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.     

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.     

Strontium Is Incorporated into the Fracture Callus but Does Not Influence the Mechanical Strength of Healing Rat Fractures

Strontium ranelate (SrR) is a new agent used in the treatment of osteoporosis and is suggested to reduce bone resorption and increase bone formation. We investigated whether SrR influences the macro- and nanomechnical properties of healing fractures in rats. A closed tibia fracture model was used to study fracture healing in rats after 3 and 8 weeks of healing. Two groups of rats were treated with SrR (900 mg/kg/day) mixed into the food, while two groups served as control animals. The healing fractures were investigated by three-point bending, dual energy X-ray absorptiometry, energy-dispersive X-ray spectroscopy (EDX), and nanoindentation. There was a 100-fold increase (P < 0.001) in serum Sr after 3 and 8 weeks of SrR treatment. The callus volume was significantly higher in the SrR-treated group than in control animals (P < 0.01) after 3 weeks of healing. This was accompanied by a significant increase in callus bone mineral content (P < 0.05). However, after 8 weeks of healing, no difference was found in either callus volume or bone mineral content. SrR did not influence maximum load or stiffness of the fractures after either 3 or 8 weeks of healing. EDX showed that Sr was incorporated into the callus; however, this did not influence the nanomechanical properties. In conclusion, SrR stimulates callus formation but has no effect on callus remodeling. Sr is incorporated into the newly formed callus tissue, but this has no deteriorating effect on the mechanical properties of rat tibial fractures at either the macroscopic or nanoscopic level after 3 or 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.     

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.     

Effect of Stabilization on the Healing Process of Femur Fractures in Aged Mice

Background: The influence of mechanical stability on fracture healing has previously been studied in adult mice, but is poorly understood in aged animals. Therefore, we herein studied the effect of stabilization on the healing process of femur fractures in aged mice. Methods: Twenty-four 18-month-old CD-1 mice were stabilized after midshaft fracture of the femur with an intramedullary screw. In another 24 18-month-old mice, the femur fractures were left unstabilized. Bone healing was studied by radiological, biomechanical, histomorphometric, and protein expression analyses. Results: After 2 and 5 weeks of healing, the callus of nonstabilized fractures compared to stabilized fractures was significantly larger, containing a significantly smaller amount of osseous tissue and a higher amount of cartilaginous tissue. This was associated with a significantly lower biomechanical stiffness during the early phase of healing. However, during the late phase of fracture healing both nonstabilized and stabilized fractures showed a biomechanical stiffness of ～40%. Of interest, Western blot analyses of callus tissue demonstrated that the expression of proteins related to angiogenesis, bone formation and remodeling, i.e. VEGF, CYR61, BMP-2, BMP-4, Col-2, Col-10, RANKL, OPG, did not differ between nonstabilized and stabilized fractures. Conclusion: Nonstabilized fractures in aged mice show delayed healing and remodeling. This is not caused by an altered protein expression in the callus but rather by the excessive interfragmentary movements.     

Dynamic Effects of the Third Generation Bisphosphonate of Risedronate on Rat Osteoporotic Fractures for Clinical Usage Guidance

ObjectiveTo better understand the risks of bisphosphonates in order to develop guidance for appropriate clinical usage, to compared femoral fracture healing at different time points and to explore the effects of Residronate on fracture healing.MethodsOsteoporosis model was achieved by ovariectomy surgery, followed by surgical incision of left femoral shaft 4 weeks after ovariectomy surgery. Three days after fracture surgery, risedronateor saline was fed by intragastric administration. X ray examination was used to check the callus formation, Bone Mineral Density (BMD), Bone Mineral Content (BMC), biomechanical, imaging and micromorphological of bone tissue as well as the trabecular bone parameters were all examined. The femoral pathology tissue of each rat was used to analyze trabecular bone parameters, including trabecular bone volume/tissue volume (Tb. BV/TV), bone surface to tissue volume ratio (BS/TV), trabecular bone mineral density (Tb. BMD), trabecular bone number (Tb. N), trabecular bone thickness (Tb. Th) and small bone Trabecular bone space (Tb. Sp).Results Via X‐ray and pathologically, risedronate treatment promoted the callus forming at the fracture site during the following 6 weeks after osteoporotic fracture by X‐ray (P < 0.01), increased the local bone mineral density (P < 0.01), and accelerated the fracture healing during the first 3 weeks (P <0.01), but delayed facture healing in the later 3 weeks (P < 0.01). Risedronate increased the bone continuity of fracture at 7th week, but this phenomenon was not found at the 10th week (P < 0.01). Delayed fracture healing occurred locally at the fracture site. At 7th week, Risedronate may promote cartilage cells proliferating at fracture site, increase the dense of bone trabeculae and the connection of bone trabeculae, thicken the bone cortex showing better fracture healing than OPF‐Saline groups (P < 0.01). However, these parameter did not continue during the 7th and 10th weeks. Comparing the first and the later 3 weeks, the rats in group Osteoporotic Fracture‐Risedronate (OPF‐RD) accelerated the local fracture healing in the first 3 weeks but not in the last 3 weeks, which is consistent for the BMD and BMC among each group (P < 0.05). Through evaluation of bone mineral density and bone mineral content, risedronate dramatically increased the BMD at the fracture site and resulted in reduction of BMC by risedronate at the fracture site (P < 0.05) among each group still exist, indicating dramatic (P < 0.05). Through load testing, Risedronate increased the structural strength and mechanical indexes of the new callus (P < 0.01).ConclusionRisedronate can improve the structural strength and mechanical index of newborn callus. Longer than 7 weeks usage of third generation bisphosphonate of risedronate does not contribute to osteoporotic fracture.     

