Journal of Orthopaedic Research 8364-371 Raven Press, Ltd., New York 0 1990 Orthopaedic Research Society

Acidic Fibroblast Growth Factor (aFGF) Injection Stimulates Cartilage Enlargement and Inhibits Cartilage Gene Expression in Rat Fracture Healing S. Jingushi, A. Heydemann, S. K. Kana, L. R. Macey, and M. E. Bolander Orthopaedic Research Unit, NIAMS, NIH, Bethesda, Maryland, U.S.A.

Summary: The effect of the administration of acidic fibroblast growth factor (aFGF) on normal fracture healing was examined in a rat fracture model. One microgram of aFGF was injected into the fracture site between the first and the ninth day after fracture either every other day or every day. aFGF-injected calluses were significantly larger than control calluses, although this does not imply an increased mechanical strength of the callus. Histology showed a marked increase in the size of the cartilaginous soft callus. Total DNA and collagen content in the cartilaginous portion of the aFGF-injected calluses were greater than those of controls, although the collagen content/DNA content ratio was not different between the aFGF-injected and control calluses. Fracture calluses injected with aFGF remained larger than controls until 4 weeks after fracture. The enlarged cartilaginous portion of the aFGF-injected calluses seen at 10 days after fracture was replaced by trabecular bone at 3 and 4 weeks. Northern blot analysis of total cellular RNA extracted separately from the cartilaginous soft callus and the bony hard callus showed decreased expression of type I1 procollagen and proteoglycan core protein mRNA in the aFGF-injected calluses when compared with controls. A slight decrease in types I and I11 procollagen mRNA expression was also observed. We concluded that aFGF injections induced cartilage enlargement and decreased mRNA expression for type I1 procollagen and proteoglycan core protein. Key Words: Wound healing-Growth factor-Acidic fibroblast growth factorCollagen-Proteoglycan-Cartilage.

Fracture healing is a multistep process consisting of many diverse cellular events. After fracture, mesenchymal cells migrate into the site, proliferate, differentiate, and synthesize various proteins (4). Precursor cells differentiate into chondrocytes and form cartilage, while bone formation proceeds by membranous ossification and by endochondral ossification of the cartilage tissue to bone union. Recent studies suggest that wound healing is reg-

ulated by growth factors, proteins that effect cell proliferation, differentiation, and protein synthesis by paracrine or autocrine mechanisms. In vitro studies have shown that growth factors affect several cell types found in the fracture callus, including capillary endothelial cells, fibroblasts, chondrocytes, and osteoblasts (18). High amounts of several growth factors including acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor, and transforming growth factor p can be extracted from demineralized bone (9). aFGF is a member of a class of growth factors that were first described as mitogens for endothelial

Received December 2, 1988; accepted October 4, 1989. Address correspondence and reprint requests to Dr. M. E. Bolander, Orthopaedic Research Unit, Bldg. 10, Rm. 9N228, NIAMS, NIH, Bethesda, MD 20892, U.S.A.

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aFGF EFFECTS ON FRACTURE HEALING cells and fibroblasts. The endothelial cell growth factors are divided into two groups: anionic and basic. Although several anionic peptides in this group have been purified, current studies revealed that each is derived from a single large precursor (5). In vitro studies have shown that aFGF can act on several cell types other than endothelial cells, including fibroblasts and osteoblasts (7,8,12,16,19,21,22). The high concentration of aFGF in bone (9) suggests that aFGF is an important factor in regulating fracture healing or bone remodeling. No previous studies, however, have demonstrated that a growth factor, including aFGF, has a role in fracture repair. We injected human recombinant aFGF into fracture calluses in a rat femoral fracture model and investigated the influence of this growth factor on the fracture healing process in vivo. We expected that aFGF injections into fracture callus in vivo would emphasize the role of this factor in the normal repair process. These studies show that aFGF injection has a dramatic effect on cartilage formation during fracture repair.

