An experimental approach to spinal fusion using sintered bovine bone in a pig model

Sintered bovine bone is a biomaterial based on calcium phosphate, an organized crystal of bone mineral that possesses a natural trabecular structure. The authors considered whether sintered bovine bone can integrate with recipient bone and adjust to the strength of recipient bone for anterior spinal fusion in an animal model. Either autologous iliac bone or sintered bovine bone was implanted for a spinal body fusion. Some pigs underwent exposure of the spinal bodies without implantation. Based on radiographic evaluation, manual palpation, biomechanical testing, and histologic examination, spinal fusion with sintered bovine bone resulted in a composition and structure similar to that of the autograft (or of no implantation). The sintered bovine bone with its moderate strength tended to adjust to the bone stiffness of the host bone in the specimens as new bone grew.     

Growth of new bone guided by implants in a murine calvarial model

New methods to increase vertical bone growth are needed to permit dental implant placement in patients with low alveolar ridge height after extended periods of tooth loss. While ectopic rodent models are typically used to evaluate new osteogenic implant surface coatings, a more relevant intramembraneous rodent model was needed to address the particular clinical need to grow a new layer of bone above an existing layer of bone. In this study we report on a novel murine calvaria model in which successful vertical bone growth around miniaturized dental implants was achieved when using non-glycosylated bone morphogenetic protein-2 (ng/rhBMP-2). Twenty CD-1 mice received two Ti implants each consisting of a Ti ring implant stabilized by a Ti screw into the occipital calvarial bone. Four groups were evaluated: control Ti, Ti+20 mug ng/rhBMP-2, hydroxyapatite (HA)-coated Ti, and HA+20 mug ng/rhBMP-2. The mice were sacrificed 21 days following implant placement. MicroCT analysis showed no new bone formation around the untreated Ti or the HA-coated implants, but demonstrated new bone growth in every dimension around and above the Ti+ng/rhBMP-2 and the HA+ng/rhBMP-2 treated implants. Histopathologic analysis showed that a thin fibrous capsule covered the untreated Ti implants. Limited bone-to-implant contact (BIC) was observed for the HA-coated implants, while in contrast both ng/rhBMP-2 treated groups exhibited extensive new supracalvarial woven bone that covered the implant and merged with the calvarial plate. Histomorphometrically, supracalvarial bone heights and bone widths and BIC were not statistically different from one another for the two ng/rhBMP-2 treated groups. However, the total supracalvarial bone surface area was significantly greater (p<0.05) for the Ti+ng/rhBMP-2 implants (7.2 mm(2)) than the HA+ng/rhBMP-2 (4.0 mm(2)) treated implants. The bone density within 1 mm around the implant was also significantly greater (p<0.05) for the Ti+ng/rhBMP-2 implants (9.9%) than the HA+ng/rhBMP-2 (4.0%) implants, indicating that HA coatings may not be required for sustained release when non-glycosylated BMP-2 is used. This new murine model is capable of discriminating between various bone augmentation strategies and may represent a clinically more relevant model for alveolar bone augmentation than the commonly used ectopic muscle pouch or long bone models.     

组织工程骨修复兔颅骨缺损的实验研究

目的　探讨组织工程化骨修复骨缺损实用价值。方法　将自体成骨样细胞即刻种植在胶原包埋的聚羟基乙酸（PGA）基质材料上,然后将该复合体或单纯基质材料移植到兔颅骨的一侧全层骨缺损区,作为实验侧Ⅰ或实验侧Ⅱ。对侧设对照,不作任何植入。将40只新西兰兔分别于术后2、6、8、12w处死,标本行大体组织学检查,X线摄片及灰量测定检查。结果　术后2w实验侧I缺损区可见骨小梁形成,且各期成骨面积明显优于实验侧Ⅱ及对照侧,另外,植入材料内的灰量测定结果及X线摄片结果也表明实验侧I成骨量明显大于实验侧Ⅱ（p<0.01）,而对照侧为纤维组织修复。结论　应用胶原包埋PGA加成骨样细胞复合体移植可修复自体的兔颅骨缺损,为组织工程化骨在颅面外科及整形外科领域的临床应用奠定了基础。     

