A single systemic dose of pamidronate improves bone mineral content and accelerates restoration of strength in a rat model of fracture repair.

Complications in fracture repair that lead to a delay in union remain clinically problematic. We believe that unwanted pre-mature catabolism of the healing callus, for example, in stress shielded situations, diminishes the rate at which strength is restored in bone repair and possibly leads to delayed union. We hypothesized that a single systemic dose of a nitrogen-containing bisphosphonate (N-BP) would increase bone mineral content (BMC), volume, and mechanical strength of union in fracture repair. We also set out to investigate local delivery to assess whether systemic exposure could be eliminated, due to concerns of bisphosphonate dosing of non-target organs. After an open osteotomy fixed with a K wire, 40 12-week old Wistar male rats were divided into four groups of 10: saline control, bolus systemic subcutaneous injection of pamidronate (3 mg/kg), local low dose of pamidronate (0.1 mg), and a local high dose of pamidronate (1.0 mg). Rats were sacrificed 6 weeks post-operatively. Operated and non-operated femora underwent radiographic evaluation, quantitative computer tomography, and biomechanical testing in torsion. The growth plates and metaphyses of the tibia of the non-operated side were assessed for evidence of systemic exposure in the local groups. Significant increases in callus BMC and volume of the bolus systemic dose group were found compared to the saline control (p< or =0.05). Further, the strength of the systemic dose callus was increased by 60% from 0.35 Nm (+/-0.11) for the saline control callus to 0.56 Nm (+/-0.25) for the systemic group (p=0.05). Local treatment did not result in increased strength. The contralateral tibial growth plates of the local groups showed evidence of systemic exposure by the presence of retained primary spongiosa. This study confirms that a single perioperative systemic dose of pamidronate leads to significant increases in the BMC, volume, and strength of healing fractures in rats, making single dose N-BP therapy an appealing candidate for further examination in fracture repair.     

Bolus or weekly zoledronic acid administration does not delay endochondral fracture repair but weekly dosing enhances delays in hard callus remodeling

INTRODUCTION: It has been widely assumed that osteoclasts play a pivotal role during the entire process of fracture healing. Bisphosphonates (BPs) are anti-catabolic agents commonly used to treat metabolic bone diseases including osteoporosis, minimizing fracture incidence. Yet, fractures do occur in these patients and the potential for negative effects of BPs on healing has been suggested. We aimed to examine the effect of different dosing regimes of the potent BP zoledronic acid (ZA) on early endochondral fracture repair and later callus remodeling in a normal bone healing environment. METHODS: Saline, a Bolus dose of 0.1 degrees mg/kg ZA or 5 weekly divided doses of 0.02 degrees mg/kg of ZA commenced 1 week post operatively in a rat closed fracture model. Samples at 1, 2, 4 and 6 weeks post fracture were used to analyze initial fracture union, and 12 and 26 weeks post fracture to investigate the progress of remodeling. RESULTS: ZA did not alter the rate of endochondral fracture union. All fractures united by 6 weeks, with no difference in the progressive reduction of cartilaginous soft callus between control and treatment groups over time. ZA treatment increased hard callus bone mineral content (BMC), volume and increased callus strength at 6 and 26 weeks post fracture. Hard callus remodeling commenced at 4 weeks post fracture with Bolus ZA treatment but was delayed until after 6 weeks in the Weekly ZA group. By 12 and 26 weeks, Bolus ZA had equivalent callus content of remodeled neo-cortical bone to the Saline controls, whereas Weekly ZA remained reduced compared to Saline controls at these times (P<0.01). Callus material properties such as peak stress were significantly reduced in both ZA groups at 6 weeks. At 26 weeks, Bolus ZA-treated calluses generated peak stress equivalent to control values, whereas Weekly ZA callus peak stress remained significantly reduced, indicating remodeling delay. CONCLUSIONS: Osteoclast inhibition with ZA does not delay endochondral fracture repair in healthy rats. Bolus ZA treatment increased net callus size and strength at 6 weeks while allowing hard callus remodeling to proceed in the long term, albeit more slowly than control. Prolonged bisphosphonate dosing during repair does not delay endochondral ossification but can significantly affect remodeling long after the drug is ceased.     

Manipulation of the anabolic and catabolic responses with OP-1 and zoledronic acid in a rat critical defect model.