jected into the fracture site. Fifty microliters of PBS was similarly injected in the contralateral limb as control. Clearance of 12’I-Labeled aFGF lZ5I-labeledpurified bovine brain aFGF was provided by R & D Systems, Inc. (Minneapolis, MN, U.S.A.). The specific activity was 160 pCi/pg. A total of 0.1 pg of labeled aFGF and 0.9 pg of cold aFGF was combined and suspended in PBS. The labeled growth factor was injected into the callus 5 days after fracture. The radioactivity remaining in the fracture site was counted using a Geiger counter and calculated as a percentage of the initial count. Histology Harvested callus specimens were fixed in 5% formalin, decalcified, and embedded in paraffin. Six micron thick sections were cut through the long axis of the femur and stained with Masson-trichrome stain (Baker Histolabs, Falls Church, VA, U.S.A.).

MATERIALS AND METHODS Fracture Model Bilateral femoral fractures were produced in male Long-Evans rats (287 k 24 g). Sodium pentobarbital, 65 mg/kg of body weight, was injected intramuscularly. Under anesthesia, rats were prepared for surgery by shaving and cleansing both legs. A medial peripatellar incision was made and the patella was dislocated laterally, exposing the femoral condyles. A Kirshner wire (1.1 mm diameter, 2.7 cm length) was introduced from the intercondylar notch into the intramedullary canal. After closing the knee joints, the middiaphysis of the pinned femur was fractured by means of a three-point bending device driven by a dropped weight as described by Bonnarens and Einhorn (3). The rats were permitted full weight-bearing and unrestricted activity after awakening from anesthesia. Injection of aFGF Human recombinant aFGF (a endothelial cell growth factor) was kindly provided by Michael Jaye of Rorer Biotechnology (King of Prussia, PA, U.S.A.) (11). The sample was stored in liquid nitrogen prior to use. Under anesthesia, 1 pg of aFGF in 50 p1 of phosphate-buffered saline (PBS) was in-

Measurement of the Callus Cross-Sectional Area Animals were killed by CO, asphyxiation. The femoral bone was harvested, dissected from the surrounding soft tissue, and the Kirshner wire was removed from the medullary canal. The maximum and minimum diameters of the callus were measured with a caliper accurate to 0.1 mm (Bel-Art Products, Pequannock, NJ, U.S.A.), and the crosssectional area was estimated as the area of an oval with these two diameters. DNA Content and Collagen Content of the Cartilaginous Soft Callus The cartilaginous soft callus was carefully dissected from the bony hard callus. Soft calluses harvested at day 10 after fractures were comprised of cartilage and very small amounts of mesenchymal tissue. Collagen content was measured as pepsinresistant material. Calluses were homogenized in 0.5 M acetic acid, digested with 1 mglml of pepsin in 4 ml of 0.5 M acetic acid at 4°C overnight, and centrifuged at 2,000 rpm for 20 min. The pellet was suspended in 0.5 M acetic acid and was redigested by pepsin. After the second digestion, both supernatant fractions were combined, and then precipitated by the addition of sodium chloride to a final

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concentration of 3 M . The total pepsin-resistant material was combined, dialyzed against 5 mM acetic acid, lyophilized, and weighed for total collagen content. DNA content was measured on the basis of absorbance at 260 nm. The supernatant fractions that remained after sodium chloride precipitation were combined, and the absorbance at 260 nm was determined. DNA content was calculated on the basis of 50 pg/ml of DNA per O.D. unit.

and Dr. T. Segui-Real in LDBA, NIH (Bethesda, MD), respectively. The alkaline phosphatase probe was a gift of Drs. G. Rodan and M. Thiede from Merck Sharp & Dohme (West Point, PA, U.S.A.). Bone gla protein probe was provided by Dr. V. Rosen, Genetics Institute (Cambridge, MA, U.S.A.). The osteonectin cDNA probe was prepared in this laboratory as previously reported (2).

