Evaluation of recombinant human bone morphogenetic protein-2 as a bone-graft substitute in a canine segmental defect model.

A study was performed in dogs to evaluate the dose-response characteristics and effectiveness of recombinant human bone morphogenetic protein-2 with a collagen sponge carrier in a segmental defect model. Twenty-seven dogs underwent bilateral radial osteotomies with creation of a 2.5-cm diaphyseal defect. All received autogenous cancellous bone graft in one defect and a collagen implant in the other. These implants contained recombinant human bone morphogenetic protein-2 at the following doses: group 1 at 0 microg (three dogs, 0 microg/ml total implant volume), group 2 at 150 microg (three dogs, 50 microg/ml), group 3 at 600 ,g (three dogs, 200 microg/ml), group 4 at 2,400 microg (three dogs, 800 microg/ml), group 5 at 0 microg (five dogs, 0 microg/ml), group 6 at 150 microg (five dogs, 200 microg/ml), and group 7 at 600 microg (five dogs, 50 microg/ml). The defects were stabilized with external fixators. The dogs in groups 1-4 were killed at 12 weeks postoperatively, and those in groups 5-7 were killed at 24 weeks postoperatively except for one dog in group 7, which was killed at 48 weeks. Evaluation included monthly radiographs, biomechanical testing, and nondemineralized histology. All 27 radii with autogenous cancellous bone graft and all 19 implants treated with recombinant human bone morphogenetic protein-2 achieved radiographic and histologic union and gross stability. The eight radii treated with collagen carrier alone went on to radiographic and histologic nonunion and were grossly unstable at death. A dose-dependent occurrence of cyst-like bone voids was noted radiographically and histologically. Biomechanical performance tended to be better at the lowest dose studied at 12 weeks, and all three doses performed better than the placebo (p < 0.05) at 12 and 24 weeks. By 24 weeks, radiolucent areas corresponding to histologic bone voids persisted radiographically, although there was evidence of early bone remodeling. This remodeling progressed to 48 weeks in the single animal followed to this time point, although bone voids remained. These radiologic findings were confirmed histologically. Recombinant human bone morphogenetic protein-2 in a collagen sponge carrier has significant osteoinductive activity in this canine segmental defect model. A dose-response relationship is evident, with heterotopic bone and cyst-like void formation at higher doses and a minimum effective dose of 0-150 microg. At 12 and 24 weeks postoperatively, biomechanical parameters achieved by defects treated with recombinant human bone morphogenetic protein-2 were comparable with those of autograft controls and were significantly stronger than those of the placebo (p < 0.05).     

Evaluation of a biodegradable graft substitute in rabbit bone defect model

Objective:

To evaluate a new biodegradable copolymer calcium sulfate/poly amino acid (CS/PAA) as a graft substitute for the repair of the surgically created cancellous bone defects in rabbits and its biological properties in vivo.

Materials and Methods:

Cancellous bone defects were created by drilling holes in the unilateral lateral aspect of the femoral condyle of New Zealand white rabbits. Three groups were assigned: Group A rabbits were grafted with 80% CS/PAA and group B rabbits were grafted with 95% CS/PAA as two treatment groups; group C was sham-operation control group. To study the osteogenic capability in vivo, specimens were harvested at 4, 8, 12, and 16 weeks after implantation and were evaluated by gross assessment, X-ray, histological examination, and histomorphometry. In order to identify the molecular mechanism of bone defect repair, the expression of bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) was detected using Western blot at 4 weeks.

Results:

Group A and group B showed more vigorous and rapid repair leading to regeneration of cancellous bone than sham-operation control group on gross observation, radiology, and histomorphometry. There was no significant difference between groups A and B. Morphological observation and histological examination showed that the copolymers degraded in sync with the new bone formation process. The expression of BMP-2 and VEGF in implantation groups was higher than that in control group by western blot.

