Establishment and effects of allograft and synthetic bone graft substitute treatment of a critical size metaphyseal bone defect model in the sheep femur

Assessment of bone graft material efficacy is difficult in humans, since invasive methods like staged CT scans or biopsies are ethically unjustifiable. Therefore, we developed a novel large animal model for the verification of a potential transformation of synthetic bone graft substitutes into vital bone. The model combines multiple imaging methods with corresponding histology in standardized critical sized cancellous bone defect. Cylindrical bone voids (10 ml) were created in the medial femoral condyles of both hind legs (first surgery at right hind leg, second surgery 3 months later at left hind leg) in three merino‐wool sheep and either (i) left empty, filled with (ii) cancellous allograft bone or (iii) a synthetic, gentamicin eluting bone graft substitute. All samples were analysed with radiographs, MRI, μCT, DEXA and histology after sacrifice at 6 months. Unfilled defects only showed ingrowth of fibrous tissue, whereas good integration of the cancellous graft was seen in the allograft group. The bone graft substitute showed centripetal biodegradation and new trabecular bone formation in the periphery of the void as early as 3 months. μCT gave excellent insight into the structural changes within the defects, particularly progressive allograft incorporation and the bone graft substitute biodegradation process. MRI completed the picture by clearly visualizing soft tissue ingrowth into unfilled bone voids and presence of fluid collections. Histology was essential for verification of trabecular bone and osteoid formation. Conventional radiographs and DEXA could not differentiate details of the ongoing transformation process. This model appears well suited for detailed in vivo and ex vivo evaluation of bone graft substitute behaviour within large bone defects.     

The effects of microporosity on osteoinduction of calcium phosphate bone graft substitute biomaterials

The effect of increasing strut porosity on the osteoinductive ability of silicate substituted calcium phosphate (SiCaP) biomaterials was investigated in an ectopic ovine model. Implants with strut porosities of 22.5%, 32.0% and 46.0% were inserted into the parapsinalis muscle. At 8, 12 and 24 weeks histological sections were prepared. Sections were examined using backscattered scanning electron microscopy and un-decalcified histology. Bone area, implant area and bone-implant contact were quantified. At 8 weeks there was no significant difference between the groups in terms of bone area and implant area. However at 12 weeks, the amount of bone formation observed was significantly greater in SiCaP-46 (6.17 ± 1.51%) when compared with SiCaP-22.5 (1.33 ± 0.84%) p=0.035. Results also showed significantly increased amounts of bone-implant contact to the SiCaP-46 scaffold (3.30 ± 1.17%) compared with SiCaP-22.5 (0.67 ± 0.52%, p=0.043) at 8 weeks and 12 weeks; (SiCaP-46 (21.82 ± 5.59%) vs SiCaP-22.5 (3.06 ± 1.89%), p=0.012). At 24 weeks, bone formation and graft resorption had significantly increased in all groups so that the level of bone formation in the SiCaP-46 group had increased 75-fold to 30.05 ± 8.38%. Bone formation was observed in pores <10 μm. Results suggest that bone graft substitute materials with greater strut porosity are more osteoinductive.     

The use of coral as a bone graft substitute

Experiments have been performed to investigate the use of coral skeletons as bone graft substitutes. Skeletal fragments of different coral genera were implanted into cortical and spongy bone defects and used to bridge transcortical resections in the femur. The implant site was monitored for up to 18 months. Radiographically, both cortical and spongy bone defects were at least partially filled by new bone after 8 weeks while the implants underwent continuous resorption. Coral resorption and replacement by new tissue was also observed in the transcortical resections. The process of resorption was attributed to the enzymatic attack, especially carboanhydrase. This was confirmed by experiments in which dogs were implanted with coral in transcortical resections and treated daily with acetazolamide, a carboanhydrase inhibitor; the absorption appeared delayed and the resections failed to heal.     