RNA Extraction and Northern Blotting Total cellular RNA was extracted separately from the cartilaginous soft callus and from the bony hard callus. Before RNA extraction, bone marrow was washed from the femoral bone in the hard calluses using PBS containing 0.1% diethylpyrocarbonate (DEPC). To prevent degradation of the RNA, specimens were frozen in liquid nitrogen immediately after harvesting. Total cellular RNA was prepared as follows: calluses were homogenized in 4 M guanidine hydrochloride containing 1.O% sarcosyl, 67.5 mM potassium acetate, and 0.1% antifoam A (Sigma Chemical Co., MO, U.S.A.). RNA was isolated by centrifugation at 32,000 rpm for 18 h over a 5.6 M cesium chloride pad. The RNA pellet was solubilized in DEPC-treated water, extracted with chloroform, and precipitated overnight with 4 M sodium acetate (pH 5.0) at -20°C. The concentration of RNA was determined by spectrophotomeric absorption at 260 nm. After agarose gel electrophoresis, RNA was stained by ethidium bromide to determine the integrity of 28 S and 18 S bands. Ethidium bromide staining also indicated that equal amounts of total RNA were loaded in each lane. Transfer to nylon membrane (Hybond-N, Amersham, Arlington Heights, IL, U.S.A.) was performed by standard methods (17). cDNA probes were labeled with 32Pby nick translation or random priming using kits (Amersham). Northern hybridization with specific cDNA probes was carried out for 16 h in 50% formamide. After hybridization, the filters were washed twice at room temperature in 2 x SSC containing 0.05% sodium pyrophosphate and twice at 65°C in 0.1 x SSC containing 0.05% sodium pyrophosphate. Autoradiograms were developed after exposure of the nylon membrane to x-ray film for variable periods of time. The cDNA probe for pro-ol2 (I) chain of type I collagen was a gift from Dr. D. Rowe, University of Connecticut, Farmington. Rat collagen I1 and 111, proteoglycan core protein, and laminin receptor cDNA probes were gifts from Dr. Y. Yamada, Dr. K. Doege,

RESULTS

J Orthop Res, Vol. 8 , No. 3, 1990

Injection into Fracture Calluses To establish that a direct injection into the fracture site would deliver a growth factor to the callus, we injected amide-black dye into the fracture callus 5 days after fracture. The dye stained the soft callus tissue at the fracture site. Significant staining was also seen in the muscle and the soft tissue surrounding the callus and the bone (data not shown). To evaluate the clearance rate of injected aFGF from the fracture callus, '251-labeled aFGF was injected into the fracture site, and the radioactivity of the callus was counted. Fifty percent of the radioactivity remained at the site after 1 h (Fig. 1). Effect of aFGF Injections on the Histology and the Size of Fracture Calluses aFGF-injected calluses were larger than controls. The cartilaginous portion of the aFGF-injected cal-

loo

01

0

I

I

I

I

60

120

180

240

TIME (rnin.) FIG. 1. Clearance of '251-labeled aFGF from the injection site in an anesthetized animal. Radioactivity was counted at the fracture site in the injected limb. The value was calculated as a percentage of the initial count.

367

aFGF EFFECTS ON FRACTURE HEALING luses was enlarged, and proliferating chondrocytes were observed (Fig. 2). Even though the growth factor penetrated a large area as indicated in the dye experiment, no histological change was noted in the soft tissue around the fracture callus. Every other day injections of aFGF increased the cross sectional area of the fracture callus from a control value of 0.375 2 0.014 to 0.435 k 0.012 cm2 (mean 2 SEM, n = 9) (p < 0.01). (Mean values and ranges for all data are listed in Table 1.) Every day injections increased the area from a control value of 0.393 k 0.021 to 0.481 f 0.048 cm2 (n = 9) 0, < 0.01). Comparison of every other day and daily injections showed a dose-response effect of aFGF (Fig. 3A). The total DNA content of the cartilaginous soft callus after aFGF injections was higher than that of controls. Daily injections of aFGF increased the DNA content from a control value of 594.9 2 82.1 to 859.9 2 72.8 Fg (n = 4) 0, < 0.06) (Fig. 3B). Injections of aFGF also increased collagen content in a dose-dependent manner, from 17.6 k 1.5 vs. 14.9 k 1.4 mg in controls after every other day injections, to 24.0 2 1 . 1 vs. 15.7 2 2.9 mg in controls (p < 0.05) after daily injections (Fig. 3C). Although the total collagen content of the cartilag-

inous portion of the aFGF-injected callus was higher than that of control calluses, the collagen content/DNA content ratio showed no significant difference between the aFGF-injected and control calluses (Fig. 2D). Late Effect of aFGF Injections on Fracture Callus

