endogenous growth factors may participate in the regulation of the bone induction process once it has been initiated by the bone inductive protein. Fibroblast growth factor(FGF) is one of the endogenous factors found in bone matrix . It is known to stimulate colony formation by anchorage-independent chondroblast in vitro . FGF is also a potent stimulator of capillary formation . Following an inductive stimulus, both chondrocyte colony formation and capillary formation are important steps in the development. Accordingly, test was made on the effect of adding recombinant human basic FGF to the suspension of bBMP.The mixture was then injected intramuscularly in mice, and the resulting amount of calcium content in the implant was determined after 3 weeks to see whether an enhancement was obtained in group A.

Material and methods

The bBMP used was prepared by different biochemical extraction of fresh bovine cortical bone . The partially purified bBMP had high osteoinductive activity in mouse thigh pouch model. 48 mg of bBMP was dissolved in 24 ml of a 6M urea solution. The solution was dialyzed against sterile distilled water at 4℃ for 24 hours, and loaded into 24 amples, 1 ml for each. The aliquots were lyophilized, sterilized with ethylene oxide gas. Lyophilized recombinant human basic FGF (Synergen, Boulder, CO, U.S.A.)was used .
In the expriment 48 adult male Kunming mice weighing 25g were classified into 4 groups: A were injected into the thigh muscles suspension of 4mg of bBMP+100ng of bFGF+0.3ml of PBS solution (PH7.2); B.suspension of 4mg of bBMP+0.3ml of PBS; C.solution of 100ng of bFGF +0.3ml of PBS; D. solution of 0.3ml of PBS. 
The mice were killed on day 21 after injection. The button-like samples were dissected from thigh muscle . Six of the harvested samples of each group were fixed in 10% neutral formalin and decalcified for 24 hours, embeded in paraffin, sectioned,stained with hematoxylin and eosin, and examined by light microscope. The other six tissues retrieved from each group were cleared of adherent tissue, weighed, and homogenized in 2ml of 0.15M NaCl. The homogenate was centrifuged at 10, 000 rpm,for 10 minutes. Calcium content of the acidsoluble fraction of the pellet was then measured with atomic absorption spectrophotometry ( Hitachi 7500), which was used as a quantitative parameter for new bone formation .

Results

A hard button-like node could be palpated in the thigh muscle of group A, which was notably larger than that in group B. Histological examinations showed the appearence of lamellar bone, trabecular bone,and bone marrow with some fibroblast cells(Fig 1.). Surrounding the newly formed bone some capillary nets occurred(Fig 1.). There was also matured new bone formed(Fig 2.) in group B, but no evidence of new bone formation in groups C, D except for fibroblast proliferation(Fig 3.).
Simply injecting bFGF+PBS solution caused no difference in calcium content compared with PBS solution only (Table 1.), whereas the bFGF+ bBMP implants yielded three times greater calcium than the bBMP+ PBS implants (P<0.01; Table 1) and 8 times more calcium than the bFGF+PBS. Calcium content in group B was also higher than in group C, D.

Discussion

The results demonstrated that addition of the exogenous bFGF to suspension of bBMP increased the amount of new bone formed by induction when the mixture was injected intramusclarly in mice.
Guacci et al found in his study on ultrastructure of human tibia nonunion tissue that the structure and function of the tissue cells seemed normal but the numbers of capillary was less than in normal callus. The results suggested that nonunion tissue had the capacity to be mineralized, but the lack of sufficient blood supply to bring calcium to the site of bone repairing was the real cause of nonunion .
FGF is a kind of protein contained mainly in brain tissue and also in other tissues such as bone and cartilage. It plays an important role in early embryonic development and in repairing of wounded tissue . FGF has been reported to promote proliferation of chondrocyte,fibroblast and capillary formation . FGF can also be released by chondrocyte at the site of injury and stimulate the differentiation of its precursors . Aspenberg et al found in his study that the amount of bone yield was significantly higher than that in the controls when FGF was added to the allograft bone matrix which was implanted into the muscle of mice .
The cellular mechanisms influenced by bFGF are not clear. Cartilage proliferation may have been directly stimulated by bFGF as demonstated in vitro and in vivo . Because FGF is a potent stimulator of capillary formation, it may have facilitated endochondral ossification, which requires vascular supply. In our experiment in group A there was markedly capillary formation adjacent to and within the new bone in which calcium content was greater than in group B.
Recent studies showed that a considerable number of endogenous growth factors involved in the process of bone cell proliferation and differentiation . Clinically, bone inductive implants may be available for patients for whom local bone formation is essential,consisting of one or several recombinant inductive proteins on an appropriate carrier. Our results suggested (1) exogenous bFGF enhanced a process of ectopic ossification elicited by implantation of bBMP, occuring in the presence of a number of endogenous growth factors.(2) possibly, bone healing process may also be influenced by other exogenous growth factors.⑶Fracture healing, in situations in whichthe host vascular bed is compromised, may be accelerated by implanting bone inductive material containing an angiogenic factor bFGF.

Fig 1 Groups and Their Treatments
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Treatments Calcium
Groups ───────────── content(mg)
bBMP(mg) bFGF(ng) PBS(ml)
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A(n=12) 4 100 0.3 1.02±0.17*
B(n=12) 4 0 0.3 0.35±0.05*
C(n=12) 0 100 0.3 0.14±0.02
D(n=12) 0 0 0.3 0.12±0.01
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* : X±SD, P<0.01, Compared to D