rhBMP-2 Modulation of Gene Expression in Infected Segmental Bone Defects

The osteoinductive capability of BMPs appears diminished in the setting of acute infection. We applied rhBMP-2 to a segmental defect in a rat femur and measured the expression of key bone formation genes in the presence of acute infection. Types I and II collagen, osteocalcin, and BMP Type II receptor mRNA expression were characterized in 72 Sprague-Dawley rats, which received either bovine collagen carrier with 200 μg rhBMP-2 plus Staphylococcus aureus, carrier with bacteria only, carrier with rhBMP-2 only, or carrier alone. Six animals from each group were euthanized at 1, 2, and 4 weeks. Total RNA was isolated and extracted, and mRNA was determined by real-time comparative quantitative PCR. Infected defects had little expression of collagen I and II and osteocalcin mRNAs, while BMP receptor II expression with infection was greater than carrier-only controls at Weeks 2 and 4. Notably, all four genes were upregulated in infected defects in the presence of rhBMP-2. Thus, in a clinical setting with a high risk of infection and nonunion, such as a compound fracture with bone loss, rhBMP-2 may increase the rate and extent of bone formation. Even if infection does occur, rhBMP-2 may allow a quicker overall recovery time.     

Vitamin K2 Promotes Bone Healing in a Rat Femoral Osteotomy Model with or without Glucocorticoid Treatment

The purpose of the present preclinical study was to determine whether vitamin K₂ would promote bone healing in a rat femoral osteotomy model with or without glucocorticoid (GC) treatment. Thirty-eight 6 week-old female Sprague–Dawley rats underwent a unilateral osteotomy of the femoral diaphysis followed by intramedullary wire fixation and then were randomized into four groups that received the following treatment schedules: vehicle, vitamin K₂, GC + vehicle, and GC + vitamin K₂. GC (prednisolone, 2.5 mg/kg) was administered subcutaneously twice a week. Vitamin K₂ (menatetrenone, 30 mg/kg) was administered orally five times a week. After 8 weeks of treatment, the wires were removed and a bone histomorphometric analysis was performed on the bone tissue inside the callus. Vitamin K₂ administration to GC-untreated rats decreased the osteoclast surface/bone surface (OcS/BS), osteoblast surface (ObS)/BS, eroded surface (ES)/BS, and bone formation rate (BFR)/BS and increased the lamellar area/bone area. Although GC treatment increased the ES/BS and decreased the ObS/BS, BFR/BS, and lamellar area/bone area, vitamin K₂ administration to GC-treated rats decreased the OcS/BS and prevented an increase in the ES/BS and a decrease in the lamellar area/bone area. These results suggested that vitamin K₂ downregulated bone turnover and stimulated lamellar bone formation in GC-untreated rats and prevented an increase in bone resorption while maintaining bone formation and prevented a decrease in lamellar bone formation in GC-treated rats. Thus, vitamin K₂ appears to be effective for promoting bone healing in a rat femoral osteotomy model with or without GC treatment.     

Guided Bone Regeneration of Femoral Segmental Defects using Equine Bone Graft: An In-Vivo Micro-Computed Tomographic Study in Rats

ABSTRACT

Background and objectives: Guided bone regeneration (GBR) is commonly used for osseous defect reconstruction. The objective of this study was to evaluate in real-time (in-vivo) the efficacy of equine bone graft for GBR in segmental critical-size defects (CSD) of the femur in a rat model. Materials and methods: Following ethical approval, 30 male Wistar-Albino rats (age 12–14 months/weight 450–500 grams) were included. Under general-anesthesia, a mid-diaphyseal segmental CSD (5 mm) was created in the femur and stabilized using titanium Miniplate(4 holes,1.0 mm thickness). Depending upon material used for GBR, animals were randomly divided into three groups(n = 10/per group). Negative control-Defect covered with resorbable collagen membrane(RCM); Positive control-Defect filled with autologous bone and covered by RCM; Equine bone-Defect filled with equine bone and covered by RCM. Real-time in-vivo Micro-CT was performed at baseline, 2, 4, 6 and 8 weeks to determine volume and mineral density of newly formed bone (NFB) and remaining bone graft particles (BGP). Results: In-vivo micro-CT revealed increase in volume and mineral density of NFB within defects from baseline to 8-weeks in all groups. At 8-weeks NFB-volume in the equine bone group(53.24 ± 13.83 mm³; p < 0.01) was significantly higher than the negative control(5.6 ± 1.06 mm³) and positive control(26.07 ± 5.44 mm³) groups. Similarly, NFB-mineral density in the equine bone group(3.33 ± 0.48 g/mm³; p < 0.01) was higher than the other (negative control–0.27 ± 0.02 g/mm³; positive control–2.55 ± 0.6 g/mm³). A gradual decrease in the BGP-volume and BGP-mineral density was observed. Conclusion: The use of equine bone for GBR in femoral segmental defects in rats, results in predictable new bone formation as early as 2-weeks after bone graft placement.     

