The effects of clodronate on increased bone turnover and bone loss due to ovariectomy in rats

The present study was carried out to investigate the ability of clodronate to inhibit ovariectomy-induced bone loss and increased bone turnover in rats. Estradiol was administered as a reference compound. Seventy Sprague-Dawley rats were ovariectomized (OVX) or sham-operated (Sham) at the age of 90 days and divided into seven groups. Two Sham and two OVX groups received subcutaneously either the vehicle of clodronate or the vehicle of estradiol. Other OVX groups were given s.c. either disodium clodronate at two dose levels (5 mg/kg or 12.5 mg/kg twice a week) or 17β-estradiol (10 μg/kg five times a week) for 8 weeks. Femur length, volume, dry weight, and ash weight were determined, and proximal ends of tibiae were used for bone histomorphometry. Markers of bone metabolism were measured from urine and serum. A significant loss of 54% of trabecular bone area of proximal tibial metaphysis was found at 8 weeks after ovariectomy. Clodronate and estradiol inhibited (p < 0.001) this osteopenia. Both drugs prevented the decrease in ash weight/volume of the femur. The inhibitory effect of clodronate and estradiol on bone resorption in OVX rats could be detected also in decreased urinary excretion of hydroxyproline and lysylpyridinoline (p < 0.001). Clodronate and estradiol decreased (p < 0.001) the ovariectomy-induced enhanced tibial endocortical mineral apposition rate (Ec.MAR) on the lateral cortex to the level of the Sham group. In contrast, periosteal MAR analyzed on the medial side of tibial cortical bone did not change significantly in the OVX/Veh group. Estradiol decreased periosteal MAR to below the level in the Sham group (p < 0.01). These results suggest that ovariectomy of growing rats resulted in tibial and femoral osteopenia two months later. Clodronate as well as estradiol can suppress bone resorption and turnover in ovariectomized rats, inhibiting the development of osteopenia. Both clodronate doses (5 and 12.5 mg/kg) had beneficial effects in ovariectomized animals.     

Long-term administration of clodronate does not prevent fracture healing in rats

Clinicians have been concerned that fractures do not heal properly in individuals exposed to bisphosphonate treatment, a treatment that strongly affects bone metabolism. The current study attempted to clarify the long-term effects of clodronate (dichloromethylene bisphosphonate) treatment on fracture healing in growing rats. Clodronate was administered subcutaneously twice a week in a dose of 2 mg/kg or 10 mg/kg. Physiologic saline served as a control. After 24 weeks of treatment, the tibiae were fractured, and the treatment was continued for another 4 weeks and 8 weeks. At both end points the cross-sectional areas of the callus, measured by peripheral quantitative computed tomography, were greater in the clodronate-treated rats than in controls, but there were no significant differences in bone mineral density. There were no significant differences between treatments in radiologic healing, histomorphometry, or in mechanical failure load of the callus with the exception of increased tensile stiffness at a dose of 2 mg/kg at 4 weeks. Clodronate treatment does not seem to prolong the fracture healing process, even when administered on a long-term basis before the fracture. Clodronate increases the size of the callus, but has only a minor effect on its biomechanical properties. The current results suggest that long-term clodronate treatment does not inhibit fracture healing.     

Traumatic late dissociation of the polyethylene articulating surface in a total knee arthroplasty--a case report

Clodronate was administered daily 28 days before and after an experimental tibial fracture in 35 male rats, and the effect on fracture healing and posttraumatic bone loss was studied. 5 groups were tested. The clodronate/clodronate group received clodronate in daily doses of 10 mg/ kg body weight for 28 days before being subjected to a standardized fracture of the right tibia, and during the fracture healing period of 28 days. The clodronate/ saline group received clodronate before fracture and saline during the healing period. The saline/ clodronate group received saline before and clodronate after fracture. The saline/saline group received saline only, while the control group served as unfractured, untreated controls. After 28 days of fracture healing, the tibias were evaluated with dual energy x-ray absorptiometry, and tested mechanically in a 3-point ventral bending test. Bone mineral content and bone mineral density were approximately 30% higher in the groups receiving clodronate during the experiment, compared to the untreated groups. The weight and cross-sectional area of the fracture callus were equal in all groups. Whether clodronate was administered before the fracture, after the fracture or both, did not affect the bone mineral. Ultimate bending moment, energy absorption, stiffness and deflection were not significantly different between the groups. Our findings suggest that clodronate increases bone mineral both when given before and after a tibial shaft fracture, without affecting fracture healing at 28 days.     