Conclusion:

These findings demonstrated that the novel biodegradable copolymers can repair large areas of cancellous bone defects. With its controllable degradation rate, it suggests that CS/PAA may be a series of useful therapeutic substitute for bone defects.     

Impaction grafting with a bone-graft substitute in a sheep model of revision hip replacement

An experimental sheep model was used for impaction allografting of 12 hemiarthroplasty femoral components placed into two equal-sized groups. In group 1, a 50:50 mixture of ApaPore hydroxyapatite bone-graft substitute and allograft was used. In group 2, ApaPore and allograft were mixed in a 90:10 ratio. Both groups were killed at six months. Ground reaction force results demonstrated no significant differences (p > 0.05) between the two groups at 8, 16 and 24 weeks post-operatively, and all animals remained active. The mean bone turnover rates were significantly greater in group 1, at 0.00206 mm/day, compared to group 2 at 0.0013 mm/day (p < 0.05). The results for the area of new bone formation demonstrated no significant differences (p > 0.05) between the two groups. No significant differences were found between the two groups in thickness of the cement mantle (p > 0.05) and percentage ApaPore-bone contact (p > 0.05). The results of this animal study demonstrated that a mixture of ApaPore allograft in a 90:10 ratio was comparable to using a 50:50 mixture.     

The use of a coral composite implant containing bone morphogenetic protein to repair a segmental tibial defect in sheep

Summary. A composite implant consisting of a coral cylinder, moose bone morphogenetic protein and type IV collagen was used to repair a segmental tibial defect in sheep. Healing, related variance in mechanical strength and immune responses were evaluated. In comparison with a coral control, a larger amount of newly formed external callus was observed in the composite group at 6 weeks. The maximal torque capacity, maximal angular deformation at failure and bone stiffness of a healed osteotomised tibia recovered 113%, 117% and 120% in the coral controls and 67%, 92% and 79% in the composite implants against the corresponding contralateral tibia at 16 weeks respectively. A significantly elevated anti-BMP antibody was detectable in the composite group at 3 and 6 weeks. Augmented bone formation at an early stage and weakened torsional performance at a later stage in the composite implants may indicate the phase-specific osteoinduction and the immune response of xenogenic BMP with time.

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Résumé. Nous avons étudié l’action d’un composite comprenant un cylindre de corail, une protéine morphogénétique xénogénique extraite de l’os de souris et du collagène de type IV, sur la consolidation la résistance mécanique et la réponse immune d’une perte de substance diaphysaire. L’étude au scanner à 6 semaines a montré une réduction significative du cal dans le groupe composite. A 16 semaines, la résistance à la torsion, la déformation angulaire maximale et la solidité des cals consolidés ont été respectivement de 113%, 117% et 120% dans le groupe controle, et de 67%, 92% et 79% dans le groupe composite, comparées au tibiai contralatéral. Dans le groupe composite, il y a eu une élévation significative des anticorps anti-mBMP. L’augmentation de la formation osseuse au stade précoce et la diminuation des propriétés mécaniques au stade tardif dans le groupe composite, pourraient être l’expression d’une induction séquentielle de l’ostéogenèse et d’une initiation immune des antigènes mBMP xénogéniques après une greffe hétérotopique.

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Accepted: 2 January 1997     

Allogeneic mesenchymal stem cells regenerate bone in a critical-sized canine segmental defect

