Calcitonin Producing Tumour: Effects on Fracture Repair and Normal Bone in Rats

The mechanical properties and the collagen metabolism of healing fractures and intact bones have been studied in rats with a transplanted, calcitonin (CT) secreting, medullary thyroid carcinoma (MCT). Sham operated animals served as controls. the MCT was transplanted beneath the kidney capsule. Seven months later, when the rats with MCT had increased circulating levels of CT, a standardized femoral fracture was produced in all the animals.

The serum levels of CT were 3-40 times higher in tumour bearing rats than in controls in the period following the fracture. the fracture strength of rats with MCT was reduced by about 60 per cent compared to controls at 16 weeks after the fracture. the strength of intact femora (ultimate torsional moment) seemed to be progressively impaired by increasing levels of circulating CT. Also the strength of bone as a material (ultimate torsional stress) was reduced in the rats with MCT. the collagen synthesis was reduced in MCT rats, but the amounts of collagen in fractured or intact bones were not changed compared to controls.

We conclude that chronic hypercalcitoninaemia due to MCT seems to have a negative influence both on fracture healing and on bone metabolism.     

Calcitonin Producing Tumour: Effects on Fracture Repair and Normal Bone in Rats

The mechanical properties and the collagen metabolism of healing fractures and intact bones have been studied in rats with a transplanted, calcitonin (CT) secreting, medullary thyroid carcinoma (MCT). Sham operated animals served as controls. the MCT was transplanted beneath the kidney capsule. Seven months later, when the rats with MCT had increased circulating levels of CT, a standardized femoral fracture was produced in all the animals.

The serum levels of CT were 3-40 times higher in tumour bearing rats than in controls in the period following the fracture. the fracture strength of rats with MCT was reduced by about 60 per cent compared to controls at 16 weeks after the fracture. the strength of intact femora (ultimate torsional moment) seemed to be progressively impaired by increasing levels of circulating CT. Also the strength of bone as a material (ultimate torsional stress) was reduced in the rats with MCT. the collagen synthesis was reduced in MCT rats, but the amounts of collagen in fractured or intact bones were not changed compared to controls.

We conclude that chronic hypercalcitoninaemia due to MCT seems to have a negative influence both on fracture healing and on bone metabolism.     

Influence of Age on Mechanical Properties of Healing Fractures and Intact Bones in Rats

Mechanical properties of fractured and intact femora have been studied in young and adult, male rats. A standardized, closed, mid-diaphyseal fracture was produced in the left femur, the right femur serving as control. The fracture was left to heal without immobilization. At various intervals, both fractured and intact femora were loaded in torsion until failure.

The fractured femora regained the mechanical properties of the contralateral, intact bones after about 4 weeks in young and after about 12 weeks in adult rats. For intact bones, both the ultimate torsional moment (strength) and the torsional stiffness increased with age of the animals, whereas the ultimate torsional angle remained unchanged. For bone as a material, however, the ultimate torsional stress (strength) and the modulus of rigidity (stiffness) increased with age only in young rats, being almost constant in the adult animals.

The various biomechanical parameters of the healing fractures did not reach those of the contralateral, intact bones simultaneously. The torsional moment required to twist a healing femoral fracture 20 degrees (0.35 radians), a deformation close to what an intact femur can resist, proved to be a functional and simple measure of the degree of fracture repair in rats.     

Congenital Coxa Vara and Perthes' Disease

Mechanical properties of fractured and intact femora have been studied in young and adult, male rats. A standardized, closed, mid-diaphyseal fracture was produced in the left femur, the right femur serving as control. The fracture was left to heal without immobilization. At various intervals, both fractured and intact femora were loaded in torsion until failure.

The fractured femora regained the mechanical properties of the contralateral, intact bones after about 4 weeks in young and after about 12 weeks in adult rats. For intact bones, both the ultimate torsional moment (strength) and the torsional stiffness increased with age of the animals, whereas the ultimate torsional angle remained unchanged. For bone as a material, however, the ultimate torsional stress (strength) and the modulus of rigidity (stiffness) increased with age only in young rats, being almost constant in the adult animals.

