Effect of nerve injury on fracture healing: Callus formation studied in the rat

In an attempt to determine the effects of peripheral nerve lesions on fracture healing, radiographic, histomorphometric and chemical methods were used to evaluate callus formation in tibial fractures of rats with sciatic denervation. Fracture union by bridging external callus was more rapid in denervated limbs than in controls. By contrast, external calluses of denervated fractures were smaller and less dense and contained less collagenous matrix (hydroxyproline) and minerals (calcium, phosphorus) than controls. The RNA/DNA ratio decreased more rapidly in denervated calluses than in controls. Mineralization of collagenous matrix (estimated from the calcium/hydroxyproline ratio) was not affected by denervation.     

Fracture healing in paraplegic rats

In paraplegic rats, histomorphometric and chemical methods were used to evaluate callus production in tibial fractures stabilized by intramedullary nails. There were no differences in the mean sizes of fracture callus between paraplegic rats and their non-weight-bearing and weight-bearing controls. However, the variance of callus size was large in paraplegic rats. The concentration of nitrogen was high in calluses of paraplegic rats during the cartilaginous stage of healing. The hydroxyproline concentration did not differ between the groups. The rate of callus ossification was more rapid in paraplegic rats than in controls, but fracture calluses of paraplegic rats showed delayed accumulation of calcium and incomplete maturation of woven new bone. The results suggest that there are both mechanical and non-mechanical factors affecting callus formation in fractures below a spinal lesion.     

Fracture healing in paraplegic rats

In paraplegic rats, histomorphometric and chemical methods were used to evaluate callus production in tibial fractures stabilized by intramedullary nails. There were no differences in the mean sizes of fracture callus between paraplegic rats and their non-weight-bearing and weight-bearing controls. However, the variance of callus size was large in paraplegic rats. The concentration of nitrogen was high in calluses of paraplegic rats during the cartilaginous stage of healing. The hydroxyproline concentration did not differ between the groups. The rate of callus ossification was more rapid in paraplegic rats than in controls, but fracture calluses of paraplegic rats showed delayed accumulation of calcium and incomplete maturation of woven new bone. The results suggest that there are both mechanical and non-mechanical factors affecting callus formation in fractures below a spinal lesion.     

Biochemical Changes in Bone Grafts Stabilized with Rigid Plates: I. Cancellous Grafts

The effect of rigid plate fixation on the chemical composition of cancellous interposition grafts was studied in rabbit tibio-fibular bones.

The concentrations of hexosamines and, to a lesser degree, of hydroxyproline and nitrogen, were high in the graft for the first 6 weeks, decreased from weeks 6 to 12, but remained higher than the corresponding values for the controls throughout the experiment (52 weeks). The ratio of hexosamines to hydroxyproline was highest for the graft at 3 weeks, indicating formation of cartilage and osteoid.

The initially low calcium concentration of the graft increased by 35 per cent from weeks 1 to 6, decreased from weeks 6 to 12, and remained below normal thereafter in comparison with corresponding values for the cortical host bone. The ratio of calcium to hydroxyproline increased throughout the experiment, reflecting maturation of the graft to lamellar bone.

Thus, biochemically the early incorporation of rigidly fixed cancellous interposition grafts resembles the healing of unimmobilized fractures by callus formation.     

Acidic fibroblast growth factor (aFGF) injection stimulates cartilage enlargement and inhibits cartilage gene expression in rat fracture healing