Bone repair involves both anabolic and catabolic responses. We hypothesized that anabolic treatment with OP-1 (BMP-7) and anti-catabolic treatment with zoledronic acid could be synergistic. In a rat critical defect, this combination therapy produced significant increases in new bone volume and strength. INTRODUCTION: When used to augment bone healing, osteogenic protein 1 (OP-1/BMP-7) and other BMPs stimulate the anabolic response, inducing osteoblast recruitment, differentiation, and bone production. However, BMPs can also upregulate catabolism by direct stimulation of osteoclasts and indirectly by osteoblasts through RANKL/RANK. We hypothesized that if such osteoclastic upregulation were modulated by zoledronic acid (ZA), the combination of OP-1 and ZA should produce increased new bone over OP-1 alone. MATERIALS AND METHODS: Rats with a surgically induced 6-mm femoral critical size defect were separated into five dosing groups: Carrier, Carrier + ZA, OP-1, OP-1 + ZA, and OP-1 + ZA administered 2 weeks after surgery (2W). Carrier +/- 50 microg OP-1 was placed in the defect, and 0.1 mg/kg ZA or saline was administered subcutaneously. Bone repair was analyzed by radiographs, QCT, mechanical testing, histology, and histomorphometry. RESULTS: Carrier alone and Carrier ZA groups did not unite by 8 weeks. Radiological union occurred in all OP-1 groups but was tenuous in some animals treated with OP-1 alone. BMC was increased by 45% in the OP-1 ZA group and 96% in the OP-1 ZA 2W group over OP-1 alone (p < 0.01). Callus volume increased over OP-1 alone by 45% and 86% in the OP-1 ZA and OP-1 ZA 2W groups, respectively (p < 0.01). The increased callus volume in the OP-1 ZA 2W group translated to increases in strength of 107% and stiffness of 148% (p < 0.05). BFR was not significantly different between OP-1 groups regardless of ZA treatment. CONCLUSIONS: ZA treatment significantly increased the BMC, volume, and strength of OP-1-mediated callus in a critical size defect in rats at 8 weeks. Thus, modulation of both anabolic and catabolic responses may optimize the amount and mineral content of callus produced, which could be of clinical benefit in obtaining bone union.     

Increases in callus formation and mechanical strength of healing fractures in old rats treated with parathyroid hormone

We studied the effects of intermittent administration of parathyroid hormone (PTH(1-34)) on callus formation and mechanical strength of tibial fractures in 27-month-old rats after 3 and 8 weeks of healing. 200 microg PTH(1-34)/kg was administered daily during both periods of healing, and control animals with fractures were given vehicle. At 3 weeks, PTH treatment increased maximum load and external callus volume by 160% and 208%; at 8 weeks, by 270% and 135%. It also enhanced callus bone mineral content (BMC) by 190% and 388% (3 and 8 weeks). From week 3 to week 8, callus BMC increased by 60% in the vehicle-injected animals, and by 169% in the PTH-treated animals. In the contralateral intact tibia, PTH treatment increased BMC by 18% and 21% (3 and 8 weeks). No differences in body weight were found between the vehicle-injected and the PTH-treated animals during the experiment. In conclusion, PTH treatment enhances fracture strength, callus volume and callus BMC after 3 and 8 weeks of healing.     

Increases in callus formation and mechanical strength of healing fractures in old rats treated with parathyroid hormone

We studied the effects of intermittent administration of parathyroid hormone (PTH(1-34)) on callus formation and mechanical strength of tibial fractures in 27-month-old rats after 3 and 8 weeks of healing. 200 &#119 g PTH(1-34)/kg was administered daily during both periods of healing, and control animals with fractures were given vehicle. At 3 weeks, PTH treatment increased maximum load and external callus volume by 160% and 208%; at 8 weeks, by 270% and 135%. It also enhanced callus bone mineral content (BMC) by 190% and 388% (3 and 8 weeks). From week 3 to week 8, callus BMC increased by 60% in the vehicle-injected animals, and by 169% in the PTH-treated animals. In the contralateral intact tibia, PTH treatment increased BMC by 18% and 21% (3 and 8 weeks). No differences in body weight were found between the vehicle-injected and the PTH-treated animals during the experiment. In conclusion, PTH treatment enhances fracture strength, callus volume and callus BMC after 3 and 8 weeks of healing.     

Transient retention of endochondral cartilaginous matrix with bisphosphonate treatment in a long-term rabbit model of distraction osteogenesis.