The various biomechanical parameters of the healing fractures did not reach those of the contralateral, intact bones simultaneously. The torsional moment required to twist a healing femoral fracture 20 degrees (0.35 radians), a deformation close to what an intact femur can resist, proved to be a functional and simple measure of the degree of fracture repair in rats.     

Mechanical Properties of Fractured and Intact Rat Femora Evaluated by Bending, Torsional and Tensile Tests

Mechanical properties of healing fractures and growing, intact bones were studied in male rats aged 8 weeks at the beginning of the study period. A standardized, closed fracture was produced in the middle of the left femur. The fracture was not immobilized. At various intervals after the fracture, the healing fractured femora and the contralateral, intact femora were subjected to bending, torsional and tensile tests.

The fractured femora regained the strength and the ultimate deformation of the contralateral, intact femora after about 8 weeks when tested in bending, and after about 13 weeks when tested in torsion. In the first phases of fracture repair, the healing fractures could resist more torsional than bending load, whereas the opposite was found for solidly consolidated fractures and intact bones.

For intact bones, the ultimate bending and torsional moments increased with increase in age and weight of the animals, whereas the ultimate angular deformation remained constant. The ultimate bending and torsional stresses (bone material strength) increased to reach a plateau when the rats were about 14 weeks old. No significant differences were observed between the bending, torsional and tensile test methods. For the evaluation of fracture repair, each test has its particular application.     

Effects of salmon calcitonin on synthesis and mineralization of collagen in rats

The effects of salmon calcitonin (CT) on collagen metabolism and mineral deposition in fractures and intact femora, and on collagen metabolism in healing skin wounds and intact skin have been studied in young male rats. Serum calcium and serum phosphorus were reduced 3 h after the daily subcutaneous CT injection (3 MRC-U/kg body weight), whereas a rebound increase in the serum levels of both minerals was observed at 24 hours after the injection. CT had an early transient inhibitory influence on the collagen synthesis, and this resulted in a reduced total content of collagen in bones and skin specimens from treated rats compared to controls. The concentration of collagen in bone and skin was, however, increased in treated animals compared to controls after prolonged CT administration. Following an early transient increase, the incorporation of strontium-85 into the fractured bones was impaired after 30 days of CT treatment. This resulted in a reduced mineral concentration in the fractures of treated rats compared to controls in the last part of the experiment. The recorded effects of CT treatment, which were most pronounced in healing fractures and intact skin specimens, may be interpreted as an inhibitory influence of CT both on synthesis, mineralization and degradation of collagen.     

Effects of Salmon Calcitonin on Synthesis and Mineralization of Collagen in Rats

The effects of salmon calcitonin (CT) on collagen metabolism and mineral deposition in fractures and intact femora, and on collagen metabolism in healing skin wounds and intact skin have been studied in young male rats. Serum calcium and serum phosphorus were reduced 3 h after the daily subcutaneous CT injection (3 MRC-U/kg body weight), whereas a rebound increase in the serum levels of both minerals was observed at 24 hours after the injection.

CT had an early transient inhibitory influence on the collagen synthesis, and this resulted in a reduced total content of collagen in bones and skin specimens from treated rats compared to controls. the concentration of collagen in bone and skin was, however, increased in treated animals compared to controls after prolonged CT administration.     

Decreased collagen organization and content are associated with reduced strength of demineralized and intact bone in the SAMP6 mouse.

To examine the link between bone material properties and skeletal fragility, we analyzed the mechanical, histological, biochemical, and spectroscopic properties of bones from a murine model of skeletal fragility (SAMP6). Intact bones from SAMP6 mice are weak and brittle compared with SAMR1 controls, a defect attributed to reduced strength of the bone matrix. The matrix weakness is attributed primarily to poorer organization of collagen fibers and reduced collagen content. INTRODUCTION: The contribution of age-related changes in tissue material properties to skeletal fragility is poorly understood. We previously reported that bones from SAMP6 mice are weak and brittle versus age-matched controls. Our present objectives were to use the SAMP6 mouse to assess bone material properties in a model of skeletal fragility and to relate defects in the mechanical properties of bone to the properties of demineralized bone and to the structure and organization of collagen and mineral. MATERIALS AND METHODS: Femora from 4- and 12-month-old SAMR1 (control) and SAMP6 mice were analyzed using bending and torsional mechanical testing of intact bones, tensile testing of demineralized bone, quantitative histology (including collagen fiber orientation), collagen cross-links biochemistry, and Raman spectroscopic analysis of mineral and collagen. RESULTS: Intact bones from SAMP6 mice have normal elastic properties but inferior failure properties, with 60% lower fracture energy versus SAMR1 controls. The strength defect in SAMP6 bones was associated with a 23% reduction in demineralized bone strength, which in turn was associated with poorer collagen fiber organization, lower collagen content, and higher hydroxylysine levels. However, SAMP6 have normal levels of collagen cross-links and normal apatite mineral structure. CONCLUSIONS: Bones from SAMP6 osteoporotic mice are weak and brittle because of a defect in the strength of the bone matrix. This defect is attributed primarily to poorer organization of collagen fibers and reduced collagen content. These findings highlight the role of the collagen component of the bone matrix in influencing skeletal fragility.     