The effect of the administration of acidic fibroblast growth factor (aFGF) on normal fracture healing was examined in a rat fracture model. One microgram of aFGF was injected into the fracture site between the first and the ninth day after fracture either every other day or every day. aFGF-injected calluses were significantly larger than control calluses, although this does not imply an increased mechanical strength of the callus. Histology showed a marked increase in the size of the cartilaginous soft callus. Total DNA and collagen content in the cartilaginous portion of the aFGF-injected calluses were greater than those of controls, although the collagen content/DNA content ratio was not different between the aFGF-injected and control calluses. Fracture calluses injected with aFGF remained larger than controls until 4 weeks after fracture. The enlarged cartilaginous portion of the aFGF-injected calluses seen at 10 days after fracture was replaced by trabecular bone at 3 and 4 weeks. Northern blot analysis of total cellular RNA extracted separately from the cartilaginous soft callus and the bony hard callus showed decreased expression of type II procollagen and proteoglycan core protein mRNA in the aFGF-injected calluses when compared with controls. A slight decrease in types I and III procollagen mRNA expression was also observed. We concluded that aFGF injections induced cartilage enlargement and decreased mRNA expression for type II procollagen and proteoglycan core protein.     

Effects of delayed stabilization on fracture healing

Previous studies have revealed that delayed internal fixation can stimulate fracture callus formation and decrease the rate of nonunion. However, the effect of delayed stabilization on stem cell differentiation is unknown. To address this, we created fractures in mouse tibiae and applied external fixation immediately, at 24, 48, 72, or 96 h after injury. Fracture healing was analyzed at 10 days by histological methods for callus, bone, and cartilage formation, and the mechanical properties of the calluses were assessed at 14 days postinjury by tension testing. The results demonstrate that delaying stabilization for 24–96 h does not significantly affect the volume of the callus tissue (TV) and the new bone (BV) that formed by 10 days, or the mechanical properties of the calluses at 14 days, compared to immediate stabilization. However, delaying stabilization for 24–96 h induces 10–40× more cartilage in the fracture calluses compared with fractures stabilized immediately. These findings suggest that delaying stabilization during the early phase of fracture healing may not significantly stimulate bone repair, but may alter the mode of bone repair by directing formation of more cartilage. Fractures that are not rigidly stabilized form a significantly larger amount of callus tissue and cartilage by 10 days postinjury than fractures stabilized at 24–96 h, indicating that mechanical instability influences chondrocytes beyond the first 96 h of fracture healing. © 2007 Orthopaedic Research Society. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:1552–1558, 2007     

Early callus of fractured rib of rat contracts and relaxes ex vivo.

PURPOSE: Wound contraction is an essential process in early soft-tissue repair, yet contraction of callus in fracture repair has not been investigated previously. Fracture callus consists of several cell types, many of which may have the capacity to contract. Accordingly, the purpose of the present study was to (i) determine whether early soft fracture calluses contract and relax ex vivo and (ii) identify and locate the contractile protein, alpha smooth muscle actin (alphaSMA) in callus. METHODS: One non-weight-bearing rib was fractured in adult male rats under anaesthesia and 10 calluses were removed 5, 7 and 9 days later for examination. Force production by calluses was measured using a sensitive force transducer when callus preparations were immersed sequentially in solutions known to either contract or relax smooth muscle preparations. Calluses and unfractured rib were analysed for the presence of alphaSMA using Western Blot and immunohistochemical techniques. RESULTS: When immersed in normal Krebs-Henseleit solution (K-H; pH 7.4, 22 degrees C) 7 callus preparations contracted and 3 relaxed. The force response was phasic (3 calluses) or tonic (7 calluses). Subsequent immersion in Ca(2+)-free K-H resulted in no change in force in 4 calluses, a decrease in force (relaxation) in 3 calluses, and an increase in force (contraction) in 2 calluses when compared to the force in the preceding solution (K-H). The final incubation in a solution having a high [K+] (64 mM) partially relaxed 6 calluses, contracted 3 and produced no change in force in 1 callus compared to the final force of the callus in the Ca(2+)-free solution. Collagen (in the form of rat Achilles tendon), the major structural protein in soft fracture callus, relaxed in K-H and continued to relax during exposure to Ca(2+)-free K-H and to solutions having a high [K+]. Western Blot and immunohistochemical studies detected the presence of alphaSMA in calluses and (in particular) in osteoprogenitor cells of fibrous callus respectively, as well as its absence from unfractured rib. CONCLUSIONS: (i) Early, soft fracture callus is capable of contracting and relaxing, (ii) the responses of callus to K-H, Ca(2+)-free and high [K+] solutions are distinctly different from the responses of smooth muscle preparations reported in the literature, (iii) the cell types in callus, particularly osteoprogenitor cells in uncalcified, collagenous matrix, have an essential contractile protein, alphaSMA, to support the observed contraction and relaxation and (iv) the contraction of soft fracture callus may facilitate fracture repair by creating tension within the callus and drawing the fracture ends together.     

