Inhibitory effects of tumor necrosis factor alpha on fracture healing in rats

Fracture healing, which involves a cascade of biological tissue responses, may be affected by various biochemical substances. One of these substances is tumor necrosis factor alpha (TNF). Studies were made on the effects of TNF on healing of fractured ribs of rats. Fracture healing was inhibited by daily administration of recombinant human TNF (400 μg/kg body weight per day, intraperitoneally) after fracture. The rate of union on day 20 was significantly lower in the TNF-treated group (4/18, 22.2%) than in the control group (14/18, 77.8%) (p < 0.001 by Chi-square test). Histological examination showed that TNF inhibited cartilagenous callus formation. On day 10, cartilage was seen in the gap zone and under the periosteum in the control group, but no cartilage formation was observed in the gap zone in 9 of 12 specimens from the TNF-treated group. On day 20, the fracture ends were united by newly formed bone in the control group, but mature fibrous tissue was seen in the gap zone, and bony or cartilagenous union was not achieved in the TNF-treated group. These results show that TNF inhibits cartilage formation in the early phase of bone induction in fracture healing and suggest that this effect of TNF is due to its inhibition of differentiation of mesenchymal cells into chondroblasts.     

Regulation of vascular endothelial growth factor on the expression of fracture healing,related factors

Objective ： To study the effect of vascular endothelial growth factor （VEGF）and anti-VEGF on the expression of fracture healing-related factors and observe pathological changes at fractured sites. Methods： Fracture models were established in 105 New Zealand white rabbits and they were randomly divided into control group, VEGF group and anti-VEGF group. The relevant factors expression at fractured sites was assayed and pathological changes were observed in decalcified samples at 8, 24, 72 hours and 1,3,5,8 weeks after fracture. Results： After application of VGEF, the expression of BMP appeared earlier and expression time lasted longer. On the contrary, anti-VEGF completely inhibited the expression of BMP. The fractured sites were filled with fibrous callus, cartilaginous callus and bony callus at the 3rd week and woven bone was constructed at the 5th week. Fracture healing was accomplished at the 8th week in VEGF group. In anti-VEGF polyclonal antibody group, cellular necrosis increased at early period. Continuous focal necrosis was seen in the fractured sites from the 1st week to 5th week. Vascularization reduced obviously at the 3rd week. Conclusions： Fracture healing is a result of mutual regulation and coordination among many factors. VEGF may be an important factor in fracture healing.     

脑源性神经营养因子在大鼠胫骨骨折愈合中的作用研究

目的通过研究脑源性神经营养因子(BDNF)对骨折愈合的影响,探讨神经营养因子在骨组织工程和临床治疗中的应用前景。方法使用SD大鼠胫骨横行骨折髓内针内固定模型,术后处理组每隔一天皮下注射BDNF,对照组注射生理盐水,分别在第2、3、4、5周取胫骨对骨折愈合行大体比较、力学测试和扫描电镜观察。结果术后2周两组骨折均为结缔组织连接;3周处理组已有编织骨愈合,对照组仍有明显的骨折端活动;4周两组均呈骨性愈合,但处理组的骨痂、成角畸形较小;5周时处理组骨折塑型好,对照组骨折端仍有较大骨痂。处理组骨折矢状面成角在第3、4、5周小于对照组(P<0.05)。各阶段处理组抗折应力均优于对照组(P<0.05)。结论早期应用BDNF对骨折愈合的各阶段均可能有促进作用。     

血管内皮生长因子对骨折愈合相关因子表达的调控

目的探讨应用和拮抗血管内皮生长因子(VEGF）对骨折愈骨相关因子表达的影响，并观察骨折愈台过程中的病理改变。方法用105只天上I=白兔制作骨折模型．随机分为对照组、应用VECF组和拮抗vEGF组，分别于伤后8、24、72b和1．3、5、8周测定各组动物骨折端相关因子的表达变化、同时取骨折端标本脱钙进行兜镜观察。结果应甩VEGF使骨折端埘BMP的表达时同提前和延良，拈抗VEGF抑制rBMP的表达，应用VEGF组伤后3J嗣时骨折端充填有纤维性骨痴，软骨骨痂和骨性骨痂．5周时新生骨以编织骨为主，8周时骨折正常愈合；括抗VH押导受伤后初期骨细胞坏死增多，1～5周各时相点骨折端均有灶性坏死出现，3同时骨折部位血管生成明显减少。结论骨折愈台过程需要多因子参与、协调，VEGF有可能是作为骨折愈台过程中的一种重要因子在发挥作用     