Bisphosphonates induce major increases in strength of callus in distraction osteogenesis in the short term. Poor understanding of the underlying mechanism, however, raises concerns about long-term consequences. In this long-term study in 32 rabbits, zoledronic acid transiently increased trabeculae by delayed temporal progression of endochondral bone remodeling but did not prevent radiographic completion of bone repair. INTRODUCTION: We hypothesized that bisphosphonate inhibition of osteoclast-mediated resorption would retain bone during repair, producing a larger callus in the short term. However, if remodeling was not restored, completion of the bone repair process in the long term could be jeopardized. MATERIALS AND METHODS: Juvenile rabbits underwent right tibial osteotomy and 2 weeks of distraction, followed by a period of consolidation. Animals received saline (controls) or zoledronic acid (ZA; 0.1 mg/kg at surgery and again 2 weeks later), and distracted tibias were examined by radiograph, DXA, histology, and histomorphometry at 2, 4, 6, 18, and 44 weeks after surgery. RESULTS: Regenerated bone in ZA-treated animals was denser than controls on radiographs at 6 weeks and had more distinct radiodense trabeculae and retention of original cortices at 18 weeks. By 44 weeks, controls and ZA-treated animals were radiographically healed and indistinguishable. Regenerate BMD and BMC increased between 2 and 4 weeks in all animals, with a greater effect in ZA. At 6 weeks, BMD and BMC in ZA-treated animals were 1.6- and 2-fold greater, respectively, than controls (p < 0.01). From 6 to 44 weeks, the control values gradually increased and approached the ZA-treated values. Regenerate bone volume and trabecular number by histomorphometry were from 1.6- to 2-fold greater in ZA-treated animals at 6 and 18 weeks (p < 0.05). Endochondral cartilaginous matrix volume was up to 2.4-fold greater in ZA-treated animals at 2 and 4 weeks (p < 0.05). TRACP+ cells in ZA-treated animals were larger with more nuclei. Mineral apposition rate and osteoblast number and surface were lower in ZA-treated animals at 6 weeks (p < 0.01) but not at later times. CONCLUSIONS: Disruption of TRACP+ cell function by ZA during bone regeneration seems to lead to an accretion of cancellous bone built on a larger endochondral cartilaginous matrix and increased bone mass, consistent with reported increases in short-term callus strength. This increase in bone mass, caused by a delay in remodeling, provided a transient advantage without preventing radiographic completion of the bone repair process in the long term. Noncontinuous treatment with nitrogen-containing bisphosphonates thus can have short-term beneficial effects without preventing long-term bone repair.     

Negative effect of zoledronic acid on tendon-to-bone healing

Background and purpose — Outcome after ligament reconstruction or tendon repair depends on secure tendon-to-bone healing. Increased osteoclastic activity resulting in local bone loss may contribute to delayed healing of the tendon–bone interface. The objective of this study was to evaluate the effect of the bisphosphonate zoledronic acid (ZA) on tendon-to-bone healing.

Methods — Wistar rats (n = 92) had their right Achilles tendon cut proximally, pulled through a bone tunnel in the distal tibia and sutured anteriorly. After 1 week animals were randomized to receive a single dose of ZA (0.1?mg/kg IV) or control. Healing was evaluated at 3 and 6 weeks by mechanical testing, dual-energy X-ray absorptiometry and histology including immunohistochemical staining of osteoclasts.

Results — ZA treatment resulted in 19% (95% CI 5–33%) lower pullout strength and 43% (95% CI 14–72%) lower stiffness of the tendon–bone interface, compared with control (2-way ANOVA; p = 0.009, p = 0.007). Administration of ZA did not affect bone mineral density (BMD) or bone mineral content (BMC). Histological analyses did not reveal differences in callus formation or osteoclasts between the study groups.

Interpretation — ZA reduced pullout strength and stiffness of the tendon–bone interface. The study does not provide support for ZA as adjuvant treatment in tendon-to-bone healing.     

