The Journal of Bone & Joint Surgery

The Journal of Bone & Joint Surgery, Volume 89, Issue 10

Scientific Articles | October 01, 2007

The Effect of Osteoclastic Activity on Tendon-to-Bone Healing: An Experimental Study in Rabbits

Scott A. Rodeo, MD¹; Sumito Kawamura, MD¹; C. Benjamin Ma, MD¹; Xiang-hua Deng, MD¹; Patrick S. Sussman, MD¹; Peyton Hays, BS¹; Liang Ying, BS¹

¹ The Hospital for Special Surgery, 535 East 70th Street, New York, NY 10021. E-mail address for S.A. Rodeo: rodeos@hss.edu

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Disclosure: In support of their research for or preparation of this work, one or more of the authors received, in any one year, outside funding or grants in excess of $10,000 from the Orthopaedic Research and Education Foundation and the National Institutes of Health (a Pilot and Feasibility Grant as part of a NIH Core Centers grant [1P30AR46121-01]; in addition, this investigation was conducted in a facility constructed with support from Research Facilities Improvement Program Grant Number C06-RR12538-01 from the National Center for Research Resources, NIH). Neither they nor a member of their immediate families received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, division, center, clinical practice, or other charitable or nonprofit organization with which the authors, or a member of their immediate families, are affiliated or associated.

Investigation performed at The Laboratory for Soft Tissue Research, The Hospital for Special Surgery, New York, NY

The Journal of Bone and Joint Surgery, Incorporated

J Bone Joint Surg Am, 2007 Oct 01;89(10):2250-2259. doi: 10.2106/JBJS.F.00409

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Abstract

Background: Healing of a tendon graft in a bone tunnel depends on bone ingrowth into the interface between tendon and bone. Excessive osteoclastic activity may contribute to bone resorption, tunnel widening, and impaired healing. We hypothesized that inhibition of osteoclastic activity by osteoprotegerin (OPG) would increase bone formation around a tendon graft in anterior cruciate ligament reconstruction in a rabbit model, while increased osteoclastic activity due to the aplication of receptor activator of nuclear factor-kappa B ligand (RANKL) would impair bone ingrowth.

Methods: Sixty skeletally mature, male New Zealand White rabbits underwent bilateral anterior cruciate ligament reconstruction. OPG (100 µg per tunnel) or RANKL (10 µg per tunnel) was delivered to the tendon-bone interface with use of a synthetic calcium phosphate carrier vehicle. Twenty animals were killed at two, four, and eight weeks after surgery. Two rabbits from each group were prepared for histological evaluation, and the other rabbits were used for biomechanical testing.

Results: A significantly greater amount of bone surrounded the tendon at the healing tendon-bone interface in the OPG-treated limbs compared with the controls and the RANKL-treated limbs at all time-points (p < 0.05). There were significantly fewer osteoclasts in the OPG-treated limbs compared with the controls and the RANKL-treated limbs (p < 0.05). The average tunnel area in the OPG group was significantly smaller than that in the RANKL group (p = 0.003 at two weeks and p = 0.004 at four weeks). The femur-anterior cruciate ligament-tibia complex of the OPG-treated limbs had significantly increased stiffness compared with RANKL-treated limbs at eight weeks (p = 0.04).

Conclusions: Osteoprotegerin significantly improves bone formation around the grafted tendon and improves the stiffness at the healing tendon-bone junction in a rabbit model.

Clinical Relevance: Inhibition of excessive osteoclastic activity may improve tendon-to-bone healing.

Figures in this Article

It is well established that injury to the anterior cruciate ligament results in knee instability and functional disability. Furthermore, in a study of 100 knees, articular cartilage damage was reported in association with 23% of forty-four knees with an acute anterior cruciate ligament injury and in 54% of fifty-six knees with chronic laxity or instability of the anterior cruciate ligament¹. The inability to return to strenuous sporting activities has also been well documented². As a result, these injuries frequently require ligament reconstruction with use of a tendon graft.