A standardized experimental fracture in the mouse tibia

The increased use of transgenic mice as experimental animals provides new opportunities to study the biology of fracture repair. We have developed a technique for the production of a standard closed experimental fracture in the mouse tibia. A 0.2 mm stainless-steel rod was introduced into the medullary cavity and the pre-nailed tibial shaft was fractured by an impact device, which resulted in a reproducible transverse or slightly oblique fracture pattern. The intramedullary rod maintained axial alignment, and the fractures united without displacement. On the basis of measurements of callus geometry, four-point bending tests, biochemical analyses, and quantitative histology, the progress of callus formation and remodeling occurred in a predictable sequence of healing phases. The ultimate bending loads of the fractures increased with time, reaching 74% of the strength of intact control tibias in 4 weeks. The stiffness values of the fractures returned to normal levels and, as determined radiographically, the fractures united by external callus in 4 weeks. Radiographically, callus size, cross-sectional callus area, and callus mass peaked at 2 weeks and decreased thereafter, indicating the start of external remodeling. Histologically, the amount of mesenchymal tissue was maximal at days 5 and 7. The callus cartilage area peaked at day 9; at its maximum, it accounted for 46% of the total callus area. Early periosteal formation of membranous new bone, followed by endochondral ossification, resulted in a linear increase of callus bone during the healing process. The healing sequence of the mouse tibial fracture was similar to that seen in the rat tibia. The major difference is the small size of the mouse, which makes the surgical technique and anesthetic procedures more demanding.     

Moderate soft tissue trauma delays new bone formation only in the early phase of fracture healing.

The aim of this study was to investigate the effect of a moderate soft tissue trauma to the course of fracture healing in a standardized animal model. Thirty-eight Wistar rats were randomly divided into a fracture group (F, n = 19) and a group with a fracture and a soft tissue trauma (F + STT, n = 19). The fracture and the soft tissue trauma were created using an impact device with a standardized energy. All fractures were stabilized by two Kirschner wires. Three rats were measured for blood flow and sacrificed at days 1, 3, 7, and 14, and seven rats at day 28, from both groups. A three-point bending test was performed on the healed tibia after 28 days. During the first 24 h there was a reduction in blood flow, which was more pronounced in the F + STT group than in the F group. From histological sections, the shape of the callus formation, as well as the tissue distribution of newly formed bone, fibrous cartilage and fibrous connective tissue were determined. Distinctly more periosteal new bone formed and a larger callus formed at days 3 and 7 in group F compared to group F + STT. However, by days 14 and 28, the ossification and overall callus size no longer showed differences between the two groups. A fast recovery of blood flow and callus formation took place in the F + STT group, which led to similar histological and biomechanical results in fracture healing observed after 28 days between the two groups.     

Fixation stability dictates the differentiation pathway of periosteal progenitor cells in fracture repair

This study compared fracture repair stabilized by intramedullary pin (IMP) or external fixation (EF) in GFP reporter mice. A modified IMP was used as control while EF utilized six needles inserted transversely through the tibia and into a segment of a syringe barrel. X‐rays taken at days 0, 14, and 35 showed that IMP resulted in significant three‐dimensional deformity with a large callus while EF showed minimal deformity and callus formation. Cryohistological analysis of IMP at day 14 confirmed a large ColX‐RFPchry+ callus surrounded by woven bone (Col3.6‐GFPcyan) and TRAP+ osteoclasts with mature bone (hOC‐GFPtpz) at the base. By day 35, cartilaginous components had been resorbed and an outer cortical shell (OCS) showed evidence of inward modeling. In contrast, the EF at day 14 showed no evidence of cartilage formation. Instead, periosteal‐derived osteoblasts (Col3.6‐GFPcyan) entered the fracture cleft and formed woven bone that spanned the marrow space. By day 35, mature bone had formed that was contiguous with the opposing cortical bone. Fracture site stability greatly affects the cellular response during repair and must be considered in the preclinical models that test therapies for improving fracture healing. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:948–956, 2015.     

Local administration of activin promotes fracture healing in the rat fibula fracture model.

