Structural and mechanical repair of diffuse damage in cortical bone in vivo.

Physiological wear and tear causes bone microdamage at several hierarchical levels, and these have different biological consequences. Bone remodeling is widely held to be the mechanism by which bone microdamage is repaired. However, recent studies showed that unlike typical linear microcracks, small crack damage, the clusters of submicron-sized matrix cracks also known as diffuse damage (Dif.Dx), does not activate remodeling. Thus, the fate of diffuse damage in vivo is not known. To examine this, we induced selectively Dif.Dx in rat ulnae in vivo by using end-load ulnar bending creep model. Changes in damage content were assessed by histomorphometry and mechanical testing immediately after loading (ie, acute loaded) or at 14 days after damage induction (ie, survival ulnae). Dif.Dx area was markedly reduced over the 14-day survival period after loading (p < 0.02). We did not observe any intracortical resorption, and there was no increase in cortical bone area in survival ulnae. The reduction in whole bone stiffness in acute loaded ulnae was restored to baseline levels in survival ulnae (p > 0.6). Microindentation studies showed that Dif.Dx caused a highly localized reduction in elastic modulus in diffuse damage regions of the ulnar cortex. Moduli in these previously damaged bone areas were restored to control values by 14 days after loading. Our current findings indicate that small crack damage in bone can be repaired without bone remodeling, and they suggest that alternative repair mechanisms exist in bone to deal with submicron-sized matrix cracks. Those mechanisms are currently unknown and further investigations are needed to elucidate the mechanisms by which this direct repair occurs.

Nitric oxide-mediated vasodilation increases blood flow during the early stages of stress fracture healing.

Despite the strong connection between angiogenesis and osteogenesis in skeletal repair conditions such as fracture and distraction osteogenesis, little is known about the vascular requirements for bone formation after repetitive mechanical loading. Here, established protocols of damaging (stress fracture) and nondamaging (physiological) forelimb loading in the adult rat were used to stimulate either woven or lamellar bone formation, respectively. Positron emission tomography was used to evaluate blood flow and fluoride kinetics at the site of bone formation. In the group that received damaging mechanical loading leading to woven bone formation (WBF), (15)O water (blood) flow rate was significantly increased on day 0 and remained elevated 14 days after loading, whereas (18)F fluoride uptake peaked 7 days after loading. In the group that received nondamaging mechanical loading leading to lamellar bone formation (LBF), (15)O water and (18)F fluoride flow rates in loaded limbs were not significantly different from nonloaded limbs at any time point. The early increase in blood flow rate after WBF loading was associated with local vasodilation. In addition, Nos2 expression in mast cells was increased in WBF-, but not LBF-, loaded limbs. The nitric oxide (NO) synthase inhibitor N(ω)-nitro-l-arginine methyl ester was used to suppress NO generation, resulting in significant decreases in early blood flow rate and bone formation after WBF loading. These results demonstrate that NO-mediated vasodilation is a key feature of the normal response to stress fracture and precedes woven bone formation. Therefore, patients with impaired vascular function may heal stress fractures more slowly than expected.

Rapid remineralization of the distal radius after forearm fracture in children.

BACKGROUND: Bone mineral content (BMC) and density (BMD) have been shown to diminish after fracture and immobilization in adults. Distal radius fractures are common in children, and unlike adults, there is a low incidence of refracture. The primary aim of this study was to assess the change in radial BMC and BMD after upper extremity fracture and casting in healthy pediatric patients. METHODS: Patients were recruited at the time of distal radius fractures casting. The nonfractured (non-Fx) distal radius was initially scanned by dual energy x-ray absorptiometry (baseline), and then both arms were scanned at the time of cast removal (CastOff), and 4, 8, 12, 24, and 52 weeks post CastOff. RESULTS: Twenty-one patients were enrolled (13 male, 13 Caucasian; 10.4±2.5 y) with an average length of casting of 38±11 days. Eighteen patients (86%) completed all protocol requirements. At CastOff, there was no significant difference in total BMC or BMD between the Fx and non-Fx arms. From CastOff to 24 weeks, the overall change in BMC and BMD for the non-Fx arm was +4.2% and +0.2%, respectively, whereas for the Fx arm, the change was +8.3% and +3.4%, respectively. By 24 weeks, the difference in the overall change in BMD between the Fx and non-Fx arms was statistically significant (greater than instrumental error; P<0.05). However, by 52 weeks, these differences were no longer significant. The increased mineralization was unrelated to age, sex, arm dominance, or calcium intake. CONCLUSIONS: These data show that there is rapid remineralization after a simple forearm fracture in children, with a transient elevation in BMD in the Fx arm after casting. This novel finding suggests that bone may be stronger around the site of fracture and could significantly change how we counsel young patients recovering from forearm fracture. Future research should focus on children immobilized for varying lengths of time and those with repeat fractures, using volumetric techniques of bone geometry and strength assessment. CASE SERIES: Therapeutic Studies-investigating the results of treatment, Level IV.