BACKGROUND: Mesenchymal stem cells from adult bone marrow are multipotent cells capable of forming bone, cartilage, and other connective tissues. In a previous study, we demonstrated that autologous mesenchymal stem cells could repair a critical-sized bone defect in the dog. The objective of this study was to determine whether the use of allogeneic mesenchymal stem cells could heal a critical-sized bone defect in the femoral diaphysis in dogs without the use of immunosuppressive therapy. METHODS: A critical-sized segmental bone defect, 21 mm in length, was created in the mid-portion of the femoral diaphysis of twelve adult dogs that weighed between 22 and 25 kg. Each defect was treated with allogeneic mesenchymal stem cells loaded onto a hollow ceramic cylinder consisting of hydroxyapatite-tricalcium phosphate. A complete mismatch between donor stem cells and recipient dogs was identified by dog leukocyte antigen typing prior to implantation. The healing response was evaluated histologically and radiographically at four, eight, and sixteen weeks after implantation. The radiographic and histological results at sixteen weeks were compared with the historical data for the control defects, which included defects that had been treated with a cylinder loaded with autologous mesenchymal stem cells, defects treated with a cylinder without mesenchymal stem cells, and defects that had been left untreated (empty). The systemic immune response was evaluated by the analysis of recipient serum for production of antibodies against allogeneic cells. RESULTS: For defects treated with allogeneic mesenchymal stem cell implants, no adverse host response could be detected at any time-point. Histologically, no lymphocytic infiltration occurred and no antibodies against allogeneic cells were detected. Histologically, by eight weeks, a callus spanned the length of the defect, and lamellar bone filled the pores of the implant at the host bone-implant interface. Fluorescently labeled allogeneic cells were also detected. At sixteen weeks, new bone had formed throughout the implant. These results were consistent with those seen in implants loaded with autologous cells. Implants loaded with allogeneic or autologous stem cells had significantly greater amounts of bone within the available pore space than did cell-free implants at sixteen weeks (p < 0.05). CONCLUSIONS: The results of this study demonstrated that allogeneic mesenchymal stem cells loaded on hydroxyapatite-tricalcium phosphate implants enhanced the repair of a critical-sized segmental defect in the canine femur without the use of immunosuppressive therapy. No adverse immune response was detected in this model.     

自体脂肪干细胞复合珊瑚修复犬颅骨标准缺损的初步研究

目的 应用自体脂肪干细胞(adipose-derived stem cells,ADSCs)复合珊瑚构建组织工程化骨,修复犬颅骨标准缺损.方法 体外扩增培养、成骨诱导Beagle犬ADSCs,将第2代细胞接种在珊瑚支架上共同培养.制造实验犬双侧颅骨全层标准缺损(20 mm×20 mm),一侧以细胞材料复合物修复作为实验组(n=7),另一侧以单纯珊瑚材料修复作为对照组(n=7).术后24周分别通过影像学、大体形态观察、生物力学检测、组织学方法检测颅骨缺损的修复效果.结果 成骨诱导的犬ADSCs体外呈现成骨特性,在珊瑚支架上生长良好.3D-CT重建显示术后12周实验组有新生骨痂形成,对照组材料大部分降解;24周时实验组为骨性愈合,对照组为骨不连.24周时实验组缺损修复百分比为(84.19±6.45)%,显著高于对照组的(25.04 ±18.82)%(P<0.01).大体观察见实验组由新生骨痂修复缺损,对照组缺损边缘可见少量骨痂形成,主要为软组织充填;24周生物力学检测修复组织能耐受的最大压力载荷,实验组为(73.45±17.26)N,为犬顶骨最大压力负荷(104.27±22.71)N的70%,两者比较差异有统计学意义(P<0.01),对照组为软组织无法完成上述检测.HE染色见实验组有较多成熟骨呈骨性愈合,对照组为纤维性愈合.结论 自体成骨诱导的ADSCs复合珊瑚形成的组织工程化骨可修复犬颅骨标准缺损.     