Determination of callus quantity in 4-week-old fractures of the rat tibia

In clinical practice the quantity of fracture callus is usually estimated on the basis of radiographs. In experimental studies on fracture healing, more exact chemical measurements (total protein, DNA, and mineral contents) have been used to quantitate the amount of callus. It is not known, however, how these two parameters correlate with each other. In this study we used different (radiographic, histomorphometric, and chemical) methods to evaluate the quantity of fracture callus in 4-week-old tibial fractures of rats stabilized by intramedullary nailing. The wet and dry weights and the nitrogen, collagen, calcium, phosphorus, and DNA contents of the calluses showed no correlation with the radiographic size of the calluses. There was a strong correlation (p less than 0.001), however, between radiographic and histomorphometric callus sizes. The mass of callus and the chemically defined parameters of callus production thus indicate different properties of the healing bone.     

Levofloxacin and trovafloxacin inhibition of experimental fracture-healing

We previously have shown that experimental fractures exposed to ciprofloxacin have diminished fracture healing. The purpose of this study was to assess the effect of levofloxacin and trovafloxacin on experimental fracture healing to test the hypothesis that diminished fracture healing is a quinolone class effect. Sixty-one male Wistar rats were divided into three groups, which received 25 mg/kg of levofloxacin twice daily for 3 weeks, 35 mg/kg of trovafloxacin twice daily for 3 weeks, or no treatment, beginning 7 days after production of closed, nondisplaced, bilateral femoral fractures. The mean peak serum concentrations of levofloxacin and trovafloxacin drawn 30 minutes after administration were 6.9 and 7.0 microg/mL, respectively. Radiographic, histologic, and biomechanical studies were used to evaluate fracture healing. Torsional strength testing of fracture callus exposed to levofloxacin and trovafloxacin revealed a decrease in strength (299 and 257 N-mm, respectively) as compared with controls (364 N-mm). Radiographs revealed significantly more advanced healing in control animals (Goldberg score of 2.1) compared with the fractures in the rats treated with levofloxacin and trovafloxacin (Goldberg score of 1.5 in both groups). Fracture calluses in the animals treated with levofloxacin and trovafloxacin showed a lower histologic grade (5.3 and 3.5, respectively) as compared with control animals (7.5) representing a less mature callus with the presence of more cartilage and less woven bone. These data suggest that experimental fractures systemically exposed to levofloxacin or trovafloxacin have diminished healing during the early stages of fracture repair. The administration of quinolones during early fracture repair may compromise fracture healing in humans.     

Improved healing of experimental long bone fractures in rabbits by delayed internal fixation.

Experiments in rabbits were conducted to test the clinical propositions that a) the risk of nonunion in certain long bone fractures can be sharply reduced by open reduction and internal fixation, and b) that there is an optimum critical period following trauma in which this procedure should be carried out to facilitate healing. Bilateral standard open fractures of rabbit radii were internally fixed by intramedullary K-wires immediately or at 5, 10, and 17 days after trauma. These bones were subjected to stress and histometric analyses at 4-56 days after the initial fractures. The observations suggested that the pace of fracture healing could be optimized by time-delay surgery. The most exuberant and strongest calluses developed when the bones were fixed 10 days after fracture, and the rate of healing was improved. Delay times of 5 or 17 days did not provide better healing than that achieved by an immediate operation. Callus quality, in terms of the proportion of fiber bone, lamellar bone, and cartilage, was similar in all groups. The tensile strengths of the bones during callus formation were highly correlated with the ratio of callus/cortical bone areas (p less than 0.01).     