Healing of non-displaced fractures produced by fatigue loading of the mouse ulna.

We developed a fatigue loading protocol in mice to produce a non-displaced ulnar fracture in vivo, and characterized the early healing response. Using adult (5 month) C57Bl/6 mice, we first determined that cyclic compression of the forelimb under load-control leads to increasing applied displacement and, eventually, complete fracture. We then subjected the right forelimbs of 80 mice to cyclic loading (2 Hz; peak force approximately 4N) and limited the displacement increase to 0.75 mm (60% of the average displacement increase at complete fracture). This fatigue protocol created a partial, non-displaced fracture through the medial cortex near the ulnar mid-shaft, and reduced ulnar strength and stiffness by >50%. Within 1 day, there was significant upregulation of genes related to hypoxia (Hif1a) and osteogenesis (Bmp2, Bsp) in loaded ulnae compared to non-loaded, contralateral controls. The gene expression response peaked in magnitude near day 7 (e.g., Osx upregulated 8-fold), and included upregulation of FGF-family genes (e.g., Fgfr3 up 6-fold). Histologically, a localized periosteal response was seen at the site of the fracture; by day 7 there was abundant periosteal woven bone surrounding a region of cartilage. From days 7 to 14, the woven bone became denser but did not increase in area. By day 14, the woven-bone response resulted in complete recovery of ulnar strength and stiffness, restoring mechanical properties to normal levels. In the future, the fatigue loading approach can be used create non-displaced bone fractures in transgenic and knockout mice to study the mechanisms by which the skeleton rapidly repairs damage.

The effect of zymosan and the protective effect of various antioxidants on fracture healing in rats.

AIM: To investigate the effects of free oxygen radicals and various antioxidants on bone healing after experimental formation of fracture. MATERIALS AND METHODS: Fifty male rats were used and divided into five groups (ten rats in each). The right forelimbs of the rats were broken by bimanual compression method. One hour before this procedure, 5 ml/kg of intraperitoneal (i.p.) physiologic saline were given to the control Group 1. All 40 rats in the experimental Groups 2, 3, 4 and 5 were treated with i.p. zymosan at a dosage of 100 mg/kg to induce the production of free radicals by stimulating NADPH oxidase in polymorphonuclear leukocytes. Zymosan induction was stopped on the fifth post-fracture day. In addition to the zymosan, i.p. 1 g/kg/day of dimethyl sulfoxide were given to the animals in Group 3, 50 mg/kg/d of Ginko biloba Extract (EGb 761) in Group 4 and 500 mg/kg/day of vitamin C in Group 5. Radiographs of the fractures of all animals were obtained to assess callus formation, remodeling and bridging bone formation under ether anesthetics on postfracture day 7, 14 and 21. All rats were euthanized on day 22, and sections of the radius and ulna were examined both histologically with light and electron microscopy and ultrastructurally. Statistical analysis was made with Kruskal-Wallis variance analyze test and comparison between groups was performed by Dunn's multiple comparison test. RESULTS: An impairment of bone healing was observed in Group 2 inducted with purely zymosan. Variable results were obtained for bone healing in the groups treated with various antioxidants. There was very significant difference of fracture healing between Groups 1 and 2 both histologically and radiologically (P < 0.001). There was significant difference between Groups 2 and 5 radiologically (P < 0.05). CONCLUSION: Free oxygen radicals demonstrate a negative effect on fracture healing and vitamin C (an antioxidant) partially prevents the negative effect of zymosan on fracture healing.

Unravelling the secrets of foetal wound healing: an insight into fracture repair in the mouse foetus and perspectives for clinical application.