Hydrogel-based Delivery of rhBMP-2 Improves Healing of Large Bone Defects Compared With Autograft

Background

Autologous bone grafting remains the gold standard in the treatment of large bone defects but is limited by tissue availability and donor site morbidity. Recombinant human bone morphogenetic protein-2 (rhBMP-2), delivered with a collagen sponge, is clinically used to treat large bone defects and complications such as delayed healing or nonunion. For the same dose of rhBMP-2, we have shown that a hybrid nanofiber mesh-alginate (NMA-rhBMP-2) delivery system provides longer-term release and increases functional bone regeneration in critically sized rat femoral bone defects compared with a collagen sponge. However, no comparisons of healing efficiencies have been made thus far between this hybrid delivery system and the gold standard of using autograft.

Questions/purposes

We compared the efficacy of the NMA-rhBMP-2 hybrid delivery system to morselized autograft and hypothesized that the functional regeneration of large bone defects observed with sustained BMP delivery would be at least comparable to autograft treatment as measured by total bone volume and ex vivo mechanical properties.

Methods

Bilateral critically sized femoral bone defects in rats were treated with either live autograft or with the NMA-rhBMP-2 hybrid delivery system such that each animal received one treatment per leg. Healing was monitored by radiography and histology at 2, 4, 8, and 12 weeks. Defects were evaluated for bone formation by longitudinal micro-CT scans over 12 weeks (n = 14 per group). The bone volume, bone density, and the total new bone formed beyond 2 weeks within the defect were calculated from micro-CT reconstructions and values compared for the 2-, 4-, 8-, and 12-week scans within and across the two treatment groups. Two animals were used for bone labeling with subcutaneously injected dyes at 4, 8, and 12 weeks followed by histology at 12 weeks to identify incremental new bone formation. Functional recovery was measured by ex vivo biomechanical testing (n = 9 per group). Maximum torque and torsional stiffness calculated from torsion testing of the femurs at 12 weeks were compared between the two groups.

Results

The NMA-rhBMP-2 hybrid delivery system resulted in greater bone formation and improved biomechanical properties compared with autograft at 12 weeks. Comparing new bone volume within each group, the NMA-rhBMP-2-treated group had higher volume (p < 0.001) at 12 weeks (72.59 ± 18.34 mm³) compared with 8 weeks (54.90 ± 16.14) and 4 weeks (14.22 ± 9.59). The new bone volume was also higher at 8 weeks compared with 4 weeks (p < 0.001). The autograft group showed higher (p < 0.05) new bone volume at 8 weeks (11.19 ± 8.59 mm³) and 12 weeks (14.64 ± 10.36) compared with 4 weeks (5.15 ± 4.90). Between groups, the NMA-rhBMP-2-treated group had higher (p < 0.001) new bone volume than the autograft group at both 8 and 12 weeks. Local mineralized matrix density in the NMA-rhBMP-2-treated group was lower than that of the autograft group at all time points (p < 0.001). Presence of nuclei within the lacunae of the autograft and early appositional bone formation seen in representative histology sections suggested that the bone grafts remained viable and were functionally engrafted within the defect. The bone label distribution from representative sections also revealed more diffuse mineralization in the defect in the NMA-rhBMP-2-treated group, whereas more localized distribution of new mineral was seen at the edges of the graft pieces in the autograft group. The NMA-rhBMP-2-treated group also revealed higher torsional stiffness (0.042 ± 0.019 versus 0.020 ± 0.022 N-m/°; p = 0.037) and higher maximum torque (0.270 ± 0.108 versus 0.125 ± 0.137 N-m; p = 0.024) compared with autograft.

Conclusions

The NMA-rhBMP-2 hybrid delivery system improved bone formation and restoration of biomechanical function of rat segmental bone defects compared with autograft treatment.

Clinical Relevance

Delivery systems that allow prolonged availability of BMP may provide an effective clinical alternative to autograft treatment for repair of segmental bone defects. Future studies in a large animal model comparing mixed cortical-trabecular autograft and the NMA-rhBMP-2 hybrid delivery system are the next step toward clinical translation of this approach.     