Despite the generally good results of contemporary techniques of anterior cruciate ligament reconstruction, the biology of healing between the grafted tendon and bone is incompletely understood. It is well established that secure graft-to-bone integration is crucial for successful anterior cruciate ligament reconstruction. Experimental studies have found that tendon-to-bone healing proceeds by formation of a fibrovascular tissue in the healing tendon-bone interface. This is followed by bone ingrowth into the tendon-bone interface^3-6. Studies from our laboratory have found that bone ingrowth plays an important role in graft-to-bone fixation^3,5,6. Because healing between tendon and bone is a slow process, necessitating a delay in weight-bearing, range of motion, and resumption of activities, there is much interest in determining methods to accelerate healing. For example, bone-growth factors such as bone morphogenetic protein (BMP) have been found to augment bone ingrowth into tendon in several studies^5-7.

A common clinical concern following anterior cruciate ligament reconstruction is expansion of the bone tunnels as seen radiographically^8-10. The etiology and clinical relevance of this phenomenon remain unclear. Possible etiologies for tunnel widening include synovial fluid cytokines and inflammatory mediators, graft-tunnel micromotion, and an immune response to allograft tissue^11-13. The final common pathway is likely to be osteoclast-mediated bone resorption. Other potential causes, such as bone resorption due to mechanical abrasion or bone loss due to necrosis, have not been found or suspected as the etiology of tunnel widening. In clinical practice, bone loss due to tunnel expansion may compromise graft fixation to bone and can complicate revision surgery. A recent study has demonstrated that increased knee laxity was associated with radiographic evidence of femoral tunnel widening in anterior cruciate ligament reconstructions with use of a hamstring tendon graft¹⁴.

Osteoclast-mediated bone resorption is seen in many disorders, including postmenopausal osteoporosis¹⁵, juvenile Paget disease¹⁶, and rheumatoid arthritis¹⁷. Recent insights into osteoclast differentiation and activation are provided by the analysis of a family of biologically related tumor necrosis factor receptor (TNFR) and/or TNF-like proteins: osteoprotegerin (OPG), receptor activator of nuclear factor-kappa B (RANK), and RANK ligand (RANKL), which together regulate osteoclast function^18-20. RANKL is the main stimulatory factor for the formation of mature osteoclasts. The effects of RANKL are counteracted by OPG, which acts as a soluble receptor antagonist. Balanced activity of the OPG-RANKL-RANK system regulates bone resorption¹⁸. The observations that mutations in the genes encoding RANK and OPG cause bone diseases in humans^16,21 suggest that inhibition of RANKL signaling may be a viable therapeutic strategy to prevent excessive bone resorption. For example, OPG gene therapy was found to effectively inhibit wear debris-induced osteoclastogenesis and osteolysis in a mouse calvarial model²². The Fc-OPG fusion molecules have demonstrated long-acting suppression of bone-turnover markers with a good safety profile²³.

On the basis of the role of the OPG-RANKL-RANK system in bone metabolism, we tested the hypothesis that OPG-induced inhibition of osteoclastic activity would increase bone interdigitation at the healing tendon-bone junction. Our corollary hypothesis was that increased osteoclastic activity due to exogenous RANKL would impair bone ingrowth into the grafted tendon.

Materials and Methods

Materials and Methods | Results | Discussion | Acknowledgements | References

Sixty skeletally mature, male New Zealand White rabbits were used to evaluate the effect of OPG and RANKL (Amgen, Thousand Oaks, California) on thehealing of a tendon graft in a bone tunnel with use of an established model of anterior cruciate ligament reconstruction⁴. The rabbits were obtained from a licensed United States Department of Agriculture dealer and were housed in the facility for the care of laboratory animals at our institution, in accordance with the standards established by the National Institutes of Health for the care and use of laboratory animals. This study was approved by the Institutional Animal Care and Use Committee.

