Vancomycin-bearing Synthetic Bone Graft Delivers rhBMP-2 and Promotes Healing of Critical Rat Femoral Segmental Defects

Background

Bone grafts simultaneously delivering therapeutic proteins and antibiotics may be valuable in orthopaedic trauma care. Previously, we developed a poly(2-hydroxyethyl methacrylate)-nanocrystalline hydroxyapatite (pHEMA-nHA) synthetic bone graft that, when preabsorbed with 400-ng rhBMP-2/7, facilitated the functional repair of critical-size rat femoral defects. Recently, we showed that pHEMA-nHA effectively retains/releases vancomycin and rhBMP-2 in vitro. The success of such a strategy requires that the incorporation of vancomycin does not compromise the structural integrity of the graft nor its ability to promote bone healing.

Questions/purposes

(1) To evaluate the ability of pHEMA-nHA-vancomycin composites in combination with 3-µg rhBMP-2 to repair 5 mm rat femoral segmental defects, and (2) To determine if the encapsulated vancomycin impairs the graft/rhBMP-2-assisted bone repair.

Methods

pHEMA-nHA-vancomycin, pHEMA-nHA, or collagen sponge control with/without 3-µg rhBMP-2 were press-fit in 5 mm femoral defects in SASCO-SD male rats (289–300 g). Histology, microcomputed tomography, and torsion testing were performed on 8- and 12-week explants to evaluate the extent and quality of repair. The effect of vancomycin on the temporal absorption of endogenous BMP-2 and stromal cell-derived factor-1 was evaluated by immunohistochemistry. These factors are important for bone healing initiation and stem cell recruitment, respectively.

Results

Partial bridging of the defect with bony callus by 12 weeks was observed with pHEMA-nHA-vancomycin without rhBMP-2 while full bridging with substantially mineralized callus and partial restoration of torsional strength was achieved with 3-µg rhBMP-2. The presence of vancomycin changed the absorption patterns of endogenous proteins on the grafts, but did not appear to substantially compromise graft healing.

Conclusions

The composite pHEMA-nHA-vancomycin preabsorbed with 3-µg rhBMP-2 promoted repair of 5 mm rat femoral segmental defects. With the sample sizes applied, vancomycin encapsulation did not appear to have a negative effect on bone healing.

Clinical Relevance

pHEMA-nHA-vancomycin preabsorbed with rhBMP-2 may be useful in the repair of critical-size long bone defects prone to infections.     

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.     

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。     

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。     

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。     

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.     

Characterization of Demineralized Bone Matrix-Induced Osteogenesis in Rat Calvarial Bone Defects: III. Gene and Protein Expression

Our previous studies of rat cranial defect repairs after the implantation of demineralized bone matrix (DBM) have demonstrated that healing occurs initially and principally by the direct induction and proliferation of osteoblasts derived principally from resident mesenchymal stem cells of the dura, and to a lesser extent by resident mesenchymal stem cells of the connective tissues beneath the skin flap. A small amount of cartilage is also synthesized after the direct process of ossification occurs. To further confirm the molecular phenotypes of the repair cells in rat cranial defects, the present study evaluated mRNA expression and synthesis of collagens I, II, and X and osteocalcin in the DBM-induced repair tissue by Northern blot analyses, autoradiography after in vivo ³H-proline labeling of collagen, and immunohistochemistry. The results demonstrated that osteocalcin mRNA appeared in small amounts by day 4 and continued to increase over the experimental period. Much lesser quantities of collagen types II and X mRNAs appeared by day 6 and day 8, respectively. Collagen type I mRNA was present at all times examined but its expression significantly increased by day 5. Autoradiographic and immunohistochemical studies showed that type II collagen was not detected whereas type I collagen was synthesized on days 3–5. The data provide definitive molecular evidence confirming that the initial and by far the major pathway of cranial defects repair induced by implantation of DBM is by the direct induction of resident mesenchymal stem cells to osteoblasts and the direct formation of bone, which is spatially and temporarily distinct from the later formation of cartilage. Received: 30 November 1999 / Accepted: 21 March 2000     

以自体诱导和无诱导的骨髓基质干细胞复合珊瑚修复犬下颌骨节段缺损的比较研究

目的分别采用诱导和无诱导的自体骨髓基质干细胞（Bone marrow stromal cells，BMSCs）复合珊瑚构建组织工程化骨，修复犬下颌骨节段性缺损，比较修复效果。方法体外扩增、成骨诱导或无诱导培养犬BMSCs，分别将第2代细胞复合珊瑚后修复犬自体右侧3cm的下颌骨节段缺损（诱导组n=6，无诱导组n=6）。术后32周，分别通过Micro-CT、大体形态观察和组织学方法检测骨缺损的修复效果。结果32周时，Micro-CT检测示诱导组骨容积率和密度均显著高于对照组（P〈0.05）；大体观察示诱导组骨愈合良好，无诱导组中的3条犬为骨不连；组织学检测诱导组有较多成熟骨形成，缺损部分均呈骨性愈合。无诱导组中的3只犬有新骨形成，但形态不完整，另3只犬的缺损部分呈纤维性愈合。结论成骨诱导的自体BMSCs复合珊瑚形成的组织工程化骨修复犬下颌骨节段缺损效果优于无诱导组。     