Healing of Membranous and Long Bone Defects

Different isolation models have been used for qualitative and quantitative studies on the individual roles of periosteum, cortical bone, endosteum and bone marrow in the repair of long bone defects. Corresponding skull defects were studied to evaluate also the dural osteogenetic capacity. A total of 53 operations were performed on the tibia and skull of 33 growing rabbits. The animals were killed 10–12 weeks after surgery. Ordinary rough histological methods were used for studying bone formation, and quantitative estimates were made using serial sections. The cortex, endosteum and bone marrow were demonstrated to play a minor part in the healing of tibial defects. Periosteum, on the other hand, had the most potent healing capacity. Calvarial periosteum, however, was found to be less bone producing and in that respect not to be superior to the dura. For a complete bony restoration combined periosteal and dural bone formation is necessary, also regarding the structural normalization.     

组织工程引导骨再生膜修复节段性骨缺损

目的　将组织工程技术和引导性骨再生技术相结合修复长骨节段性缺损。方法　兔 2 7只 ,动物模型均为桡骨 1 2cm节段性骨 骨膜缺损 ,分成 3组 ,A组 :利用体外构建的细胞 材料复合物膜修复 ;B组 :利用单纯的膜材料进行修复 ;C组 :断端不作任何处置作空白对照。分别观察 3、 6、 12周后进行X线观察和组织学观察。结果　A组的骨愈合优于B组 ,在 12周时已经完成骨缺损的修复 ,B组在术后 12周仍处于塑形改建之中 ;C组 12周形成典型的骨不连。结论　将组织工程的技术与引导性骨再生技术相结合 ,可以比单纯的引导性骨再生更好地修复长骨节段性缺损。     

Vitamin-D Binding Protein Does Not Enhance Healing in Rat Bone Defects: A Pilot Study

Vitamin D-binding protein (DBP) has an anabolic effect on the skeleton and reportedly enhances bone ingrowth. We used an in vivo critical bone defect model to determine whether local administration of DBP promotes bone defect healing. We created a 5-mm segmental bone defect in the radial shaft in a rat model. Forty-eight rats were assigned to eight groups: local application of 1 μg, 5 μg, 10 μg, or 50 μg DBP (DBP-1, DBP-5, DBP-10, DBP-50), autogenous bone marrow mononuclear cells with or without 10 μg DBP (BM-DBP-10, BM), 80 μg BMP-2 delivered in gelatin sponge (BMP-2), and the sham operated group. Radiographic evaluation, histological stains, and epifluorescence microscopy were performed. Grossly, all bone gaps of the BMP-2 group were solidly bridged by callus, while all those in the sham operated group remained unhealed by 9 weeks. Only one specimen of the BM-DBP-10 and DBP-50 groups and three specimens of the BM group were solidly healed; pseudarthroses occurred in all of the other specimens. Histological study and radiographs of the specimens showed similar results. We did not observe the enhanced bone healing reported in a previous study.     

pHEMA-nHA Encapsulation and Delivery of Vancomycin and rhBMP-2 Enhances its Role as a Bone Graft Substitute

Background

Bone grafts are widely used in orthopaedic procedures. Autografts are limited by donor site morbidity while allografts are known for considerable infection and failure rates. A synthetic composite bone graft substitute poly(2-hydroxyethyl methacrylate)-nanocrystalline hydroxyapatite (pHEMA-nHA) was previously developed to stably press-fit in and functionally repair critical-sized rat femoral segmental defects when it was preabsorbed with a single low dose of 300 ng recombinant human bone morphogenetic protein-2/7 (rhBMP-2/7).

Questions/purposes

To facilitate clinical translation of pHEMA-nHA as a synthetic structural bone graft substitute, we examined its ability to encapsulate and release rhBMP-2 and the antibiotic vancomycin.

Methods

We analyzed the compressive behavior and microstructure of pHEMA-nHA as a function of vancomycin incorporation doses using a dynamic mechanical analyzer and a scanning electron microscope. In vitro release of vancomycin was monitored by ultraviolet-visible spectroscopy. Release of rhBMP-2 from pHEMA-nHA-vancomycin was determined by ELISA. Bioactivity of the released vancomycin and rhBMP-2 was examined by bacterial inhibition and osteogenic transdifferentiation capabilities in cell culture, respectively.

