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High Doses of OP-1 Inhibit Fibrous Tissue Ingrowth in Impaction Grafting

Hannink, Gerjon; Aspenberg, Per; Schreurs, B, Willem; Buma, Pieter

Author Information
Clinical Orthopaedics and Related Research: November 2006 - Volume 452 - Issue - p 250-259
doi: 10.1097/01.blo.0000229340.18974.dc


Aseptic loosening is the main reason for total hip arthroplasty (THA) component failure.32 The loosening process often is accompanied by progressive bone-stock loss around the prosthesis. Several reconstruction techniques to deal with this problem have been suggested.1,41 One of these techniques, bone impaction grafting, has become a popular method to restore bone-stock deficiencies. The intent of this technique is to obtain stable implant fixation, to restore normal hip biomechanics, and in the long term, to revitalize bone with restoration of normal bone anatomy. Several authors report satisfactory clinical results on the acetabular and femoral sides.20,21,40,49

The chance of a reconstruction failure is greatest immediately after the reconstruction when the bone graft is not yet incorporated. If the incorporation of bone grafts can be facilitated by growth factors, the critical period after a reconstruction with impacted bone grafts might be shortened.30,31,42

Growth factors, such as BMP-2, PDGF, and OP-1 (BMP-7) are well known stimulators of bone formation.2,7,14 However, BMP-2 and OP-1 also stimulate osteoclasts in vitro23,26,27 and PDGF has been associated with aseptic loosening of prostheses.38,55 The role of OP-1 in stimulation of osteoclasts has not been studied in vivo. Preliminary results of a trial in humans with unstable thoracolumbar spine fractures, treated with transpedicular OP-1 transplantation, suggest increased bone resorption as a primary event.28 Salkeld et al,39 using autograft and allograft with OP-1, found an accelerated resorption of the graft material but also substantial formation of new bone. Moreover, a study by Jensen et al,24 using impacted mixtures of allograft and OP-1 in a gap model around implants in dogs, showed an extensive effect on new bone formation by OP-1, but also accelerated graft resorption. The results from these studies suggest OP-1 initially enhances osteoclast activity.

Impacted morselized allografts around prostheses serve not only as a bone conductor, but also as a mechanical support for the prostheses. Accelerated bone graft resorption before the formation of bone may cause loss of stability of the prostheses, resulting in micromotion and ultimately failure. An often suggested solution would be an osteoconductive material providing initial stability after reconstruction.6,18,34,35,47,53 Synthetic ceramic calcium phosphate-based materials such as TCP, HA, and biphasic mixtures of these two components are considered promising materials. Tricalcium phosphate and HA are incorporated in a way similar to human bone grafts. However, the remodeling process, as being part of the incorporation process, generally takes place much slower in TCP/HA.19 The role of BMPs in hip revision surgery then may be to serve as a promoter of bone formation when used in combination with slow resorbing or unresorbable graft materials, such as TCP/HA particles or bone grafts pretreated with bisphosphonates.3,5

We attempted to determine if (1) OP-1 promotes the incorporation of impacted morselized allografts and TCP/HA into host bone, (2) bone formation is preceded by an initial process of accelerated resorption, and (3) a dose- related bone graft remodeling response to OP-1 exists.


We performed two experiments, an allograft experiment and a TCP/HA experiment (Table 1). In both experiments, we tested three doses of OP-1 combined with allograft or TCP/HA. In each experiment an allograft control was included. For each experiment, 12 mature Dutch milk goats (Capra Hircus Sana) (range, 46-55 kg) were obtained from the Central Animal Laboratory, University of Nijmegen, The Netherlands. The goats received three bone chambers at each side in the cortical bone of the proximal medial tibia (Fig 1). The position of implantation among the six chambers and the side for each type of chamber were randomized. We observed the animals for 4 weeks. All procedures were approved by the Animal Ethics Committee of the University of Nijmegen, The Netherlands. In the TCP/HA experiment, one of the groups was used for testing another osteoconductive material. Because the outcome was outside the scope of our hypothesis and the outcome of the other groups was not influenced by this particular group, it was excluded from the statistical analysis.