Bovine bone implant with bovine bone morphogenetic protein in healing a canine ulnar defect

Xenograft is considered an alternative material for bone transplantation, but its bone healing capacity is inferior compared to that of autografts and allografts. Here, we tested whether bone morphogenetic protein (BMP) addition enhances the suitability of demineralized xenogeneic bovine bone for bone grafting in dogs, and whether xenogeneic bone is a suitable carrier material for BMPs. The capacity of demineralized bovine bone implants, with and without native partially purified bovine BMP, to heal a 2-cm ulnar defect was determined in six dogs over a follow-up time of 20 weeks. No instances of bone union were seen, but there was slightly more bone formation in the xenografts with BMP, though the difference was not statistically significant. The ulnas treated with an implant with BMP were also mechanically stronger, but the difference was not significant. Computed tomography scans showed no differences in the implant area in bone density, bone mineral content, or bone cross-sectional area. It is concluded that native, partially purified BMP does not sufficiently improve the suitability of bovine demineralized xenografts as a bone substitute material for dog. Demineralized xenogeneic bone does not seem to be a feasible carrier material for BMP.     

骨蛋白强化脱钙骨基质板块修复犬长骨节段性骨缺损

目的 :研究骨蛋白 (boneprotein ,BP)强化脱钙骨基质 (demineralizedbonematrix ,DBM) (BP/DBM )板块在修复节段性骨缺损中的作用。方法 :在犬双侧桡骨中段各做一 1 5cm的骨膜骨缺损 ,分别植入板块状BP/DBM ,DBM ,自体髂骨块及留置空白 ,观察时间为 4个月。结果 :BP/DBM植入组有 3例完全骨愈合 (3 / 5 ) ,自体骨移植组只有 3例部分骨愈合 ,单纯DBM组及空白组未见骨愈合。生物力学测试 :术后 4个月BP/DBM组新生骨极限压缩强度值最高 ,已达到正常桡骨组织的 48%。BP/DBM组新生骨为成熟的板状骨。结论 :BP/DBM板块可促进节段性骨缺损的修复。     

仿生活性人工骨修复兔桡骨节段性骨缺损的研究

目的 研制在成分和结构上与天然骨基质高度仿生的新型材料，应用于节段性骨缺损的修复。方法 以Ⅰ型胶原蛋白分子为模板，引入钙磷盐生成体重，合成纳米相羟基磷灰石/胶原（NHAC）并以之为主体，以1：1的质量比加入聚乳酸（PLA）制备多孔框架，与重组人骨形态发生蛋白2（rh-BMP2)复合后植入兔桡骨15mm缺损，观察骨修复情况。结果 所合成的材料具有纳米级的层装排列结构。层间距为11.7nm，实验组中10只兔术后12周骨缺损均完全愈合。结论 NHAC可能成为修复骨缺损的较理想材料，具有较大的临床应用潜力。     

Porous hydroxyapatite as a bone-graft substitute in metaphyseal defects. A histometric study

Porous hydroxyapatite (Interpore 500) formed by conversion of the Porites goniopora coral exoskeleton has pores averaging 600 micrometers and pore interconnections averaging 260 micrometers in diameter. In the proximal tibial metaphysis of eight dogs, a defect one cubic centimeter in size was created unilaterally and was fitted with a block of Interpore 500. Both proximal tibial metaphyses were retrieved at two, four, six, and twelve months. Stained undecalcified sections were examined by light microscopy and quantitated by histometric methods. The implant-side specimens contained compact bone along the external surface and trabecular bone interiorly. The interior of these specimens was composed of 51.9 +/- 1.3 per cent soft tissue, 13.0 +/- 1.2 per cent bone, and 35.1 +/- 1.2 per cent Interpore 500 (mean and standard error). The interior of the normal specimens was composed of 79.7 +/- 1.4 per cent soft tissue and 20.2 +/- 1.4 per cent bone. The allocation of implant pore space between bone and soft tissue was proportional to that of bone and soft tissue in the normal tibiae. The stereological distribution of regenerated bone in the porous hydroxyapatite was also the same as in the normal tibiae. The appositional process of incorporation of the implant was confirmed by the finding that 66.5 per cent of the surface of the Interpore 500 was covered with bone ingrowth at twelve months.     