Experimental Design

Recombinant mouse osteoprotegerin (OPG)-Fc and recombinant soluble human RANKL were provided by Amgen. Mouse OPG was fused with the Fc portion of human IgG1 (immunoglobulin isotype) by means of a polypeptide linker. Synthetic calcium phosphate matrix (ETEX, Cambridge, Massachusetts) containing bicarbonate was used as a carrier material for both OPG and RANKL. This is a synthetic bone substitute material that has a chemical composition and crystalline structure essentially identical to the calcium phosphate component of natural bone²⁴. OPG and RANKL were dissolved in phosphate-buffered saline solution (pH 7.3) and then mixed with calcium phosphate matrix. The final concentration of these agents was adjusted to 2 mg/mL (OPG) and 0.2 mg/mL (RANKL) paste (liquid-powder ratio = 0.64).

All animals underwent bilateral reconstruction of the anterior cruciate ligament. Each animal was assigned to one of three groups: control (twenty-four rabbits), OPG (100 µg per tunnel in eighteen rabbits), and RANKL (10 µg per tunnel in eighteen rabbits). The control group knees received calcium phosphate matrix (CPM⁺) in the left knee and tendon transplantation alone (CPM^—) in the right knee. We used these two controls to determine whether calcium phosphate matrix alone had an effect on healing. Animals in the OPG-treated and RANKL-treated groups received the experimental material in both knees. Twenty animals were killed at two, four, and eight weeks after surgery. Two rabbits from each group were prepared for histological evaluation, and the other rabbits were saved for biomechanical testing.

Surgical Procedure

The animals were anesthetized with use of ketamine hydrochloride (60 mg/kg) combined with acetylpromazine (0.5 mgkg) in a single syringe and were maintained on inhalation anesthesia (isoflurane, nitrous oxide, and oxygen). Ampicillin (25 mg/kg) was used for antibiotic prophylaxis. A medial parapatellar incision was used to expose the knee. The semitendinosus tendon was identified and transected at its musculotendinous junction⁴. The native anterior cruciate ligament was resected, and then tunnels (2.4 mm in diameter) were drilled in the lateral femoral condyle and the medial aspect of the tibia, entering the joint at the anatomic origin and insertion of the native anterior cruciate ligament. The graft was routed through the tunnels. We then used an 18-gauge spinal needle to inject 50 µL of the calcium phosphates matrix (prepared as previously described) to the interface between the grafted tendon and the bone tunnel (Fig. 1). The trocar of the spinal needle was used to push the material out of the needle to ensure that all of the material was delivered into the tunnel. The right knee of the control animals did not receive any calcium phosphate matrix (CPM^— limbs). Care was taken to distribute the calcium phosphate matrix evenly throughout the tendon-bone interface. After the injection, the ends of the graft were sutured under tension to the soft tissues at the tunnel exits (the fibular collateral ligament outside the femoral tunnel and the medial collateral ligament outside the tibial tunnel) with use of 4-0 Ethibond suture (Ethicon, Somerville, New Jersey). The retinacular incision and wound were closed in layers in a standard fashion. The limbs were not immobilized postoperatively, and the rabbits were allowed normal activity in individual cages. The animals were assessed twice per day for signs of pain, and they were given 0.01 to 0.05 mg/kg of Buprenex (buprenorphine) intramuscularly up to twice per day for postoperative analgesia. The rabbits typically required analgesia for three days postoperatively. The animals were killed at two, four, and eight weeks following surgery by a dose of barbiturates, administered intravenously.