Natural coral exoskeleton as a bone graft substitute: a review

Natural coral graft substitutes are derived from the exoskeleton of marine madreporic corals. Researchers first started evaluating corals as potential bone graft substitutes in the early 1970s in animals and in 1979 in humans. The structure of the commonly used coral, Porites, is similar to that of cancellous bone and its initial mechanical properties resemble those of bone. The exoskeleton of these high content calcium carbonate scaffolds has since been shown to be biocompatible, osteoconductive, and biodegradable at variable rates depending on the exoskeleton porosity, the implantation site and the species. Although not osteoinductive or osteogenic, coral grafts act as an adequate carrier for growth factors and allow cell attachment, growth, spreading and differentiation. When applied appropriately and when selected to match the resorption rate with the bone formation rate of the implantation site, natural coral exoskeletons have been found to be impressive bone graft substitutes. The purpose of this article is to review and summarize all the pertinent work that has been published on natural coral as a bone graft including in vitro, animal and clinical human studies. Preliminary report of our own experiments as well as our recommendations on the use of coral are also included.     

In vitro characterization of a calcium sulfate/PLLA composite for use as a bone graft material

A study was conducted to characterize the dissolution, morphology, and chemical composition of a calcium sulfate/poly (L-lactic acid) (CS/PLLA) composite material before and after immersion in simulated body fluid (SBF). Twelve groups of experimental samples were prepared by coating CS pellets 1, 2, 3, or 4 times with one of three concentrations of a PLLA solution and wrapping them in mesh; CS pellets for use as controls were similarly prepared but not coated. The PLLA coating added from 1 to 22% to the weight of experimental pellets; scanning electron microscopy revealed that the coating thickness ranged from 2 to 50 microm depending on the concentration of the coating solution and the number of coatings. All samples were immersed in SBF for up to 97 days. After immersion, the experimental coatings thinned out, small cracks and holes formed in the coating, and the coating became roughened. Mean dissolution rates for each of the 12 CS/PLLA groups were significantly lower than those of uncoated CS pellets; among CS/PLLA groups, dissolution rates varied according to concentration of the coating solution and number of coatings. The half-life of pure CS pellets was 19 days whereas the half-life of CS/PLLA composite pellets ranged from 30 to 70 days. X-ray microprobe analysis of experimental pellets after immersion in SBF revealed that mineralization occurred in the CS portion of these pellets as well as on the coating; most of the mineral was calcium phosphate, most of which was on the coating. Further studies will be required to confirm this composite's promise as a clinically effective osteoconductive material.     

Impaction grafting with morsellised allograft and tricalcium phosphate-hydroxyapatite: incorporation within ovine metaphyseal bone defects

An ovine model was used to investigate the in vivo properties of impacted tricalcium phosphate-hydroxyapatite (TCP-HA) aggregates, varying in chemical composition (ratio of TCP to HA) and particle size distribution (8 versus 3 particle size ranges). All pellets were impacted to a standard compactive effort. Eight sheep underwent implantation of pellets in 4 metaphyseal defects in both rear limbs. Treatment groups consisted of: (1) allograft (clinical control). (2) 50/50 allograft/80% HA/20% TCP in 8 particle size ranges, (3) 50/50 allograft/80% TCP/20% HA in 8 sizes and (4) 50/50 allograft/80% HA/20% TCP in only 3 sizes of particles. Healing of defects was evaluated at 14 weeks with computed tomography, histology and histomorphometry. The computer tomography (CT) density measured in all defects containing synthetic agents was higher than in defects filled with allograft alone (p<0.01). Defects containing 8 sizes of 80% HA/ 20% TCP granules (group 2) achieved lower histological scores and contained less bone than the clinical control (p<0.05), whereas groups 3 and 4 did not differ from the control. Although all synthetic agents were osteoconductive, our results suggest that increasing the ratio of TCP over HA and limiting the number of particle size ranges to 3 instead of 8 improve the performance of impacted aggregates as graft expanders. Evaluation under loading conditions of morsellised allograft expanded with 80% TCP/20% HA (BoneSave) in 3 particle size ranges is warranted.     