After injecting aFGF every other day until day 9, the fracture calluses were harvested at 2, 3, and 4 weeks. The large cartilaginous callus seen on day 11 had been replaced by trabecular bone at 3 and 4 weeks after fracture by a process undistinguishable from the normal endochondral ossification seen in controls (Fig. 4). aFGF-injected calluses remained significantly larger than controls until 4 weeks after fractures (Fig. 5). Effect of aFGF Injections on mRNA Expression

Total cellular RNA was extracted separately from the cartilaginous soft callus and the osseous hard callus. mRNA for structural and extracellular matrix proteins was detected by Northern blot with cDNA probes for a2(I), al(II), and al(II1) procol-

FIG. 2. Histology of an aFGF-injected callus. One rnicrogram of aFGF was injected into a fracture site every other day after fracturing the femur. After 11 days, the aFGFinjected callus (A) was larger than the control callus (B). The cartilaginous portion of the aFGF-injected callus was enlarged, and proliferating chondrocytes were observed (C). Masson-trichrorne staining. Bars are 1 m m (A, B) or 0.2mm (C).

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TABLE 1. Mean value, SEM, and range for callus area, DNA content, and collagen content Every other day Callus area (an2)(n = 9) Control aFGF DNA content (mg) (n = 4) Control aFGF Collagen content (mg) (n = 4) Control aFGF Collagen contentDNA content (n = 4) Control aFGF

* p < 0.05,

**p

Range

Mean i SEM

Range

0.375 f 0.014** 0.435 ? 0.035**

0.327-0.389 0.40-0.460

0.393 t 0.021** 0.481 i 0.017**

0.325-0.415 0.445-0.542

617.3 f 24.8 683.0 ? 72.6

549.8-661.3 487.5-824.O

594.9 2 82.1 859.8 f 72.8

499.6-840.0 665.7-986.0

14.88 f 1.38 17.58 ? 1.49

10.91-17.23 14.03-20.91

15.72 f 2.85* 24.00 2 1.13*

10.71-23.57 22.59-27.32

23.97 f 1.50 25.98 2 0.90

19.84-26.28 24.62-28.79

26.26 28.35

20.05-32.27 24.5633.94

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30

C

p

< 0.05

I-

z W Iz

20

8 z

; 10 4 0

f ?

2.60 3.97

< 0.01.

lagens, proteoglycan core protein, osteonectin, alkaline phosphatase, bone gla protein, and laminin receptor (Fig. 6). Genes for type I1 collagen and proteoglycan core protein were expressed at high levels in the soft callus, but expression for type I1 procollagen was not limited to these tissues since low levels of mRNA were detected in hard callus. aFGF injections had an effect on type I1 procollagen and proteoglycan core protein gene expression, where both every other day and daily injections decreased mRNA levels for these proteins. Daily aFGF injections caused a 76 and 61% decrease of mRNA expression for type I1 procollagen and proteoglycan core protein, respectively.