Fig 1
Fig 1:
A radiograph shows implanted bone chambers in the proximal medial tibia of the goat.
Overview of Experimental Design

We used the bone conduction chamber (BCC), which is a model for membranous ossification (Fig 2).4 The BCC consists of a titanium screw with a cylindrical interior space. It is made up of two threaded half-cylinders held together by a hexagonal closed screw cap. The interior of the chamber has a diameter of 2 mm, and a length of 7.5 mm. There are two ingrowth openings for bone ingrowth located at the bone end of the chamber. Therefore, the ingrowing tissues enter the cylindrical space from the bone compartment. The chamber extends far out into the subcutaneous region and the ingrown bone-derived tissue can fill the chamber without competing with other tissues. Thus, the tissue ingrowth distance from the holes toward the other end of the chamber can be used to estimate tissue regeneration. The shape of the chamber makes it easy to distinguish areas for histomorphometry.52 The tissue ingrowth distance can be increased by placing an osteoconductive material, such as a bone graft, in the chamber. Originally developed as a rat model, the BCC was adjusted for use in goats.50 The threaded end of the implant is screwed into the bone, so that the ingrowth openings are in direct contact with the endosteal transition from marrow into bone. To accomplish this in goats, a 1-mm thick plate (a disk) was inserted into the cap to lower the ingrowth openings through the cortex.

Fig 2
Fig 2:
A schematic drawing shows the bone conduction chamber. A 1-mm thick plate (D) was inserted into the cap to lower the ingrowth openings through the cortex. New bone (NB) and fibrous tissue (FT) are shown growing into the graft. Unremodeled graft (GR) is seen at the top of the chamber (Modified with permission from Jeppson C, Astrand J, Tagil M, Aspenberg P. A combination of bisphosphonate and BMP additives in impacted bone allografts. Acta Orthop Scand. 2003;74:483-489 .)

We obtained cancellous allografts from the sternum of six donor goats. Familial bands between donor and recipient goats were excluded. To prevent bias from different immunologic reactions, the allografts were pooled. Most blood and marrow were removed by rinsing the grafts with saline for approximately 1 minute, leaving only a white bone structure. Rinsing was done using a high-pressure pulsatile lavage system (SurgiLav® Plus, Stryker Nederland BV, Waardenburg, The Netherlands). The grafts were in a sieve during rinsing, and then were stored at −80°C until use. Cultures from the grafts were negative. Before implantation, the grafts were thawed at room temperature and cut in 2 x 2 x 1-mm pieces by using a rongeur.

The TCP/HA particles were composed of 20% HA [Ca10(PO4)6(OH)2] and 80% TCP [Ca3(PO4)2] (BoneSave™, Stryker Howmedica Osteonics, Limerick, Ireland). We used granules with a diameter of 2 to 4 mm. The TCP/HA granules have a 50% noninterconnected macroporosity (range, 300-600 μm), which is produced by burning sacrificial carbonaceous filler during sintering. The granules are also microporous (range, 5-80 μm) (porosity values derived from Stryker Orthopaedics). Before use, the TCP/HA particles were crushed to fit in the BCC and subsequently soaked in saline for 30 minutes.

The recombinant human osteogenic protein-1 (rhOP-1) device (Stryker Biotech, Hopkinton, MA) was supplied sterile for implantation and consisted of 3.5 mg rhOP-1 combined with 1000 mg highly purified bovine bone-derived Type I collagen. Immediately after warming to room temperature, the rhOP-1 device was mixed with a preweighed amount of allograft chips before impaction. The BCC volume was 23.56 mm3, which allowed 0.0325 g of allograft chips and 0.0163 g of TCP/HA particles to be impacted in each implant.

We tested three doses of the rhOP-1 device, a low-dose OP-1 (0.83 μg/implant), a medium-dose OP-1 (2.5 μg/implant), and a high-dose OP-1 (25 μg/ implant). The medium dose (2.5 μg/ implant) would correspond to approximately one OP-1 device combined with one ordinary femoral head. According to the instructions of the manufacturer, this is the intended dose for bone impaction grafting. We tested two doses of collagen carrier, a low-dose collagen carrier (0.24 mg/implant) and a high-dose collagen carrier (7.22 mg/implant), which equals the amount of carrier used in the low-dose OP-1 and high-dose OP-1 groups described above.

