Bone morphogenetic protein-7 selectively enhances mechanically induced bone formation

Bone Vol. 31, No. 5 November 2002:570 –574

Bone Morphogenetic Protein-7 Selectively Enhances Mechanically Induced Bone Formation A. J. CHELINE, A. H. REDDI, and R. B. MARTIN Orthopaedic Research Laboratory, Center for Tissue Regeneration and Repair, University of California at Davis Medical Center, Sacramento, CA, USA

Introduction The responses of bone cells to skeletal loading are clearly an important factor in bone biology, but much remains to be learned about the role of these responses in skeletal development, maintenance, and tissue repair. Bone morphogenetic proteins (BMPs) are key regulators of bone formation. We examined the effect of BMP-7 on periosteal and endosteal bone formation in response to increased mechanical loading using the rat tibial bending model. Female Sprague-Dawley rats were divided into four groups of six rats each. Three groups received four point bending loading at 60 N force; the fourth group received sham loading at the same force. The right tibia received 36 cycles of loading on Monday, Wednesday, and Friday for 2 weeks; the left tibia served as a nonloaded control. Just prior to loading, the three loaded groups were injected intraperitoneally with vehicle only or 10 ␮g/kg or 100 ␮g/kg of recombinant human BMP-7. Half the sham group received vehicle, and half were given 100 ␮g/kg of BMP-7. Bone forming surfaces were labeled twice in vivo with calcein, and histomorphometry was performed to quantify periosteal and endosteal bone formation in the loaded and control tibiae. BMP-7 had no effect on periosteal or endosteal bone formation in control or sham-loaded tibiae. Loading produced significantly more woven bone on the periosteal surface than sham loading, but BMP-7 treatment had no effect on this response. Endosteal bone formation was entirely lamellar, and loading (but not sham loading) increased the endosteal mineral apposition and bone formation rates. The higher BMP-7 dose more than doubled the loadinduced increase in endosteal lamellar bone formation rate, primarily by increasing the amount of bone forming surface. (Bone 31:570 –574; 2002) © 2002 by Elsevier Science Inc. All rights reserved.

Knowledge concerning the responses of bone cells to skeletal loading is important for understanding the biology of a healthy skeleton and for discovering corrective measures for a wide variety of bone diseases.1,13,14,22,32 These responses take several forms. One of the most important of these is increased bone formation on the periosteal and/or endosteal surfaces of long bones in response to increased mechanical loading, particularly in growing individuals. This response has been demonstrated in several animal models,9,18,19,23,26,33 as well as in humans.3,10,15 A better understanding of such responses may clarify the processes of bone development during growth, and the factors leading to larger bones for a given amount of loading, thus mitigating against osteoporosis later in life. Alternatively, it may be possible to induce bone formation on the endosteal or periosteal surfaces of the bones of adults susceptible to osteoporosis, reducing fracture risk. An established method for studying such responses is the rat tibial bending model originally developed by Turner et al.29 In this model, dose-response relationships have been demonstrated between bone formation and the amount of bending transcutaneously applied to the tibia. The periosteal and endosteal responses typically involve woven and lamellar bone formation, respectively.30 This distinction may be due to the fact that bending produces larger strains on the periosteal surface than on the endosteal surface, simply due to the mechanics of the situation. The dissimilar biological situations at these two bone surfaces may also be important in causing such distinctly different responses; that is, the endosteal surface is populated by bone lining cells in a marrow environment, whereas the periosteal surface is covered by the periosteal membrane. Finally, direct pressure from the bending apparatus may influence the periosteal response, and the model incorporates a sham loading mode of treatment to assess this, as described in what follows. The details of these responses are presumably mediated by a number of molecules moving through several biological pathways, and work is underway to discover and describe these pathways on each bone envelope. For example, Turner et al.31 found that, although periosteal woven bone formation in this model arose from proliferating osteoblast precursor cells, most of the bone forming cells on the endosteal surface had nonproliferative origins—that is, as existing osteoblasts, bone lining cells, or preosteoblasts. Also, Forwood6 obtained evidence that the endosteal osteoblastic response is inflammatory, involving the production of cyclooxygenase-2 (COX-2) and prostaglandins, but the periosteal response is not.

Key Words: Bone morphogenetic protein-7/osteogenic protein-1 (BMP-7/OP-1); Bone formation; Rat; Bending; Mechanical adaptation.

Address for correspondence and reprints: Dr. R. Bruce Martin, Orthopaedic Research Laboratory, Research Facility, 4635 Second Avenue, UC Davis Medical Center, Sacramento, CA 95817. E-mail: rbmartin@ ucdavis.edu © 2002 by Elsevier Science Inc. All rights reserved.