Histology and Histomorphometry

At the time that the animals were killed, each specimen was grossly examined for evidence of cartilage degeneration, synovial inflammation, and effusion. The entire joint, including the femoral and tibial bone tunnels, was harvested. The tissues were fixed in 10% neutral buffered formalin for three days at 4°C and then were decalcified in Immunocal (Decal Chemical, Congers, New York) for two days at 4°C. The decalcified specimens were dehydrated with ethanol and embedded in paraffin. Sections were made from the middle of both the femoral and tibial tunnels by cutting the length of the tunnel in half (tunnel cross section) and then embedding each half. Care was taken to ensure that the tissue was embedded so that the sections were exactly perpendicular to the tunnel. The sections were then made from the cut surface, in order to examine the middle of the length of the tunnel. Consecutive 5-µm-thick sections were cut perpendicular to the bone tunnel and were stained with hematoxylin-eosin and Alcian blue for routine histological evaluation. Tartrate-resistant acid phosphatase (TRAP) histochemical staining was used to localize osteoclasts. Histomorphometric measurements were made of the width of new bone around the grafted tendon. Histological cross sections of the tendon in the bone tunnel were viewed on a light microscope (Nikon Optiphot; Nikon USA, Melville, New York) at eighty times magnification. These histological sections were then scanned into a desktop computer with use of a charge-coupled device video camera system (Nikon DXM1200; Nikon USA). A computerized image-analysis system (ImageJ software; National Institutes of Health, Bethesda, Maryland) was then used to directly measure the width of the bone around the grafted tendon. The width of bone was measured at a minimum of nine sites around the circumference of the bone tunnel. The number of osteoclasts per millimeter of the bone tunnel was counted on the TRAP-stained slides. An osteoclast was defined as a multinucleated cell (>3 nuclei) that was TRAP-positive and adjacent to a resorbed bone surface²⁵.

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Fig. 1: Surgical transplantation of the tendon graft into the bone tunnel. The graft was routed through 2.4-mm-diameter bone tunnels. The drawing shows the calcium phosphate matrix (CPM) delivered through an 18-gauge spinal needle. The experimental limbs (except the CPM^— group) received 50 µL of the calcium phosphate matrix (containing OPG, RANKL, or no agent) applied to the interface between the grafted tendon and bone tunnel. LCL = lateral collateral ligament, and MCL = medial collateral ligament.

Biomechanical Testing

The limbs that were used for biomechanical analysis were frozen at —80°C until testing. At the time of biomechanical testing, the limbs were thawed and all soft tissue except the grafted tendon was removed. All scar tissue and sutures at the tunnel exits were carefully removed. The femur-anterior cruciate ligament graft-tibia complexes were fixed in specially designed clamps allowing tensile loading along the long axis of the graft in a materials testing machine. A preload of 1 N was applied for sixty seconds. Following cyclic preconditioning of the constructs between elongation limits of 0 and 0.75 mm at a rate of 10 mm/min, a load-to-failure test was performed at an elongation rate of 10 mm/min. Stiffness (N/mm) was calculated from the slope of the linear region of the load elongation curve between 1.5 and 2 mm of elongation.

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Fig. 2-A: New bone formation around the grafted tendon. Representative photomicrographs from the CPM⁺ limbs (upper row), OPG-treated limbs (middle row), and RANKL-treated limbs (lower row) at two, four, and eight weeks after surgery (hematoxylin and eosin, ×80). Bar = 100 µm. T = tendon, B = bone, IF = interface tissue, and NB = new bone.

Radiographic Evaluation

After biomechanical testing, 3-mm-thick cross sections were cut from the middle of the tunnels, and high-resolution radiographs were made at 26 kVp/10-sec exposure with use of the Faxitron Specimen Radiography System (model MX-20; Faxitron X-Ray, Wheeling, Illinois). Computerized image analysis (ImageJ software; National Institutes of Health) was used to measure the bone tunnel area and the area of newly formed bone around the grafted tendon. Although microscopic damage to the bone tunnel could have occurred from the biomechanical testing, no such damage was evident by gross inspection of the cut specimens. Furthermore, on careful inspection, no bone was found on the edge of the tendons after pullout from the tunnel.

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Fig. 2-B: There were no significant differences between the control groups (CPM^— compared with CPM⁺) in terms of the volume of bone surrounding the tendon graft; however, the OPG-treated limbs exhibited a significantly greater volume of bone surrounding the tendon graft compared with the controls (p = 0.004 at two weeks and p = 0.033 at eight weeks) and the RANKL-treated limbs (p = 0.02 at two weeks, p = 0.002 at four weeks, and p = 0.001 at eight weeks).

