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.     

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.     

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.     

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.     

An experimental study on fracture healing in paraplegic rats

Fracture healing in denervated limbs was studied using paraplegic rats of Wistar stain. Femoral fractures were made at the same time as spinal cord injury or at regular intervals after spinal cord injury, for roentgenological and histological observation. In the former, proliferation and differentiation of osteogenic cells derived from the periosteum was almost the same as controls, with earlier bone union than controls. In the latter, with longer intervals between spinal cord injury and fracture, osteogenic cells were less proliferated and differentiated resulting in scant callus or delayed union. The environment of paralytic limbs was evidently altered substantially from 2 to 3 weeks after spinal cord injury, because thereafter fracture healing seemed to become poor. Circulatory disturbance plays a major role in fracture healing in paralytic limbs. Although healing is accelerated by increased circulatory volume at the acute phase of spinal cord injury, this potentiality is gradually decreased because of the regressive degeneration of long-term vasomotor nerve insufficiency.     

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。     

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.     

Bolus or weekly zoledronic acid administration does not delay endochondral fracture repair but weekly dosing enhances delays in hard callus remodeling

INTRODUCTION: It has been widely assumed that osteoclasts play a pivotal role during the entire process of fracture healing. Bisphosphonates (BPs) are anti-catabolic agents commonly used to treat metabolic bone diseases including osteoporosis, minimizing fracture incidence. Yet, fractures do occur in these patients and the potential for negative effects of BPs on healing has been suggested. We aimed to examine the effect of different dosing regimes of the potent BP zoledronic acid (ZA) on early endochondral fracture repair and later callus remodeling in a normal bone healing environment. METHODS: Saline, a Bolus dose of 0.1 degrees mg/kg ZA or 5 weekly divided doses of 0.02 degrees mg/kg of ZA commenced 1 week post operatively in a rat closed fracture model. Samples at 1, 2, 4 and 6 weeks post fracture were used to analyze initial fracture union, and 12 and 26 weeks post fracture to investigate the progress of remodeling. RESULTS: ZA did not alter the rate of endochondral fracture union. All fractures united by 6 weeks, with no difference in the progressive reduction of cartilaginous soft callus between control and treatment groups over time. ZA treatment increased hard callus bone mineral content (BMC), volume and increased callus strength at 6 and 26 weeks post fracture. Hard callus remodeling commenced at 4 weeks post fracture with Bolus ZA treatment but was delayed until after 6 weeks in the Weekly ZA group. By 12 and 26 weeks, Bolus ZA had equivalent callus content of remodeled neo-cortical bone to the Saline controls, whereas Weekly ZA remained reduced compared to Saline controls at these times (P<0.01). Callus material properties such as peak stress were significantly reduced in both ZA groups at 6 weeks. At 26 weeks, Bolus ZA-treated calluses generated peak stress equivalent to control values, whereas Weekly ZA callus peak stress remained significantly reduced, indicating remodeling delay. CONCLUSIONS: Osteoclast inhibition with ZA does not delay endochondral fracture repair in healthy rats. Bolus ZA treatment increased net callus size and strength at 6 weeks while allowing hard callus remodeling to proceed in the long term, albeit more slowly than control. Prolonged bisphosphonate dosing during repair does not delay endochondral ossification but can significantly affect remodeling long after the drug is ceased.     

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.     

Site-specific CGRP innervation coincides with bone formation during fracture healing and modeling: A study in rat angulated tibia.

Sensory neuropeptide involved in local bone turnover is known, but poorly understood. In the present study, we analyze the occurrence of neuronal CGRP during healing and modeling of straight and angular tibial fractures in 74 rats. Bone healing and modeling was assessed by radiography and reinnervation by semi-quantitative immunohistochemistry method at fracture site between 1-12 weeks postfracture. The regenerating nerve fibers containing CGRP were observed in fracture callus as well as in close proximity to chondrocytes, with woven bone in both fractures already at week 1. Notably, it located predominantly on the concave side of angulated fracture in the manner of sprouting into bone from weeks 3 to 5 postfracture. In both fractures, fracture calluses peaked radiographically at week 3 postfracture. In angulated fracture, a reduction of 11% in callus thickness on convex side and an increase of 365% on concave side were noted from weeks 3 to 12. A 27-fold increase in total neuronal CGRP in straight fracture and 38-fold increases in angular fracture compared to intact bone was observed at week 3. In both types of fracture, neuronal CGRP was greater on the concave side than the convex; this difference was more pronounced in the angulated fracture. CGRP immunoreactivity clearly coincides with amount of new bone formation especially on the concave side of angulated fracture. The combined results suggest that fracture evokes an intense, localized in-growth of new nerve fibers containing CGRP, which may prove to be a prerequisite of fracture healing and modeling.     