We performed impaction in the chambers by gradually filling the BCC with the allograft bone/OP-1 mixes or TCP/HA particles/OP-1 mixes. We used a piston, slightly in smaller diameter, for impaction. The piston was guided by low-friction bearings, strictly limiting it to vertical movement.52 The BCC was clamped into a cylindrical holder. A constant force of 40 N was kept on the free end of the piston for 2 minutes. During this time, fluid could escape between the piston and the wall of the bone chamber and the ingrowth openings. The applied pressure was calculated to be 12.5 MPa. The plate was placed in the hexagonal closed screw cap and the cap was screwed on the two threaded half-cylinders.

The goats were anesthetized by intravenous administration of sodium pentobarbital (Nembutal, CEVA Santé Animale, Maasluis, The Netherlands) (0.5 mL/kg) and maintained after intubation with nitrous oxide, oxygen, and isoflurane (1.5% - 2%). Under aseptic conditions, a longitudinal incision was made in the skin and fascia over the medial side of the proximal tibia. After raising the periosteum, a hole was drilled through the medial cortex approximately 4 cm from the joint cleft using a 3.1-mm drill. The hole was tapped and bone debris from drilling was removed. The bone chamber was screwed in manually. The other bone chambers were placed 10 mm from the others. This was repeated for the other side.

The subcutaneous layer and the skin were sutured. All animals were allowed unrestricted movement in their cages and had free access to water and food after the operation. After the implantation procedure the animals received subcutaneous ampicillin (Albipen LA, Intervet International BV, Boxmeer, The Netherlands) (15 mg/kg/48 hours) three times.

After 4 weeks all goats were sacrificed with an overdose of sodium pentobarbital (Nembutal, 1 mL/kg). Tibiae were removed, and the bone chambers with surrounding cortex were fixed in 4% buffered formalin. After one day, the contents were removed from the chambers and fixed in 4% buffered formaldehyde, dehydrated using ethanol, and embedded in polymethylmethacrylate (PMMA). Specimens from the TCP/HA experiment were decalcified using 25% ethylenedinitrilo tetraacetic acid (EDTA) in 0.1 mol/L phosphate buffer (pH 7.4) before dehydration. The specimens were cut with a microtome (Leica RM 2155, Leica Microsystems Nederland BV, Rijswijk, The Netherlands) parallel to the longitudinal axis of the chamber. Sections were taken at 0, 300, and 600 μm from the center of the BCC specimens,4 each section 5 μm thick. All sections in each experimental group were investigated in random order. The tests were blinded, but it was possible to see whether a specimen contained allograft or TCP/HA. The sections were stained with hematoxylin and eosin, Goldner-Masson trichrome, and tartrate resistant acid phosphatase (TRAP) for routine histology.

Histomorphometric analysis was performed by using interactive computer-controlled image analysis (analySIS®, Soft Imaging System GmbH, Münster, Germany). The bone ingrowth distance in each slide was calculated by dividing the new bone area by the width of the specimen. In all specimens, marrow cavities surrounded by bone were included in the bone area. The mean of the three sections at 0, 300, and 600 μm from the center yielded a value for each specimen. The total tissue ingrowth distance, which is the distance from the ingrowth end to the fibrous ingrowth frontier, was measured in the same way as bone ingrowth.4

Statistical analysis was performed using a univariate analysis of variance (SPSS Inc, Chicago, IL) with the factors goat, side, position, and graft type. To isolate the different groups, we used Tukey's multiple comparison procedure. Normality and homogeneity of variance were tested using Kolmogorov-Smirnov's and Levene's tests. When the assumption of normality or homogeneity of variance was not met, a Friedman Repeated Measures ANOVA on Ranks (nonparametric test) was performed. In both experiments an allograft control was included. Therefore, we were able to compare the difference in bone and fibrous tissue ingrowth between equal OP-1-dosed groups by comparing the differences between the OP-1 groups and their allograft controls.


No intraoperative complications occurred during surgery. All goats recovered fully after surgery, were standing within 1 day, and had a normal gait pattern within 3 days after surgery. There were no signs of inflammation, skin ulceration, or wound healing problems. All bone chambers were strongly fixed into the tibia. In most cases, the bone chambers were surrounded with a layer of callus and covered with fibrous tissue, regardless of the contents of the chamber. No new bone formation was seen at the endosteal surface of the tibial cortex.

