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.     

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.     

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.     

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.     

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.     

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.     

The Healing of Experimental Fractures by Compression Osteosynthesis: I. Torsional Strength

Biomechanical properties of osteotomized rabbit tibio-fibular bones fixed with 6-hole stainless steel AO/DCP plates were investigated with torsional loading 3 to 24 weeks postoperatively.

During the first 9 weeks maximum torque capacity, energy absorption and torsional rigidity increased, reflecting progressive bony union between the fractured bone ends. From 9 to 24 weeks the values of torque capacity and energy absorption decreased, whereas torsional rigidity seemed to reach a steady state without further significant changes. For the three parameters considered, the mean percentage differences between the osteotomized plated bones and their paired sham-operated controls were 69, 64 and 80 per cent, respectively.

The results suggest that internal fixation of fractured bones provides conditions for undisturbed fracture healing, but that subsequently the rigid nature of the implant has an adverse effect on the cortical bone, which slowly loses strength. Thus the optimal time for removal of the plate seems to be shortly after the fracture has healed and before the bony tissue has been weakened by secondary changes, such as cancellous transformation and spatial rearrangement of the tubular bone.     

Increase in Plasma Calcitonin Following Femoral Fracture in Rats

The secretion of calcitonin (CT) has been studied in male rats following a standardized, left femoral fracture after administration of subcutaneous fluanisone and fentanyl citrate anaesthesia. Twenty-five minutes after the fracture, the plasma concentrations of CT were increased by about 20 per cent in young and by about 60 per cent in adult rats compared to prefracture levels of the hormone. Three weeks later, plasma CT had decreased and was not significantly different from prefracture levels.

Anaesthesia combined with femoral fracture did not influence plasma calcium significantly, whereas the plasma concentrations of calcium increased in young control rats during 35 minutes of anaesthesia alone. Plasma CT, however, remained unchanged in these control rats in the same period.

In rats with a transplanted, CT-secreting, medullary thyroid carcinoma, femoral fracture did not alter the already high plasma concentrations of CT. It is suggested that increase in CT secretion is part of a general response to trauma.     

Studies on Mechanical Strength of Bone: I. Torsional Strength of Normal Rabbit Tibio-Fibular Bone

A method is described for testing torsional strength in small cortical bones at low velocities. The linearity and precision of the testing equipment, and the effect of the dimensions and chemical composition of the bone on torsional properties are analysed.

Rabbit tibio-fibular bones exhibited right-to-left differences in energy absorption capacity (up to 10.0 per cent), torsional rigidity (7.1 per cent), torque moment at fracture (6.3 per cent) and angular deformation (5.7 per cent). The scatter in energy absorption was more dependent on differences in angular deformation (strain) than on differences in torque moment (stress). No statistically significant dominance of either side could be observed.

The biomechanical properties of the bones were dependent on the body weight of the test animal and the transverse dimensions of the bone, but were not influenced by the small variations measured in the chemical composition of normal bone.     

Studies on Mechanical Strength of Bone: II. Torsional Strength of Cortical Bone after Rigid Plate Fixation with and without Compression

Biomechanical properties of intact rabbit tibio-fibulae were investigated with torsional loading 1 day to 36 weeks alter fixation using 4-hole stainless steel ASIF/DCP plates with and without compression.

During the first 12 weeks energy absorption and torque moment increased slightly as a result of subperiosteal new bone formation following application of the plates. There was a concomitant increase in the yielding properties of the bone, reflecting progressive porotic transformation. Thereafter, up to 36 weeks postoperatively, the values for energy absorption, torque moment and angular deformation gradually declined in both groups of bones. The differences between normal control bones and plated bones at 36 weeks were 70.1, 53.0 and 26.3 per cent, respectively. The decay of torsional strength was the same whether or not compression was used in the plate fixation.

The results suggest that even in normal bone rigid plates induce a considerable loss of strength due to the cancellous transformation they are known to cause in cortical bone.     

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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Restoration of bone flow following fracture and reaming in rat femora

In rats, bilateral closed femoral fracture was produced after intramedullary reaming to 1.6 mm on the left side and 2.0 mm on the right side. The fractures were fixed with 1.6 and 2.0 mm steel pins. Radioactive microspheres were used to determine bone blood flow at 30 min, 1 day, 3 days and 9 days after fracture. 8 rats were used to estimate normal bone blood flow, and an additional 8 rats to examine the vascular effects of fracture only.