Effects of acetylsalicylic acid on heterotopic bone resorption and formation in rats

The effects of acetylsalicylic acid (ASA) on bone metabolism have been studied in a bone transplantation model using radioisotopic and biochemical parameters. Isografts (femora) from infant inbred rats, extensively prelabeled with collagen and mineral-tracing radioisotopes, were transplanted to muscle pouches in young male rats. Bones from the opposite side of the donor rats served as nonimplanted reference bones. The recipients were given 150 mg/kg/12 h of ASA by gavage for 18 days. The serum concentrations obtained were comparable with the recommended anti-inflammatory levels in humans. Twenty-four hours before being killed the animals were labeled again with other collagen and mineral radioisotopes. After 18 days of medication the resorption of the transplanted bone was inhibited by about 15% in the ASA treated rats compared with controls, as measured by the losses of collagen (14C-hydroxyproline) and mineral (strontium-85). Also, the net gains of mineral and collagen in the ASA-treated transplants were reduced by about 15% and 11% respectively compared with controls during the medication period. During the last 24 h of the study the rates of mineral incorporation (calcium-47 uptake) and collagen synthesis (3H-hydroxyproline) were reduced to an even greater degree in the ASA-treated transplants. These results indicate an inhibitory effect of ASA on bone metabolism.     

Type 1 Diabetes in Young Rats Leads to Progressive Trabecular Bone Loss,Cessation of Cortical Bone Growth,and Diminished Whole Bone Strength and Fatigue Life

People with diabetes have increased risk of fracture disproportionate to BMD, suggesting reduced material strength (quality). We quantified the skeletal effects of type 1 diabetes in the rat. Fischer 344 and Sprague‐Dawley rats (12 wk of age) were injected with either vehicle (Control) or streptozotocin (Diabetic). Forelimbs were scanned at 0, 4, 8, and 12 wk using pQCT. Rats were killed after 12 wk. We observed progressive osteopenia in diabetic rats. Trabecular osteopenia was caused by bone loss: volumetric BMD decreased progressively with time in diabetic rats but was constant in controls. Cortical osteopenia was caused by premature arrest of cortical expansion: cortical area did not increase after 4–8 wk in diabetic rats but continued to increase in controls. Postmortem μCT showed a 60% reduction in proximal tibial trabecular BV/TV in diabetic versus control rats, whereas moments of inertia of the ulnar and femoral diaphysis were reduced ～30%. Monotonic bending tests indicated that ulna and femora from diabetic animals were ～25% less stiff and strong versus controls. Estimates of material properties indicated no changes in elastic modulus or ultimate stress but modest (～10%) declines in yield stress for diabetic bone. These changes were associated with a ～50% increase in the nonenzymatic collagen cross‐link pentosidine. Last, cyclic testing showed diminished fatigue life in diabetic bones at the structural (force) level but not at the material (stress) level. In summary, type 1 diabetes, left untreated, causes trabecular bone loss and a reduction in diaphyseal growth. Diabetic bone has greatly increased nonenzymatic collagen cross‐links but only modestly reduced material properties. The loss of whole bone strength under both monotonic and fatigue loading is attributed mainly to reduced bone size.     