Treatment with Parathyroid Hormone hPTH(1-34), hPTH(1-31), and Monocyclic hPTH(1-31) Enhances Fracture Strength and Callus Amount After Withdrawal Fracture Strength and Callus Mechanical Quality Continue to Increase

The influence of intermittent hPTH(l-34)NH₂, hPTH(1-31)NH₂, and monocyclic [Leu²⁷]cyclo(Glu²²-Lys²⁶)hPTH(1-31)NH₂ treatment on callus formation, mechanical strength, and callus tissue mechanical quality of tibial fractures in rats was investigated after 8 and 16 weeks of healing. In the 8 weeks of healing animals, the PTH-peptides were injected subcutaneously during the entire observation period (15 nmol/kg/day [hPTH(1-34)NH₂: 15 nmol = 60 µg]), and control animals with fractures were given vehicle. In the 16 weeks of healing animals, the PTH-peptides were injected only during the first 8 weeks of healing (15 nmol/kg/day), after which the animals were left untreated during the rest of the healing period. After the first 8 weeks of healing, increased fracture strength and callus volume were seen in the PTH-treated rats (ultimate load 66%, ultimate stiffness 58%, callus volume 28%), and the three peptides were equally effective. No difference in callus tissue mechanical quality was found between PTH and vehicle animals. After 16 weeks of healing, no differences in fracture strength, callus volume, or callus tissue mechanical quality were seen between PTH and vehicle. When comparing PTH-treated animals at 8 and 16 weeks, fracture strength and callus tissue mechanical quality continued to increase after the withdrawal of PTH (ultimate load 23%, ultimate stress 88%, elastic modulus 87%) and external callus volume declined during this period (27%).     

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.     

Low‐magnitude high‐frequency vibration enhances gene expression related to callus formation,mineralization and remodeling during osteoporotic fracture healing in rats

Low magnitude high frequency vibration (LMHFV) has been shown to improve anabolic and osteogenic responses in osteoporotic intact bones and during osteoporotic fracture healing; however, the molecular response of LMHFV during osteoporotic fracture healing has not been investigated. It was hypothesized that LMHFV could enhance osteoporotic fracture healing by regulating the expression of genes related to chondrogenesis (Col‐2), osteogenesis (Col‐1) and remodeling (receptor activator for nuclear factor‐ κ B ligand (RANKL) and osteoproteger (OPG)). In this study, the effects of LMHFV on both osteoporotic and normal bone fracture healing were assessed by endpoint gene expressions, weekly radiographs, and histomorphometry at weeks 2, 4 and 8 post‐treatment. LMHFV enhanced osteoporotic fracture healing by up‐regulating the expression of chondrogenesis‐, osteogenesis‐ and remodeling‐related genes (Col‐2 at week 4 (p = 0.008), Col‐1 at week 2 and 8 (p < 0.001and p = 0.008) and RANKL/OPG at week 8 (p = 0.045)). Osteoporotic bone had a higher response to LMHFV than normal bone and showed significantly better results as reflected by increased expression of Col‐2 and Col‐1 at week 2 (p < 0.001 for all), larger callus width at week 2 (p = 0.001), callus area at week 1 and 5(p < 0.05 for all) and greater relative area of osseous tissue (p = 0.002) at week 8. This study helps to understand how LMHFV regulates gene expression of callus formation, mineralization and remodeling during osteoporotic fracture healing. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1572–1579, 2014.     

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.     