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Figure 1. Rat tibia showing locations of the inner and outer loading supports (black bars) and the three histological sections. The tibiofibular junction lies between the distal loading supports.

Bone morphogenetic proteins (BMPs) may also play a role in these responses. Under certain conditions, BMPs induce de novo bone development1 in vivo.25 For example, the hypothesis that BMP-7 (also called osteogenic protein-1, or OP-1) has a capacity to induce bone formation in an environment of tissue damage is supported by its capacity to aid the healing of large segmental bone defects.4,5,8 More specifically, BMP-7 has been shown to stimulate osteoblast proliferation and to induce differentiation of fibroblasts into osteoblasts.4,5 As noted earlier, bone formation by these two pathways has also been postulated in response to mechanical loading in the rat tibial bending model. To learn more about the possibility that BMP-7 may play a role in, or interact with, mechanically induced bone formation, we studied the effect of systemically administered BMP-7 in the rat tibial bending model. More specifically, we hypothesized that BMP-7 would increase mechanically induced bone formation on the periosteal surface by promoting osteoblast proliferation, and on the endosteal surface by its ability to transduce bone lining cells, and perhaps other cells of mesenchymal origin, into osteoblasts. Methods Twenty-four virgin female Sprague-Dawley rats, initially weighing 284 ⫾ 12 g, were used in this study. The experimental protocol was approved by the University of California, Davis, Animal Use and Care Administrative Advisory Committee. Rats were caged individually with a 12 h light-dark cycle and fed water and a commercial diet (Lab Diet 5001, PMI Nutrition International, Inc., Brentwood, MO) ad libitum. The apparatus we used for controlled tibial bending has been described previously by Forwood et al.7 Briefly, it consists of a small electromagnetic oscillatory loader with a device to hold the rat’s leg during the loading procedure. The tibial diaphysis is bent transcutaneously by four padded cylindrical supports. Following the usual practice for this model, we placed the inner supports 11 mm apart on the lateral side of the leg, with the outer supports symmetrically disposed 23 mm apart on the leg’s medial side (Figure 1). Alternatively, sham loading was produced by positioning the medial pads 11 mm apart and directly opposed to the lateral supports (see also Figure 1 in Forwood et

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al.7). This results in similar pressures being applied to the soft tissues and bone surfaces, but with negligible bending moments in the tibia. The rat’s right tibia was loaded, with the left tibia serving as an internal control. The right foot was situated in a stirrup to secure the leg in a consistent position such that the loading points produced compression on the lateral surface and tension on the medial surface of the tibia. The rats were anesthetized for the loading procedure by halothane inhalation followed by an intramuscular injection of ketamine (60 mg/kg) and xylazine (6 mg/kg) at the nape of the neck. We wanted the experiment to induce endosteal bone formation while keeping periosteal strains in the physiological range of 2000 –3000 microstrain (␮ε).27 By instrumenting the tibiae of female Sprague-Dawley rats weighing 324 ⫾ 6 g with periosteal strain gauges, Forwood et al.7 found that a peak load of 60 N produced slightly more than 2500 ␮ε. Therefore, we also applied a load that varied sinusoidally between 0 and 60 N. However, because our rats weighed about 9% less than Forwood’s, it is likely that we induced tibial surface strains somewhat greater than 2500 ␮ε, but well below 3000 ␮ε. The applied load varied sinusoidally at 2 Hz, and each loading bout consisted of 36 cycles. The loader was calibrated between experimental sessions and consistently found to be within ⫾ 1 N of the desired load. The effects of BMP-7 were studied by dividing the rats into four groups of six rats each. Three groups received bending loading to the right tibia on Monday, Wednesday, and Friday for 2 weeks. These groups were, respectively, treated with recombinant human (rh)BMP-7 at three dose levels: phosphate-buffered saline vehicle only (Veh-Bend); 10 ␮g/kg (Low-Bend); and 100 ␮g/kg (HighBend). These systemically administered doses (approximately 3 and 30 ␮g/rat, respectively) were about 30 and 300 times the amount (100 ng) that has been shown to produce bone formation when injected subcutaneously or in muscle.11 The fourth group received sham loading and was divided into subgroups of three rats each, one receiving vehicle only (Veh-Sham), and the other 100 ␮g/kg of rhBMP-7 (High-Sham). Injections were given intraperitoneally following anesthesia and 5 min before loading. Bone forming surfaces were labeled by intraperitoneal injections of calcein (7 mg/kg) given on the first and second Friday of the treatment regimen. Rats were killed by CO2 inhalation 3 days after the last loading session. Body weight was measured on the first day of loading and at time of killing. Immediately after killing, the right and left tibiae were cleaned of soft tissue, and each end of the bone was removed to expose the marrow for embedding and staining. The specimens were progressively dehydrated for 12 h each in 70%, 80%, 90%, and 100% ethanol, embedded in methylmethacrylate, and 80-␮m-thick transverse sections were cut using a Leitz Sawing Microtome (Leica, Inc., Deerfield, IL). Three sections were made from each tibia, beginning 5 mm proximal to the tibiofibular junction (TFJ) and moving proximally (Figure 1). The sections were placed in Villanueva bone stain (Polysciences, Inc., Warrington, PA) for 12 h; washed in water, dehydrated through a quick progression of 70%, 80%, 90%, and 100% ethanol, and mounted on glass slides using Eukitt (Calibrated Instruments, Inc., Hawthorne, NY) to attach the coverslips. One section from each tibia was selected for analysis, based on section quality, with preference given to sections closest to the TFJ, and assigned a blind code. For each rat, equivalent sections were chosen from the left and right tibiae (e.g., the first, second, or third section from the TFJ). Sections were imaged and analyzed using a Zeiss Laser Scanning Microscope (LSM 510 Carl Zeiss, Inc., Thornwood, NY) with a krypton-argon laser and version 2.3 analysis software (IBM Pentium Computer, Windows NT 4.0 operating system). Histomorphometric measurements were made by tracing the desired features in computer images and using the LSM software