Manipulation of the anabolic and catabolic responses with BMP-2 and zoledronic acid in a rat femoral fracture model

Bone repair involves a complex set of regulated signaling pathways that control the formation of new bone matrix and the resorption of damaged bone matrix at the fracture site. It has been reported that the optimal time point for single-dose zoledronic acid (ZA) administration systemically increased the strength of bone morphogenetic protein (BMP)-7-mediated callus. However, its repair mechanism during bone fracture healing remains unknown. We aimed to investigate the synergic effect of recombinant human (rh) BMP-2 and ZA in a rat femoral fracture model. Fifty-eight rats were divided into 4 groups. Group I (n = 14) animals were implanted with a carrier alone. Group II (n = 15) animals were implanted with a carrier containing 1-μg rhBMP-2. Group III (n = 14) animals were implanted with a carrier and a subcutaneous systemic ZA injection 2 weeks after surgery. Group IV (n = 15) animals were implanted with a carrier containing 1-μg rhBMP-2 and ZA subcutaneous injection 2 weeks after surgery. The rats were euthanized after 6 weeks and their fractured femurs were explanted and assessed by manual palpation, radiographs, and high-resolution micro-computerized tomography (micro-CT) and were subjected to biomechanical and histological analysis. The fusion rates in Group IV (93.3%) were considerably higher than those in Groups I (28.6%), II (53.3%), and III (57.1%). Additionally, the radiographic scores of Group IV were higher than those in Groups I, II, and III. In micro-CT analysis, the tissue volume (TV) of the callus was higher in Group IV than in Groups I and II (p < 0.05). New bone volume (BV) and trabecular spacing (Tb.Sp) also showed essentially the same trend as that of TV. The ratio of BV to TV (BV/TV), the trabecular number (Tb.N), and the trabecular thickness (Tb.Th) was higher in Groups III and IV than in Groups I and II (p < 0.05). In biomechanical analysis, the ultimate loads at failure and stiffness in Groups III and IV were on average higher than those in Groups I and II (p < 0.05), while the energy absorption of Group IV was higher than those of Groups I and II (p < 0.05). The synergic effect of rhBMP-2 and ZA given systemically as a single dose at the optimal time was efficacious for fracture repair and significantly enhanced bone fusion. Our results suggest that this combination facilitates bone healing and has potential clinical application.     

Skeletal self-repair: stress fracture healing by rapid formation and densification of woven bone.

Stress fractures of varying severity were created using a rat model of skeletal fatigue loading. Periosteal woven bone formed in proportion to the level of bone damage, resulting in the rapid recovery of whole bone strength independent of stress fracture severity. INTRODUCTION: A hard periosteal callus is a hallmark of stress fracture healing. The factors that regulate the formation of this woven bone callus are poorly understood. Our objective was to produce stress fractures of varying severity and to assess the woven bone response and recovery of bone strength. MATERIALS AND METHODS: We used the forelimb compression model to create stress fractures of varying severity in 192 adult rats. Forelimbs were loaded in fatigue until the displacement reached 30%, 45%, 65%, or 85% of fracture. The osteogenic responses of loaded and contralateral control ulnas were assessed 7 and 14 days after loading using pQCT, microCT, mechanical testing, histomorphometry, and Raman spectroscopy. RESULTS: Loading stimulated the formation of periosteal woven bone that was maximal near the ulnar midshaft and transitioned to lamellar bone away from the midshaft. Woven bone area increased in a dose-response manner with increasing fatigue displacement. Whole bone strength was partially recovered at 7 days and fully recovered at 14 days, regardless of initial stress fracture severity. The density of the woven bone increased by 80% from 7 to 14 days, caused in part by a 30% increase in the mineral:collagen ratio of the woven bone tissue. CONCLUSIONS: Functional healing of a stress fracture, as evidenced by recovery of whole bone strength, occurred within 2 wk, regardless of stress fracture severity. Partial recovery of strength in the first week was attributed to the rapid formation of a collar of woven bone that was localized to the site of bone damage and whose size depended on the level of initial damage. Complete recovery of strength in the second week was caused by woven bone densification. For the first time, we showed that woven bone formation occurs as a dose-dependent response after damaging mechanical loading of bone.     

Mechanisms for the enhancement of fracture healing in rats treated with intermittent low-dose human parathyroid hormone (1-34).