Osteogenic activities of activin, a member of the transforming growth factor (TGF)-beta superfamily, have been demonstrated in both in vitro and in vivo studies. The present study investigates the effects of topical application of activin on fracture healing using rat fibula fracture models. Activin (0.4-10 microg/day) was injected locally to the fracture once a day for 2 weeks. Activin promoted callus formation in a dose-dependent manner and both callus volume and callus weight were significantly increased with doses of 2-10 microg/day activin. Also, 3 weeks of activin treatment increased the mechanical strength of the healing bone in addition to the callus mass. Histological study 2 weeks after the fracture revealed that activin promoted endochondral bone formation. Immunohistochemical examination of the fractured tibia revealed that activin was localized to osteoblasts and chondrocytes in the region ossified both endochondrally and intramembranously. These findings suggest that activin expressed during fracture healing promotes the healing process through an autocrine/paracrine mode of action.     

Topical application of nerve growth factor improves fracture healing in rats

The aim of the present study was to examine the effects of nerve growth factor on the healing of unsplinted fractured ribs. After fracture of a rib in male rats, nerve growth factor was delivered by a miniosmotic pump to the fracture site for 7 days at the rate of 1.4 μg/day. Callus catecholamine concentrations, hone callus size, histomorphometry, and biomechanical properties of the repairing rib were measured at 7,21, and 42 days after fracture. After 21 days, concentrations of norepinephrine and epinephrine were significantly increased in the group treated with nerve growth factor compared with those in the control group (211% norepinephrine and 322% epinephrine). Also, the midline longitudinal area of non osseous (fibrous tissue and cartilage) callus of the fracture was significantly smaller (54%) and had a higher proportion of cartilage in the treated group than in the controls. By 42 days, there was only bony callus between the fracture ends in both the control group and the treated group. The treated group, however, again showed significantly elevated concentrations of norepinephrine and epinephrine (286 and 382%, respectively) and significantly elevated breaking stress (50%) and Young's modulus (51%), together wilh a reduction in the transverse cross-sectional area of the repair site (57%). The resultant increases in effectiveness and rate of repair of bone with administration of nerve growth factor suggest that it may play an important role in the healing processes of fractured bone.     

Expression of connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) during fracture healing

Localization and expression of connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) during fracture healing in mouse ribs were investigated. In situ hybridization demonstrated that CTGF/Hcs24 mRNA was remarkably expressed, especially in hypertrophic chondrocytes and proliferating chondrocytes, in the regions of regenerating cartilage on days 8 and 14 after fracture. CTGF/Hcs24 mRNA was also expressed in proliferating periosteal cells in the vicinity of the fracture sites on days 2 and 8, and in cells in fibrous tissue around the callus on day 8. Northern blot analysis showed that expression of CTGF/Hcs24 mRNA was 3.9 times higher on day 2 of fracture healing than that on day 0. On day 8, it reached a peak of 8.6 times higher than that on day 0. It then declined to a lower level. Immunostaining showed that CTGF/Hcs24 was localized in hypertrophic chondrocytes and proliferating chondrocytes in the regions of regenerating cartilage, and in active osteoblasts in the regions of intramembranous ossification. Although CTGF/Hcs24 was abundant in the proliferating and differentiating cells (on days 8 and 14), immunostaining decreased as the cells differentiated to form bone (on day 20). CTGF/Hcs24 was also detected in cells in fibrous tissue, vascular endothelial cells in the callus, and periosteal cells around the fracture sites. These results suggest that CTGF/Hcs24 plays some role in fracture healing.     

Localization of nitric oxide synthases during fracture healing.

Previously, we have reported that nitric oxide synthases (NOSs), which generate NO, modulate fracture healing. However, the cellular sources of the NOS isoforms during the course of fracture healing have not been studied systematically. The purpose of this study was to localize the cellular distribution of NOS isoforms (inducible NOS [iNOS], endothelial NOS [eNOS], and neuronal NOS [bNOS]) by in situ hybridization and immunohistology after femoral fractures in rats. The iNOS signal was detected during the initial stages (on day 4 and day 7) of fracture healing in 52 +/- 2% (mean +/- SE, n = 7) of cells within the intramembranous region, along the edge of the periosteal callus. The iNOS signal in callus cells declined to an undetectable level on day 14. eNOS was detected during the middle stages (on day 7 and day 14) of fracture healing in cells lining the blood vessels and also in 49 +/- 3% of cells in the chondral region. The bNOS signal was found to be increased at the later stages (day 14 and day 21) of fracture healing in 51 +/- 3% of cells at the junction between fibrous tissue and cartilage within the fibrochondral region. In summary, the,expression of NOS isoforms during fracture healing was time dependent and cellular distinctive.     