Long-term follow-up study of bioactive bone cement for repairing a segmental defect in a canine femur

We report on a 7-year long-term follow-up study of a bioactive bone cement (BA cement) that was used to repair a segmental defect in a canine femur. Bilateral femoral segmental defects were repaired with metallic implants that were fixed to the femur using two kinds of bone cement. The BA cement used in this study consists of an apatite- and wollastonite-containing glass ceramic (AW-GC) with a bis-phenol-alpha-glycidyl methacrylate (bis-GMA)-based resin. The bone-cement interface was examined histologically. Previous short-term studies have shown that using BA cement for segmental replacement of the canine femur produced excellent biomechanical and histological results. The BA cement maintained the fixation of a metallic implant to the femur very well. In contrast, the PMMA cement did not maintain alignment under long-term weight-bearing conditions. The results of histological examinations showed direct bonding between the BA cement and bone, while an intervening soft tissue layer was observed at the bone-cement interface with the PMMA cement. The BA cement bonded to the bone through a Ca-P-rich reactive layer, which was twice as thick after 7 years than it was at 26 weeks. No adverse effects of BA cement were observed during the 7-year observation period.     

Endothelial progenitor cells promote fracture healing in a segmental bone defect model

The objective of this study was to evaluate the effects of local endothelial progenitor cell (EPC) therapy on bone regeneration in a rat model. A segmental bone defect (5 mm) was created in the femur and fixed with a mini‐plate. There were two groups: EPC‐treated (N = 28) and control (N = 28). Seven animals were sacrificed from each group at 1, 2, 3, and 10 weeks postoperatively. Healing of the defect was evaluated with radiographic, histological, and quantitative micro‐computed tomography (micro‐CT) scans. Radiographically, mean scores of the EPC and control groups were, respectively, 1.16–0.61 (p < 0.05) at 1 week, 2.53–1.54 (p < 0.05) at 2 weeks, and 4.58–2.35 at 3 weeks (p < 0.05). At 10 weeks, all the animals in the EPC‐treated group had complete union (7/7), but in the control group none achieved union (0/7). Histological evaluation revealed that specimens from EPC‐treated animals had abundant new bone and vessel formation compared to that in controls. Micro‐CT assessment of the samples from the animals sacrificed at 10 weeks (N = 14) showed significantly improved parameters of bone volume (36.58–10.57, p = 0.000), bone volume density (0.26–0.17, p = 0.000), model index ?2.22–2.79, p = 0.000), trabecular number (1.28–0.91, p = 0.063), trabecular thickness (0.21–0.15, p = 0.001), trabecular spacing (0.63–1.07, p = 0.022), bone surface (353.75–152.08, p = 0.000), and bone surface to bone volume ratio (9.54–14.24, p = 0.004) for the EPC group compared to control, respectively. In conclusion, local EPC therapy significantly enhanced bone regeneration in a segmental defect model in rat femur diaphysis. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1007–1014, 2010     

The monocortical critical size bone defect as an alternative experimental model in testing bone substitute materials.

BACKGROUND: Reproducibility and comparability are prerequisites in testing bone substitute materials in an animal study. Various animal test models are used. The standard in testing bone substitute materials is the so-called critical size defect (CSD) model described by Schmitz and Hollinger. In most studies a bicortical (full thickness defect) CSD is used. OBJECTIVE: In this study, monocortical defects (10 x 10 mm) in calvarias of adult pigs were created and compared to examine the spontaneous physiologic healing process and the required criteria for a CSD. STUDY DESIGN: Regeneration of defects, either filled using autogenous bone (group 1) or unfilled (group 2), was evaluated by means of microradiography and light microscopy after 2, 4, 12, 26, and 52 weeks. RESULTS: No complete osseous regeneration was found microradiographically and light microscopically in the control group at week 52 (nonmineralized area: 30% +/- 1.7%; mineralized area: 55% +/- 1.7%; bone marrow area: 15% +/- 1.7%). Defects treated with autogenous bone showed a 100% osseous defect filling after 26 weeks. CONCLUSION: The monocortical CSD fulfills the requirements for a CSD and represents a procedure which can be handled simply for investigations focusing on bone regeneration and testing of bone substitute materials.     