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Fig. 3-A: The number of osteoclasts per millimeter along the edge of the bone tunnel was compared among the groups with use of tartrate-resistant acid phosphatase (TRAP)-stained sections. Osteoclasts were defined as multinucleated cells (>3 nuclei) that were TRAP-positive and adjacent to a resorbed bone surface. Representative photomicrographs from the CPM⁺ limbs (upper row), OPG-treated limbs (middle row), and RANKL-treated limbs (lower row) at two, four, and eight weeks after surgery (×80). Bar = 100 µm. T = tendon, B = bone, and IF = interface tissue.

Statistical Methods

We compared the histomorphometric, radiographic, and biomechanical measures between the control, OPG-implanted, and RANKL-implanted limbs. We first examined the tibial and femoral tunnel data separately and found no differences between the two tunnels. Thus, the data for the tibial and femoral tunnels were combined for further analysis. Each limb was counted as an independent measurement for the biomechanical analyses. Since the histomorphometric data were not normally distributed, a Mann-Whitney test was used to compare the measurements of the width of the new bone and the number of osteoclasts between the control group and the experimental groups, and a Kruskal-Wallis one-way analysis of variance on ranks was used to compare the width of new bone and the number of osteoclasts between different time-points. For the biomechanical data, a two-way analysis of variance was performed and a post hoc test for multiple comparisons between the three groups was conducted with the Fisher protected least-significant-difference method. The bone tunnel area (measured radiographically) was correlated with ultimate load-to-failure for all specimens that failed by pullout from the femoral tunnel and for the specimens that failed by pullout from the tibial tunnel, with use of a Pearson product correlation. All data are presented as the mean and the standard deviation, unless otherwise noted. The differences were considered significant at p < 0.05 unless otherwise noted.

Results

Materials and Methods | Results | Discussion | Acknowledgements | References

New Bone Formation Around the Grafted Tendon

On the basis of both gross and histological inspection, we could not identify any remaining calcium phosphate matrix carrier material by two weeks in any group. There were no significant differences between the control groups (CPM^— compared with CPM⁺) in the histomorphometric measurements throughout the study; thus, we report only the comparisons to the CPM⁺ control group. Healing occurred by progressive new matrix formation and bone ingrowth, leading to the establishment of collagen fiber continuity between the tendon and bone (Fig. 2-A). The OPG-treated limbs had a significantly greater amount of bone surrounding the tendon graft compared with RANKL-treated limbs at all time-points (p = 0.02 at two weeks, p = 0.002 at four weeks, and p = 0.001 at eight weeks) (Fig. 2-B). There were significant differences between OPG-treated limbs and the CPM⁺ group at two weeks and eight weeks after surgery (p = 0.004 and p = 0.033, respectively). There were no significant differences in the amount of bone surrounding the tendon graft between the RANKL-treated limbs and the controls at two weeks and eight weeks. There were no significant differences in the width of bone around the tendon graft between each time-point for both the OPG-treated limbs and the RANKL-treated limbs.

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Fig. 3-B: The graph depicts the number of osteoclasts along the edge of the bone tunnel at each time-point. The OPG-treated limbs had significantly fewer osteoclasts compared with the RANKL-treated limbs at two weeks (p = 0.025) and four weeks (p = 0.01). The OPG-treated limbs also had significantly fewer osteoclasts compared with CPM⁺ group at two weeks (p = 0.014) and four weeks after surgery (p = 0.02). The RANKL-treated limbs had a significantly greater number of osteoclasts compared with the CPM⁺ control group at two weeks after surgery (p = 0.034). A single asterisk indicates a significant difference between the RANKL-treated limbs at two and eight weeks (p = 0.001). Double asterisks indicate a significant difference between RANKL-treated limbs at four and eight weeks (p < 0.001).