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.     

Healing of a tibial double osteotomy is modified by clodronate administration

We studied the healing of tibial double osteotomies in rats during clodronate administration 10 mg/kg daily subcutaneously for 6 weeks. Clodronate treatment did not inhibit the endochondral bone formation in callus or in the epiphyseal plate. The differentiation of osteoblasts into mature osteocytes was disturbed, and the bone structure around the osteocytes was not normal. In the clodronatetreated group, the amount of newly formed bone matrix seemed increased, but mineralization of the bone matrix was delayed. The bundle bone trabeculae in callus were not so spatially well organized as in the controls. No delay in the union of the bone segment by new bone was seen during clodronate administration.     

Osteoclast depletion with clodronate liposomes delays fracture healing in mice

Osteoclasts are abundant within the fracture callus and also localize at the chondro‐osseous junction. However, osteoclast functions during fracture healing are not well defined. Inhibition of osteoclast formation or resorptive activity impairs callus remodeling but does not prevent callus formation. Interestingly, though anti‐osteoclast therapies differentially affect resolution of callus cartilage into bone. Treatments that inhibit osteoclast formation or viability tend to impair callus cartilage resolution, while treatments that target inhibition of bone resorption generally do not affect callus cartilage resolution. Here, we tested whether depletion of osteoclasts by systemic treatment with clodronate liposomes would similarly impair callus cartilage resolution. ICR mice were treated by intraperitoneal injections of clodronate‐laden liposomes or control liposomes and subjected to closed femur fracture. Femurs were resected at multiple times after fracture and analyzed by radiography, histology, and mechanical testing to determine effects on healing. Clodronate liposome treatment did not prevent callus formation. However, radiographic scoring indicated that clodronate liposome treatment impaired healing. Clodronate liposome treatment significantly reduced callus osteoclast populations and delayed resolution of callus cartilage. Consistent with continued presence of callus cartilage, torsional mechanical testing found significant decreases in callus material properties after 28 days of healing. The results support a role for osteoclasts in the resolution of callus cartilage into bone. Whether the cartilage resolution role for osteoclasts is limited to simply resorbing cartilage at the chondro‐osseous junction or in promoting bone formation at the chondro‐osseous junction through another mechanism, perhaps similar to the reversal process in bone remodeling, will require further experimentation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1699–1706, 2017.

     

Effect of trabecular bone contour on ultimate strength of lumbar vertebra after bilateral ovariectomy in rats