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Table 1. Body weights and weight loss of the experimental groups (mean ⫾ standard deviation) Experimental group

N

Initial weight (g)

Final weight (g)

Weight loss (g)

Veh-Bend Low-Bend High-Bend Veh-Sham High-Sham

6 6 6 3 3

285 ⫾ 16 285 ⫾ 15 287 ⫾ 12 282 ⫾ 10 282 ⫾ 7

278 ⫾ 17 277 ⫾ 19 280 ⫾ 16 274 ⫾ 10 272 ⫾ 9

6.7 ⫾ 5.0 7.3 ⫾ 7.3 7.0 ⫾ 10.1 8.0 ⫾ 4.0 9.7 ⫾ 3.2

The final weights are significantly less than the initial weights (p ⬍ 0.0001), but there were no significant differences between groups for any of these variables.

to calculate distances or enclosed areas. Lamellar bone was distinguished from woven bone by its highly organized collagen fibers and parallel fluorochrome labels. New bone formation on the periosteal surface was almost entirely woven bone. The portion of the periosteal perimeter covered by woven bone was expressed as a percentage of the periosteal surface (Wb.S [%]). The area of periosteal woven bone was expressed as a percentage of cortical area (Wb.Ar [%]). On the endosteal surface, new bone formation consisted almost entirely of lamellar bone. Here, working at 60⫻ magnification, we measured total endosteal bone surface length, and the proportion of this surface that was double-labeled, and expressed the ratio as percent mineralizing surface (MS/BS [%]). Greater magnification (400⫻) was used to accurately measure the distance between double labels approximately every 200 ␮m along the double-labeled surfaces. The mean of these distances (in ␮m) was calculated for each specimen, and divided by the 7 day interval between the calcein labels to obtain the mineral apposition rate (MAR [␮m/day]). Also, the total area of lamellar bone formed on the endosteal surface in (␮m2) was measured by tracing the area contained between the two labels. This was divided by the interval between labels (now expressed in years) and the endosteal surface perimeter to obtain the surface-based endosteal bone formation rate, BFR/BS (in ␮m3 per ␮m2). STATVIEW for Windows version 5.01 (SAS Institute, Inc., Cary, NC) was used for analysis of the five histomorphometric variables just described. Because we were interested in the effect of BMP-7 on load-induced endosteal and periosteal bone formation, the net mechanical effect was calculated by subtracting the value for the control (left) tibia from that for the loaded (right) tibia, and designating this difference by the prefix “m” (e.g., mMAR). Analysis of variance was then used to study the effects of loading and BMP-7 treatment on endosteal and periosteal bone formation, with Fisher’s PLSD as the post hoc analysis. The criterion for statistical significance was p ⬍ 0.05, and variability expressed as ⫾ standard deviation. Results The rats lost a small but statistically significant amount of weight (2.7%, 7.5 ⫾ 6.5 g, p ⬍ 0.0001 by paired t-test) during the experiment. However, there were no significant differences in initial or final body weight, or weight loss, among the experimental groups (Table 1). Other than those described in what follows, no effects of systemic BMP-7 administration were observed. Both calcein labels were present in all sections. We first addressed direct pressure effects by comparing the responses of bending and sham loading on the periosteal and endosteal surfaces. Measures of bone formation in the five experimental groups were tested using one-way analysis of variance (ANOVA) with loading method (sham or bending) as the factor.