Effect of increased strut porosity of calcium phosphate bone graft substitute biomaterials on osteoinduction

The effect of increasing strut porosity on the osteoinductivity of porous calcium phosphate (CaP) and silicate-substituted calcium phosphate (SiCaP) bone substitute materials was investigated in an ovine ectopic model. One to two millimeter-sized granules or block implants with strut porosities of 10, 20, or 30% were inserted into the left and right paraspinalis muscle. At 12 weeks, histological sections were prepared through the center of each implant and bone contact, bone area and implant area quantified. Backscattered scanning electron microscopy (bSEM) was used to visualize bone within small pores in the struts of the scaffolds. Increased bone formation was measured in the SiCaP with 30% strut porosity (5.482% ± 1.546%) when compared with the nonsilicate CaP with the same morphology (1.160% ± 0.502%, p = 0.02), indicating that silicate substitution may increase osteoinduction. Greater bone formation was seen in scaffolds with increased strut porosity. No bone growth was found in any of the SiCaP scaffold with 10% porosity. There was no significant difference between block and granule specimens. Scanning electron microscopy and EDX in combination with histology demonstrated bone formation within pores <5 μm in size. The use of silicate-substituted CaP material with increased strut porosity may further augment repair and regeneration in bony sites.     

Use of a novel bone graft substitute in peri-articular bone tumours of the knee

We treated three patients with aneurysmal bone cysts and seven with giant cell tumours who presented with a large osteolytic lesion in peri-articular areas of the knee. The patients age ranged from 8 to 49 years (mean 25 years). The average tumour volume was measured at 39.8 cm³ (range 18 to 65 cm³). The tumour cavities were treated with curettage, phenolisation and application of a composite bone graft substitute containing 35% calcium-sulphate hemihydrate and 65% hydroxyapatite granules. All patients were followed up for 4.0 to 5.2 years (mean 4.5 years). Tumour recurrence was noted in two cases. In the remaining patients consolidation of the lesion was considered complete at a mean of 4.5 months (3–6.5 months). During follow-up no deformities developed and no radiological signs of joint degeneration were noted. All patients regained close to normal function, with a mean Musculoskeletal Tumour Society Rating Score of 95.1%.

Due to their good osteoconductive abilities, composite synthetic bone graft substitute combining porous hydroxyapatite with calcium-sulphate appears to be an effective alternative to autologous cancellous bone graft in the treatment of large osteolytic lesions in peri-articular areas around the knee joint. They bear major advantages through ubiquitous availability and the avoidance of morbidity associated with iliac crest harvest. Concerns remain as the radio-opaque appearance of the bone graft substitute may potentially delay the detection of tumour recurrence.     

骨替代材料的生物相容性评价研究进展

生物安伞性的评价在于预测生物材料或医疗器械在与人体接触使用过程中的潜在危害性,保证牛物材料的应用安全性.骨替代材料的应用越来越广泛,需要进行一系列实验证明材料的安全性.文章对骨替代材料的生物相容性实验研究方法 进行了详细的论述,包括细胞相容性实验、全身毒性实验、血液相容性实验、皮肤致敏实验、刺激实验、遗传毒理学实验、体内植入实验.     