The incorporation process of impacted morselized allo- grafts and TCP/HA was not promoted by OP-1. A layer of necrotic, nonvascularized graft remnants or TCP/HA granules were present in the top of the chamber, with fibrous tissue infiltration or only graft material as was inserted. In the allograft and TCP/HA groups, a well- vascularized loose mesenchymal-like tissue with numerous blood sinusoids and capillaries penetrated the graft material. In the OP-1 and carrier (collagen particles) groups, a denser fibrous tissue was observed. These groups showed a sharply defined ingrowth frontier, where the fibrous tissue was organized more loosely at the transition with the allograft and TCP/HA remnants. However, in general, no accumulation of plasma cells, lymphocytes, or polymorphonuclear cells was seen in OP-1 and nonOP-1 groups. After 4 weeks, newly formed bone was present in part of the bone chambers (Table 2). New bone was formed by intramembranous ossification, mainly at the level of the ingrowth openings, sometimes growing upward halfway into the chamber. If the resorption of the graft remnant was not complete (as seen with the TCP/HA cases), new bone was deposited on these remnants (Fig 3). Active osteoblasts and osteoid were seen. No cartilage was seen. Regardless of group or OP-1 dose, in some sparse cases, we found a direct contact between newly formed bone and collagen particles. In the allograft experiment, bone ingrowth was higher in the allograft with low-dose OP-1 (p = 0.019) and the allograft with medium-dose OP-1 (p = 0.038) compared with the allograft with high- dose collagen carrier (Table 3; Fig 4). However, we observed no difference in bone ingrowth between the groups tested in the TCP/HA experiment (Table 3; Fig 5).

Fig 3
Fig 3:
A typical example of remodeling and incorporation of TCP/HA granules is shown. Newly formed bone (NB) was deposited on TCP/HA granules (TCP/HA) (Stain, hematoxylin and eosin; original magnification, x100). FT = fibrous tissue
Fig 4
Fig 4:
The incorporation process of impacted morselized allografts was not promoted by OP-1. Fibrous tissue ingrowth into the graft decreased with an increase in OP-1 or carrier concentration. A boxplot shows the histomorphometric results of the allograft experiment including the ingrowth distances of fibrous tissue and bone for each treatment group. The OP-1 device doses included: (1) 0.83 μg OP-1 combined with 0.24 mg collagen carrier per implant; (2) 2.5 μg OP-1 combined with 0.72 mg collagen carrier per implant; and (3) 25 μg OP-1 combined with 7.2 mg collagen carrier per implant. The collagen carrier doses included: (1) 0.24 mg collagen carrier per implant; and (3) 7.2 mg collagen carrier per implant.
Fig 5
Fig 5:
The incorporation process of impacted TCP/HA granules was not promoted by OP-1. Fibrous tissue ingrowth into the granules decreased with an increase in OP-1 concentration. A boxplot shows the histomorphometric results of the TCP/HA experiment including the ingrowth distances of fibrous tissue and bone for each treatment group. The OP-1 device doses included: (1) 0.83 μg OP-1 combined with 0.24 mg collagen carrier per implant; (2) 2.5 μg OP-1 combined with 0.72 mg collagen carrier per implant; and (3) 25 μg OP-1 combined with 7.2 mg collagen carrier per implant.
Number of Bone Chambers Showing Bone Formation after 4 Weeks

Bone formation was not preceded by an initial process of accelerated resorption. At the ingrowth front mainly osteoclasts were present as indicated by the TRAP staining. Thus, osteoclasts invaded the grafts clearly ahead of bone ingrowth. After the passage of the ingrowth front, resorbing cells disappeared from the stroma. Multinucleated giant cells were seen only occasionally, close to the resorption front, in the fibrous tissue. In the OP-1 and collagen carrier groups, the osteoclasts were aligning the sharp defined ingrowth front, where in the nonOP-1 groups (controls) the osteoclasts were distributed over the more loosely organized ingrowth front (Fig 6). However, the number of osteoclasts per bone-resorbing surface seemed similar. In most cases, allografts totally resorbed behind the ingrowth front. In some sparse cases, regardless of group or OP-1 dose, allograft was not totally resorbed and new bone was deposited on the graft remnants. The TCP/HA granules showed no signs of resorption in the form of presence of resorption pits. However, TCP/HA granules/particles were surrounded by TRAP-positive cells (osteoclasts and multinucleated giant cells) (Fig 6). In addition, very small TCP/HA particles (< 1 mm) were engulfed in mononuclear macrophage-like cells. At the level of the ingrowth front, we found resorption of the collagen particles as indicated by the presence of osteoclasts adjacent to the particles. The collagen particles did not seem to behave different than the bone grafts.