Figure 2. Graphs showing results for periosteal surface woven bone formation: mWb.S (%) above, and mWb.AR (%) below. The experimental groups are identified as VB (Veh-Bend), LB (Low-Bend), HB (High-Bend), VS (Veh-Sham), and HS (High-Sham). Bars sharing the same letter (A, B, etc.) are significantly different, with post hoc p values shown at right.

The effect of BMP-7 treatment was controlled by making its dose (0, 10, or 100 ␮g/kg) a correlate. Bending produced significantly more bone formation than sham loading on both the periosteal and endosteal surfaces, as measured by the five variables (mWb.S, p ⫽ 0.012; mWb.Ar, p ⫽ 0.0043; mMAR, p ⫽ 0.017; mMS/BS, p ⫽ 0.0403; mBFR.BS, p ⫽ 0.0027). Sham loading did result in some periosteal woven bone formation: 41 ⫾ 22% of the surface in the combined Veh-Sham/High-Sham group exhibited some woven bone. However, the amount of bone formed in the sham-loaded groups was relatively inconsequential: mWb.Ar was only 9% of that in the rats receiving bending loads. Subsequent analysis addressed the effects of BMP-7 using single-factor ANOVA to compare the five treatment groups. All tibiae subjected to bending loading expressed substantial amounts of periosteal woven bone covering more than half this surface (Figure 2, top), but treatment with BMP-7 had no significant effect on mWb.S or mWb.Ar. Bone formation on the endosteal surface was exclusively lamellar. Bending loading, but not sham loading, increased endosteal bone formation (Figure 3). BMP-7 treatment did not affect mMAR, but mMS/BS exhibited significant stepwise increases with increasing BMP-7 dosage. The lower dose of BMP-7 did not increase mBFR/BS, but it was nearly doubled by the higher dose. In animals receiving sham loading, BMP-7 had no effect on any of the three endosteal bone formation variables. Two rats in the Veh-Sham group and one rat in the High-Sham group showed no new bone formation on the endosteal surface. Discussion We hypothesized that systemic administration of BMP-7 would increase the osteogenic response to mechanical loading both periosteally and endosteally in the rat tibial bending model. The results support the latter but not the former hypothesis; that is, systemic administration of BMP-7 differentially enhanced only lamellar bone formation on endosteal surfaces of bones receiving

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Figure 3. Graphs showing results for endosteal surface lamellar bone formation: mMAR (␮m/day), top; mMS/BS (%), middle; and mBFR/BS (␮m3/␮m2 per year), bottom. See caption of Figure 2 for further information.

bending loads. Furthermore, it is noteworthy that BMP-7 had no effect on the tibiae that were not loaded nor on those that were sham loaded. Thus, the osteogenic response to BMP-7 in this model seems to depend on both the nature of the mechanical stress or strain in the adjacent bone tissue and the characteristics of the two skeletal envelopes. At this point, we cannot determine whether the lack of response to BMP-7 on the perioseal surface was related to its membraneous anatomy, or to the fact that the cellular response produced woven rather than lamellar bone. This could be further studied by repeating the experiment using lower loads that would result in lamellar bone formation on the periosteal surface. Other limitations in our experiment should be noted. First, the rats lost a slight amount of weight during the experiment, suggesting that they experienced some physiological stress. Second, the dosages of BMP-7 and the timing of their administration relative to mechanical loading were chosen carefully but rather arbitrarily. The concentrations of the injected BMP-7 at the endosteal and periosteal surfaces of the tibia over time are unknown, and may have been different at the two surfaces. In addition, the histomorphometric analyses did not include variables at the cellular level. It should also be noted that there have been differences of opinion as to the interpretation of periosteal responses observed using the rat tibial bending model. Of the four experimental reports we found that tested periosteal sham loading effects, three stated that bending loading produced significantly more woven bone formation on the periosteal surface than did sham loading.7,23,29 These results have supported the use of the model to study periosteal as well as endosteal responses to mechanical