掺锶硫酸钙复合骨修复材料体内组织相容性研究

目的初步验证掺锶硫酸钙复合骨修复材料的体内组织相容性旨在为后续的进一步研究提供基础。方法取含锶量为0．5％的掺锶硫酸钙材料,采用新西兰兔背脊肌内植入模型。分别于手术后1周,2周,4周,8周四个时间点各处死3只兔子取材。HE染色,光镜观察细胞和肌肉组织的形态学变化。植入材料周围组织冰冻切片（厚度约51am）。选择细胞色素氧化酶（CCO）和乳酸脱氢酶（LDH）两种2种主要细胞器的标志酶进行酶组织化学染色。镜下观察得出计数资料,整理成等级资料。染色强度分为4个等级：阴性（O）,阳性（1）,强阳性（2）,和极强阳性（3）。结果所有实验动物术后1～3小时恢复活动,1天后进食正常。实验动物伤口均一期愈合。组织学结果显示掺锶硫酸钙材料植入1w后,植入体周围组织内有较多中性粒细胞浸润,由成纤维细胞组成厚薄不均、结构疏松的纤维囊壁。随着植入时间的延长,材料周围组织的炎症反应逐渐消失,炎细胞浸润越来越少,包膜先变厚再变薄,纤维包囊较薄。术后8w,植入体周围组织内未见炎性细胞浸润,纤维囊腔壁进一步变薄,主要成分为胶原纤维和成纤维细胞。其组织学改变与对照材料α-半水硫酸钙相比无明显区别。酶组织化学染色结果显示掺锶硫酸钙组植入材料周边肌细胞在1w时CCO和LDH活性均减弱（P〈0．05）,2w时CCO和LDH活性均恢复正常（P〉0．05）,其后活性无明显变化（P〉0．05）。掺锶硫酸钙组与a．CSH组和单纯创口组之间两两比较无明显差异（P〉0．05）。结论掺锶硫酸钙复合骨修复材料对体内组织细胞无毒性作用,具有与α-CSH相似的良好体内组织相容性。     

Porous single-phase NiTi processed under Ca reducing vapor for use as a bone graft substitute

Porous nickel-titanium alloys (NiTi, nitinol) have recently attracted attention in clinical surgery because they are a very interesting alternative to the more brittle and less machinable conventional porous Ca-based ceramics. The main remaining limitations come from the chemical homogeneity of the as-processed porous nickel-titanium alloys, which always contain undesired secondary Ti- and Ni-rich phases. These are known to weaken the NiTi products, to favor their cavitation corrosion and to decrease their biocompatibility. Elemental nickel must also be avoided because it could give rise to several adverse tissue reactions. Therefore, the synthesis of porous single-phase NiTi alloys by using a basic single-step sintering procedure is an important step towards the processing of safe implant materials. The sintering process used in this work is based on a vapor phase calciothermic reduction operating during the NiTi compound formation. The as-processed porous nickel-titanium microstructure is single-phase and shows a uniformly open pore distribution with porosity of about 53% and pore diameters in the range 20-100 microm. Furthermore, due to the process, fine CaO layers grow on the NiTi outer and inner surfaces, acting as possible promoting agents for the ingrowth of bone cells at the implantation site.     

Development of a calcium phosphate-gelatin composite as a bone substitute and its use in drug release.

This study was carried out to develop a calcium phosphate-gelatin composite implant that would mimic the structure and function of bone for use in filling voids or gaps and to release bioactive compounds like drugs, growth hormones into the implant site to assist healing. XDS analysis of the synthesized calcium phosphate revealed a calcium to phosphorus molar ratio of ca. 2.30, implying a less erodible material than hydroxyapatite (1.67). Release of the antibiotic gentamicin from the implant was with a burst, whether in situ or in vivo, followed by an almost constant release for about three months. It was found that the release rate could be decreased by increasing the density of the gelatin membrane. Upon implantation into rabbit tibia the release duration was substantially shortened (to about 4 weeks) with respect to the in situ tests basically due to the degradation of gelatin. In vivo studies with rabbits confirmed this degradation. The composite was perfectly biocompatible as shown by the histological studies. It, thus, has a great potential as a bone substitute material.     