-

Every day

Mean t SEM

A

D

Type I and I11 procollagen gene expression was detected in both hard and soft callus. Every other day injections of the callus with aFGF did not alter expression of these genes. Daily injections of aFGF did not change expression in the hard callus, but steady-state levels of mRNA for both genes in soft callus were slightly decreased. Expression of genes for osteonectin, alkaline phosphatase, and laminin receptor were detected at equal levels in both hard and soft callus. No differences were noted in the expression of these genes after aFGF injections. Bone gla protein gene expression was limited to hard callus. aFGF treatment had no apparent effect on the expression of the gene for this protein.

r L

T

111 EVERY OTHER DAY

EVERY DAY

INJECTION

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EVERY EVERY OTHER DAY DAY INJECTION

FIG. 3. Size and DNA and collagen content of the cartilaginous portion of aFGF-injected calluses. aFGF was injected every other day or every day after fracturing femurs, and the calluses were harvested on day 10. The aFGF-injected calluses were larger than controls (A). Comparison of every other day and daily injections demonstrates a doseresponse effect of aFGF. Although DNA content (B) and total collagen content (C) were higher in aFGF-injected calluses than in controls, the collagen content/DNA content ratio was not significantly different (D). Values are means ? SEM for nine (A) or four (B, C, D) calluses.

aFGF EFFECTS ON FRACTURE HEALING

aFGF-injected

369

control

3 wks

4 wks

FIG. 4. Late effect of aFGF injections on callus histology. After making fractures, aFGF was injected every other day until day 9. The cartilaginous portion was substituted by trabecular bone at 3 and 4 weeks after fracture in both aFGF-injected and control calluses. Masson-trichrome staining. Bars are 1 mm.

DISCUSSION We found that the major effect of multiple high doses of human recombinant aFGF on fracture healing was an increase in the cartilaginous tissue of the fracture callus. The DNA content of the soft callus in aFGF-injected fractures was higher than 0.5I

I

0.2

3

2

4

5

WEEKS AFTER FRACTURE FIG. 5. Late effect of aFGF injections on the callus size. After injecting aFGF on days 1, 3, 5, 7, and 9, the fracture calluses were harvested at 2, 3, 4, and 5 weeks. aFGF-injected calluses were larger than control until 4 weeks after fracturing. Values are means SEM for eight calluses.

*

that of controls, while the collagen content/DNA content ratio was not different between aFGFinjected and control calluses. This increase in area was not due to hypertrophy of the chondrocytes. These data indicate that the increase in cartilaginous area is due to an increase in cartilaginous cell number and not to an increase in intracellular volume or extracellular matrix. The administration of exogenous aFGF to the fracture calluses, therefore, causes proliferation of chondrocytes in the fracture callus and/or induces migration of chondrocyte progenitor cells into the fracture site. A significant amount of aFGF is found in bone matrix (9), and several in vitro experiments have shown a direct effect of aFGF on DNA synthesis of bone cells (7,19). bFGF, a growth factor structurally and functionally related to aFGF, has been shown to have a proliferative effect on chondrocytes as well as osteoblasts (6,10,14). These studies suggest that aFGF may have a role in bone remodeling or repair, although no significant effect of aFGF on bone was seen in this experiment. Recently, we detected an aFGF mRNA signal in the early phase of the fracture healing, possibly during the time when chondroprogenitor cells are migrating into the callus and cartilaginous cells are proliferating (Jingushi et al. , unpublished results). Although in our in vivo study it is hard to demonstrate that the increase in cartilage was a direct effect of aFGF injection, the data suggest a potential role for aFGF in the process of fracture repair.

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370 HARD CALLUS

SOFT CALLUS

m m

Every Other

Every

Every Other

Every

Day

Day

Day

Day

nn

nn B e

$$? Type II Collagen

PG Core Protein

Type I Collagen

Type 111 Collagen

Osteonectin

ALP

BGP

LR

P e

&$?

-

5.5 kb

-

9.5

C-

5.7 5.1

-

5.4

2.2

c- 2.5

-

0.6

1.2

Ethidine Bromide Staining

FIG. 6. Northern blot of RNA extracted from the cartilaginous soft callus or the hard callus. Calluses were injected with aFGF every other day or every day and were harvested 10 days after fracture. mRNA was detected using cDNA probes for type I, II, and 111 collagen, proteoglycan core protein (PG core protein), osteonectin, alkaline phosphatase (ALP), bone gla protein (BGP), and laminin receptor (LR). mRNA for type II collagen and PG core protein was decreased in the cartilaginous portion of aFGF-injected calluses. Ethidium bromide staining of the RNA demonstrates that an equal amount of RNA was loaded into each lane.