Fig 6A
Fig 6A:
F. (A,C,E) Typical examples of bone chamber content after 4 weeks are shown. (B,D,F) The details show the presence and position of TRAP-positive cells. (A) Loosely organized fibrous tissue (FT) can be seen penetrating the graft material (GR) in the allograft groups. (B) Osteoclasts (arrows) were distributed over the loosely organized ingrowth front. (C) In contrast, a sharply defined ingrowth front was shown in the OP-1 and carrier groups in allograft and TCP/HA groups. (D) Osteoclasts (arrows) aligned the sharply defined ingrowth front. (E) Loosely organized fibrous tissue penetrated the TCP/HA control group granules (TCP). (F) The TCP/HA granules (TCP) were surrounded by TRAP-positive cells (arrows) (Stain, hematoxylin and eosin; original magnification, x12.5 [A,C,E]; Stain, TRAP; original magnification, x100 [B,D,F]).

We observed a dose-related remodeling response to OP-1. An increase in OP-1 dose resulted in an inhibition of fibrous tissue formation. Fibrous tissue penetration into the graft decreased with an increase in OP-1 or carrier concentration. Depending on the OP-1 dose tested, more or less collagen carrier particles were entrapped in the spaces between the impacted grafts or TCP/HA particles (Fig 7). Fibrous tissue penetration into the graft seemed to be the highest in the allograft and TCP/HA groups without OP-1 added. This was confirmed by our histomorphometric data. The TCP/HA with the high-dose OP-1 (25 μg) showed less (p < 0.001) fibrous tissue ingrowth compared with the TCP/HA with the medium-dose OP-1 (2.5 μg). The TCP/HA with the medium-dose OP-1 showed less (p = 0.003) fibrous tissue ingrowth than the TCP/HA with the low-dose OP-1 (0.833 μg). The allograft, TCP/HA, and TCP/HA with the low-dose OP-1 (0.833 μg) showed similar fibrous tissue ingrowth (Table 3; Fig 4). Similar results were found in the allograft experiment. Fibrous tissue ingrowth in the allograft with medium-dose OP-1 did not differ from the allograft with low-dose OP-1 (p = 0.176), whereas the allograft with the high-dose OP-1 showed less (p < 0.001) fibrous tissue ingrowth compared with the allograft with low-dose OP-1. The OP-1 carrier groups and their OP-1-supplemented counterparts were similar, as was the fibrous tissue ingrowth between allo- graft, allograft with low- and medium-dose OP-1, and allograft with low-dose collagen carrier (Table 3; Fig 5). We observed no differences in bone and fibrous tissue in- growth for allograft and TCP/HA with equal OP-1 doses.

Fig 7A
Fig 7A:
B. A dose-dependent response to OP-1 on the incorporation process was found. Depending on the OP-1 dose tested, more or less collagen carrier was entrapped in the spaces between the impacted graft. Type I collagen carrier particles (CC) are shown trapped between the impacted allo- grafts. Typical examples of (A) low-dose OP-1 and (B) high- dose OP-1 are shown (Stain, Goldner-Masson trichrome; original magnification, x50).
Histomorphometric Results


The addition of OP-1 to allograft bone may hasten incorporation and remodeling, including an initial process of accelerated resorption. Accelerated resorption before the formation of bone may have negative consequences for bone impaction grafting because this may compromise implant fixation. An osteoconductive material, resistant to resorption and able to provide initial stability after reconstruction combined with growth factors would be a solution. We investigated the early effect of an OP-1 device on the incorporation process of impacted morselized allo- grafts and TCP/HA. Because BMP-7 is combined with a collagen carrier in the OP-1 device, we also studied the effects of the carrier on the incorporation process.