Calcitonin and fracture healing. An experimental study on rats

The effects of systemically administered calcitonin (CT) on fracture healing were analyzed in an experimental study on rats. The healing of a fracture was followed from 3 days up to 9 weeks postoperatively. Half of the rats in each age group were given daily CT 10 MRC-U/kg body wt s.c. Mechanical properties of the healing tibial fractures (tension strength) as well as various connective tissue components of the callus tissue were analyzed. No difference in the radiological or microscopical appearance of the fractures was detectable between the animals receiving CT and the controls. In the biochemical analysis matrix production as assessed from the concentrations of nitrogen, hexosamines, and hydroxyproline within the callus followed the usual lines of undistributed fracture union without any difference between the groups with and without CT. No differences could be detected in the mineralization of the callus between the specimens from animals receiving CT and those without. The tensile strength values of the fractures increased almost linearly up to 9 weeks. At 1 week the tensile strength values for fractures union in the animals without CT were approximately 50% higher, but later on no differences could be detected between the groups. These results indicate that although in the early phases of long-term CT therapy collagen synthesis may be impaired, there will be no effect on the net content of collagen or calcifying tissue in the callus or on the mechanical strength of healing fractures.     

Accelerated fracture healing in the geriatric,osteoporotic rat with recombinant human platelet‐derived growth factor‐bb and an injectable beta‐tricalcium phosphate/collagen matrix

Aging and osteoporosis contribute to decreased bone mass and bone mineral density as well as compromised fracture healing rates and bone repair quality. Consequently, the purpose of this study was to determine if recombinant human platelet‐derived growth factor‐BB (rhPDGF‐BB) delivered in an injectable beta‐tricalcium phosphate/collagen matrix would enhance tibial fracture healing in geriatric (>2 years of age), osteoporotic rats. A total of 80 rats were divided equally among four groups: Fracture alone; Fracture plus matrix; Fracture plus matrix and either 0.3 mg/mL or 1.0 mg/mL rhPDGF‐BB. At 3 and 5 weeks, rats were euthanized and treatment outcome was assessed histologically, radiographically, biomechanically, and by micro‐CT. Results indicated rhPDGF‐BB‐treated fractures in osteoporotic, geriatric rats caused a statistically significant time‐dependent increase in torsional strength 5 weeks after treatment. The healed fractures were equivalent in torsional strength to the contralateral, unoperated tibiae. Data from the study are the first, to our knowledge, to underscore rhPDGF‐BB efficacy in an injectable beta‐tricalcium phosphate/collagen matrix accelerated fracture repair in a geriatric, osteoporotic rat model. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J. Orthop Res 26:83–90, 2008     

Biomechanical evaluation of early fracture healing in normal and diabetic rats.

Diabetes mellitus has been shown to alter the properties of bone and impair fracture healing in both humans and animals. The objective of this study was to document changes in the structural and material properties of intact bone and bone with healed fractures in diabetic rats compared with nondiabetic controls after 3 and 4 weeks of healing. Rods were inserted in the right femurs of control rats and rats with streptozotocin-induced diabetes, and the femurs were fractured in a standardized procedure and then allowed to heal for 3 and 4 weeks. After death, all femurs were mechanically tested to failure in torsion. The degree of healing was quantified for each animal by normalizing mechanical parameters for the femur with a healed fracture with those for the intact contralateral femur. At both time points of healing, diabetic rats exhibited inferior healing compared with that of control animals in terms of failure torque, failure stress, structural stiffness, and material stiffness of the femur with the healed fracture relative to the intact contralateral femur (p < 0.05). Our results demonstrate that the recovery of structural and material strength in femurs with healed fractures in diabetic rats is delayed by at least 1 week compared with that in controls.     

Effects of acetylsalicylic acid and naproxen on the mechanical properties of intact femora in rats

The influence of acetylsalicylic acid (ASA) and naproxen on growing bones was studied. Young male rats were used. The drugs were administered via gastric gavage twice a day for 9 or 18 days. Drug doses giving serum concentrations corresponding to ordinary anti-inflammatory steady-state levels in humans were used. There was a drug-related influence on the strength of the growing femur. After 9 days the ultimate bending moment of the distal femoral epiphyseal plate and ultimate torsional moment and stress of the femoral diaphysis increased by about 10% in the rats treated with 150 mg/kg/12 h of ASA as compared with controls. After 18 days there were no differences. The ultimate metaphyseal bending moment of the distal femur was not influenced after 9 days with this dose, but was reduced by about 10% compared with controls after 18 days. Doses of 100 mg/kg/12 h of ASA and 20 mg/kg/12 h of naproxen did not change the bone strength. The doses used were well tolerated and did not influence the bone growth or body weight of the rats. A naproxen dose of 40 mg/kg/12 h was lethal; rats that received this dose succumbed to jejunal perforations. The results indicate that ASA influences the remodeling of normally growing bones.     