Results

Up to 4.8 wt% of vancomycin was incorporated into pHEMA-nHA without compromising its structural integrity and compressive modulus. Encapsulated vancomycin was released in a dose-dependent and sustained manner in phosphate-buffered saline over 2 weeks, and the released vancomycin inhibited Escherichia coli culture. The pHEMA-nHA-vancomycin composite released preabsorbed rhBMP-2 in a sustained manner over 8 days and locally induced osteogenic transdifferentiation of C2C12 cells in culture.

Conclusions

pHEMA-nHA can encapsulate and deliver vancomycin and rhBMP-2 in a sustained and localized manner with reduced loading doses.

Clinical Relevance

The elasticity, osteoconductivity, and rhBMP-2/vancomycin delivery characteristics of pHEMA-nHA may benefit orthopaedic reconstructions or fusions with enhanced safety and efficiency and reduced infection risk.     

云南白药促进骨缺损修复及引导性骨再生的实验研究

目的：阐明云南白药促进骨缺损修复及引导性骨再生的愈合机制，为临床上治疗骨折提供理论依据。方法：36只新西兰兔制作桡骨缺损不愈合模型和引导性骨再生模型，随机均分用药组及对照组，手术后3d、1、3、5、10、12周取材，观察骨痂愈合程度的差异及骨痂组织学变化，结果：实验组有明显的促进骨缺损修复作用，并在引导性骨再生中的膜内成骨作用强于对照组，结论：云南白药对骨缺损修复及引导性骨再生有明显的促进作用。     

带旋股外侧血管大转子骨瓣移植治疗成年股骨颈骨折

目的：探讨带旋股外侧血管大转子骨瓣移植治疗成年股骨颈骨折的方法和疗效。方法：采用带旋股外侧血管横支为蒂大转子骨瓣加松质骨镙钉内固定,治疗成年股骨颈骨折8例。结果：术后随访10个月～3年,8例骨折均愈合,未发生骨折不愈和股骨头缺血性坏死,患者髋关节功能恢复良好。结论：该术式操作相对简便,可提高成年股骨颈骨折的愈合率,避免发生骨不连和股骨头缺血性坏死等严重并发症。     

Functional Restoration of Critically Sized Segmental Defects With Bone Morphogenetic Protein-2 and Heparin Treatment

Background  

Bone defects and fracture nonunions remain a substantial challenge for clinicians. Grafting procedures are limited by insufficient volume and donor site morbidity. As an alternative, biomaterial scaffolds functionalized through incorporation of growth factors such as bone morphogenetic proteins (BMPs) have been developed and appear to regenerate the structure and function of damaged or degenerated skeletal tissue.     

rhBMP-2/卵磷脂复合材料修复犬长骨节段性骨缺损的实验研究

目的：观察rhBMP-2／卵磷脂复合材料修复犬长骨节段性骨缺损的能力，检验rhBMP-2的诱导成骨活性，方法：手术造成20mm桡骨中上段骨缺损，实验组植入rhBMP-2／卵磷脂的复合材料片，对照组植入单纯卵磷脂片，通过影像学、组织学观察及骨密度测定，评价rhBMP-2／卵磷脂复合材料修复犬长骨节段性骨缺损的效果，结果：影像学检查示实验组术12周骨痂桥接缺损，术后24周皮质骨连接；对照组无骨痂形成，组织学检查示实验组术后12周组织和肌组织充填，骨密度测定示术后12周骨痂密度达到正常值的76％，24周达正常值的85％，结论：rhBMP-2具有良好的诱导成骨活性，rhBMP-2／卵磷脂复合材料能够很好的修复犬桡骨20mm的骨缺损。     

块形重组合异种骨修复犬桡骨骨缺损

为使异种骨种移植应用于骨干节段性骨缺损治疗。作者将经去抗原处理的牛松质骨骨块与牛骨形态发生蛋白复合形成块形重组合异种骨，通过犬桡骨干中上段２０ｍｍ，骨－骨膜缺损修复模型，综合评价ＭＲＢＸ修复大型动物长骨节段性骨缺损的能力。方法：６只成年犬左侧桡骨节段性骨骨膜缺损处理ＭＲＢＸ，右侧不做修复，为空白对照。     

Tubular Remodeling of Massive Cancellous Bone Graft in the Treatment of Long Bone Defects