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loading, provided that sham-loaded animals are used to control for the effect of direct pressure on the periosteal tissue. However, Robling et al.26 reported an experiment in which rats receiving 360 cycles/day of 54 N loading over a 3 day period exhibited no differences in periosteal response to sham and bending loading. This result has again called into question the effect of direct pressure, and the present results should be viewed in that light. Nevertheless, our analysis found that, when the effects of BMP-7 treatment dose were accounted for, bending loading produced significantly more bone formation than sham loading on the periosteal as well as the endosteal surface. Regarding the endosteal effects of systemic BMP-7 administration, the following observations can be made. BMP-7 signaling is dependent on types I and II BMP receptors, which are membrane-associated protein kinases phosphorylating serine and threonine in signaling substrates called Smads 1 and 5.25 Phosphorylated Smads 1 and 5 enter the nucleus in partnership with co-Smad, Smad 4, to initiate the transcription of BMP-response genes. It is postulated that strain-generated signals in the osteocytic syncytium are transduced via cell processes in the canalicular network and transmitted to bone lining cells and preosteoblasts.2,17 BMP-7 may enhance this pathway to further increase the strain-mediated endosteal lamellar bone formation rate. It has been demonstrated that strain-mediated endosteal bone formation depends initially on expansion of the osteoblast population by nonproliferative means. Using sustained release bromodoxyuridine labeling, Turner and coworkers31 found that ⬎95% of the osteoblasts responsible for the initial response to loading derived from differentiation of bone lining cells or committed preosteoblasts into functional osteoblasts. This process took ⬍2 days. After 4 days, 30%– 40% of the endosteal osteoblasts had originated from proliferating osteoprogenitor cells, indicating that the availability of cells committed to bone formation was exhausted after a relatively brief period of time, and ongoing bone formation required the generation of osteoblasts from stem cells or other uncommitted progenitors. In contrast, mechanically induced woven bone formation on the periosteal surface seemed to be entirely due to the proliferative generation of osteoblasts from stem cells or uncommitted progenitor cells. In our experiment, endosteal bone formation was measured between calcein labels given 4 and 11 days after loading began. Assuming that the initial cohort of osteoblasts was nonproliferative in origin, and that it took about 3 days for these cells to begin forming bone, a substantial portion of the bone formation we measured must have been accomplished by differentiation without proliferation. It seems clear, however, that a significant portion of the bone formation we measured occurred later and depended on proliferative recruitment of osteoblasts from stem cells. Comparing our experimental results with those of Turner et al.,31 we see that the surface on which BMP-7 had no effect (i.e., the periosteal surface) was the one where osteoblasts were produced entirely by proliferation. This suggests that systemic BMP-7 acted to increase the differentiation of bone lining cells, fibroblasts, or other committed cells to osteoblasts rather than to increase their proliferative generation from putative stem cells. One of the paracrine factors produced in bone subjected to elevated strain in organ culture is prostaglandin E2 (PGE2).24 The inducible enzyme, cyclooxygenase-2 (COX-2), is associated with inflammatory responses and apparently mediates the production of PGE2 in bone cells subjected to elevated strain.12,16 Through the use of the specific COX-2 inhibitor, NS-398, in the rat tibial bending model, Forwood6 showed that COX-2 is required for the endosteal lamellar bone formation response, and has postulated that this molecule is produced by bone cells in

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response to fluid flow induced within the bone matrix by mechanical strain. Presumably, the responsible cells are the osteocyte-bone lining cell syncytium.2,21 Osteopontin (OPN) is a key noncollagenous protein in bone, existing in two different forms.28 A 44 kDa OPN of high phosphorylation that is secreted by osteoblasts, osteocytes, and bone lining cells is thought to be an important component of bone matrix and inactive bone surfaces, whereas a 55 kDa OPN of low phosphorylation expressed by preosteoblasts is a key constituent of bone cement lines. This expression of two different forms of OPN by cells at different points in the osteoblastic differentiation sequence has been useful in identifying factors responsible for the control of bone formation. Li et al.20 concluded from in vitro studies of fetal rat calvarial cells that BMP-7 increases bone formation primarily by promoting preosteoblast differentiation, as manifest by upregulation of 55 kDa OPN expression. They noted that BMP-7 acts “to stimulate the growth of pre-osteoblastic cells and their differentiation into osteoblasts, without having a significant effect on the production of bone matrix by individual osteoblasts.” Our finding, that systemically administered BMP-7 acted differentially to enhance endosteal but not periosteal bone formation in the rat tibial bending model, is consistent with their hypothesis.

Acknowledgments: This work was partially supported by the Lawrence J. Ellison Endowed Chair in Musculoskeletal Molecular Biology (held by A.H.R.) and the Doris Linn Chair of Bone Biology (held by R.B.M.). The authors gratefully acknowledge the valuable advice of Dr. Mark Forwood, Dr. Susan Stover, and Dr. Neil Willets.

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Date Received: January 2, 2002 Date Revised: May 4, 2002 Date Accepted: July 5, 2002