Healing of fractures with freeze-dried cortical bone plates. Comparison with compression plating

The healing of fractures of the radius with internal fixation by stainless-steel compression plates was compared with fractures fixed with freeze-dried bone-plate allografts. Fractures fixed with metallic plates gained slightly less than half the biomechanical strength of the contralateral control bone and healed without noticeable external callus formation. Bone-plated fractures regained three-fourths of the biomechanical strength of controls and healed by forming an external callus. Bone-plate allografts were eventually incorporated in the host bone. Allograft plates were vascularized and remodeled into cancellous bone in the process of incorporation in the host bones.     

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.     

Influence of systemic bisphosphonate treatment on mechanical properties of BMP‐induced calluses in a rat fracture model: Comparison of three‐point bending and twisting test

The combination of autograft, BMP and bisphosphonate has been shown to produce strong calluses. In this study, without autograft we investigate the effect of bisphosphonate treatment on BMP‐induced calluses, both in bending and in rotation. Sprague–Dawley rats (n = 42) underwent femoral osteotomy and BMP‐7 treatment. At 2 weeks an injection of saline or bisphosphonate was administered. The animals were sacrificed after 6 weeks. Both femurs were tested in either three‐point bending or twisting. All femurs healed. BMP + bisphosphonate‐treatment led to larger calluses (p < 0.05) and in three‐point bending, higher ultimate force (p < 0.01) and greater stiffness (p < 0.05) than BMP alone. The BMP + bisphosphonate group was nearly 60% stronger than controls, while the BMP group did not reach the strength (p < 0.05) nor stiffness (p < 0.01) of the controls. In the twisting test, similar trends were found but less pronounced. Three‐point bending produced transverse callus associated fractures, whereas the twisting test produced spiral fractures, located in the structurally weaker distal femur. BMP + bisphosphonate‐treatment produces calluses that are mechanically superior to calluses induced by BMP alone, when tested both in three‐point bending and in twisting. For the mechanical evaluation of pharmacologically enhanced calluses with breaking strengths exceeding the native bone, the bending test is recommended. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:721–726, 2014.     

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.     

Comparison of growth-induced resorption and denervation-induced resorption on the release of [3H]tetracycline, 45calcium, and [3H]collagen from whole bones of growing rats

The major effect of immobilization during growth is a smaller bone mass induced by either an increased bone resorption or a decreased bone formation. Using a method of analyzing radioisotopic loss of [3H]tetracycline and [3H]collagen from bone prelabeled in vivo, we compared the amount of bone resorption due to immobilization with bone resorption induced by growth. One hind limb was denervated in growing male rats, 6 weeks of age, that had been chronically prelabeled with [3H]tetracycline, 45calcium, and [3H]proline. The total radioactivity of the whole femur and tibia/fibula from the denervated limb was compared with that from bones of the control limb at 0, 1, 2, 4, and 8 weeks after denervation. The effect of growth on bone formation was measured by net increases in bone length, volume, and mass of matrix and mineral. Experimental bones had a significantly smaller volume and mass. Bone resorption was much greater during growth modeling than during denervation. The additional bone resorption induced by denervation was a small fraction (one-fourth) of the resorption induced by growth. Denervation during growth resulted in less bone being formed due to a smaller gain in matrix and mineral mass as a result of a reduction in bone formation.     