Expression of metalloproteinase-13 (Collagenase-3) is induced during fracture healing in mice.

In fracture healing, a large amount of cartilage is formed, then rapidly replaced by osseous tissue. This process requires the transition of extracellular matrix component from type II to type I collagen. We investigated the expression of matrix metalloproteinase-13 (MMP-13), which has a high potential to cleave type II as well as type I collagen, during fracture repair in mouse ribs. In situ hybridization demonstrated that MMP-13 mRNA was present throughout the healing process. It was detected in the cells of the periosteum at day 1. As fracture callus grew, strong MMP-13 mRNA signals were detected in cells of the cartilaginous callus. In the reparative and remodeling phases, both hypertrophic chondrocytes and immature osteoblastic cells in the fracture callus expressed MMP-13 mRNA strongly. These cells were located adjacent to tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts at the sites of cartilage/bone transition. In osteoclasts, MMP-13 expression was not detected. The level of MMP-13 mRNA peaked at day 14 postfracture by northern blotting. Immunohistochemical staining showed that MMP-13 was detected primarily in hypertrophic chondrocytes. These results indicate that MMP-13 is induced during fracture healing. The site- and cell-specific expression of MMP-13 and its enzymatic property suggest that MMP-13 initiates the degradation of cartilage matrix, resulting in resorption and remodeling of the callus. In conclusion, MMP-13 plays an important role in the healing process of fractured bone in mice.     

Expression of smooth muscle actin in connective tissue cells participating in fracture healing in a murine model

The role of alpha-smooth muscle actin (SMA)-expressing fibroblasts in the contraction of skin wounds has been known for three decades. Recent studies have demonstrated that osteoblasts can also express the gene for this contractile muscle actin isoform and can contract a collagen-glycosaminoglycan analog of extracellular matrix in vitro. These findings provided rationale for the hypothesis that SMA-expressing cells contribute to fracture healing by drawing the bone ends together. To begin to test this hypothesis, immunohistochemistry was employed to evaluate the distribution of connective tissue cells expressing SMA in a mouse model of successful fracture healing. The results demonstrated that the majority of the cells comprising the mesenchymal tissue interposed between the fracture ends contained SMA after 7 and 21 days, supporting the working hypothesis. Most of the osteoblasts lining the surfaces of newly forming bone and the chondrocytes comprising the cartilaginous callus also expressed this contractile actin isoform. The maximal SMA expression extended from 7 to 21 days postfracture. The finding of high levels of SMA expression in connective tissue cells participating in fracture healing suggests that SMA-enabled contraction may be playing a role in the healing process. These results warrant further study of the specific SMA-dependent cell behavior.     

Effect of COX-2-specific inhibition on fracture-healing in the rat femur

BACKGROUND: Nonsteroidal anti-inflammatory medications have been shown to delay fracture-healing. COX-2-specific inhibitors such as celecoxib have recently been approved for human use. Our goal was to determine, mechanically, histologically, morphologically, and radiographically, whether COX-2-specific inhibition affects bone-healing. METHODS: A nondisplaced unilateral fracture was created in the right femur of fifty-seven adult male rats. Rats were given no drug, indomethacin (1 mg/kg/day), or celecoxib (3 mg/kg/day) daily, starting on postoperative day 1. Fractures were analyzed at four, eight, and twelve weeks after creation of the fracture. Callus and bridging bone formation was assessed radiographically. The amounts of fibrous tissue, cartilage, woven bone, and mature bone formation were determined histologically. Morphological changes were assessed to determine fibrous healing, callus formation, and bone-remodeling. Callus strength and stiffness were assessed biomechanically with three-point bending tests. RESULTS: At four weeks, only the indomethacin group showed biomechanical and radiographic evidence of delayed healing. Although femora from rats treated with celecoxib appeared to have more fibrous tissue than those from untreated rats at four and eight weeks, radiographic signs of callus formation, mechanical strength, and stiffness did not differ significantly between the groups. By twelve weeks, there were no significant differences among the three groups. CONCLUSIONS: Postoperative administration of celecoxib, a COX-2-specific inhibitor, did not delay healing as seen at twelve weeks following fracture in adult rat femora. At four and eight weeks, fibrous healing predominated in the celecoxib group as compared with the findings in the untreated group; however, mechanical strength and radiographic signs of healing were not significantly inhibited. Clinical Relevance: Many orthopaedists rely on narcotic analgesia for postfracture and postoperative pain, despite deleterious side effects and morbidity. Traditional nonsteroidal anti-inflammatory medications have been shown to delay fracture union. This effect may be smaller with COX-2-specific inhibitors.     