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.     

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     

Calcium-dependent neutral proteinase (calpain) in fracture healing in rats

Calpain refers to Ca²⁺-dependent neutral cysteine proteinase, which originally was thought to be an intracellular proteinase but recently has been shown to function extracellularly as well. This report describes the immunohistochemical demonstration of calpain and biochemical changes in the amount of calpain during fracture healing in rats. The tibiae of 6-week-old Wistar rats were fractured, and calluses were obtained 5–28 days after fracture. A frozen section of the fracture callus was stained by the immunoperoxidase method with use of polyclonal antibodies of calpains I and II. Positive staining was noted with the anti-calpain II antibody in the perivascular areas, chondrocytes, and cartilage matrix in calluses at 5, 7, and 10 days. Less intense staining was seen in older calluses. The caseinolytic activity of calpain II reached its maximum on the 5th day, was high on the 7th and 10th days, and decreased rapidly thereafter. The quantity of calpain II was dependent on the process of fracture healing. It was concluded that calpain was working as one of the matrix proteinases in fracture callus.     

Spatial and temporal gene expression in chondrogenesis during fracture healing and the effects of basic fibroblast growth factor.

Chondrogenesis is an essential component of endochondral fracture healing, though the molecular and cellular events by which it is regulated have not been fully elucidated. In this study, we used a rat model of closed fracture healing to determine the spatial and temporal expression of genes for cartilage-specific collagens. Furthermore, to determine the effects of basic fibroblast growth factor (bFGF) on chondrogenesis in fracture healing, we injected 100 microg recombinant human bFGF into the fracture site immediately after fracture. In normal calluses, pro-alpha1(II) collagen mRNA (COL2A1) was detected in proliferative chondrocytes beginning on day 4 after the fracture, and pro-alpha1(X) collagen mRNA (COL10A1) in hypertrophic chondrocytes beginning on day 7. In FGF-injected calluses, the cartilage enlarged in size significantly. On day 14, both COL2A1- and COL10A1-expressing cells were more widely distributed, and the amounts of COL2A1 and COL10A1 mRNAs were both approximately 2-fold increased when compared with uninjected fractures. Temporal patterns of expression for these genes were, however, identical to those found in normal calluses. The number of proliferating cell nuclear antigen-positive cells was increased in the non-cartilaginous area in the bFGF-injected calluses by day 4. The present molecular analyses demonstrate that a single injection of bFGF enhances the proliferation of chondroprogenitor cells in fracture callus, and thus contributes to the formation of a larger cartilage. However, maturation of chondrocytes and replacement of the cartilage by osseous tissue are not enhanced by exogenous bFGF, and this results in the prolonged cartilaginous callus phase. We conclude that, in the healing of closed fractures of long bones, exogenous bFGF has a capacity to enlarge the cartilaginous calluses, but not to induce more rapid healing.     

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.     

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.     

Programmed removal of chondrocytes during endochondral fracture healing

This investigation tested the hypothesis that the removal of chondrocytes during endochondral fracture healing involves an ordered process of programmed cell death. To accomplish this, unilateral closed fractures were created in the femora of 36 Sprague-Dawley rats. The rats were killed in groups of four on days 1, 3, 7, 14, 21, 28, 42, 49, and 56 after fracture. The femora were embedded in paraffin and tested for expression of specific markers of fragmented DNA with use of a terminal deoxyuridyl transferase-mediated deoxyuridine triphosphatebiotin nick end labeling (TUNEL) technique. To determine the potential for trans–differentiation of chondrocytes to osteoblasts calluses were also hybridized to detect expression of osteocal in mRNA. Cell proliferation was assessed by an immunohistochemical detection method for proliferating cell nuclear antigen. A separate group of four rats was killed on day 28 to represent the later stage of the endochondral ossification, and the calluses were examined for cellular morphology with transmission electron microscopy. The results showed a coordination in both time and space of the activities of cellular proliferation and programmed cell death. Cell proliferation was most active in the earlier phases of fracture healing (days 1 through 14) although TUNEL expression was apparent in hypertrophic chondrocytes on day 14 after fracture and persisted until day 28. In the later stages of fracture healing (days 14 through 28), proliferating cell nuclear antigen was no longer synthesized in hard callus (intramembranous bone) and cell removal was the dominant activity in soft callus chondrocytes. Expression of osteocalcin mRNA was detected in osteoblasts but not in hypertrophic chondrocytes or in any other nonosteoblastic cell type. These findings support the hypothesis that the removal of chondrocytes during endochondral fracture healing is part of an ordered transition of tissue types in which the cellular mechanisms are genetically programmed to involve proliferation, maturation, and apoptotic cell death.     