Cancellous bone adaptation to in vivo loading in a rabbit model

Biophysical stimuli are important to the development and maintenance of cancellous bone, but the regulatory mechanisms need to be understood. We investigated the effects of mechanical loading applied in vivo to native cancellous bone in the rabbit on bone formation and trabecular realignment. A novel device was developed to apply controlled compressive loads to cancellous bone in situ. The effect of loading on cancellous bone volume fraction and architecture was quantified. A 4-week experiment was performed in rabbits with devices implanted bilaterally. Cyclic 1 MPa pressures were applied daily to the right limb for 10, 25, or 50 cycles at 0.5 Hz, and the left limb served as the control without any applied loading. Microcomputed tomography and histomorphometry were used to characterize the cancellous tissue within a 4-mm spherical volume located below the loading core. In vivo cyclic loading significantly increased the bone volume fraction, direct trabecular thickness, mean intercept length, and mineral apposition rate in the loaded limbs compared with contralateral limbs. Insufficient evidence was found to demonstrate an effect of number of cycles on the cancellous adaptation between loaded and control limbs. Using a rabbit model, we demonstrated that mechanical loading applied to cancellous bone in situ increased bone formation and altered trabecular morphology. This in vivo model will allow further investigation of cancellous functional adaptation to controlled mechanical stimuli and the influence of mechanical loading parameters, metabolic status, and therapeutic agents.     

An in vivo evaluation of bone response to three implant surfaces using a rabbit intramedullary rod model

Our study was designed to evaluate osseointegration among implants with three surface treatments: plasma-sprayed titanium (P), plasma-sprayed titanium with hydroxyapatite (PHA), and chemical-textured titanium with hydroxyapatite (CHA). Average surface roughness (Ra) was 27 microns for the P group, 17 microns for the PHA group, and 26 microns for the CHA group. Bilateral distal intramedullary implants were placed in the femora of thirty rabbits. Histomorphometry of scanning electron microscopy images was used to analyze the amount of bone around the implants at 6 and 12 weeks after implantation. Greater amounts of osseointegration were observed in the hydroxyapatite-coated groups than in the noncoated group. For all implant surfaces, osseointegration was greater at the diaphyseal level compared to the metaphyseal level. No significant differences were seen in osseointegration between the 6 and 12 week time points. Although the average surface roughness of the P and the CHA groups was similar, osseointegration of the CHA implants was significantly greater. The results of this in vivo lapine study suggest that the presence of an hydroxyapatite coating enhances osseointegration despite similarities in average surface roughness.     

腺病毒介导NELL1促进骨溶解后的高质量骨再生

[目的]研究具有特异性成骨作用的NELL1因子对磨屑所致骨溶解的影响,为骨溶解病的治疗提供一种新方案.[方法]建立聚乙烯( Polyethylene,PE)颗粒诱导的颅骨骨溶解模型,68只Balb/c小鼠随机分成4组,即假手术组:术后1d颅顶皮下注射0.1 ml生理盐水;颗粒对照组:植入10 mg PE颗粒,术后1d注射0.1ml生理盐水;实验组(Ad - GFP - NELL1组):植入10 mg PE颗粒,术后1d注射0.1 ml( 109 pfu) NELL1荧光重组腺病毒载体( Ad - GFP - NELL1);安慰剂组(Ad - GFP组):植入10 mg PE颗粒,术后1d注射0.1ml (109 pfu)荧光重组腺病毒载体( Ad - GFP).术后10 d采用活体荧光成像检测NELL1及GFP表达情况,并于处死后进行Micro - CT、组织学及生物力学的检测.[结果]活体荧光证实实验组与安慰剂组小鼠颅顶有NELL1及GFP的高度表达,Micro - CT 3D图像观察实验组小鼠的颅骨骨溶解所致骨量丢失明显减少,所选兴趣区(ROI)的骨体积(BV)、骨密度(BMD)、骨小梁厚度(Tb.Th.3D)、骨表面积体积比(BS/BV)均明显优于对照组及安慰剂组.组织学切片观察到实验组的骨再生明显增多.另外,实验组小鼠的颅骨弹性模量[(2.50±0.12)MPa]也明显优于对照组[(0.60±0.20)MPa]及安慰剂组[ (0.82±0.09) MPa](OP＜0.01).[结论]腺病毒介导表达的成骨因子NELL1能通过促进高质量的骨再生平衡磨屑所致骨溶解的骨量丢失,是治疗骨溶解的一种潜在性解决方案.     