感觉神经(元)对失神经骨骼肌超微结构保护作用的实验研究

目的观察感觉神经（元）对失神经骨骼肌超微结构的保护作用。方法60只SD大鼠,按手术先后顺序随机分成10组,每组6只。A组(对照组)肱二头肌完全失神经支配。B～E组(实验组)B组为失神经支配加感觉神经种植组,C组为失神经支配加感觉神经寄养组,D组为失神经支配加背根神经节种植组,E组为节前撕脱的感觉神经种植组。术后1、3个月取材,各组又分为二个时间组。用透射电镜观察肌肉超微结构的变化。结果与对照组相比,各实验组肌肉退变核数少、线粒体肿胀及肌质网扩张程度轻、毛细血管/肌纤维数比值大、间质胶原纤维和成纤维细胞少、肌丝肌节排列整齐。结论感觉神经（元）对失神经骨骼肌超微结构有保护作用。     

Mechanisms for the enhancement of fracture healing in rats treated with intermittent low-dose human parathyroid hormone (1-34).

Recent reports have demonstrated that intermittent treatment with parathyroid hormone (1-34) [PTH(1-34)] increases callus formation and mechanical strength in experimental fracture healing. However, little is known about the optimal dose required for enhancement of fracture repair or the molecular mechanisms by which PTH regulates the healing process. In this study, we analyzed the underlying molecular mechanisms by which PTH affects fracture healing and tested the hypothesis that intermittent low-dose treatment with human PTH(1-34) can increase callus formation and mechanical strength. Unilateral femoral fractures were produced and a daily subcutaneous injection of 10 microg/kg of PTH(1-34) was administered during the entire healing period. Control animals were injected with vehicle solution alone. The results showed that on day 28 and day 42 after fracture, bone mineral content (BMC), bone mineral density (BMD), and ultimate load to failure of the calluses were significantly increased in the PTH-treated group compared with controls (day 28, 61, 46, and 32%; day 42, 119, 74, and 55%, respectively). The number of proliferating cell nuclear antigen (PCNA)-positive subperiosteal osteoprogenitor cells was significantly increased in the calluses of the PTH-treated group on day 2, and TRAP+ multinucleated cells were significantly increased in areas of callus cancellous bone on day 7. The levels of expression of type I collagen (COLlA1), osteonectin (ON), ALP, and osteocalcin (OC) mRNA were increased markedly in the PTH-treated group and accompanied by enhanced expression of insulin-like growth factor (IGF)-I mRNA during the early stages of healing (days 4-7). The increased expression of COL1A1, ON, ALP, and OC mRNA continued during the later stages of healing (days 14-21) despite a lack of up-regulation of IGF-I mRNA. These results suggest that treatment of fractures with intermittent low dose PTH(1-34) enhances callus formation by the early stimulation of proliferation and differentiation of osteoprogenitor cells, increases production of bone matrix proteins, and enhances osteoclastogenesis during the phase of callus remodeling. The resultant effect to increase callus mechanical strength supports the concept that clinical investigations on the ability of injectable low-dose PTH(1-34) to enhance fracture healing are indicated.     

强骨胶囊联合鲑鱼降钙素针剂辅助治疗老年骨质疏松性压缩骨折临床观察

目的探讨强骨胶囊联合鲑鱼降钙素针剂辅助治疗老年骨质疏松性压缩骨折临床疗效及影响。方法 257例胸腰椎骨质疏松性骨折患者随机分为3组,观察组口服强骨胶囊同时肌肉注射鲑鱼降钙素注射液,对照I组口服强骨胶囊,对照Ⅱ组肌肉注射鲑鱼降钙素针剂。观察临床疗效,测定治疗前和治疗后骨密度(BMD),血骨钙素(BGP)、血钙、血磷浓度及碱性磷酸酶。尿液中尿钙,尿羟脯氨酸,尿肌酐的含量,并计算尿钙/尿肌酐与羟脯氨酸/尿肌酐比值。结果观察组总有效率明显优于对照I组和对照II组(P0.05)。治疗后,3组BMD明显升高,痛觉评分均明显降低(P0.01),尤其观察组更加明显(P0.05)。治疗后,3组骨钙素较治疗前有明显的升高(P0.05或0.01),碱性磷酸酶和羟脯氨酸/尿肌酐较治疗前有明显的下降(P0.05或0.01),尤其治疗组明显优于对照I组和对照II组。治疗后对照组I组和观察组血钙较治疗前有明显的下降(P0.05),但3组组间无差异性(P0.05)。治疗后,3组血磷和尿钙/尿肌酐较治疗前变化不明显,组间比较无差异性(P0.05)。结论骨胶囊和鲑鱼降钙素治疗老年骨质疏松性骨折疗效肯定,能缓解疼痛,促进愈合和降低骨折的再发生,无明显不良反应。     