BMP2Gene Therapy on the Repair of Bone Defects of Aged Rats

Age-related decline in the number of mesenchymal stem cells (MSCs) and their reduced capability to differentiate osteogenically, along with diminished availability of growth factors, may be major factors accounting for reduced bone formation in the aging mammalian body. In the first part of the study, we compared the number of MSCs in bone marrow (BM) and the content of bone morphogenetic protein 2 (BMP2) in cortical bone tissue in juvenile, adult, and aged (1, 9, and 24 months, respectively) male rats. To assay the influence of aging on osteogenic differentiation ability, MSCs from the three age groups were transduced with the BMP2 gene. Following gene transduction, the production of BMP2 in culture media, expression of osteogenic proteins (e.g., alkaline phosphatase, type Iα1 collagen, osteopontin, and bone sialoprotein), as well as ectopic bone formation in athymic mice were compared. Results showed that the number of MSCs in BM as well as the content of BMP2 in cortical bone tissue decreased with age, but no significant differences between the three age groups were found with regard to production of BMP2 or capability of BMP2 gene-modified MSCs to differentiate osteogenically. The second part of the study applied BMP2 gene-modified autologous MSCs/β-tricalcium phosphate for repair of bone defects in aged rats with positive results. Our data indicate that the osteogenic potential of MSCs of aged rats can be restored following BMP2 gene transduction and that this technique may be a useful approach in the future planning of gene therapy for age-related osteoporotic fractures.     

吻合血管节段性腓骨移植修复邻近关节巨大腔性骨缺损

目的：邻近关节骨肿瘤或肿瘤样病变切除后的修复。方法：仿据排骨血供的特性,设计吻合血管腓骨多节段移植,修复胫骨上端、股骨下端及股骨近端骨缺损。结果：临床应用10例,经术后3个月随访,移植骨愈合良好,关节功能恢复令人满意。结论：采用该方法,修复邻近关节骨肿瘤切除后巨大腔性骨缺损,远优于吻合血管或单纯松质骨植入,从根本上避免或替代了异体半关节移植。     

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.     

碱性成纤维细胞生长因子增强人重组骨形态发生蛋白-2诱导成骨的调节机理

目的：对ｂＦＧＦ增强ｒｈＢＭＰ－２诱导成骨的调节机理进行探讨。方法：２１０只ＢＡＬＢ／ｃ小鼠随机分为３组,每组７０只,试验侧均位于右后肢。设立ｒｈＢＭＰ－２／牛松质骨载体、单纯牛松质骨载体为对照组,分别于术后１２ｈ～２１ｄ共１１个时间点取材,观察其诱导成骨过程。结果：ｒｈＢＭＰ－２／ｂＦＧＦ／聚乙烯吡咯啉酮／牛松质骨载体组在诱导间充质细胞增殖、分化,软骨细胞、新生骨形成方面均早于ｒｈＢＭＰ－２／牛松质骨载体组,成骨量优于ｒｈＢＭＰ－２／牛松质骨载体组,而单纯牛松质骨载体组在２１ｄ仅出现了少量增殖的间充质细胞。结论：ｒｈＢＭＰ－２和ｂＦＧＦ在诱导成骨调节中存在着协同作用。     

表面脱钙骨基质明胶修复长骨骨缺损9例报告

采用表面脱钙骨基质明胶（ＳＤＢＭＧ）移植治疗长骨大块骨缺损９例，结果，除１例因适应证选择不当而失败外，其它８例均得到治愈，患健肢等长。说明ＳＤＢＭＧ具有良好的成骨作用和可靠的生物力学性能，是修复长骨大块骨缺损的理想移植材料，可作为自体骨合适的替代材料推广使用，但对急性感染性骨缺损应慎用     

Evolution and Development of Ilizarov Technique in the Treatment of Infected Long Bone Nonunion with or without Bone Defects

The treatment of infected bone nonunion and bone defects is a considerable challenge in the orthopedics field. The standard clinical therapy methods include local free bone transplantation, vascularized bone graft, and the Ilizarov technique; the first two are controversial due to the iatrogenic self‐injury. The Ilizarov bone transport technique has been widely used to treat infected bone nonunion and bone defects, and good clinical effect has been demonstrated. Yet, it brings many related complications, which exerts additional suffering to the patient. The best treatment is to combine bone defect rehabilitation with infection control, intramedullary nail fixation, appropriate time for bone grafts, beaded type scaffold slippage and new Taylor fixation, reducing the external fixation time and the incidence of complications, thereby reducing the occurrence of patients'' physical and psychological problems. This review focuses on the induction, summary and analysis of the Ilizarov bone transport technique in the treatment of infected long bone nonunion with or without bone defects, providing new ideas and methods for orthopedic disease prevention and treatment by the Ilizarov technique, which is following the development direction of digital orthopedics.     

骨髓间充质干细胞复合骨形态发生蛋白和纤维蛋白修复节段性骨缺损

目的　用自体骨髓间充质干细胞 (MSCs)、骨形态发生蛋白 (BMP)和纤维蛋白的复合物修复骨缺损。方法　抽取兔自体骨髓并分离和大量培养MSCs。在 12只MSCs供体兔的两侧桡骨中段造成 1 5cm缺损 ,一侧植入自体MSCs、 15mgBMP和纤维蛋白的复合物 (B +M +F) ,另一侧植入自体MSCs与纤维蛋白的复合物 (M +F)。另一组等数量实验兔造成相同缺损后 ,一侧植入 15mgBMP与纤维蛋白的复合物 (B +M ) ,另一侧留作空白对照。术后第 2 ,4 ,8周做放射学、组织学和ECT检查。结果　B +M +F组在术后 2周即产生填满缺损区的骨痂影 ,8周时骨缺损得到良好修复。B +M组所产生骨痂量和修复效果均不如B +M +F组 ,但优于M +F组。结论　用自体骨髓间充质干细胞复合骨形态发生蛋白和纤维蛋白修复骨缺损是一种切实可行和有效的方法。