Recent reports have demonstrated that intermittent treatment with parathyroid hormone (1-34) [PTH(1-34)] increases callus formation and mechanical strength in experimental fracture healing. However, little is known about the optimal dose required for enhancement of fracture repair or the molecular mechanisms by which PTH regulates the healing process. In this study, we analyzed the underlying molecular mechanisms by which PTH affects fracture healing and tested the hypothesis that intermittent low-dose treatment with human PTH(1-34) can increase callus formation and mechanical strength. Unilateral femoral fractures were produced and a daily subcutaneous injection of 10 microg/kg of PTH(1-34) was administered during the entire healing period. Control animals were injected with vehicle solution alone. The results showed that on day 28 and day 42 after fracture, bone mineral content (BMC), bone mineral density (BMD), and ultimate load to failure of the calluses were significantly increased in the PTH-treated group compared with controls (day 28, 61, 46, and 32%; day 42, 119, 74, and 55%, respectively). The number of proliferating cell nuclear antigen (PCNA)-positive subperiosteal osteoprogenitor cells was significantly increased in the calluses of the PTH-treated group on day 2, and TRAP+ multinucleated cells were significantly increased in areas of callus cancellous bone on day 7. The levels of expression of type I collagen (COLlA1), osteonectin (ON), ALP, and osteocalcin (OC) mRNA were increased markedly in the PTH-treated group and accompanied by enhanced expression of insulin-like growth factor (IGF)-I mRNA during the early stages of healing (days 4-7). The increased expression of COL1A1, ON, ALP, and OC mRNA continued during the later stages of healing (days 14-21) despite a lack of up-regulation of IGF-I mRNA. These results suggest that treatment of fractures with intermittent low dose PTH(1-34) enhances callus formation by the early stimulation of proliferation and differentiation of osteoprogenitor cells, increases production of bone matrix proteins, and enhances osteoclastogenesis during the phase of callus remodeling. The resultant effect to increase callus mechanical strength supports the concept that clinical investigations on the ability of injectable low-dose PTH(1-34) to enhance fracture healing are indicated.     

Implications for Fracture Healing of Current and New Osteoporosis Treatments: An ESCEO Consensus Paper

Osteoporotic fracture healing is critical to clinical outcome in terms of functional recovery, morbidity, and quality of life. Osteoporosis treatments may affect bone repair, so insights into their impact on fracture healing are important. We reviewed the current evidence for an impact of osteoporosis treatments on bone repair. Treatment with bisphosphonate in experimental models is associated with increased callus size and mineralization, reduced callus remodeling, and improved mechanical strength. Local and systemic bisphosphonate treatment may improve implant fixation. No negative impact on fracture healing has been observed, even after major surgery or when administered immediately after fracture. Experimental data for denosumab and raloxifene suggest no negative implications for bone repair. The extensive experimental results for teriparatide indicate increased callus formation, improved biomechanical strength, and greater external callus volume and total bone mineral content and density. Case reports and a randomized trial have produced mixed results but are consistent with a positive impact of teriparatide on clinical fracture healing. Studies with strontium ranelate in models of fracture healing indicate that it is associated with improved bone microstructure, callus volume, and biomechanical properties. Finally, there is experimental evidence for a beneficial effect of some of the agents currently being developed for osteoporosis, notably sclerostin antibody and DKK1 antibody. There is currently no evidence that osteoporosis treatments are detrimental for bone repair and some promising experimental evidence for positive effects on healing, notably for agents with a bone-forming mode of action, which may translate into therapeutic applications.     

Effects of strontium on bone strength, density, volume, and microarchitecture in laying hens.

Strontium has been reported to have beneficial effects on bone. Treatment of laying hens, which are susceptible to osteoporosis and bone fracture, with strontium increased DXA measurements of BMD and BMC and microCT measurements of bone volume and microarchitecture and improved the mechanical performance of whole bone, but had no effect on the estimated material properties of the bone tissue. INTRODUCTION: Strontium (Sr) has been reported to dissociate bone remodeling and have positive influences on bone formation. We supplemented the diet of laying hens, which are susceptible to osteoporosis and bone fracture, with Sr to study the capacity of the element to improve bone mechanical integrity and resistance to fracture. MATERIALS AND METHODS: Increasing dosages of Sr (0, 3000, 4500, and 6000 ppm) were fed to 196 13-week-old pullets for 11 months. BMD and BMC, as measured by conventional and DXA methods, microarchitectural parameters derived from microCT, and structural and material properties as determined by three-point bending test, were studied. Calcium (Ca), phosphorus (P), and Sr levels in plasma and bone, as well as egg output, shell quality, and composition, were assessed. RESULTS: Sr concentrations in plasma and bone increased in a dose-dependent manner without affecting Ca and P. Treatment with Sr increased BMD and BMC as measured by DXA, increased cortical and medullary bone volume, trabecular thickness, number, and surface, and improved whole bone ultimate load, but had no effect on the estimated material properties of diaphyseal bone. Sr also increased the ash content of eggshells and did not affect egg output and shell quality. CONCLUSIONS: Sr supplementation induced large positive effects on bone density, volume, and microarchitecture as measured by radiographic methods. Sr treatment also improved the structural strength of diaphyseal bone but had no effect on the estimated material properties of the bone tissue.     

