JOURNAL OF BONE AND MINERAL RESEARCH Volume 6, Number 11, 1991 Mary Ann Liebert. Inc., Publishers

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TGF-P, Induces Bone Closure of Skull Defects L. STEVEN BECK,' LEO DEGUZMAN,' WYNE P. LEE,' YVETTE XU,' LORRIE A. McFATRIDGE,' NANCY A. GILLETT,2 and EDWARD P. AMENTO'

ABSTRACT Transforming growth factor PI (TGF-0,) is a multifunctional regulatory protein. It is capable of inducing site-specific healing responses by increasing collagen synthesis and deposition as well as remodeling at sites of soft tissue repair. Large bony defects in the skull heal by fibrous connective tissue and never form bone unless osteoinductive bony fragments or powders are placed in the defect. We have found, however, that the single application of human recombinant TGF-0, in a simple 3% methylcellulose gel to skull defects induced a dose-dependent increase in intramembranous bone formation. Complete bony bridging of defects occurred within 28 days after treatment with 2 pg TGF-0,. Sites treated with vehicle alone did not heal with bone formation but rather contained dense fibrous connective tissue between the defect margins.

INTRODUCTION

nonhealing bone defect induces complete bone closure within 4 weeks.

B

ONE IS A COMPLEX MINERALIZED CONNECTIVE TISSUE

under endocrine and paracrine control that normally heals without scar formation. Under conditions in which a large gap exists between fragments of bone, however, osseous union does not occur, but the separation fills with fibrous connective tissue. This is especially evident in defects of the adult skull. Demineralized bone powder or bone fragments are used to induce closure of bony defects.'l) Bone is the largest reservoir for transforming growth factor$, (TGF-@,),'" a 25 kD homodimeric protein, that stimulates migration and proliferation of mesenchymal cells as well as matrix synthesis by these cells (see review by Sporn and Roberts, 1990).'31TGF-P also stimulates osteoblast-like cells to proliferate and synthesize collagen in c u l t ~ r e ' ~and - ~ ) to increase bone thickness when applied adjacent to periosteum in V ~ V O . " - 'It~ )has been assumed that specific bone morphogens in complex matrices("-'8) are required to bridge large defects. To date, no purified growth factor has been shown to induce bone in a defect in the absence of bone powder. We report here that a single application of recombinant human TGF-PI to a

MATERIALS AND METHODS

Source and preparation of TGF-0, Human recombinant TGF-BI was a product of transfected CHO cells and purified as previously described.'") Individual samples of the active portion of TGF-PI were prepared under sterile conditions in 3% methylcellulose containing 20 mM sodium acetate buffer at pH 5.0. Vehicle was formulated in a similar manner without TGF-8,. The material was stored at 5°C until use.

Animal surgery and treatment All studies were performed in accordance with the American Association for the Accreditation of Laboratory Animal Care (AAALAC) guidelines. A group of 28 male New Zealand white rabbits (2.8-3.2 kg; Elkhorn Rabbitry, Watsonville, CA) were anesthetized with 0.75 ml/kg of Hypnorm (Jenssen Pharmaceutica, Beersa, Belgium). The top of the head and base of the ears were shaved and asep-

'Inflammation, Bone and Connective Tissue Research, Developmental Biology, Genentech Inc., South San Francisco, California. 'Department of Safety Evaluation, Genentech Inc., South San Francisco, California.

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tically prepared for surgery. An elliptical incision was made over the skull, reflecting the skin flap anteriorly. Similarly, the periosteum was reflected anteriorly as a flap, exposing the top of the skull. Both skin and periosteal flaps were covered with sterile, moistened gauze. A 12 mm skull defect was selected since, in the absence of treatment, bone does not bridge the gap but rather a fibrous tissue nonunion of the skull persists."O' A sterile trephine attached to an electric drill was used to produce the defect at the point of intersection between the sutures of the right and left parietal and frontal bones. The site was liberally irrigated with physiologic saline during the drilling to prevent overheating of the bone margins. Care was taken not to puncture or damage the underlying dura. A precut, sterile, saline-moistened piece of Gelfilm (Upjohn, Kalamazoo, MI) was inserted through the defect overlying the dura to function as a barrier between the dura and the edges of bone. Sterile vehicle (3% methylcellulose) or recombinant human TGF-0, (0. I , 0.4, or 2 pg) in 3% methylcellulose was applied to the defect at a constant volume (0.1 ml) filling the defect. The periosteal flap was sutured back in place'with 6-0 proline sutures and the skin flap was closed with 4-0 silk. Rabbits were returned to their cages and allowed to recover. After 28 days rabbits were euthanized with an overdose of barbiturate and the defect sites were removed with adjacent normal bone. The defect sites were rinsed in physiologic saline and transillumination photographs taken. Sites were fixed in 10% neutral buffered formalin and an x-ray representative of each group was taken using a Faxitron x-ray system and X-omat AR-2 film exposed at 25 kV, 10 s. The fixed tissue samples were then acid decalcified (Easy-cut; American Histology Reagent Co., Modesto, CA). A cross section at the center of the defect parallel to the frontaVparietal suture was taken and processed by routine histologic methods and 4 pm sections were stained with hematoxylin and eosin. The hematoxylin and eosin-stained sections taken from the midsection of the defect were examined at x 40 magnification using an ocular grid eyepiece to measure the defect size.