To test the hypothesis that the effect of trabecular microarchitecture on bone strength varies with the duration of estrogen loss, we evaluated the relationship between three-dimensional (3D) parameters for trabecular microarchitecture and bone minerals with the compressive load of the lumbar vertebra in rats. Female Sprague-Dawley rats (n = 190) were divided into 19 groups. Ten rats were killed at day 0. Half of the remaining rats underwent bilateral ovariectomy (ovx), and the others were subjected to sham surgery. Ten rats from each group were killed at 3, 7, 11, 14, 28, 42, 56, 70, and 84 days postsurgery. Urinary deoxypyridinoline and serum osteocalcin increased significantly in the ovx group from days 28 and 11, respectively, compared with the sham group. Bone mineral content (BMC) and bone mineral density (BMD) of the fifth lumbar body diminished from days 42 and 84, respectively, compared with the sham group. In ovx rats, trabecular bone volume (BV/TV), measured using 3D images of microcomputed tomography, diminished from day 28 compared with both baseline control and sham. The trabecular bone pattern factor (TBPf) and structure model index (SMI) increased from day 28 in the ovx group compared with both baseline control and sham. Ultimate compression loads diminished at day 28 compared with baseline control and decreased progressively thereafter. Neither of these parameters changed in the sham group during the same period. Within 4 weeks post-ovx, TBPf, SMI, and BV/TV correlated with load (p < 0.01). BMC and BMD correlated with load from 6 weeks post-ovx (p < 0.01). Stepwise regression analysis showed that TBPf was the most significant determinant of load within 4 weeks post-ovx (coefficient of determination [R²] = 0.669; p < 0.01). SMI correlated with TBPf (R² = 0.968; p < 0.01). Moreover, R² for ultimate load indicated higher values of 0.975 with TBPf and SMI. However, BMC was the most significant determinant of load from 6 weeks post-ovx (R² = 0.511; p < 0.01), as it was in the sham group. These data suggest that changes in trabecular bone contour with increased bone turnover are critical for reducing lumbar bone strength during the early post-ovx period in rats.     

Subclinical hypervitaminosis A causes fragile bones in rats

Excessive intake of vitamin A has been associated with an increased risk of hip fracture in humans. This finding has raised the question of whether long-term intake of relatively moderate doses (“subclinical” hypervitaminosis A) contributes to fracture risk. Although it has been known for more than half a century that toxic doses of vitamin A lead to spontaneous fractures in rats, the lowest intake that induces adverse effects is not known, and the result of exposure to excessive doses that do not cause general toxicity has been rarely investigated. In this study, mature female rats were fed a standard diet with 12 IU vitamin A/g pellet (control, C), or standard diet supplemented with either 120 IU (“10 × C”) or 600 IU (“50 × C”) vitamin A/g pellet for 12 weeks. Fifteen animals were included in each group. The supplemented diets correspond to a vitamin A intake of approximately 1800 IU/day and 9000 IU/day, respectively. The latter dose is about one third of that previously reported to cause skeletal lesions. At the end of the study, serum retinyl esters were elevated 4- (p < 0.01) and 20-fold (p < 0.001) and the total amount of liver retinoid had increased 3- (p < 0.001) and 7-fold (p < 0.001) in the 10 × C and 50 × C group, respectively. The animals showed no clinical signs of general toxicity, and there were no significant bone changes in the 10 × C group. However, in the 50 × C group, a characteristic thinning of the cortex (cortical area −6.5% [p < 0.001]) and reduction of the diameter of the long bones were evident (bone cross-sectional area −7.2% [p < 0.01] at the midshaft and −11.0% [p < 0.01] at the metaphysis), as measured by peripheral quantitative computed tomography. In agreement with these data and a decreased polar strength strain index (−14.0%, p < 0.01), the three-point bending breaking force of the femur was reduced by 10.3% (p < 0.01) in the 50 × C group. These data indicate that the negative skeletal effects appear at a subchronic vitamin A intake of somewhere between 10 and 50 times the standard diet. This level is considerably lower than previously reported. Our results suggest that long-term ingestion of modest excesses of vitamin A may contribute to fracture risk.     

Biological and clinical assessment of a new bisphosphonate, (chloro-4 phenyl)thiomethylene bisphosphonate, in the treatment of Paget's disease of bone

Several Biophosphonates have been used as therapeutic agents for Paget's bone disease. (Chloro-4 phenyl)thiomethylene-bisphosphonate (CIPsMBP) has recently been shown to have significant antiosteoclastic activity while an affect of CIPsMBP on mineralization was only observed at high doses. We tested this drug for 6 months in 23 pagetic patients distributed in three groups. Gr 1 (n = 5) receiving 200 mg/day showed a decrease of serum alkaline phosphatase (SAP) to 42 ± 4% (p < 0.01) of initial value (100%) while hydroxyprolinuria/creatinuria ratio (OH/Cr) dropped to 69 ± 8% of baseline. In 4 patients receiving 400 mg/day, SAP improved to 48 ± 9% of initial value (p < 0.01) and OH/Cr to 40 ± 3% (p < 0.01). In the last group (n = 14) receiving 200 mg/day for 3 months, and 400 mg/day thereafter up to the 6th month SAP decreased to 53 ± 4% and OH/Cr to 62 ± 6% of initial value (p < 0.01).