Osteogenic growth peptide modulates fracture callus structural and mechanical properties

The osteogenic growth peptide (OGP) is a key factor in the mechanism of the systemic osteogenic response to local bone marrow injury. Recent histologic studies have shown that OGP enhances fracture healing in experimental animals. To assess the effect of systemically administered OGP on the biomechanical and quantitative structural properties of the fracture callus, the present study used an integrated approach to evaluate the early stages (up to 4 weeks) of healing of unstable mid-femoral fractures in rats, which included biomechanical, micro-computed tomographic (microCT) and histomorphometric measurements. During the first 3 weeks after fracture, all the quantitative microCT parameters increased in the OGP- and vehicle-treated animals alike. After 4 weeks, the volume of total callus, bony callus, and newly formed bone was approximately 20% higher in animals administered with OGP, consequent to a decrease in the controls. The 4-week total connectivity was 46% higher in the OGP-treated animals. At this time, bridging between the fracture ends by newly formed bone was observed predominantly in the OGP-treated fractures. After 3 and 4 weeks, the OGP-treated animals showed higher biomechanical toughness of the fracture callus as compared to the PBS controls. Significant correlations between structural and biomechanical parameters were restricted to the OGP-treated rats. These data imply that the osteogenic effect of OGP results in enhanced bridging across the fracture gap and consequently improved function of the fracture callus. Therefore, OGP and/or its derivatives are suggested as a potential therapy for the acceleration of bone regeneration in instances of fracture repair and perhaps other bone injuries.     

Impaired angiogenesis, early callus formation, and late stage remodeling in fracture healing of osteopontin-deficient mice.

OPN is an ECM protein with diverse localization and functionality. The role of OPN during fracture healing was examined using wildtype and OPN(-/-) mice. Results showed that OPN plays an important role in regulation of angiogenesis, callus formation, and mechanical strength in early stages of healing and facilitates late stage bone remodeling and ECM organization. INTRODUCTION: Osteopontin (OPN) is an extracellular matrix (ECM) protein with diverse localization and functionality that has been reported to play a regulatory role in both angiogenesis and osteoclastic bone remodeling, two vital processes for normal bone healing. MATERIALS AND METHODS: Bone repair in wildtype and OPN(-/-) mice was studied using a femoral fracture model. microCT was used for quantitative angiographic measurements at 7 and 14 days and to assess callus size and mineralization at 7, 14, 28, and 56 days. Biomechanical testing was performed on intact bones and on fracture specimens at 14, 28, and 56 days. Histology and quantitative RT-PCR were used to evaluate cellular functions related to ECM formation and bone remodeling. RESULTS: OPN deficiency was validated in the OPN(-/-) mice, which generally displayed normal levels of related ECM proteins. Intact OPN(-/-) bones displayed increased elastic modulus but decreased strength and ductility. Fracture neovascularization was reduced at 7 but not 14 days in OPN(-/-) mice. OPN(-/-) mice exhibited smaller fracture calluses at 7 and 14 days, as well as lower maximum torque and work to failure. At 28 days, OPN(-/-) mice had normal callus size but a persistent reduction in maximum torque and work to failure. Osteoclast differentiation occurred normally, but mature osteoclasts displayed reduced functionality, decreasing late stage remodeling in OPN(-/-) mice. Thus, at 56 days, OPN(-/-) fractures possessed increased callus volume, increased mechanical stiffness, and altered collagen fiber organization. CONCLUSIONS: This study showed multiple, stage-dependent roles of OPN during fracture healing. We conclude that OPN deficiency alters the functionality of multiple cell types, resulting in delayed early vascularization, altered matrix organization and late remodeling, and reduced biomechanical properties. These findings contribute to an improved understanding of the role of OPN in vivo and provide new insight into mechanistic control of vascularization and bone regeneration during fracture repair.     

Increases in callus formation and mechanical strength of healing fractures in old rats treated with parathyroid hormone

We studied the effects of intermittent administration of parathyroid hormone (PTH(1-34)) on callus formation and mechanical strength of tibial fractures in 27-month-old rats after 3 and 8 weeks of healing. 200 μg PTH(1-34)/kg was administered daily during both periods of healing, and control animals with fractures were given vehicle. At 3 weeks, PTH treatment increased maximum load and external callus volume by 160% and 208%; at 8 weeks, by 270% and 135%. It also enhanced callus bone mineral content (BMC) by 190% and 388% (3 and 8 weeks). From week 3 to week 8, callus BMC increased by 60% in the vehicle-injected animals, and by 169% in the PTH-treated animals.