Statistical analysis Data were analyzed by one-factor analysis of variance and Scheffe's F test to determine differences between groups. The test of significance was performed at the 95% confidence interval compared to vehicle. Each group contained six to eight rabbits.

RESULTS

Transillumination and radiography Grossly observable differences were noted between the vehicle- and TGF-@,-treated defect sites at 28 days. Vehi-

cle-treated defect sites were covered by a soft, flexible thin fibrous membrane. Defects treated with TGF-0, were harder and more rigid, containing varible amounts of fibrous tissue, and were generally thicker than vehicletreated sites. Light was transmissible through the vehicletreated, healed sites, and a decrease in light transmission correlated with the application of increasing concentrations of TGF-0, (Fig. I). The vehicle-treated defect sites appeared pale yellow centrally and dense red peripherally (Fig. IA). In contrast, defect sites treated with 2 pg TGF0,appeared dark red and were dense, with little or no transillumination through the defect sites (Fig. ID). The presence of bone within the defect was confirmed radiographically (Fig. 2). New bone, which could be demonstrated histologically, was observed at sites treated with 0.1 and 0.4 pg TGF-0, as fine radiodense areas deposited in an irregular manner along the margins of the original defect (Fig. 2B and C). The radiodensity of bone from sites treated with 2 pg TGF-0, was greater than that of bone treated with the other doses and approximated the density of surrounding bone (Fig. 2D). Even the bone adjacent to the original site of the defect appeared more radiodense in groups treated with TGF$,. In contrast, bone adjacent to the defect in vehicle-treated sites appeared radiolucent, with little indication of new bone formation along the defect margins (Fig. 2A).

Histology and planimetry Histologic examination of the bone repair sites also confirmed that the defects were bridged by cancellous bone (Fig. 3). Sites that received 0. I , 0.4, or 2 pg TGF-0, healed by bony ingrowth from the margins of the skull defect. These sites contained islands of bone that appeared to be actively secreting matrix, separated by loose connective tissue. Although the lower doses of TGF-0, did not induce complete closure of the defect by day 28 (Fig. 3A), the osteoblasts lining the newly formed bone spicules appeared columnar and active (Fig. 3A, inset), indicating that active bone formation was continuing. Defects treated with 2 pg TGF-0, were completely closed in five of seven sites and were similar histologically to the bone of the adjacent normal skull. These sites contained spicules of woven and lamellar bone interspersed with numerous marrow cavities lined by columnar, active osteoblasts (Fig. 3B). The periosteum was intact and lined by osteoblasts and contained numerous blood vessels (Fig. 3B, inset). Bone formed only as a direct extension of the calvarial margins; no bone foci formed adjacent to periosteum, nor was a cartilagenous intermediate detected. Defects treated with vehicle alone did not form a bony union but were only partially bridged by new bone formation from the margins of the defect. The remainder closed by a dense fibrous connective tissue mem-

FIG. 1. TGF-0, induces closure of skull defects at 28 days: transillumination. Light was transmitted through a dissecting microscope at constant intensity and x 7.5 magnification: (A) vehicle control; (B) 0.1 pg TGF-@,;(C) 0.4 pg TGF-0,; (D) 2 pg TGF-0,. The transillumination photographs shown are representative of the findings in six to eight rabbits per group. The average decrease in light transmission for each group was correlated directly with the decrease in bone gap measured histologically (see Fig. 4).

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FIG. 2. TGF-0, induces closure of skull defects: radiographs. Radiographs of the skull defects and adjacent normal skull were used to evaluate bony ingrowth after 28 days. New bone formation occurred along the margin of the original site of the defect and filled toward the center, The defect size was reduced and the radiodensity of the filled defect and adjacent bone was increased in a dose-dependent manner: (A) vehicle control; (B) 0.1 pg TGF-0,; (C) 0.4 pg TGF-PI; (D) 2 pg TGF-PI. The radiographs shown are representative of the findings in six to eight rabbits per group. The average decrease in the defect observed radiographically for each group correlated directly with the decrease in bone gap measured histologically. (See Fig. 4.) FIG. 3. (Opposite and following pages) TGF-PI induces closure of skull defects: histology. Sections of skull defects removed at 28 days demonstrated that cancellous bone formation occurred from the edges of the defects. (A) Bone formation from the margins of the defect as well as islands of bone (open arrows) interspersed between fibrous connective tissue was observed at sites treated with 0.1 or 0.4 pg TGF-0, ( x 2.6). Residual gel film (closed arrow) adjacent to islands of cancellous bone could be seen upon higher magnification (see inset; x 40). (B) Normal cancellous bone bridged the gap between the edges of the defect of sites treated with 2 pg TGF-0, ( x 2.6). Bone spicules lined by osteoblasts and marrow cavities were evident (arrowhead). The periosteum (open arrow) was intact and lined by osteoblasts adjacent to the bony surface (see inset; x 40). Residual gel film was present underlying the new bone and could be seen at low (closed arrow) and high magnification (*). (C) The sites treated with 3 % methylcellulose alone contained cancellous bone only at the margins of the defect, with the remainder of the defect closed with dense fibrous connective tissue (closed arrow). Islands of bone were present only at the margins of the defects (open arrow; x 2.6). The bone adjacent to the fibrous tissue was rounded at the edges (see inset; x 40). The approximate edge of the original defect is demarcated by the vertical bars. The photomicrographs are representative of the changes in six to eight rabbits per group.