Following fracture, total bone blood flow was reduced to about 50 percent and cortical flow to about 40 percent of that in intact bones. Fracture and reaming to 1.6 mm reduced total bone flow to 40 percent and reaming to 2.0 mm reduced the total bone flow to approximately one third of normal flow. Cortical flow decreased to about one third and one quarter in the 2 groups. On Day 1, total flow was practically normalized in both groups. Cortical flow in the 1.6 mm group was about equal to that of intact bones, while it was about one third of normal flow in the 2.0-mm group, and significantly less than the 1.6-mm group. On Day 3, total bone flow was more than double that of intact bones and cortical flow 3 times greater in both groups. Flow continued to increase to Day 9 when a threefold increase in total bone blood flow and approximately a fivefold increase in cortical flow were found. On Day 9, a separate callus area was defined and flow measurement revealed a rich vascularized callus in both groups, but no differences between the groups were found.

Following fracture, neither moderate nor aggressive intramedullary reaming seem to create any further impairment in bone flow. Following fracture and reaming, blood flow is rapidly restored, however, extensive reaming results in a delayed restoration of cortical bone blood flow. After 9 days, rich vascularized callus areas were found irrespective of the initial degree of reaming.     

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 graded reaming on fracture healing: Blood flow and healing studied in rat femurs

In 30 rats, closed bilateral fractures of the femur were produced. On the left side intramedullary reaming was performed to 1.6 mm, and the fracture fixed with a steel pin with a diameter of 1.6 mm. On the right side the femoral canal was reamed to 2.0 mm and a hollow steel tube with a diameter of 2.0 mm was used for fixation. An additional 8 rats were used to obtain mechanical, dimensional and flow data on intact femurs, and another 10 rats were used to study the acute flow changes caused by fracture and different degrees of reaming and fracture.

Fracture and reaming reduced total bone and cortical bone blood flows to about one third of normal flow, with no differences between the 1.6-mm and the 2.0-mm reamed bones. At 4 weeks, total bone flow was about double and cortical bone flow about 4 times increased in the 1.6-mm group. In the 2.0-mm reamed bones increases of approximately 5 times in total bone flow and of about 7 times in cortical flow were found. Callus flow was about twice the size of the respective cortical flow in both groups. Both total and cortical flows gradually subsided, without differences between the 2 groups. At 12 weeks, the callus area in the 2.0-mm group was greater than in the 1.6-mm group, while bone dimensions were greater in the 2.0-mm group at 4 and 12 weeks. Bending moment and rigidity were greater in the 1.6-mm group than in the 2.0-mm one at every time interval; no differences were found in fracture energy.

We conclude that, in terms of healing, modest reaming is preferable to extensive reaming. The adverse effect of extensive reaming is not due to excessive flow derangement at the acute stage or to impaired vascularity at the phase of remodeling.     

Torsional Strength of Cortical and Cancellous Bone Grafts After Rigid Plate Fixation

Mechanical properties of cortical and cancellous interposition grafts in rabbit tibio-fibular bones fixed with 6-hole DCP/ASIF plates were tested with torsional loading after intervals of 3 to 52 weeks postoperatively.

In the cortical grafts maximum torque moment at fracture, energy absorption capacity and rigidity increased from 3 to 12 weeks, while the cancellous grafts were more plastic with lower rigidity, higher angular deformation and higher energy absorption.

From 12 to 52 weeks maximum torque moment at fracture, energy absorption, rigidity and angular deformation decreased in grafts of both types, the respective means at 36 weeks being 39, 34, 57 and 82 per cent of the control values for the cortical grafts, and 26, 17, 42 and 58 per cent of the control values for the cancellous grafts. The differences between the torsional properties of the two graft types decreased with time.     

Effect of Instability on Experimental Fracture Healing

Bilateral tibial osteotomy with fracturing of the fibula was performed on ten Wistar rats weighing 300-350 g. Intramedullary nailing was performed with 1.4 mm nails after reaming. On the left side solid stainless rods were used, while on the right side the nails had a middle part made of titanium-nickel wire covered with polyvinyl-chloride (PVC), giving the nail a high degree of flexibility.

After 8 weeks, nine of the ten flexible nails showed fracture of the central wire. The continuity was, however, maintained by the PVC tube. The bones with flexible nailing always showed hypertrophic callus while there was only scanty callus on the side with rigid nailing.

Strength, deformation at fracture and stiffness were measured in a three-point bending test after removal of the solid nails and the fibulae. The strength of the tibiae was greatest on the side with flexible nailing, as was the deformation at fracture. The mean stiffness was higher in the bones with rigid nails, but the difference here was not statistically discernible.     

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.     

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