In the contralateral intact tibia, PTH treatment increased BMC by 18% and 21% (3 and 8 weeks). No differences in body weight were found between the vehicle-injected and the PTH-treated animals during the experiment. In conclusion, PTH treatment enhances fracture strength, callus volume and callus BMC after 3 and 8 weeks of healing.     

Intermittent ibandronate preserves bone quality and bone strength in the lumbar spine after 16 months of treatment in the ovariectomized cynomolgus monkey.

The dose-dependent effect of ibandronate treatment on bone mass and architecture was assessed in a large animal study of OVX monkeys using microCT for quantitative bone morphometry and biomechanical testing for measures of bone strength. The study showed that intermittent ibandronate preserved lumbar spine bone quality and strength in these animals after 16 months of treatment. INTRODUCTION: Ibandronate is a bisphosphonate, which is a class of compounds that, in pharmacologically active doses, not only suppresses bone resorption and turnover but also prevents loss of bone mass and strength in the ovariectomized (OVX) rat. MATERIALS AND METHODS: We evaluated the effects of ibandronate on bone mass and architecture in the OVX cynomolgus macaque. Sixty-one adult female macaques were divided into five groups (N = 11-15): sham control, OVX control, and OVX low- (10 microg/kg), medium- (30 microg/kg), and high- (150 microg/kg) dose ibandronate. Treatment was administered by intravenous bolus injection every 30 days for 16 months starting at ovariectomy. This dosing schedule is equivalent to a 3-monthly dosing regimen in human subjects over 4 years. Animals were killed at the conclusion of the study, and excised bone specimens of the first lumbar vertebra (L1) were evaluated for quantitative bone densitometry, morphometry, and mechanical properties. Architectural parameters were assessed by microCT including direct 3D bone morphometry. A measure of specimen strength was obtained using destructive compression testing. RESULTS AND CONCLUSIONS: A significant loss of bone mass and related changes in bone architecture after ovariectomy resulted in a reduction of whole bone strength as expressed by high correlations between architectural and mechanical properties. In this analysis, BMC was the best single predictor of whole bone strength (r2 = 67%). Nevertheless, including architectural indices in a multiple linear regression analysis increased that prediction to 88%. With respect to the treatment, the medium- and high-dose groups were not significantly different from the sham group for all bone mineral and structural parameters. Additionally, significant differences were seen for all measured parameters between the high-dose group and the OVX group, and for some parameters, between the medium-dose group and the OVX group. Intermittent ibandronate treatment effectively and dose-dependently prevented bone loss, architectural deterioration, and strength reduction in the lumbar spine of OVX monkeys.     

No effect of systemic administration of somatomedin C on bone repair in rats

The effect of somatomedin C on the bone repair process was studied in a rat femoral osteotomy. We used continuous systemic administration of human recombinant somatomedin C (110 micrograms/100 g per day). Radiographic evaluation after 4 weeks showed no effect of somatomedin C on the healing. There was no effect on mechanical strength, and no effect was found on callus vascularization or mineralization. The weight of the callus was slightly reduced in the somatomedin C treated rats. The results were not suggestive of any acceleration with systemic somatomedin C treatment in the early phase of cortical bone repair.     

Intermittent parathyroid hormone (1-34) treatment increases callus formation and mechanical strength of healing rat fractures.

The influence of intermittent parathyroid hormone (PTH(1-34)) administration on callus formation and mechanical strength of tibial fractures in rats was investigated after 20 and 40 days of healing. A dose of 60 microg of PTH(1-34)/kg/day and 200 microg of PTH(1-34)/kg/day, respectively, was administered during the entire periods of healing, and control animals with fractures were given vehicle. The dose of 200 microg of PTH(1-34)/kg/day increased the ultimate load and the external callus volume of the fractures by 75% and 99%, respectively, after 20 days of healing and by 175% and 72%, respectively, after 40 days of healing. The dose of 60 microg of PTH(1-34)/kg/day did not influence either ultimate load or external callus volume of the fractures after 20 days of healing, but the ultimate load was increased by 132% and the external callus volume was increased by 42% after 40 days of healing. During the healing period, the callus bone mineral content (BMC) increased in all groups. After 40 days of healing, the callus BMC was increased by 108% in the 200 microg of PTH(1-34)/kg/day group and by 76% in the 60 microg of PTH(1-34)/kg/day group. Both doses of PTH(1-34) steadily augmented the contralateral intact tibia BMC (20 days and 40 days: 60 microg of PTH (1-34)/kg/day 9% and 19%, respectively; 200 microg of PTH (1-34)/kg/day 12% and 27%, respectively) and bone mineral density (20 days and 40 days: 60 microg of PTH(1-34)/kg/day 11% and 12%, respectively; 200 microg of PTH(1-34)/kg/day 11% and 15%, respectively).     