The effect of PTH(1‐34) on fracture healing during different loading conditions

Parathyroid hormone (PTH) and PTH(1‐34) have been shown to promote bone healing in several animal studies. It is known that the mechanical environment is important in fracture healing. Furthermore, PTH and mechanical loading has been suggested to have synergistic effects on intact bone. The aim of the present study was to investigate whether the effect of PTH(1‐34) on fracture healing in rats was influenced by reduced mechanical loading. For this purpose, we used female, 25‐week‐old ovariectomized rats. Animals were subjected to closed midshaft fracture of the right tibia 10 weeks after ovariectomy. Five days before fracture, half of the animals received Botulinum Toxin A injections in the muscles of the fractured leg to induce muscle paralysis (unloaded group), whereas the other half received saline injections (control group). For the following 8 weeks, half of the animals in each group received injections of hPTH(1‐34) (20 µg/kg/day) and the other half received vehicle treatment. Fracture healing was assessed by radiology, dual‐energy X‐ray absorptiometry (DXA), histology, and bone strength analysis. We found that unloading reduced callus area significantly, whereas no effects of PTH(1‐34) on callus area were seen in neither normally nor unloaded animals. PTH(1‐34) increased callus bone mineral density (BMD) and bone mineral content (BMC) significantly, whereas unloading decreased callus BMD and BMC significantly. PTH(1‐34) treatment increased bone volume of the callus in both unloaded and control animals. PTH(1‐34) treatment increased ultimate force of the fracture by 63% in both control and unloaded animals and no interaction of the two interventions could be detected. PTH(1‐34) was able to stimulate bone formation in normally loaded as well as unloaded intact bone. In conclusion, the study confirms the stimulatory effect of PTH(1‐34) on fracture healing, and our data suggest that PTH(1‐34) is able to promote fracture healing, as well as intact bone formation during conditions of reduced mechanical loading. © 2013 American Society for Bone and Mineral Research.     

Increased mechanical strength of healing rat tibial fractures treated with biosynthetic human growth hormone

The effects of biosynthetic human growth hormone on the biomechanical properties of healing tibial fractures and intact bones in the rat were studied after 20 and 40 days of healing. Growth hormone, 2.0 mg per kg per day, was given subcutaneously in two daily doses. Control animals were injected with a corresponding volume of saline. After 20 days of fracture healing, there were no differences in mechanical properties between the healing fractures and intact bones. After 40 days, the ultimate load and maximum stiffness of the fractures of the b-hGH injected animals had increased to more than 400% of the corresponding values of the saline injected animals, and ultimate stress and energy absorption at ultimate load had increased to 270% compared with the saline injected animals. Ultimate load, stiffness, and energy absorption of the intact bones increased in the b-hGH injected animals, but no differences were found in ultimate stress values or normalized energy, indicating that the changes in the intact bones were quantitative phenomena.     

The mineral and mechanical properties of bone in chronic experimental diabetes

The long-term effects of experimentally induced diabetes on bone were studied in eight male Lewis rats, intravenously (i.v.) injected with 65 mg/kg of streptozocin (STZ) and maintained for 12 months. Eight untreated age-matched rats served as controls. In the STZ-treated rats, experimentally induced diabetes was documented by the presence of hyperglycemia at 24 h and at 3 and 12 months. Significantly less weight was gained and less growth occurred in the STZ-treated rats despite careful attention to feeding and hydration. Mineral alterations were detected in the bones of the animals with experimental diabetes. Decreased hydroxyapatite crystal perfection, decreased Ca/P of the ash, and decreased ash content in the tibial metaphyses with increased ash content in the tibial diaphyses, was noted relative to controls. Bone osteocalcin content was increased in the metaphyses of the STZ-treated rats. While absolute measures of stiffness, torsional strength and energy absorption were decreased in the bones of the STZ-treated animals, when torsional strength and stiffness were normalized for differences in both growth and geometry, the normalized stiffness values for the diabetic bones were increased. The results suggest that in experimental diabetes certain aspects of bone mineralization are adversely affected and lead to reduced strength-related properties. However, a compensatory increase in stiffness occurs. The reason for this increase, although not known, may be related to changes in bone crystal structure.     