Abstract Background: This case report describes the clinical and radiological result at the 4.5-year follow-up after an extensive reconstruction of the femoral diaphysis using autologous cancellous bone graft. The radiological study including axial tomography demonstrates secondary remodelling to form tubular diaphyseal bone. Methods: A patient with an existing hip fusion, who sustained a fracture of the proximal femur 12 years later, was treated by open internal fixation using a plate and screws. Infection followed which became chronic, causing bone resorption and necrosis and producing a septic non-union. Reconstruction in two stages was performed: open radical debridement which ended with a 14.5 cm diaphyseal defect of the femur, temporary alloplastic spacer interposition and secondary de-arthrodesis of the hip with massive autologous cancellous grafts into the induced foreign body membrane left by the spacer. Fixation was provided by a plate and screws. Results: The femur was free of infectious recurrence at 4.5 years. The patient walks without crutches with a shortened lower limb using a leg length compensation shoe and a painfree sine-sine hip arthroplasty. The former bone defect is fully remodelled into new cortical bone. X-ray and CT-scan demonstrate the tubular form of the reconstructed bone. Conclusion: This clinical case demonstrates the restoration of a medullar cavity after massive cancellous bone grafting of a diaphyseal defect of the femur. The question remains open as to whether the foreign body membrane has only a simple passive protective function against extraosseous bone resorbing factors or whether it functions actively by producing growth factors or other beneficial bone inducing factors.     

Cem-OsteticTM人工骨浆修复掌骨缺损

目的C em-O steticTM人工骨浆为多种无机钙盐与纯净水混合物,观察C em-O steticTM人工骨浆修复掌骨缺损的临床疗效及不良反应。方法2004年1月至2004年4月对6例6处掌骨缺损进行修复,年龄20~32岁,平均26岁。骨缺损范围为0.2 cm×1 cm~0.5 cm×1 cm,均为陈旧性。缺损的部位为掌骨干中1/3,修整骨断端,断端露出正常骨质,骨缺损部位植入C em-O steticTM人工骨浆。结果手术均获成功,术后随访5~8个月,平均6个月。全部患者未见任何不良全身反应及局部反应,X线片显示固化后的C em-O steticTM人工骨浆与受区骨直接愈合,手术后测定血钙、磷值与术前对比无明显升高。结论C em-O steticTM人工骨浆适用于掌骨缺损修复。     

不同植骨材料对跟骨骨折切口愈合的影响

目的研究两种不同植骨材料（白体髂骨、同种异体骨）对跟骨骨折术后切口愈合的影响。方法回顾性研究安徽医科大学第一附属医院自2009年1月至2012年12月采用切开复位AO钢板内固定加植骨术治疗的76例单侧跟骨骨折患者,其中男53例,女23例,平均年龄39．5岁。自体髂骨植骨40例（A组）,同种异体骨植骨36例（B组）。应用统计学方法对患者年龄、伤后至手术时间、切口干燥时间、切口愈合时间、术后24h切口引流量等进行评价。结果A组平均年龄（39．43±11．71）岁,伤后至手术平均时间（10．27±2．54）d,切口平均干燥时间（6．02±1．37）d,切口平均愈合时间（14．08±1．27）d,术后24h平均引流量（78．58±13．92）mL;B组平均年龄（39．53±110．52）岁,伤后至手术平均时间（9．14±3．19）d,切口平均干燥时间（8．08±1．02）d,切口平均愈合时间（16．14±1．84）d,术后24h平均负压引流量（96．69±13．57）mL。A组与B组的切口干燥时间、愈合时间、术后24h引流量的比较差异有统计学意义（P〈0．05）。结论自体髂骨植骨较同种异体骨植骨会缩短跟骨骨折切口的愈合时间。     

带旋髂深血管蒂髂骨转位移植治疗股骨头坏死

目的探讨采用改进后手术器械带血管蒂髂骨移植治疗股骨头坏死的长期随访效果,为股骨头坏死治疗提供一种有效的手术方法。方法自1983年11月至2006年5月,采用沿股骨颈纵轴开窗减压,带旋髂深血管蒂髂骨转位移植治疗股骨头坏死。术后仍有部分病例股骨头出现二次塌陷。因此,特殊设计了手术器械,沿股骨颈前下方向股骨头外上方开窗减压植骨。结果患者髋关节疼痛均明显缓解或消失,术后股骨头未出现二次塌陷,关节功能恢复。平均随访13．6年（10418年）。手术前与随访时髋关节功能评价,采用髋关节创伤后的功能评分（Sanders）,手术前平均35．69,手术后增加到末次随访时的52．89,总分明显增加,优良率94．1％。结论手术器械及术式改进后,移植骨支撑股骨头负重区,更加符合生物力学要求,能有效防止股骨头塌陷。