Gene therapy with human osteoprotegerin decreases callus remodeling with limited effects on biomechanical properties

Osteoprotegerin (OPG) is a naturally occurring protein, which prevents bone resorption by inhibition of osteoclastogenesis, function, and survival. Therefore, recombinant OPG may be an attractive drug in the treatment of chronic bone resorptive diseases such as osteoporosis. Gene therapy has the potential to achieve long-term treatment by delivering genes of anti-resorptive proteins to the recipient. The effects of OPG gene therapy on fracture healing have not been described previously.

The influence of OPG gene therapy on callus formation, callus tissue structural strength, apparent material properties, and histology of tibia fractures in rats was investigated after 3 weeks and 8 weeks of healing. Intramuscular administration of adeno-associated virus (AAV) vector-mediated OPG resulted in increased levels of OPG in serum of approximately 100 ng/ml throughout the study period. Control animals with fractures received transduction with an AAV reporter gene construct (AAV-enhanced green fluorescent protein (eGFP)), and in this group serum OPG levels remained at baseline (<10 ng/ml). After 3 weeks of healing, AAV-OPG treatment reduced the number of osteoclasts in the callus tissue (33%, P < 0.001). However, AAV-OPG treatment did not influence callus dimensions, callus bone mineral content (BMC), fracture structural strength, or apparent callus tissue material properties. After 8 weeks of healing, AAV-OPG treatment reduced the number of osteoclasts in the callus tissue (31%, P < 0.001) compared with AAV-eGFP fractures. Furthermore, deposition of new woven bone at the fracture line of the original cortical bone was hampered (new woven bone present: in all AAV-eGFP animals, in 41% of AAV-OPG-treated animals, P < 0.001). AAV-OPG treatment also increased callus BMC (18%, P = 0.023) compared with AAV-eGFP fractures. AAV-OPG did not influence callus dimensions, structural strength of the fractures, or ultimate stress, whereas elastic modulus was reduced in the AAV-OPG groups (37%, P = 0.039). The experiment demonstrates that AAV-OPG gene therapy decreases the fracture remodeling, but this does not influence the structural strength of healing fractures.     

Osteoprotegerin Treatment Impairs Remodeling and Apparent Material Properties of Callus Tissue without Influencing Structural Fracture Strength

The influence of osteoprotegerin (OPG) treatment on callus formation, callus tissue structural strength, apparent material properties, and histology of tibia fractures in rats was investigated after 3 weeks and 8 weeks of healing. OPG was given intravenously (10 mg/kg twice weekly) during the entire observation period, and control animals with fractures received vehicle only. When compared with control fractures after 3 weeks of healing, OPG treatment reduced the number of osteoclasts in the callus tissue (93%, P < 0.001) and hampered resorption of genuine cortical bone in the fracture line; OPG treatment did not influence callus dimensions, callus bone mineral content (BMC), fracture structural strength, or callus tissue apparent material properties. When compared with control fractures after 8 weeks of healing; OPG treatment reduced the number of osteoclasts in callus tissue (92%, P < 0.001), augmented callus dimensions (anteriorposterior diameter: 12%, P = 0.034, mediolateral diameter: 13%, P = 0.013), and increased callus BMC (50%, P = 0.007); OPG treatment hampered deposition of new woven bone at the fracture line of the genuine cortical bone (new woven bone present in all vehicle animals, but only in 13% of the OPG-treated animals (P < 0.001)); OPG treatment did not influence structural strength of the fractures, but decreased apparent material properties of the callus tissue (ultimate stress: 51%, P < 0.001; elastic modulus: 42%, P = 0.033). The experiment demonstrates that OPG treatment does not influence the early callus expansion and fracture strength. However, during the subsequent period of remodelling, OPG treatment impairs the normal remodeling and consolidation processes.