Clinical improvement was significant from the first month of treatment. No resistance (mean decrease of SAP lower than 30%) was recorded and no radiological or clinical evidence of mineralization defect appeared. The clinical and biological tolerance was excellent throughout the study.     

Clodronate increases the calcium content in fracture callus

Summary Eighty-eight rats underwent intramedullary pin fixation and fracture of both tibiae. Half of the animals were given clodronate 50 mg/kg s. c. weekly. Clodronate treatment did not affect the growth of fibrocartilage or the endochondral and membranous new bone formation. The regaining of tensile load capacity of fractured bone remained unaffected by the drug. Calluses were remodeled to lamellar bone in both groups. However, although the total area invaded by mineralized tissue in callus remained unaffected by the drug, the areas of hematopoietic bone marrow tissue within mineralized callus were observed to be markedly smaller in clodronate-treated animals than in controls. The calluses in the clodronate group were significantly heavier and contained more calcium at 2 months after fracture than those in the controls.     

Effects of nail rigidity on fracture healing

The present study was undertaken to assess the effects of the rigidity of nails on the healing of fractures as evaluated by mechanical strength and rate of bone mineralisation. Ten rats supplied biomechanical data at the start of the experiment and another 100 rats were randomly assigned to 5 groups. In 20 rats no intervention was performed, and they served as a reference group of normal values. Thus, 80 rats underwent a standardised partial osteotomy in the left femur diaphysis prior to manual fracture, reaming and intramedullary nailing. One group received rigid steel nails (group 1) and another, cannulated steel nails (group 2). Group 3 received semi-rigid titanium nails and group 4, soft polyethylene nails. Ten rats in each group were evaluated at 6 and 12 weeks after fracture, and radiographs and callus diameter measurements were performed. Dual-energy X-ray absorptiometry (DEXA) of the bones was performed, and the degree of mineralisation in the callus segment, distal diaphysis and total femur was calculated by the scanner. Subsequently, the bones were tested mechanically by a three-point bending test in a Mini Bionix (MTS) testing system. Radiographs revealed clearly visible fractures at 6 weeks in the intervention groups. At 12 weeks there were no signs of bridging callus in group 4, while the other groups presented scarcely visible osteotomies. The callus area in group 4 was significantly larger at both 6 and 12 weeks than in groups 1–3. In these groups there was a significant decrease in callus area between 6 and 12 weeks. Both maximum bending load, bending rigidity and fracture energy increased significantly in groups 1–3 between 6 and 12 weeks, while bending load and fracture energy increased in group 4. At 6 weeks no significant differences were detected between groups 1–3 in mechanical strength. Group 4 had a significantly reduced maximum bending load and fracture energy at 6 and 12 weeks as well as bending rigidity at 12 weeks. At 12 weeks group 3 had a significantly increased maximum bending load and fracture energy compared with groups 1 and 2. Bone mineral count (BMC) in the callus region in group 4 was significantly reduced at both 6 and 12 weeks. BMC in the callus showed no significant differences between groups 1–3, either at 6 or at 12 weeks. BMC in the distal diaphysis was significantly reduced in groups 1 and 4 compared with group 2 at 12 weeks. Bone mineral density (BMD) in the callus region revealed no significant differences between groups 1–3 at 6 weeks, while at 12 weeks BMD was higher in group 3 than in group 1. BMD in the callus region and total femur in group 4 was significantly reduced at 6 weeks. This study demonstrates that diaphyseal fractures treated with titanium nails with a bending rigidity similar to the intact femur have a higher maximum bending load and fracture energy at 12 weeks than both rigidly or softly nailed fractures. BMD in the callus region was also highest after titanium nailing at 12 weeks. BMD in the distal diaphysis and total femur indicates that the degree of stiffness of the nails have little influence on total bone mineralisation at 12 weeks.     