This study was designed to investigate the nature of mammalian foetal fracture healing in utero. A compound 'pinch' fracture was created in the foetal mouse ulna at the end of the second trimester, prior to mineralisation, and healing observed in whole mount limbs and in histological section. Cartilaginous ends gained initial contact within a perichondrial sleeve by 24 h. Bony union was achieved within 48 h by cartilage remodelling during the phase of primary endochondral ossification in the limb, a process to which adult fracture healing aspires. The molecular response to wounding was investigated using a whole mount in situ hybridisation technique and antisense mRNA probes to three target genes (BMP-2, BMP-4 and GDF-5). These experiments failed to identify altered expression in wounded limbs compared with controls.

Systemic hormonal, electrolyte, and substrate changes after non-thermal limb injury in children.

Relatively little is known regarding the hormonal changes after injury in children. Adult protocols are often applied to children, although the latter often have different physiological responses to trauma. Twenty children with an angulated displaced fracture of the radius and/or ulna (injury severity score 9) were studied prospectively for changes in adrenaline, noradrenaline, cortisol, angiotensin II, arginine vasopressin, urea, electrolytes, and glucose. Two blood samples were taken: one an arrival at the accident and emergency department and one preoperatively several hours later. There were marked increases in adrenaline, noradrenaline, cortisol, and arginine vasopressin above the normal range. Five (25%) cases demonstrated greater early increases in adrenaline than those reported for adult injuries of similar severity. Early hypokalaemia in four cases had corrected towards normal within a few hours, without potassium supplementation.

[Several indicators of oxidative processes in fractures of the long bones]. / O nekotorykh pokazateliakh okislitel'nykh protsessov pri perelomakh dlinnykh kostei.

The authors have observed 55 patients with fresh fractures, with disturbances in consolidation (delayed consolidation, non-union of fractures, false joints) and with defects of diaphyses of the long bones. In the fracture area the dynamics of oxidation-reduction potential (ORP) was studied and volarographic investigations (determination of the volumetric speed of the local blood flow by hydrogen, a test with oxygen load) were carried out. Certain correlation between the quantity and the dynamics of the values under study and the state of the consolidation process was established. Coincidence of the dynamics of ORP and the local blood flow, presence of the quantity of volumetric speed of the local blood flow of 25-30 ml/min per 100 g of tissue with rather high ORP and a positive test under oxygen load in the polarographic study were prognostically favourable. The applied methods allow to control the course of the process of consolidation of the fragments and to a certain extent prognosticate the results of treatment of the patients with fractures of the long bones.

The effect of calcitonin on fracture healing.

Corrective osteotomy of the long bones may be required in patients with Paget's disease. Experiments on rabbits suggest that the safety of the procedures is of concern while the patient receives Calcitonin. Twenty-four rabbits were divided into 4 groups of 6 animals. Half of each group received maintenance doses of Calcitonin subcutaneously. In all animals the left and right ulnae were osteotomized through middiaphysis. The right ulna was fixed with a "mini" dynamic compression plate. The left ulna was not immobilized. Each group of animals was sacrificed at successive intervals of 3 weeks. Histologically, no difference was found in the healing of the fracture of test or control animals. Radiographically, the fracture line persisted longer in animals receiving Calcitonin (6 weeks vs 3 weeks). All fractures healed by 9 weeks. The fracture line disappeared more rapidly in all the immobilized bones. Based on these observations, administration of Calcitonin is not a contraindication for corrective osteotomy in Paget's disease. Osteotomies should be rigidly fixed to maintain the correction achieved surgically until healing is established.

Bone mineral content in the forearm after fracture of the upper limb.

The bone mineral content was measured by gamma absorptiometry in patients who had sustained fractures of the upper limb. Measurements were taken on the shafts of the forearm and in the trabecular bone immediately proximal to the wrist. Fractures of the surgical neck of the humerus and fractures of the shaft of the humerus did not significantly influence the bone mineral content of the ipsilateral forearm. Fracture of the radius and the ulna, however, caused a 15% loss of the bone mineral content in the trabecular bone proximal to the wrist, whereas the mineral content on the shafts of the fractured forearms increased or decreased depending on the fracture site. The findings suggest that measurements of bone mineral content of the forearm are not necessarily invalidated if there has been a fracture of the same limb. Only if the forearm itself is fractured should measurements on this site be avoided.