Cranial reconstruction using autologous split calvarial bone combined with calcium phosphate bone cement: a case report

We report a case of cranial reconstruction using autologous split calvarial bone combined with calcium phosphate bone cement (CPC). A 19-years-old man suffered from cranium defect and rhinorrhea originating from frontal skull base fracture in a traffic accident. After CSF hydration treatment had finished, continuously we performed cranial reconstruction with autologous split calvarial bone so that the patient could return to work at an early stage. The use of autologous split calvarial bone with CPC was able to increase stability of the construct and provide excellent cosmetic result in our short follow up period. The combination use of these two materials may be useful for cranial reconstruction in patients with cranium defect.     

A free full-thickness calvarial bone graft to reconstruct a post-mandibular fracture deformity

Summary A case of free calvarial bone grafting to reconstruct a post-mandibular fracture deformity with a 19 years' follow-up is reported. The grafted membranous calvarial bone has remained solid and stable with little resorption. The patient can wear a denture. This is the first successful and longest follow-up case report of a free calvarial bone graft in mandibular reconstruction. Requests for reprints: Prof. T. Fujino, M.D.     

Craniofacial reconstruction after tumor resections using vascularized outer table calvarial bone flaps

The outer table of calvarium is a useful donor site for facial reconstruction after resection of tumors. Large defects, especially when associated with multiple operations and radiotherapy, are poor recipient beds for nonvascularized bone grafts. A technique for the transfer of vascularized outer table has been developed and used in 11 patients for reconstruction of tumor defects. Satisfactory correction of both functional and esthetic problems has been achieved with satisfactory long-term stability and low morbidity.     

Effect of parathyroid hormone on amino acid transport by cultured neonatal mouse calvarial bone cells

The effect of synthetic bovine parathyroid hormone [bPTH-(1-34)] on amino acid uptake by confluent primary cultures of osteoblast-like cells isolated from neonatal mouse calvaria was studied. The uptake of proline and leucine by membrane transport Systems A, ASC, and L was discriminated on the basis of their sodium dependency and sensitivity to the system-specific amino acid analogs 2-(methylamino)-isobutyric acid (MeAIB) for System A and 2-amino-(2,2,1)-heptane-2-carboxylic acid (BCH) for System L. Treatment with 24 nM bPTH-(1-34) in serum-free EBSS for 4 hr increased the initial uptake rate of proline by 50-80% but had no effect on the uptake of leucine. Temporally, the increase in proline uptake was preceded by a 2-hr lag period and plateaued after 5-6 hr. A 5-min exposure to the hormone was sufficient to cause a significant increase in proline uptake measured 4 hr later. The magnitude of the increase was dose-related from 0.24 to 240 nM bPTH-(1-34), with the half-maximal effect occurring at 2.4 nM. Only the sodium-dependent, MeAIB-inhibitable component of proline uptake was elevated. Eadie-Hofstee analysis indicated that bPTH-(1-34) increased Vmax without changing the Km. Actinomycin D and cycloheximide prevented the hormone-stimulated increase, suggesting that RNA and protein synthesis were required. Treatment with either inhibitor alone caused a 30-35% decrease in proline transport that was not observed in the presence of bPTH-(1-34), indicating an effect not dependent on macromolecular synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