In vitro studies have shown a direct effect of aFGF on cell proliferation at low concentrations (7,8,12,16,19,21,22). We have injected 100 ng doses of aFGF into the fracture site. However, we saw no signifcant change in the callus when evaluated by histology. One microgram injections of aFGF into the peritoneal cavity were also not effective in altering fracture repair (Jingushi et al., unpublished results). The need for high doses of aFGF may represent the rapid clearance of the exogenously administered factor from the fracture site, or may be

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the result of the large volume of distribution of the injected material. Northern blot analysis of RNA extracted separately from cartilaginous soft callus and bony hard callus demonstrated several interesting facts about the expression of extracellular matrix genes in the callus. mRNAs for type I and I11 collagens were detected in both bone and cartilage, while type I1 collagen mRNA was detected primarily in cartilage. mRNAs for osteonectin and alkaline phosphatase, which are presumed to have a role in mineralization, were detected at equivalent levels in both hard and soft callus. Osteonectin is a glycoprotein involved in linking mineral to collagen fibrils, and the protein is detected in osteoblast and osteoprogenitor cell immunocytochemically (13). Our results are consistent with those of Leboy et al., who showed that significant amounts of mRNA for osteonectin were present in precalcified cartilage of chicken epiphyseal cartilage as well as in endochondral bone (15). Bernard et al. detected a Ca2+-binding glycoprotein that has alkaline phosphatase activity in the cytoplasm of resting chondrocytes and in the intercolumnar matrix between the proliferating and hypertrophic region (1). These results indicate that mRNAs for alkaline phosphatase and osteonectin are expressed in cartilaginous region or in fibrous tissue of the fracture callus. Although bone gla protein is also believed to be an important protein for mineralization, mRNA for this protein was expressed only in the hard callus, not in the cartilaginous callus. Low levels of mRNA for type I1 collagen were detected in the hard callus. Using in situ hybridization techniques, Sandberg et al. (20) observed a few scattered cells that contained type I1 collagen mRNA in the mineralizing zone of the growth plate. RNA extracted from the cartilaginous soft callus contained mRNA for type I and I11 collagen. Presumably, these mRNAs are derived from mesenchymal cells in the soft callus, or may be expressed in the cartilage tissue. The administration of exogenous aFGF to the fracture callus altered the gene expression for several functional proteins in the soft callus. mRNA for the cartilage proteins, type I1 collagen, and proteoglycan core protein were decreased, suggesting that the stimulation for chondrocyte mitogenesis in the fracture callus is distinct from the stimulation of mRNA expression and protein synthesis. This dissociated effect of aFGF is consistent with several in vitro experiments. aFGF has been shown to induce

aFGF EFFECTS ON FRACTURE HEALING osteoblast proliferation, while inhibiting collagen synthesis by these cells (7). Horton et al. showed that aFGF decreased type I1 procollagen synthesis by chondrocytes in cell cultures (11). The related growth factor bFGF also has a dissociated effect on osteoblasts in cultured rat calvaria (6). mRNA levels for osteonectin, alkaline phosphatase, and laminin receptor in the soft calluses were not affected by aFGF treatment in this study. In preliminary experiments, the mechanical properties of the injected fracture callus were investigated. The aFGF-injected calluses were found to be weaker intension testing than controls (Jingushi et al., unpublished results); aFGF, therefore, was not effective in increasing the strength of normal fracture repair. This is not unexpected, since the mechanical strength is dependent on the organization of collagen fibrils, not merely the cross-sectional area, and calluses were mechanically tested prior to significant organization of collagen fibers. aFGF administration, however, changed the fracture repair process and increased cartilage tissue. aFGF treatment might be beneficial in abnormal fracture repair if chondrogenesis is impaired. Acknowledgment: We would like to thank Dr. M. Jaye for generously providing recombinant aFGF for these experiments, and Dr. K. Doege and Mr. A. Romero for skillful technical assistance. This work was supported by grants from the American Academy of Orthopaedic Surgeons and the Orthopaedic Research and Education Foundation.