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brane (Fig. 3C). The bone margins of the vehicle-treated sites contained few, primarily inactive, osteoblasts and appeared quiescent at 28 days (Fig. 3C, inset). The gap between the bone edges was measured by microplanimetry of histologic cross sections of bone. The size of the vehicle-treated defect sites was reduced by approximately 40%. The mean defect size of the sites treated with 0.1 or 0.4 pg TGF-P, was reduced 62 and 50%, respectively. The application of 2 pg TGF-0, resulted in complete bridging in five of seven sites, with an average reduction in defect size of 94% (Fig. 4).

DISCUSSION The healing response induced by the local application of TGF-P, may be directly related to the committed cell phenotype at the repair site. This multifunctional, site-specific activity has been demonstrated in numerous tissues. Since most cells in the body produce TGF-P and in turn bear receptors for the factor, a central regulatory role would be anticipated (see review by Sporn and Roberts 1990).(3’In addition, TGF-P, is secreted in latent form and is presumably activated at specific tissue sites before binding to its receptor.‘”) In soft tissues, we and others have demonstrated that TGF-0, accelerates healing of both normal(7.2-24) and impaired healing(25’dermal wounds. The committed precursor cells at the site of exposure determine the response elicited by TGF-PI. Repeated subperiosteal injection of TGF-0, in the skull of normal neonatal rats induces bone f ~ r m a t i o n . ~ ’ , ~In, ’ addition, ~) multiple injections of TGF-0, under the periosteum of the femur of rats induces cartilage and bone.(s) As shown here, however, a single application of TGF-0, to a skull defect is sufficient to induce the cascade of events that result in bone formation. Indeed, bone formation continues until complete clo-

sure occurs without evidence of a cartilagenous intermediate. In contrast, bone morphogenetic proteins (BMPs) admixed with demineralized bone powders and implanted at soft tissue sites or sites of bony defects induce bone with a cartilagenous intermediate.‘15.17~zb) Induction of bone at soft tissue sites indicate that BMPs may be stimulating an earlier stage of cell differentiation than TGF-PI. Implant materials used to close osseous defects vary from mineralized or demineralized autologous t o allogeneic bone.‘”’ These implant materials function as a template for osseous ingrowth and are resorbed locally, as remodeling occurs. The harvesting of autologous bone fragments requires a second surgical procedure, the site varying with the type of bone required. In other circumstances, bone substitutes, such as hydroxylapatite, ceramics, titanium, acrylic resin mesh, or combinations of substitute materials, have been used with variable success.~28~*9~ The results reported here support the potential role of TGF-6, in normal bone formation and repair. Thus, TGF-0, applied once to a nonhealing skull defect is sufficient to induce a reparative cascade that culminates in complete bone closure. These observations support the hypothesis that TGF-0, is a potent osteoinductive factor in vivo.

ACKNOWLEDGMENTS We thank Stan Hansen and Robin Taylor for processing tissues for histologic examination and morphometric determinations. In addition, we thank Dr. Daniel Ladin for discussions on surgical materials for internal implantation. A portion of this work was published in abstract form in J. Cell. Biochem. Suppl. 1991 15F:192.

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FIG. 4. TGF-0, induces closure of skull defects at 28 days. The distance between advancing edges of bone were measured with an ocular grid eye piece using hematoxylin and eosin-stained sections examined at x 40 magnification. Data represent the mean 2 SEM of measurements from six to eight rabbits. *p < 0.01.

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L.. Steven Beck Inflammation, Bone and Connective Tissue Research Developmental Biolog-v Genentech, Inc. 460 Point Sun Bruno Boulevard South San Francisco, CA 94080 Received for publication June 24, 1991; in revised form July 23, 1991; accepted July 25, 1991.

Rapid publication. TGF-beta 1 induces bone closure of skull defects.

Transforming growth factor beta 1 (TGF-beta 1) is a multifunctional regulatory protein. It is capable of inducing site-specific healing responses by i...
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