Comparison of accuracy of three-dimensional spiral computed tomography, standard radiography, and direct measurements in evaluating facial fracture healing in a rat model

Complex maxillofacial fractures demand a detailed understanding of the three-dimensional (3D) pattern of injury. Evaluation of the outcome of the facial fracture repair additionally requires optimal demonstration of fracture gap, bony union, fibrous callus or incorporation of fracture ends, presence of incomplete fusion, or pseudoarthrosis. Although 3D computed tomography (CT) is reliably used for the diagnosis of complex facial fractures, its value in facial fracture healing is unknown. An experimental study was conducted to determine the accuracy of 3D spiral CT scans in evaluating facial fracture healing during the early and late postoperative periods. In 10 adult Wistar Albino rats, a standardized bone defect (3 mm) was created within the mid portion of each zygomatic arch (n = 20). At 10 and 20 weeks postfracture, gap distance displayed by 3D CT and plane radiography (posteroanterior) were measured. At 20 weeks postfracture, intraoperative measurement was also performed. A comparison between 3D CT, radiography, and intraoperative findings was performed. At 10 weeks postfracture, the fracture sites displayed larger bone defects in imaging with 3D CT than with plane radiography. The difference between groups was statistically significant (P < 0.05). The mean defect size imaged by 3D CT was 0.91 +/- 0.82 mm (standard deviation) and by plane radiography was 0.42 +/- 0.16 mm. At 20 weeks postfracture, the mean defect size imaged by 3D CT was 0.56 +/- 0.64 mm, and by plane radiography was 0.38 +/- 0.22 mm. The difference between groups was not statistically significant (P > 0.05). The defect size imaged by both plane radiography and 3D CT was significantly less than the measurement obtained from the intraoperative assessment (P < 0.05). It was concluded that 3D CT has limited benefit in the detection of newly formed bone at week 10 and in the detection of fibrous callus, which can eventually give rise to the bony tissue. Plane radiography is more valid during the early postoperative period (week 10), because it can detect the fibrous callus and newly formed bone more precisely. Gap distance between fracture ends could be determined by 3D CT accurately at week 20, although there was a tendency, which was not statistically significant, to overestimate the amount of bone defect measured by 3D CT when compared with that of plane radiography.     

Repair of segmental bone defects in the rat: an experimental model of human fracture healing.

Bone repair models in animals may be considered relevant to human fracture healing to the extent that the sequence of events in the repair process in the model reflect the human fracture healing sequence. In the present study, the relevance of a recently developed segmental defect model in rat fibula to human fracture healing was investigated by evaluating temporal progression of rigidity of the fibula, mineral content of the repair site, and histological changes. In this model, a surgically created 2-mm-long defect was grafted with a 5-mm-long tubular specimen of demineralized bone matrix (DBM) by inserting it over the cut ends of the fibula. The temporal increase in rigidity of the healing fibula demonstrated a pattern similar to biomechanical healing curves measured in human fracture healing. This pattern was characterized by a short phase of rapidly rising rigidity during weeks 4-7 after surgery, associated with a sharp increase in the mineral content of the repair tissue. This was preceded by a phase of nearly zero rigidity and followed by a phase of slow rate of increase approaching a plateau. Histologically, chondroblastic and osteoblastic blastema originating from extraskeletal and subperiosteal (near fibula-graft junction) regions, infiltrated the DBM graft during the first 2 weeks. The DBM graft assumed the role of a "bridging callus." By weeks 6-8, most of the DBM was converted to new woven and trabecular bone with maximal osteoblastic activity and minimal endochondral ossification. Medullary callus formation started with direct new bone formation adjacent to the cortical and endosteal surfaces in the defect and undifferentiated cells in the center of the defect at 3 weeks. The usual bone repair process in rodents was altered by the presence of the DBM graft to recapitulate the sequential stages of human fracture healing, including the formation of a medullary callus, union with woven and lamellar bone, and recreation of the medullary canal.     