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acidic fibroblast growth factor. Proc Nut1 Acad Sci USA 83:7216-7220, 1986 6. Canalis E, Centrella M, McCarthy T: Effect of basic fibroblast growth factor on bone formation in vitro. J Clin Invest 81~1572-1577, 1988 7. Canalis E, Lorenzo J, Burgess WH, Maciag T: Effects of endothelial cell growth factor on bone remodeling in vitro. J Clin Invest 7952-58, 1987 8. Globus RK, Patterson-Buckendahl P, Gospodarowicz D: Regulation of bovine bone cell proliferation by fibroblast growth factor and transforming growth factor beta. Endocrinology 123:98-105, 1988 9. Hauschka PV, Mavrakos AE, Iafrati MD, Doleman SE, Klagsbrun M: Growth factors in bone matrix. J Biol Chem 261 :12665-12674, 1986 10. Hiraki Y, Inoue H, Kato Y, Fukuya M, Suzuki F: Combined effects of somatomedin-like growth factors with fibroblast growth factor or epidermal growth factor in DNA synthesis in rabbit chondrocytes. Mol Cell Biochem 76:185-193, 1987 1 1 . Horton WE, Higginbotham JD, Harvey AK, Chandrasekhan S: TGFp and FGF inhibition of collagen I1 synthesis by chondrocytes involves regulatory DNA sequences. Second International Conference on Molecular Biology and Pathology of Matrix, Philadelphia, 1988, p 11-2 12. Jaye M, Burgess WH, Shaw AB, Drohan WN: Biological equivalence of natural bovine and recombinant human aendothelial cell growth factors. J Biol Chem 262: 1661216617, 1987 13. Jundt G, Berghauser K-H, Termine JD, Schultz A: Osteonectin-a differential marker of bone cells. Cell Tissue Res 248:409415, 1987 14. Kato Y, Iwamoto M, Koike T: Fibroblast growth factor stimulates colony formation of differentiated chondrocytes in soft agar. J Cell Physiol 133:491498, 1987 15. Leboy PS, Shapiro IM, Uschmann BD, Oshima 0, Lin D: Gene expression in mineralizing chick epiphyseal cartilage. J Biol Chem 263:8515-8520, 1988 16. Maciag T, Mehlman T, Friesel R, Schreiber AB: Heparin binds endothelial cell growth factor, the principal endothelial cell mitogen in bovine brain. Science 225:932-934, 1984 17. Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning. A Laboratory Manual. New York, Cold Spring Harbor Laboratory, 1982, pp 382-389 18. Nemeth GG, Bolander ME, Martin GR: Growth factors and their role in wound and fracture healing. In: Growth Factors and Other Aspects; Biological and Clinical Implications, ed by A Barbul, E Pines, M Caldwell, TK Hunt. New York, Alan R. Liss, Inc., 1988, pp 1-17 19 Rodan SB, Wesolowski G, Thomas K, Rodan GA: Growth stimulation of rat calvaria osteoblastic cells by acidic fibroblast growth factor. Endocrinology 121:1917-1923, 1987 20. Sandberg M, Vuorio E: Localization of types I, 11, and I11 collagen mRNAs in developing human skeletal tissues by in situ hybridization. J Cell Biol 104:1077-1084, 1987 21. Thomas KA, Rios-Candelore M, Gallego GG, DiSalvo J, Bennett C, Rodkey J, Fitzpatrick S: Pure brain-derived acidic fibroblast growth factor is a potent angiogenic vascular endothelial cell mitogen with sequence homology to interleukin 1 . Proc Nut1 Acad Sci USA 82:64094413, 1985 22. Unsicker K, Rwichert-Preibsch H, Schmidt R, Pettmann B, Labourdette G, Sensenbrenner M: Astroglial and fibroblast growth factors have neurotrophic functions for cultured peripheral and central nervous system neurons. Proc Natl Acad Sci USA 84:545%5463, 1987

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