组织工程化人工骨修复颅骨缺损的研究

目的应用同种异体兔成骨细胞研究组织工程化人工骨对骨缺损的修复能力.方法取4周龄新西兰幼兔的颅骨组织,应用胰酶及Ⅰ型胶原酶消化,经分离、培养而获得的成骨细胞种植到可降解聚合物(PLGA)泡沫材料上,体外培养1周后,将两者的复合体移植到兔颅骨缺损区域,细胞接种浓度为4×1010个/L.分别于术后4、8、12周取材,进行大体、X线、组织学及免疫组织化学观察.以缺损区单纯植入泡沫材料或单纯种植成骨细胞为对照组.结果成骨细胞-支架复合体组颅骨缺损修复明显,两对照组颅骨缺损无明显修复.术后12周,对各组免疫组织化学染色结果进行图像分析,其中实验组灰度值为92.6,单纯泡沫材料组与单纯种植成骨细胞组灰度值分别为131.7及119.3.结论本实验构建的组织工程化人工骨具有较强的颅骨缺损修复能力,PLGA泡沫材料是成骨细胞较适宜的支架材料.     

掺锶硫酸钙及DBM复合材料骨修复能力评价

[目的]制备一种掺锶硫酸钙与DBM复合的骨修复材料,为临床提供一种成骨活性优良的骨修复材料。[方法]制备掺锶硫酸钙与DBM复合的活性骨修复材料,其中锶元素的量为钙含量的6%,掺锶硫酸钙与DBM按体积比1∶1复合。观察兔股骨髁骨缺损模型修复效果,利用X线片观察缺损区域的新骨形成情况,采用MicroCT评价局部的骨量和骨质变化,对组织切片予以Van Gieson(VG)复合染色法染色进行组织形态学观察,荧光双标观测计算成骨速率。[结果]术后动物无死亡,所有兔子切口无渗血或出现分泌物状况,术后活动正常。从MicroCT的BV/TV结果看出实验组在12周时缺损区域已经恢复;掺锶CaSO_4复合材料组缺损部位已经基本修复,CaSO_4组未完全修复。VG组织学观察显示各组均有新生骨小梁生成,荧光双标观测得到的矿化沉积率显示复合材料组新骨生长突出,矿化沉积率为(2.31±0.13)μm/d。[结论]通过MicroCT、VG组织学染色以及荧光双标观测对兔子股骨髁缺损修复效果进行评价,发现复合材料具有较好的骨缺损修复能力。     

Development of a new method in promoting fracture healing: multiple cryopreserved bone marrow injections using a rabbit model

Introduction It was hypothesized that multiple injections of cryopreserved bone marrow (BM) would promote fracture healing.Materials and methods Experiments were performed to select a method for freezing BM nucleated cells based on the recovery rate, and to investigate the effects of frozen BM nucleated cells on fracture healing in a rabbit fracture nonunion model. Radiographic and histological evidences of bone union and new bone growth were compared between the experimental group and the control.Results Storage of BM nucleated cells in 10% DMSO plus 90% autologous serum showed the optimal rate of cell recovery, cell viability, and osteoprogenitor cell recovery. Consecutive injections directly to the bone defect site with cryopreserved BM nucleated cells significantly increased the incidence of union, the radiographic volume and bone mineral density of repaired bone as compared with the control (p<0.05).Conclusion A new method was developed to efficiently freeze BM nucleated cells, and cells cryopreserved with this method were found to promote fracture healing by multiple injections. It is suggested that cryopreserved BM injection provides an alternative approach for grafting in delayed union or nonunion.     