可控性微结构磷酸钙支架作为成骨细胞载体修复兔尺骨节段性缺损

目的采用可控性微结构磷酸钙支架作为成骨细胞载体修复兔尺骨节段缺损，观察载体微结构在新骨生成及缺损愈合中的作用并探讨其机制。方法应用快速成型（RP）间接制造技术制备可控性微结构自固化磷酸钙（CPC）支架，将兔颅骨源性成骨细胞与实验组（可控性微结构支架）及对照组（单纯CPC材料）复合培养，倒置显微镜及扫描电镜（SEM）观察细胞生长情况；培养7d后植入兔尺骨节段性缺损，术后4、8、12周取材，分别行大体、X线、组织学观察和新生骨定量分析，评价新骨生长及缺损愈合情况。结果培养7d后支架表面及管道内有大量细胞分布。实验组新骨生长、改建及材料降解均沿管道结构进行，术后12周形成新生骨与支架相互嵌合的复合体；对照组新骨生长主要发生在骨断端与材料结合处，术后12周两端骨组织长人材料，CPC出现少量降解。术后12周实验组新生骨量高于对照组，差异有统计学意义（P〈0．05）。结论可控性微结构促进支架内新骨生成及兔尺骨节段性缺损的愈合，对支架微结构的深人研究和优选是增强组织工程化骨移植物成骨效能的有效途径。     

Evaluation of bone-grafting materials in a new canine segmental spinal fusion model

This paper describes a new canine segmental spinal fusion model for the comparison of bone-grafting materials. The test sites in the model are three separate posterior interfacet-interlaminar fusion sites in the lumbar spine (L1-2, L3-4, and L5-6). The outcome was assessed by scoring of the fusion sites for union and by mechanical testing of fused segments. The results from two experiments are presented. Autogenous cancellous bone was the most effective material tested and had a statistically superior score for union compared with all other materials (p = 0.01). The results with an osteoconductive matrix of collagen and ceramic alone were no better than those with the controls (no graft). However, addition of an extract of matrix-derived proteins (15–30 kDa) to the collagen-ceramic carrier appeared to improve the score for union. The inclusion of nonresorbed ceramic granules had no evident effect on the mechanical properties of fusions, with a comparable score for union. This model appears to be a sensitive and efficient method for the comparison of graft materials. Advantages over previously described models are discussed.     

磷酸钙骨水泥作为骨形成蛋白载体修复节段性骨缺损及相关研究

目的 制备CPC/BMP复合人工骨，通过动物实验研究其对骨缺损的修复作用及相关问题，探讨临床应用的可能性。方法 参考有关文献方法合成CPC，并将其作为BMP的载体制成CPC/BMP复合物，植入兔桡骨15mm骨缺损处，术后不同时间处死动物。通过生物力学测定，组织学染色分析，电镜扫描及X射线电子能谱分析。X线摄片，无机质含量测定以及骨密度测定等手段观察新骨形成和材料降解情况。同时以单纯的CPC及空白组作为对照研究。综合评价CPC/BMP对骨缺损的修复能力及对机体的影响。结果 术后CPC/BMP和CPC两组动物均无毒性反应。随着时间的延长，血清中碱性磷酸酶浓度逐渐升高，尤以CPC/BMP组显著，提示CPC/BMP复合物和单纯的CPC均可以促进新骨形成，前者新骨形成量大，骨修复能力明显好于后者。CPC/BMP植入2周时可见大量间充质细胞分化，在材料与骨端之间出现一层软骨细胞。4周时软骨细胞向编织骨分化，16周时板骨层骨长人材料并与之相互分割包裹，24周时骨缺损初步修复，新骨密度明显高于CPC组，说明BMP的加入不仅有效地促进了新骨的形成，同时也加速了新骨的钙化。24周组标本生物力学测定结果表明，新骨形成的同时伴随材料的降解，CPC组材料降解速度缓慢，CPC/BMP组降解速度优于CPC组，但24周时仍有部分材料残存。在新骨形成和材料降解过程中可出现血清钙浓度的一过性升高。结论 CPC是BMP的理想载体。CPC/BMP生物活性人工骨对骨缺损有较强的修复能力，可望成为新型的骨缺损修复材料。     