Osteoclasts at the Tendon-Bone Interface

No significant difference in osteoclast numbers was detected between the CPM^— and CPM⁺ control groups through the study period; thus, we report only the comparisons with the CPM⁺ control group. The number of osteoclasts around the bone tunnel in the control groups decreased significantly between four weeks and eight weeks (p = 0.015) (Figs. 3-A and 3-B). The OPG-treated limbs had significantly fewer osteoclasts compared with the RANKL-treated group at two weeks (p = 0.025) and four weeks (p = 0.01). Similarly, the OPG-treated limbs had significantly fewer osteoclasts compared with the CPM⁺ groups at two weeks (p = 0.014) and four weeks after surgery (p = 0.02). The RANKL-treated limbs had a significantly greater number of osteoclasts compared with the CPM⁺ control group at two weeks after surgery (p = 0.034). The RANKL-treated limbs had significantly fewer osteoclasts at eight weeks after surgery compared with two weeks (p = 0.001) and four weeks (p < 0.001).

Biomechanical Testing

There were no significant differences among the groups in the ultimate load-to-failure during the study period (Fig. 4-A). All of the specimens from the experimental and control groups failed by tendon pullout from the bone tunnel at two weeks. However, at eight weeks, all of the OPG-treated limbs failed by rupture in the midsubstance of the intra-articular tendon, while the RANKL-treated limbs and control limbs still failed by tendon pullout from the bone tunnel. Therefore, the actual fixation strength of the tendon in the tunnel was likely higher than measured for the limbs in which failure occurred outside the tunnel. The stiffness was significantly higher in the OPG group (21.7 ± 7.5 N/mm) compared with the controls (10.6 ± 3.7 N/mm; p = 0.017) and the RANKL group (10.7 ± 6.3 Nmm; p = 0.038) at eight weeks (Fig. 4-B). A post hoc power analysis found that we would need twenty-nine specimens per group to demonstrate a significant difference in load to failure because of the large standard deviations.

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Fig. 4-A: There was no significant difference in ultimate load-to-failure among the groups.

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Fig. 4-B: The stiffness in the OPG group (21.7 ± 7.5 N/mm) was significantly higher than that in the control group (10.6 ± 3.7 N/mm) and the RANKL group (10.7 ± 6.3 N/mm) at eight weeks. A single asterisk indicates a significant difference between the OPG and the control groups (p = 0.017). Double asterisks indicate a significant difference between the OPG and RANKL groups (p = 0.038).

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Fig. 5-A: The bone tunnel area was measured on high-resolution radiographs of 3-mm-thick sections taken from the middle of the bone tunnel. Representative photomicrographs from the CPM⁺ limbs (upper row), OPG-treated limbs (middle row), and RANKL-treated limbs (lower row) at two, four, and eight weeks after surgery.

Radiographic Evaluation

The margins of the original tunnel and newly formed bone were easily discerned on the high-resolution radiographs. The margins of the original tunnel were sclerotic, while newly formed bone had a fine trabecular pattern to distinguish it from the edge of the tunnel. The RANKL-treated limbs had increased bone tunnel area compared with the original bone tunnel area (4.52 mm²) at two weeks after surgery (Figs. 5-A and 5-B). The average bone tunnel area in the OPG group was significantly smaller than that in the RANKL groups at two weeks (p = 0.003). A significant difference between the OPG-treated limbs and the RANKL-treated limbs continued to be evident at four weeks (p = 0.004); however, there was no significant difference between the OPG-treated limbs and the RANKL-treated limbs at eight weeks (p = 0.067). No significant difference was detected between the OPG-treated limbs and the controls at four weeks. There was no significant difference between the CPM⁺ control group and the OPG-treated limbs at eight weeks (p = 0.24).

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Fig. 5-B: The graph depicts the bone tunnel areas measured on high-resolution radiographs. The average bone tunnel area of the OPG group was significantly smaller than that of the RANKL groups at two weeks (p = 0.003). A significant difference continued to be seen between the OPG-treated limbs and the RANKL-treated limbs at four weeks (p = 0.004).