Low‐magnitude high‐frequency vibration accelerates callus formation,mineralization, and fracture healing in rats

Fracture healing is a biological regenerative process that follows a well‐orchestrated sequence. Most healing is uneventful and enhancement of normal fracture healing is not commonly done, although it is clinically important in the recovery and regain of functions after fracture. This study investigated the osteogenic effect of low‐magnitude high‐frequency vibration (LMHFV, 35 Hz, 0.3 g) on the enhancement of fracture healing in rats with closed femoral shaft fracture by comparing with sham‐treated control. Assessments with plain radiography, micro‐CT as well as histomorphometry showed that the amount of callus was significantly larger (p = 0.001 for callus area, 2 weeks posttreatment); the remodeling of the callus into mature bone was significantly faster (p = 0.039, 4 weeks posttreatment) in the treatment group. The mechanical strength of the healed fracture in the treatment group at 4 weeks was significantly greater (p < 0.001). The results showed the acceleration of callus formation, mineralization, and fracture healing in the treatment group. It is concluded that LMHFV enhances healing in the closed femoral shaft fracture in rats. The potential clinical advantages shall be confirmed in the subsequent clinical trials. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 458–465, 2009     

The influence of compression on the healing of experimental tibial fractures

Purpose

Experimental studies of the effects of various mechanical conditions and stimuli on bone healing have disclosed an improvement potential in bone fracture mineralization and biomechanical properties. We therefore evaluated the effect of a clinically practicable application of a mechanical compressive interfragmentary stimulus on the healing of experimental tibial diaphyseal fractures.

Methods

Sixty Male rats received a standardized tibial shaft osteotomy stabilized with a unilateral external fixator with a zero interfragmentary distance, and then randomly assigned to the compression (N = 20), control (N = 20) or distraction (N = 20) group. From days 4 to day 14, the external fixator was either tightened (compression group) or loosened (distraction group) once daily to gradually induce a total axial displacement of the external fixator pin clamps of 1.25 mm. Evaluation at 30 and 60 days post-osteotomy included radiography, dual-energy X-ray absorptiometry (DXA), quantitative CT and mechanical testing.

Results

All fractures healed radiographically with sparse callus. At 60 days, the compression and control groups exhibited significantly less amount of mineralized callus in terms of DXA measured callus area and bone mineral content (BMC) compared to the distraction group. These groups also demonstrated a smaller volume of low-mineralized bone tissue (callus) and a larger volume of highly mineralized bone tissue (cortical bone) measured by QCT than in the distraction group. Both mechanical strength and stiffness was significantly higher in the compression and control groups than in the distraction group at 60 days.

Discussion

Compression did not enhance fracture healing in terms of mineralization, bending strength, or stiffness at the time of union, compared with the control condition. The compression and control groups exhibited improved healing in terms of mechanical strength and stiffness and a more mature callus mineralization compared with the distraction group.     

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.     

Bone fracture and bone fracture repair

Fracture healing is a multistage repair process that involves complex, well-orchestrated steps initiated in response to tissue injury. The early upregulation of IL-6, osteoprotegerin (OPG), VEGF, and BMPs indicates a central role for these factors in the initiation of cartilage and periosteal woven bone formation. In both callus fracture repair and stress fracture repair, the RANKL/OPG ratio is initially reduced, but peaks earlier in stress fracture healing than callus fracture healing. Though the understanding of the biological processes and molecular signals that coordinate fracture repair has advanced, the cause of variability observed in fracture repair is poorly understood.     

Effect of low-level laser therapy on the fracture healing process

Low-level laser therapy (LLLT) is a biophysical form of intervention in the fracture-repair process, which, through several mechanisms, accelerates the healing of fractures and enhances callus formation. The effect of laser on fracture healing is controversial. Some authors affirm that LLLT can accelerate bone formation by increasing osteoblastic activity. The objective of our study was to evaluate the effect of laser therapy on fracture healing. Thirty rabbits were subjected to tibial bone open osteotomies that were stabilized with external fixators. The animals were divided into two study groups: laser group and control group. Callus development and bone mineral density were quantitatively evaluated by CT; the animals were then killed and the fractures were assessed for biomechanical properties. The results demonstrated that the increasing rate of bone mineral density was higher in the laser (L) group than in the control (C) group. CT at 5 weeks revealed a mean callus density of 297 Hounsfield units (HU) for the control group and 691 HU for the L group, which was statistically significant (P?=?0.001). In the L group, the mean recorded fracture tension was 190.5 N and 359.3 N for healed and intact bones, respectively, which was statistically significant (P?<?0.001). The result of the study showed that the use of laser could enhance callus development in the early stage of the healing process, with doubtful improvement in biomechanical properties of the healing bone; therefore, laser therapy may be recommended as an additional treatment in non-union fractures in humans.     