VEGF producing bone marrow stromal cells (BMSC) enhance vascularization and resorption of a natural coral bone substitute

Bone graft substitutes often exhibit poor bone regeneration in large defects because of inadequate vascularization. Studies have shown that if blood supply is compromised, application of osteogenic factors alone could not induce successful healing. This study was to evaluate the effects of vascular endothelial growth factor, which combined with a coralline scaffold, on vascularization, scaffold resorption and osteogenesis in a rabbit radius critical size defect model. The scaffold was either coated with a control-plasmid DNA (group 1), coated with VEGF-plasmid DNA (group 2), loaded with mesenchymal stem cells (BMSC) transfected with control plasmid (group 3) or with both stem cells and the VEGF plasmid (group 4). X-rays were taken every 4 weeks up to week 16, when animals were euthanized. The volume of new bone was measured by mu-CT scans and blood vessels were counted after anti-CD31 staining of endothelial cells. The results from the solitary VEGF- and VEGF-transfected cells (groups 2 and 4) demonstrated significantly enhanced vascularization, osteogenesis and resorption of the carrier when compared to the control group. The highest degree of osteogenesis was found when the carrier was loaded with BMSC (group 3), whereas VEGF-transfected cells led to the highest vascularization and fastest resorption of the bone substitute. Additionally, VEGF-transfected BMSC led to a more homogenous vascularization of the defect. The results indicate that VEGF can be a helpful factor to improve healing in large bone defects, in which bone substitutes will otherwise not be vascularized and replaced by fresh bone.     

The induced membrane technique: Optimization of bone grafting in a rat model of segmental bone defect

IntroductionThe induced membrane technique (IMT) is a two-stage surgical procedure used to treat fracture nonunion and bone defects. Although there is an increasing number of animal studies investigating the IMT, few have examined the outcomes of bone healing after a second stage grafting procedure. This study aimed at comparing two bone grafting procedures, as part of the IMT, in order to establish a rat model providing consistent healing outcomes.MethodsIn male Fischer 344 rats, we created a 5 mm defect in the right femur, stabilized the bone with a plate and screws, and inserted a polymethylmethacrylate spacer into the defect. Four weeks later, the spacer was removed. Bone graft was harvested from a donor rat and placed into the defect, followed by membrane and wound closure. Experiments were conducted in two groups. In group 1 (n = 11), the bone graft contained a variable amount of cortical and cancellous bone, the time from donor euthanasia to grafting was up to 240 min, and one donor rat provided graft for 5-6 recipients. In group 2 (n = 12), we reduced the contribution of cortical bone to the graft, included bone marrow, and kept donor euthanasia to grafting time under 150 min. One donor was used per 3-4 recipients. The volume of graft per recipient and all other elements of the protocol were the same across groups. Bone healing at 12 weeks post grafting was compared radiographically by two orthopaedic surgeons in a blinded fashion, based on union status and a modified Lane & Sandhu score.ResultsHealing rates improved from 36.4% in Group 1 to 91.6% in Group 2. There was a significant relationship between the methods and resulting union status (p = 0.004). The odds of achieving full union were significantly higher in group 2 compared to group 1 (odds ratio=19.25, 95% confidence interval [1.77-209.55]; p = 0.009). The average radiographic score was also significantly higher in group 2 (p = 0.005).ConclusionThe revised bone grafting method significantly improved the healing outcomes and contributed to establishing a consistent rat model of the IMT. This model can benefit preclinical investigations by allowing for reliable and clinically-relevant comparisons.