The effect of growth factors on bone conductive materials usually is seen at an early stage during bone ingrowth. It is difficult to find the right time to measure these effects, if new ingrown bone rapidly fills the defect. Therefore, we used the bone conduction chamber, a bone chamber model that is never completely filled with ingrown bone.4,46,50 The final amount of ingrown bone in the chambers can be used to measure the effects of growth factors on bone formation. Extrapolation of results of studies with bone conduction chambers to clinical recommendations should be made carefully, because factors like vascularity and composition of the host bone bed, its interaction with the bone graft, loading conditions, and surrounding tissue are not included in this study. However, the chamber with its limitations has been used extensively, therefore we think it is valid for detecting the effects of bone substitutes and signaling molecules that arise under unloaded conditions. It has been suggested that the BCC can be regarded as a bone tissue culture in vivo.43,54 In our opinion, the BCC should be interpreted at that level in the hierarchy of experimental models.

Osteogenic proteins require a viable cell source, vascularity, and mechanical stability to induce bone formation and remodeling.12 In several studies with positive results from BMP-2 and BMP-7, considerable surgical trauma was present before the introduction of the BMP.10,11,13,16,37,39 Trauma such as a fracture or an osteotomy will release endogenous factors that activate migration of inflammatory cells and cells of mesenchymal origin, which can respond to the applied BMP and stimulate bone formation.17 Our approach involved minimal surgical trauma which may explain the absence of this effect. This explanation also was suggested by Jeppsson and Aspenberg25 and Franke Stenport et al,15 in similar studies using titanium implants. In addition, Bostrom et al,8 showed trauma (micromotion) switched the response to BMP-2 from inhibition toward stimulation of bone formation.

The migration of cells into the impacted allograft is compromised and vascularization is delayed for several weeks.41 This delayed ingrowth after impaction may be related to physical factors, such as the size and numbers of pores for tissue intrusion into the material.44 The available volume for tissue ingrowth might have been decreased by the impaction of TCP/HA particles, creating small TCP/HA particles between the larger ones.36 Furthermore, by using higher doses of the OP-1 device, more collagen carrier material was impacted between the graft material, also filling up the space and thereby delaying tissue in- growth and remodeling.

If the collagen carrier needs extensive exposure to macrophages and other cells to release the active substance in a proper way,15 the compromised migration of cells into the impacted allograft and the delayed vascularization might have delayed the release of OP-1 from the carrier. This might explain the lack of difference in bone and tissue ingrowth between the bovine Type I collagen carrier groups without OP-1 and their OP-1 added counterparts. An alternative explanation could be that a considerable amount of OP-1 was released from the carrier by the process of impaction. The amount of early release may be dependent on the carrier system used.48 This makes it interesting to compare the results of this study with results of studies in which OP-1 was combined with graft without a carrier system. In a similar bone chamber model in rats, using a dose similar to our lowest dose without a collagen carrier (OP-1 solution) in impaction grafting, Tagil et al46 found an increase in bone formation. In contrast, studies using an OP-1 device (with collagen carrier) in combination with impaction grafting showed no or a very moderate effect on bone ingrowth.29,33 Moreover, using an OP-1 solution in a weightbearing rabbit knee impaction grafting model, Tagil et al47 showed no augmentation of morselized impacted bone graft. In addition, the influence of OP-1 on impacted allograft implants recently was investigated experimentally in loaded, primary, and impaired revision settings.42 Under primary conditions, using OP-1 solution decreased mean implant fixation; in contrast, under the impaired healing conditions of the revision setting, OP-1 increased incorporation and fixation of the implants. In addition to the dose and carrier system, setting and/or application seems to be important.

In the impaction grafting setting, grafts also seem able to withstand the forces acting on them during revascularization and remodeling. Histologic studies show that full replacement of the grafts by new bone does not always take place.9,22,51 Instead, a mixed pattern of living bone and areas of dead graft in a fibrous tissue stroma often is seen. The addition of ingrowing fibrous tissue, with collagen fibers winding between the graft chips might lead to a higher resistance to shear and affect the rate of prosthetic migration. It seems the delayed or reduced new bone ingrowth seen in experiments with impaction therefore, is less important and even may be beneficial, as long as the graft/fibrous tissue composite remains strong enough to withstand forces acting on it during remodeling.45 In our case, using an OP-1 device also delayed the fibrous tissue ingrowth. Our data suggest the observed early failures in impaction grafting in combination with high doses of OP-1 device could be attributable to the lack of reinforcement by fibrous tissue and not necessarily because of an increase in resorption and remodeling.


We thank Leon Driessen for technical assistance.


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