Transforming growth factor-β enhances fracture healing in rabbit tibiae

The ability of exogenous Transforming Growth Factor-β (TGF-β) to stimulate bone formation in fracture healing was investigated. TGF-β was continuously applied in doses of 1 and 10 μg/day for 6 weeks to 2 groups of adult rabbits with unilateral plated midtibial osteotomies. A group receiving solvent without TGF-β served as control. Fracture healing was evaluated by mechanical tests, bone morphometry and bone densitometry. Increased maximal bending strength and callus formation were demonstrated in the groups receiving TGF-β. TGF-β had no effect on bending-stiffness, bone mineral content, cortical thickness or haversian canal diameter. We conclude that local application of exogenous TGF-β may enhance fracture healing in rabbits.     

Effects of torsional rigidity on fracture healing: strength and mineralization in rat femora

OBJECTIVE: To assess the effects of torsional rigidity and dynamization on fracture healing in a medullary nailed rat femoral model. STUDY DESIGN: Randomized study in male Wistar rats with a diaphyseal osteotomy/fracture. METHODS: Reamed cannulated nailing was performed in sixty rats. One group (twenty rats) received unlocked nails (UL group) and another group received nails with proximal and distal locking that was dynamized after twenty days (DL group). The third group was statically locked (SL group). A fourth group of twenty rats served as the control group. After randomization, the femurs of ten rats in each group at six and twelve weeks were studied clinically, radiologically, and biomechanically, and bone mineralization was measured by dual-energy x-ray absorptiometry (DEXA). RESULTS: Radiographs in two planes revealed clearly visible fractures at six weeks, and at twelve weeks bridging callus was apparent in all three treatment groups. The callus area in the UL group was significantly larger at six weeks than in the other groups, and at twelve weeks the UL and DL groups had larger callus areas than the SL group. Biomechanically, UL nails had reduced maximum bending load at six and twelve weeks, while DL nails had increased fracture energy at six weeks compared with SL nails. Bone mineral content and bone mineral density in the callus segment and diaphysis were increased with DL nails at six weeks. CONCLUSION: This animal study indicates that (a) interlocked nailing has a beneficial effect on bone healing and (b) although dynamization may have a beneficial effect on the quality of early bone healing, (c) dynamization does not increase the rate of bony union. The clinical implication is that routine early dynamization of locked femoral fractures may not be indicated, reserving dynamization to cases of delayed union.     

Low dose of propranolol does not affect rat osteotomy healing and callus strength

Experimental studies suggest that the β‐blocker propranolol stimulates bone formation but little work has investigated its effect on fracture healing. In this study, we examined if a low dose of propranolol, previously shown to be preventive against bone loss in rats, improves bone repair. Female Wistar rats were injected with saline or propranolol (0.1 mg/kg/day) (n = 20/group), 5 days a week for 8 weeks. Three weeks after the beginning of treatment, all rats underwent a mid‐diaphyseal transverse osteotomy in the left femur. Radiographic analysis of ostetomy healing was performed 2 and 5 weeks after osteotomy. Rats were sacrificed at 5 weeks and femora collected for measurements of fracture strength by torsional testing, callus volume, and mineral content by micro‐CT analysis and histology of fracture callus. Eighty nine percent of osteotomies achieved apparent radiological union by 5 weeks in both groups. Propranolol treatment did not significantly alter the torsional strength of the fractured femur compared with controls. The volume and mineralization of fracture callus at 5 weeks were not significantly different in both groups. Histology showed that endochondral ossification was not affected by propranolol. Altogether, our results demonstrate that propranolol using the regimen described does not significantly improve or inhibit rat osteotomy healing and mechanical strength. © 2014 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. J Orthop Res 32:887–893, 2014.     

Optimal time for cardiomyocyte transplantation to maximize myocardial function after left ventricular injury

Background. This study was designed to determine the optimal time for cell transplantation after myocardial injury.