Efficacy of monitoring long-bone fracture healing by measurement of either bone stiffness or resonant frequency: numerical simulation.

Development of noninvasive mechanical tests to monitor fracture healing has been hindered because relationships between bone geometry, measurement conditions, and fracture callus strength are not well understood. Beam theory was used to analyze the effects of fracture length, fracture location, end conditions, and fracture callus stiffness on mechanical properties (resonant frequency, bending stiffness, and torsional stiffness) of healing bone. Actual bone mineral geometry from a human tibia, quantified every 1 mm, was used in the beam analysis. Geometry of the fracture callus segment was uniformly scaled from the values for intact bone. Experimental tests on multisegmented machined rods were used to verify analytical methods. Mechanical properties of the healing bone initially increased very rapidly to 30-70% of the stiffness of intact bone, depending on the configuration. The increases then tapered off dramatically. Lateral bending stiffness was sensitive to changes in callus properties for a larger portion of the healing process than was either torsional stiffness or resonant frequency. Because callus strength increases at half the rate of callus stiffness, measures of whole-bone mechanical properties can provide insight into changes in callus strength until a maximum of less than one-half the strength of intact bone is regained. The analytical method presented is proposed for clinical use to develop individualized models of bone, fracture, and fixation conditions to identify early stages of healing. Because increases in whole-bone mechanical properties are small in the later stages of fracture healing, however, such measures must be used prudently beyond the initial stages.     

Bone grafting of cryosurgically treated bone defects: experiments in goats

It is hypothesized that cryosurgically treated bone defects are inappropriate host sites for cancellous bone grafting. The influence of autologous cancellous bone grafting on the healing of cryosurgically treated gap defects of long bones was investigated. A unilateral in vivo experiment was done to study bone strength and graft incorporation in the goat. The lining of a cylindrical defect of the femoral diaphysis was treated with a closed liquid nitrogen cryoprobe in 62 goats. Thirty-one animals received an impacted, morselized, cancellous bone graft harvested from the sternum. The other 31 animals served as controls. At 0, 4, 7, 10, 13, 16, and 26 weeks animals were euthanized and the femurs were evaluated for torsional strength, computed tomography, and histologic assessment. Specimens with a bone graft showed no significant increase in torsional strength with time compared to the controls. In all goats euthanized at 10 weeks or later, the graft was resorbed. The amount of bone apposition at the site of the cryosurgical lesion and the time at which the defect was bridged were similar in both groups. Autologous cancellous bone grafting does not accelerate healing of cryosurgically treated, stable, diaphyseal defects in the goat.     

Intact fibula improves fracture healing in a rat tibia osteotomy model.

Rat tibia fractures are often used in fracture healing studies. Usually the fracture is stabilized with an intramedullary pin, which provides bending stiffness, but little torsional stiffness. The objective of this research was to determine the in vitro torsional rigidity of an osteotomized tibia with and without the fibula, and to determine if this difference influences the healing process in vivo. In vitro eleven rat tibias received an osteotomy, were stabilized with an intramedullary pin, and were tested in internal rotation to determine the torsional rigidity. The fibula was then manually broken and the torsional rigidity measured again. In vivo 18 rats received a tibial osteotomy, eight of which had an additional fractured fibula. After three weeks, the rats were sacrificed and the tibias were analyzed. Bone density in the fracture callus was measured with qCT. Bending rigidity and maximum breaking moment were determined in three-point bending. In vitro testing demonstrated that the torsional rigidity with an intact fibula was nearly two times higher than when the fibula was fractured. Though the torsional rigidity was still small in comparison with an intact bone, it resulted in a significantly different healing process in vivo. Rats with intact fibulas had significantly higher bone mineral density, bending rigidity, and maximum breaking moment compared to rats with a fractured fibula. These results indicate that torsional stability considerably affects the healing process. In a fracture model, it is critical to characterize the mechanical environment of the fracture.