Osteogenic growth peptide modulates fracture callus structural and mechanical properties

The osteogenic growth peptide (OGP) is a key factor in the mechanism of the systemic osteogenic response to local bone marrow injury. Recent histologic studies have shown that OGP enhances fracture healing in experimental animals. To assess the effect of systemically administered OGP on the biomechanical and quantitative structural properties of the fracture callus, the present study used an integrated approach to evaluate the early stages (up to 4 weeks) of healing of unstable mid-femoral fractures in rats, which included biomechanical, micro-computed tomographic (microCT) and histomorphometric measurements. During the first 3 weeks after fracture, all the quantitative microCT parameters increased in the OGP- and vehicle-treated animals alike. After 4 weeks, the volume of total callus, bony callus, and newly formed bone was approximately 20% higher in animals administered with OGP, consequent to a decrease in the controls. The 4-week total connectivity was 46% higher in the OGP-treated animals. At this time, bridging between the fracture ends by newly formed bone was observed predominantly in the OGP-treated fractures. After 3 and 4 weeks, the OGP-treated animals showed higher biomechanical toughness of the fracture callus as compared to the PBS controls. Significant correlations between structural and biomechanical parameters were restricted to the OGP-treated rats. These data imply that the osteogenic effect of OGP results in enhanced bridging across the fracture gap and consequently improved function of the fracture callus. Therefore, OGP and/or its derivatives are suggested as a potential therapy for the acceleration of bone regeneration in instances of fracture repair and perhaps other bone injuries.     

雌激素对去势SD雌性大鼠骨折愈合影响的实验研究

为初步探讨雌激素与骨折愈合关系及其作用机制,采用SD雌性大鼠,分为A、B、C三组,后两组去势,21d后造成动物左胫腓骨闭合骨折,C组用雌激素处理,于不同时期分别行AKP测定、组织学检查、X线检查及红外光谱测定。结果显示: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显著差异,骨痂形成后期B组骨痂疏松,而C组小梁粗大,呈编织、板层化趋势。可见,雌激素在SD雌性大鼠骨折愈合早期起重要调节作用,骨痂形成早期轻度抑制软骨骨痂形成,骨痂形成后期促进小梁骨增生及其向编织骨的转化。     

Inhibition of endochondral ossification during fracture repair in experimental hypothyroid rats.

Using a rat fracture model, we investigated the effects of a decrease in serum levels of thyroid hormone on the fracture-repair process. Rats were divided into the following groups: (a) controls, (b) those treated with methimazole for the duration of the experiment, and (c) those treated with methimazole and L-thyroxine, receiving both for the same duration. Three weeks after the initiation of pharmacologic treatment, closed femoral fractures were produced. The formation of cartilage tissue in the fracture callus in all rats was not obviously different on day 7 after fracture. In the rats treated with methimazole, differentiation from proliferating to hypertrophic chondrocytes in the fracture callus was less advanced and vascular invasion was clearly inhibited on day 12. Gene expression of alkaline phosphatase and osteocalcin in the callus was significantly lower in these rats than in the controls on days 10, 12, and 14. The mechanical properties of the fracture callus were also significantly weaker in these animals than in the controls on day 21, resulting in impaired fracture repair. These results demonstrate that hypothyroidism inhibits endochondral ossification, resulting in an impaired fracture-repair process. L-thyroxine replacement in the rats treated with methimazole caused the impaired repair process to revert to normal. These results indicate that thyroid hormone is one of the critical systemic factors for fracture repair.