Methods. The left ventricular free wall of adult rat hearts was cryoinjured and the animals were sacrificed at 0, 1, 2, 4, and 8 weeks for histologic studies. Fetal rat cardiomyocytes (transplant) or culture medium (control) were transplanted immediately (n = 8), 2 weeks (n = 8), and 4 weeks (n = 12) after cryoinjury. At 8 weeks, rat heart function, planimetry, and histologic studies were performed.

Results. Cryoinjury produced a transmural injury. The inflammatory reaction was greatest during the first week but subsided during the second week after cryoinjury. Scar size expanded (p < 0.01) at 4 and 8 weeks. Cardiomyocytes transplanted immediately after cryoinjury were not found 8 weeks after cryoinjury. Scar size and myocardial function were similar to the control hearts. Cardiomyocytes transplanted at 2 and 4 weeks formed cardiac tissue within the scar, limited (p < 0.01) scar expansion, and had better (p < 0.001) heart function than the control groups. Developed pressure was greater (p < 0.01) in the hearts with transplanted cells at 2 weeks than at 4 weeks.

Conclusions. Cardiomyocyte transplantation was most successful after the inflammatory reaction resolved but before scar expansion.     

Lipopolysaccharide impairs fracture healing: an experimental study in rats

Background It has been shown that trauma causes translocation of lipopolysaccharide (LPS) endotoxins from the gut. LPS has been identified as a major bacterial bone resorbing factor. The effects of LPS on bone healing are therefore of clinical interest, as trauma involving fractures followed by sepsis is a clinical scenario. We investigated the effects of systemic and local administration of LPS on the healing of femoral fractures in rats.

Animals and methods In 3 groups, each consisting of 9 rats, a mid-diaphyseal osteotomy/fracture of the femoral bone was performed and then nailed. In one group of animals, LPS was applied intraperitoneally (systemically), and in another group, LPS was applied locally at the fracture site. The third group served as a control. The animals were killed after 6 weeks, and the mechanical characteristics of the healing osteotomies were evaluated.

Results We found that LPS induced a hypertrophic and immature callus, as evaluated by bone mineral content and density. In the rats given LPS intraperitoneally, the mechanical strength characteristics were reduced, as evaluated by bending moment, rigidity, and energy absorption.

Interpretation The rats given LPS intraperitoneally reflect a clinical situation with fracture trauma and endotoxinemia. Our findings indicate that endotoxinemia may impair the fracture healing processes.

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Human parathyroid hormone (1-34) accelerates natural fracture healing process in the femoral osteotomy model of cynomolgus monkeys

Several studies in rats have demonstrated that parathyroid hormone accelerates fracture healing by increasing callus formation or stimulating callus remodeling. However the effect of PTH on fracture healing has not been tested using large animals with Haversian remodeling system. Using cynomolgus monkey that has intracortical remodeling similar to humans, we examined whether intermittent treatment with human parathyroid hormone [hPTH(1–34)] accelerates the fracture healing process, especially callus remodeling, and restores geometrical shapes and mechanical properties of osteotomized bone.

Seventeen female cynomolgus monkeys aged 18–19 years were allocated into three groups: control (CNT, n = 6), low-dose PTH (0.75 μg/kg; PTH-L, n = 6), and high-dose PTH (7.5 μg/kg; PTH-H, n = 5) groups. In all animals, twice a week subcutaneous injection was given for 3 weeks. Then fracture was produced surgically by transversely cutting the midshaft of the right femur and fixing with stainless plate. After fracture, intermittent PTH treatment was continued until sacrifice at 26 weeks after surgery. The femora were assessed by soft X-ray, three-point bending mechanical test, histomorphometry, and degree of mineralization in bone (DMB) measurement. Soft X-ray showed that complete bone union occurred in all groups, regardless of treatment. Ultimate stress and elastic modulus in fractured femur were significantly higher in PTH-H than in CNT. Total area and percent bone area of the femur were significantly lower in both PTH-L and PTH-H than in CNT. Callus porosity decreased dose-dependently following PTH treatment. Mean DMB of callus was significantly higher in PTH-H than in CNT or PTH-L. These results suggested that PTH decreased callus size and accelerated callus maturation in the fractured femora.