The effects of testosterone propionate and methenolone enanthate on the healing of humeral osteotomies in the Wistar rat

A randomized blind prospective study was carried out to determine if an anabolic androgenic steroid with a high anabolic/androgenic ratio, Group A, (1/0.05) methenolone enanthate (me), compared to an anabolic/androgenic agent with a low anabolic/androgenic ratio, Group B, (1.0/1.0) testosterone propionate (tp), compared to a control, Group C, cottonseed oil (co), affected midhumeral osteotomy healing in 100 two-month-old female Wistar rats. The rats received 4 mg/kg me, 4 mg/kg te, and equal volumes of co weekly. The rats were sacrificed at 2, 4, and 6 weeks. The entire humerus with the healing osteotomy was carefully dissected until all soft tissue attachments were stripped. The healing callus was then subjected to (1) biochemical analysis (hexosamine, hydroxyproline, and calcium), (2) biomechanical testing (progressive distraction of the callus at 1 mm/min on an electrohydraulic materials test system, model 1331, Instron Corp, Canton, MA, and (3) histology. Results of the biochemical testing demonstrated that the percentage of calcium in the healing callus at 2 weeks in group B (tp) was 7.3 +/- 1.0, and this value was greater than that in group C (co), 4.8 +/- 1.6 (p greater than .01), and greater than that in group A (me), 5.6 +/- 0.6 (p greater than .01). At 4 weeks, the percentage of calcium in the callus in group B (tp) was 6.8 +/- 1.9, in group A (me) 7.3 +/- 3.7, and these values were both greater than that in group C (co), 3.9 +/- 2.2 (p greater than .02 and .01, respectively). At 6 weeks the percentage of calcium in the callus in group B (tp) was 11.7 +/- 3.9 and in group A (me) 12.7 +/- 3.9, and again these values were both greater than that in group C (co), 6.7 +/- 2.6 (p greater than .02 and .01, respectively). The remainder of the biochemical analysis, hexosamine and hydroxyproline content, did not show a statistical difference in groups A, B, and C at 2, 4, and 6 weeks. The biomechanical studies and histology also failed to show statistical differences between the three groups at 2, 4, and 6 weeks. The conclusion of this study is that an agent with a low androgenic activity does not increase calcium callus concentrations early in the course of fracture healing compared to an agent with higher androgenic activity. As healing progresses, both agents increase the concentration of calcium in osteotomy healing. The clinical significance of this study is that agents with low androgenic activities favorably influence osteotomy healing and may be clinically useful because they lack unwanted virilizing activity.     

雌激素对去势SD雌性大鼠骨折愈合早期骨痂形成影响的实验研究

目的：初步探讨雌激素在雌性ＳＤ大鼠骨折愈合早期的作用及其机制。方法：将ＳＤ大鼠分为Ａ、Ｂ、Ｃ３组,后２组摘除卵巢,２１ｄ后定支点折骨造成动物左胫腓骨闭合骨折,Ｃ组应用雌激素,分别于骨折后１、２、３、５周采取骨痂标本行ＡＫＰ测定,Ｘ线及组织学检查。结果：骨痂形成早期Ｂ组软骨骨痂较另两组稍多,但各组骨痂成熟程度无明显差异,骨痂形成后期Ｂ组骨痂结构疏松,而Ｃ组小梁致密,有编织、板层化趋势。结论：雌激素在ＳＤ大鼠骨痂形成早期轻度抑制软骨骨痂形成,骨痂形成后期有利于小梁骨形成、堆积及向编织骨的转化。