The bone tunnel area was correlated with ultimate load-to-failure for all specimens that failed by pullout from the femoral and tibial tunnels (combining animals from all groups), with use of a Pearson correlation coefficient. There was a modest inverse correlation between failure load and femoral bone tunnel area for femoral tunnel failures (r² = 0.33), but no correlation was detected between failure load and tibial bone tunnel area for tibial tunnel failures (r² = 0.0008). Because of the insufficient sample size, the power to detect a correlation between tibial tunnel area and failure load was only 5%.

Discussion

Materials and Methods | Results | Discussion | Acknowledgements | References

Despite generally favorable results with anterior cruciate ligament reconstruction, instrumented testing of knee stability demonstrates frequent increased anterior laxity, which may lead to symptomatic instability²⁶. The site of the attachment of the anterior cruciate ligament tendon graft to bone is the weak link in the early healing period, necessitating a delay in the return to functional activities. Studies have found increases in joint laxity over time^27-29. Although many factors determine the outcome of anterior cruciate ligament reconstruction, including the graft fixation site, initial graft tension, and graft material, one of the most important factors is graft healing to bone. Failure of secure healing of the tendon in the bone tunnel prior to the return to activity can result in graft slippage in the tunnel with resultant increased knee laxity. Several studies have demonstrated the inability of current reconstruction techniques to restore normal knee joint homeostasis and function. We believe this inability to restore normal knee function may be related to poor tendon-to-bone healing. As a result, there is great interest in discovering methods to improve tendon-to-bone healing.

Previous studies have shown improved healing between tendons and bone due to treatment with osteoinductive agents, such as rhBMP-2^5,6. However, to our knowledge, no previous study has examined the role of osteoclastic bone resorption in tendon-to-bone healing both histologically and biomechanically. (A portion of the histological data from our investigation was previously reported in another study³⁰.) In the current study, we report a significantly greater amount of bone surrounding the tendon graft in the OPG-treated limbs. OPG delivery to the tendon-bone interface significantly decreased the number of osteoclasts along the edge of the bone tunnel. The role of osteoclasts was further demonstrated by the finding of less bone surrounding the tendon graft in the limbs treated with RANKL, an osteoclastogenic factor, compared with the limbs treated with OPG. The lack of significant difference in bone formation in the RANKL group compared with controls suggests that RANKL alone is not sufficient to cause significant osteoclast activation. Osteoclast differentiation and activation is clearly a complex process and likely requires expression of multiple cellular and molecular signals. For example, granulocyte-macrophage colony-stimulating factor (GM-CSF) is also required for osteoclast stimulation and maturation in combination with RANKL³¹. In vivo models of osteoclastogenesis have demonstrated that RANKL-induced osteoclast stimulation is mediated by means of expression of the GM-CSF gene³². These studies from our laboratory are the first, as far as we know, to examine the role of antiresorptive agents in tendon-to-bone healing.

Osteoclast-mediated bone resorption may impair tendon-to-bone healing. In a recent study, we measured the relative motion between the graft and the bone tunnel (i.e., the graft-tunnel motion) in anterior cruciate ligament reconstruction in a rabbit model and related those measurements to bone ingrowth into the graft and to osteoclast numbers³³. We found that osteoclasts were most numerous in the area with the greatest graft-tunnel motion, suggesting that micromotion of the graft may trigger osteoclastic resorption. Graft-to-bone healing was slowest in the areas with greater graft-tunnel motion and greater osteoclast accumulation. We postulated that graft-tunnel micromotion activates osteoclast development and accumulation by means of the activation of the RANK-RANKL system. The findings from these studies are relevant to the clinical problem of widening of the bone tunnel, which may compromise graft fixation to bone and can complicate revision surgery. It is likely that tunnel widening is due to osteoclastic bone resorption, which is supported by our finding of wider tunnels in the RANKL-treated limbs. Of note, we found a weak inverse correlation between ultimate load-to-failure and femoral bone tunnel area for specimens that pulled out of the femoral tunnel during biomechanical testing. Since bone tunnel expansion is seen in the first three to six months following anterior cruciate ligament reconstruction, application of an antiresorptive agent such as OPG in the early healing phase may diminish tunnel expansion and improve bone formation around the tendon graft.