Effect of strontium ranelate on fracture healing in the osteoporotic rats

The aim of this study was to evaluate the effect of strontium ranelate (SrR) on fracture healing in the osteoporotic rat model. Forty female Sprague–Dawley rats aged 3 months were enrolled in the study. Osteoporosis was induced by bilateral ovariectomy and subsequent daily heparin injection started 1 week after surgery and lasted for 4 weeks. Osteoporosis was confirmed by a reduction of bone mineral density (BMD). Twenty of the osteoporotic rats were assigned to the SrR group and the remaining 20 to the control group. An open right tibial midshaft transverse fracture was created and then an intramedullary fixation was performed. SrR group was treated by 450 mg/kg/day SrR per oral. Six weeks after surgical induction of fracture, all animals were sacrificed. One animal from each group died after ovariectomy. Two tibiae from the control group failed to unite. SrR‐treated group showed higher mechanical strength and fracture stiffness when compared to the control group (p = 0.006, p = 0.001, respectively). SrR‐treated group had mature woven bone or predominantly woven bone compared with osteoporotic control group (p = 0.038). SrR‐treated group's callus maturity was significantly higher than control group (p = 0.001). SrR is associated with better fracture healing in the osteoporotic rat model. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:138–142, 2011     

Effect of Dosing Interval Duration of Intermittent Ibandronate Treatment on the Healing Process of Femoral Osteotomy in a Rat Fracture Model

The effects of bisphosphonate treatment schedule on fracture healing have not previously been tested. We evaluated the effect of ibandronate dosing interval duration on healing following surgical “fracture” (osteotomy) using a rat femoral fracture model. Six-week-old rats (n = 160) underwent osteotomy and were then allocated into vehicle control (CNT) or an ibandronate treatment group: 5 μg/kg daily (DAY, 5 days/week), 75 μg/kg once every 3 weeks (I-3), 150 μg/kg once every 6 weeks (I-6), resulting in the same total ibandronate dose over the study. Rats were killed after 6 or 18 weeks. At 18 weeks, all fracture lines had disappeared in the CNT and I-6 groups; approximately 10% of fracture lines remained in the DAY and I-3 groups. Ibandronate-treated groups showed large callus areas around the fractures, which shrank between 6 and 18 weeks after surgery; the extent of shrinkage decreased with shorter dosing interval. In histomorphometry, callus remodeling was suppressed by ibandronate; this became more apparent at shorter dose intervals. The structural properties of osteotomized femora were increased in the DAY group compared with CNT, but intrinsic material properties reduced inversely and became closer to those of CNT in response to increased dosing interval. Ibandronate induced formation of large calluses around osteotomies but delayed woven bone remodeling into lamellar bone and reduced intrinsic material properties in a rat fracture model. Extending the dosing interval of intermittent ibandronate treatment appeared to reduce the suppression of callus remodeling caused by ibandronate, which would have delayed healing after osteotomy.     

Histomorphometric analysis of fracture healing cascade in acute osteoporotic vertebral body fractures

BACKGROUND: While fracture healing has been well characterised in long bones, there is scant data relating to this process in acute vertebral body fractures. AIM: To characterise the histological process of fracture healing in acute osteoporotic vertebral body fractures using qualitative and quantitative bone histomorphometry. SUBJECTS AND METHODS: Transpedicular bone biopsy was performed in patients undergoing percutaneous vertebroplasty. Undecalcified biopsy specimens were prepared from cores of cancellous bone harvested from vertebral bodies with MRI evidence of bone marrow oedema. These were analysed by light microscopy using grid analysis and defined using bone histomorphometry criteria. Normative data obtained from 5 age-matched volunteers without evidence of metabolic bone disease or osteoporosis was used for comparison. RESULTS: Adequate biopsy specimens were obtained in 72 of 90 patients (15 men and 57 women), mean age 75.6 years. All biopsies confirmed severe osteoporosis with reduced cancellous bone volume (mean of 13.5%; P<0.001 compared to controls). The timing of biopsies varied from 1 to 24 weeks (median of 6 weeks) after the fracture event. There were 4 stages of fracture callus healing observed: Stage I in 17 (24%) patients, Stage II in 16 (22%), Stage III in 22 (30%) and Stage IV in 17 (24%). An overlap between the various stages was evident with 55 (76%) patients demonstrating at least 2 or more of the stages of fracture healing in the same biopsy specimen. The time interval since fracture event was the most important predictor of the stage of the fracture callus (R=0.32; P<0.001). CONCLUSION: Our data demonstrates a mixed fracture callus with overlapping of the various stages of fracture healing. This suggests that individual vertebra may be susceptible to multiple fractures over the course of the healing process.