VEGF、TGF-β1在骨缺损不愈合中表达的实验研究

目的观察骨缺损不愈合和骨折愈合过程中血管内皮细胞生长因子(vascularendotheligrowthfactor,VEGF)和转化生长因子β1(transforminggrowthfactor,TGF-βl)的表达差异,研究骨缺损不愈合的分子机理,进一步探讨VEGF和TGF-β1对骨折愈合的影响。方法建立兔桡骨干骨缺损不愈合模型和骨折愈合模型,术后1、2、4、6、8、12周取标本,进行组织学观察,采用免疫组织化学的方法(SP法)检测两种模型中VEGF和TGF-β1的表达和变化。结果组织学观察发现,骨缺损组骨折断端只有少量膜内成骨过程,形成的血肿和肉芽组织明显增多,只有靠近骨折断端的一部分形成软骨细胞,其中的一部分在分化到肥大软骨细胞阶段时则停止分化,出现肥大软骨细胞区的增加,并向纤维结缔组织转化,未转化成软骨细胞的肉芽组织在后期也形成了纤维结缔组织。与骨折愈合模型相比,软骨内成骨过程中血管生成的量也明显减少,而且在骨缺损组的各时段,骨折断端间都有肌纤维侵入。免疫组织化学检查发现,VEGF和TGF-β1在骨缺损不愈合中仍有表达,但与正常骨折愈合相比,表达的量明显减少(P<0.05),VEGF和TGF-β1的表达呈同步关系,即在TGF-β1强表达时,VEGF的表达程度也很强,TGF-β1的表达弱时,VEGF的表达也明显减少。结论由于造成较大段的骨缺损,骨膜缺损很多,造成膜     

Oxidant status increased during fracture healing in rats

We evaluated oxidant status during bone healing in 50 rats. In 40 rats, the right tibia was fractured and fixed intramedullarly (study leg) and the left tibia was pinned but not fractured (control leg). Rats were killed on days 1, 3, 7, 14, 28 and malondialdehyde (MDA) levels were determined in tibial bone tissue. The MDA levels of study and control legs were compared with basal MDA levels in bone in 10 rats. There was no apparent difference between the basal level and control legs, but the study legs showed a statistically significant increase in MDA levels on days 7 and 14. We conclude that no oxidative stress injury occurs during the ischemic period of fracture healing, but it may be significant during inflammation and the formation of callus.     

Oxidant status increased during fracture healing in rats

We evaluated oxidant status during bone healing in 50 rats. In 40 rats, the right tibia was fractured and fixed intramedullarlyy (study leg) and the left tibia was pinned but not fractured (control leg). Rats were killed on days 1, 3, 7, 14, 28 and malondialdehyde (MDA) levels were determined in tibial bone tissue. The MDA levels of study and control legs were compared with basal MDA levels in bone in 10 rats. There was no apparent difference between the basal level and control legs, but the study legs showed a statistically significant increase in MDA levels on days 7 and 14. We conclude that no oxidative stress injury occurs during the ischemic period of fracture healing, but it may be significant during inflammation and the formation of callus.     

Histochemical examination of ectopic bone formation induced in rat bone marrow

We examined the rapid formation and subsequent resorption of woven bone induced by partial ablation of rat bone marrow. On the 1st day after ablation, masses of clots occupied the region from which marrow was eliminated. On the 3rd day, alkaline phosphatase-(ALPase-) positive osteoblastic cells appeared in the vicinity of the marrow-eliminated region, forming woven bone. Other ectopic woven bone extended from the endosteal surface toward the bone marrow. Therefore, the newly formed bone originated in two different sites, the endosteal bone surface and the marrow tissues near the marrow-eliminated region. On the 7th day, numerous tartrate-resistant acid phosphatase- (TRAPase-) positive osteoclasts and ALPase-positive osteoblasts expressing the osteonectin gene indicated high activity in both formation and resorption of ectopic woven bone. On the 10th day, the ectopic bone had been markedly resorbed and replaced by bone marrow tissue as the ectopically formed woven bone had not been dynamically maintained, probably because of reduced bone formation activity. Immunoreactivity for basic fibroblast growth factor (bFGF) was indistinctly observed on osteoblastic and preosteoblastic cells on the 1st day after ablation. The fibroblastic cells in the marrow-eliminated region on the 3rd day, and both osteoblasts and preosteoblasts in the woven bone on the 7th day, showed strong immunoreactivity for bFGF. Unlike fractured cortical bone, no chondrogenesis was observed. This model appears to provide convenient material and an important clue for investigation of imbalanced bone formation and subsequent resorption.