In this study, we utilized a synthetic calcium phosphate carrier to deliver OPG and RANKL to the tendon-bone interface. Although we did not examine the pharmacokinetics of OPG and RANKL release from this calcium phosphate carrier, this material has been used to deliver rhBMP-2 in several different animal models (rabbit ulnar osteotomy and monkey fibular osteotomy) and has been reported to slowly release rhBMP-2 for up to three weeks²⁴. Release of the OPG may have been slower than RANKL release since the calcium phosphate carrier requires osteoclast resorption to release the drug. Further study would be necessary to determine the kinetics of drug release from the calcium phosphate matrix carrier, especially in the setting of OPG-induced osteoclast inhibition.

Although our studies suggest that modulation of osteoclastic activity may improve tendon-to-bone healing, it is important to note that, in normal bone remodeling, osteoblastic bone formation follows osteoclastic bone resorption. Although the characteristics of this coupling phenomenon have not been determined completely in vivo and in vitro, recent data have shown that the elevated osteoblast function in OPG-deficient (OPG(^—/—) mice was sharply decreased after suppression of bone resorption by daily injection of risedronate, suggesting that bone formation is coupled with bone resorption at local sites³⁴. In the present study, we did not quantify the number of osteoblasts; however, we often found osteoblasts adjacent to osteoclasts (Figs. 3-A and 3-B). Prolonged inhibition of osteoclast-mediated resorption by administration of OPG may negatively affect osteoblastic function and ultimate bone formation around the grafted tendon.

The lack of improvement in attachment strength despite greater bone formation may be due to important structural and compositional factors. Osteoclasts play an important role in bone remodeling. Inhibition of osteoclast activity may negatively affect remodeling of the newly formed bone around the tendon graft, resulting in inadequate quality and/or organization of the newly formed bone. The newly formed bone may not yet have "anchored" the collagen fibers in the fibrous interface tissue. This bone is also likely to be incompletely remodeled and may not yet be fully mineralized. Clearly, the formation of new bone is only one part of the healing process. Integration with the interface tissue and outer tendon is required to improve the structural properties of the femur-anterior cruciate ligament-tibia complex. All of these factors could account for the lack of improvement in load-to-failure in this study. Further studies are required to examine the role of OPG-RANKL signaling in tendon-to-bone healing.

In conclusion, osteoclast inhibition resulted in a significantly greater volume of bone surrounding a tendon graft, with increased stiffness of the attachment site. Since tendon-to-bone healing requires new bone formation and bone ingrowth around a tendon graft, OPG treatment may improve biologic graft fixation to bone. Furthermore, these preliminary results suggest that activated RANK-RANKL signal transduction may contribute to tunnel expansion after anterior cruciate ligament reconstruction. Further study is needed to understand the role of bone resorption compared with bone formation in bone-to-tendon healing. Future investigations may examine the effect of other inhibitors of bone resorption and will be required to determine the optimal parameters for clinical application. ?

Acknowledgements

Materials and Methods | Results | Discussion | Acknowledgements | References

Note: The authors thank Dr. Colin Dunstan (Amgen Inc.) for providing the OPG and RANKL.

References

Materials and Methods | Results | Discussion | Acknowledgements | References

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Topics

calcium phosphates ; limb ; osteoclasts ; oryctolagus cuniculus ; tendon ; anterior cruciate ligament reconstruction ; trance protein ; tendon transplantation

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Scott A. Rodeo, Sumito Kawamura, C. Benjamin Ma, Xiang-hua Deng, Patrick S. Sussman, Peyton Hays, Liang Ying; The Effect of Osteoclastic Activity on Tendon-to-Bone Healing: An Experimental Study in Rabbits. The Journal of Bone & Joint Surgery. 2007 Oct;89(10):2250-2259.

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