PTH accelerates the natural fracture healing process by shrinking callus size and increasing degree of mineralization of the fracture callus, thereby restoring intrinsic material properties of osteotomized femur shaft in cynomolgus monkeys although there were no significant differences among the groups for structural parameters.     

Effects of oral clodronate on bone mineral density in patients with relapsing breast cancer

The high prevalence of bone metastases in breast cancer and the risk that spinal and femoral osteoporosis may add further morbidity provide a rationale for bisphosphonate therapy in patients with skeletal metastases from mammary carcinoma. We investigated the effects of oral clodronate given during 9 months, with a 24-month follow-up, on bone mineral density (BMD), on biochemical markers of bone remodeling, and on osseous complications in 67 women with documented relapsing breast cancer, aged 58.7 ± 1.5 years (x ± SEM). Patients with active cancer disease were randomly allocated to two groups, with or without clodronate treatment (1600 mg/day, orally). Twenty-six women considered in complete remission (52.4 ± 2.4 years) were also studied. Expressed in deviation from gender- and age-matched normals (z score), base-line BMD at the levels of lumbar spine (LS), femoral neck (FN), and midfemoral shaft (FS) was +0.10 ± 0.22 vs. −0.12 ± 0.25, +0.03 ± 0.19 vs. −0.54 ± 0.24, and +0.08 ± 0.14 vs. −0.02 ± 0.22, in patients with active breast cancer and in subjects in remission, respectively. After 9 months of treatment, fasting urinary calcium to creatinine ratio was lower (0.26 ± 0.04 vs. 0.40 ± 0.04 mmol/mmol creatinine, p < 0.02) and serum osteocalcin was stabilized (−2.1 ± 1.1 vs. +7.0 ± 3.3 μg/L, as compared with pretreatment values, p < 0.02), in the clodronate-treated group. The rate of osseous complications (pathological fracture, hypercalcemic episode, scintigraphic or radiological evidence of metastasis development, chemo- or radiotherapy for bone disease progression) was 28.8 events per 100 patient-year in the clodronate-treated group vs. 39.0 in controls, and 31.5 vs. 40.5, after 9 and 15 months of follow-up, respectively. In 15 women without evident LS bone metastasis (7 clodronate-treated and 8 controls), LS BMD increased in the clodronate-treated group by +5.2 ± 2.5% vs. −0.3 ± 1.4%, and +8.1 ± 4.7 vs. −0.9 ±1.7, after 10.3 ± 0.4 and 17.3 ± 1.2 months, respectively (p < 0.01), as compared with pretreatment values. These results indicate that clodronate treatment decreased bone turnover and attenuated cancer-related bone morbidity. In addition, clodronate increased LS BMD in apparently unaffected bone of women with relapsing breast cancer.     

Mechanical effects of function on bone healing. Nonweight bearing and exercise in osteotomized rats

The effects of different degrees of function and weight bearing on the healing of femoral osteotomies were studied in rats. A transverse osteotomy of the left femur was stabilized by intramedullary pinning. The rats were allocated to three groups: (1) tenotomy of the left achilles tendon to avoid weight bearing, (2) unrestricted weight bearing, and (3) a 4-week training programme 4 weeks after the osteotomy. After 8 weeks, the rats were killed; and callus production, bending moment, bending rigidity, and fracture energy at the osteotomy site were evaluated. There were no differences in the area of external callus. The bending moment was less in the nonweight-bearing rats. Bending rigidity and fracture energy were marginally less in the nonweight-bearing rats. There were no significant differences between the weight bearing and exercised rats. The results indicate that normal weight bearing stimulates bone healing, whereas activity above normal neither accelerates nor impairs this process.     

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%).