Erythropoietin augments bone formation in a rabbit posterolateral spinal fusion model

We tested the hypothesis that erythropoietin (EPO) enhances bone formation after posterolateral spinal fusion (PLF) in a rabbit model. Thirty-four adult rabbits underwent posterolateral intertransverse arthrodesis at the L5-L6 level using 2.0 g autograft per side. The animals were randomly divided into two groups receiving subcutaneous daily injections of either EPO or saline for 20 days. Treatment commenced 2 days preoperatively. Hemoglobin was monitored at baseline and 2, 4, and 6 weeks after fusion surgery. After euthanasia 6 weeks postoperatively, manual palpation, radiographic, and histomorphometric examinations were performed. Bone volume of the fusion mass was estimated by CT after 6 weeks. EPO increased bone fusion volume to 3.85 ccm (3.66-4.05) compared with 3.26 ccm (2.97-3.55) in the control group (p<0.01). EPO treatment improved vascularization of the fusion mass and increased hemoglobin levels (p<0.01). Fusion rate tended to be higher in the EPO group based on manual palpation, CT, and radiographic examinations. For the first time EPO has shown to augment bone formation after autograft PLF in a rabbit model. Increased vascularization provides a partial explanation for the efficacy of EPO as a bone autograft enhancer.     

Systemic corticosteroids inhibit bone healing in a rabbit ulnar osteotomy model

Prolonged systemic administration of corticosteroids causes osteoporosis and increased risk of fracture. Despite this well documented side effect of systemic corticosteroids, the effect of these compounds on fracture healing is not well defined. The goal of this study was to test the hypothesis that systemic corticosteroid therapy adversely affects fracture healing in a rabbit ulnar osteotomy model. Non-critical sized (1 mm) defects were created bilaterally in 18 adult female New Zealand White rabbits. Starting 2 months before operative intervention and continuing for 6 weeks during healing of the osteotomies, a subcutaneous dose of either sterile saline or prednisone (0.15 mg/kg) was administered daily. Serial radiographs of the forelimb were taken immediately postoperatively and weekly beginning the second week postoperatively. After killing at 6 weeks, only 3 of 20 limbs from animals treated with prednisone achieved radiographic union while 13 of 16 control limbs achieved union. The radiographic density of bone in the defect as well as callus size were greater in the control limbs than in the limbs from prednisone-treated animals. DEXA confirmed that the bone mineral content was lower in the ulnae of prednisone-treated rabbits both within the defect and in adjacent ulnar bone. Mechanical data indicated that osteotomies from rabbits chronically treated with prednisone were weaker than in controls. In this rabbit ulnar osteotomy model, chronic prednisone treatment clearly inhibited bone healing.     

Systemic corticosteroids inhibit bone healing in a rabbit ulnar osteotomy model

Prolonged systemic administration of corticosteroids causes osteoporosis and increased risk of fracture. Despite this well documented side effect of systemic corticosteroids, the effect of these compounds on fracture healing is not well defined. The goal of this study was to test the hypothesis that systemic corticosteroid therapy adversely affects fracture healing in a rabbit ulnar osteotomy model. Non-critical sized (1 mm) defects were created bilaterally in 18 adult female New Zealand White rabbits. Starting 2 months before operative intervention and continuing for 6 weeks during healing of the osteotomies, a subcutaneous dose of either sterile saline or prednisone (0.15 mg/kg) was administered daily. Serial radiographs of the forelimb were taken immediately postoperatively and weekly beginning the second week postoperatively. After killing at 6 weeks, only 3 of 20 limbs from animals treated with prednisone achieved radiographic union while 13 of 16 control limbs achieved union. The radiographic density of bone in the defect as well as callus size were greater in the control limbs than in the limbs from prednisone-treated animals. DEXA confirmed that the bone mineral content was lower in the ulnae of prednisone-treated rabbits both within the defect and in adjacent ulnar bone. Mechanical data indicated that osteotomies from rabbits chronically treated with prednisone were weaker than in controls. In this rabbit ulnar osteotomy model, chronic prednisone treatment clearly inhibited bone healing.