Hydroxyapatite as a bone substitute in the jaws

Densely sintered calcium hydroxyapatite has previously been shown to be biocompatible and stable. Its possible role in jaw surgery was investigated using a clinically analogous animal model. The apatite was implanted into the mandible of twelve dogs for 12 weeks, and the healing assessed radiologically and histologicauy. The new bone which was deposited directly onto the surface of the implants bonded them firmly to the adjacent tissues. There was no fibrous tissue between implant and bone. This material appears to be a suitable substitute for autogenous bone when used as an inert ‘space filler’ in maxillo-facial surgical procedures.     

经皮球囊扩张复位结合硫酸钙植骨治疗跟骨骨折的研究

目的探讨经皮球囊扩张复位、注射硫酸钙植骨治疗SanderⅡ、Ⅲ型跟骨骨折的临床疗效。方法选取本院2011年1月~2013年1月收治的21例sanderⅡ、Ⅲ型跟骨骨折患者,采用经皮球囊扩张复位结合注射用硫酸钙植骨治疗,根据美国足踝外科协会(AOFAS)后足评分系统进行评分综合评估最终疗效。结果患者均获得随访,术后随访12~18个月,平均随访13.2个月,术后骨折愈合平均时间为3.2个月,术前测量Bohler角(6.7±2.8)°,Gissane角(85.9±6.3)°,跟骨高度(丘部)(27.3±4.4)mm,跟骨宽度(35.8±4.0)mm,术后1年测量Bohler角(30.0±2.8)°,Gissane角(130.0±6.4)°,跟骨高度(丘部)(40.1±3.2)mm,跟骨宽度(32.3±1.2)mm,与术前相比均有明显改善,差异均具有统计学意义(0.05)。结论经皮球囊扩张复位、注射用硫酸钙植骨治疗跟骨骨折,创伤小,临床效果好,无需二次取内固定,是一种较为理想的治疗方法。     

The ultrastructure of anorganic bovine bone and selected synthetic hyroxyapatites used as bone graft substitute materials.

The objective of this study was to investigate the morphology and organization of apatite crystallites in mature mammalian bone. Anorganic bovine bone was studied in this investigation to allow for the examination of the mineral crystallites after removal of the organic phase. Field-emission low-voltage scanning electron microscopy (FE-LVSEM) was employed to obtain images at nanometer resolution without the application of a conductive coating. Transmission electron microscopy (TEM) of the samples was also performed to confirm the identification of features observed in the SEM and to allow for comparison with earlier studies of bone mineral architecture. For comparison, in order to demonstrate how the interaction of collagen and apatite results in the architecture and crystal structure of bone mineral, two synthetic hydroxyapatite materials were also analyzed: OsteoGen and OsteoGraf/LD300. FE-LVSEM revealed distinctive features of bone mineral: a fibrillar organization of crystallites, a periodic spacing of crystallites along the fibrils consistent with the banding pattern of collagen, inter-fibrillar bridging crystallites, and a plate-like habit of the crystallites. These findings supported the hypothesis, derived from the earlier TEM data of others, that the mineralization of collagen comprising osteoid proceeds by the formation of apatite crystallites within the fibers at selected periodic sites along their length. Moreover, the very presence in this anorganic material of distinct fibers comprised of the crystallites is demonstration of inter-crystallite bonding. The crystallites of the synthetic hydroxyapatite materials did not display any of these ultrastructural features.     

Resorbable calcium phosphate particles as a carrier material for bone marrow in an ovine segmental defect

Resorbable calcium phosphate ceramics are only osteoconductive; therefore, their combination with osteogenic substances may lead to stimulation of bone healing. In the present study this combination, using autologous bone marrow, was investigated. In 31 sheep, a 3-cm tibial segmental defect was created and stabilized with an intramedullary nail. The animals were divided into four groups: empty defects (group 1, n = 7), and defects filled with 10-mL dense resorbable calcium phosphate particles (group 2, n = 8), with 10-mL particles soaked in bone marrow (group 3, n = 8), or with 10-mL autologous bone (group 4, n = 8). On evaluation after 12 weeks, significantly higher values were seen in group 3 than in group 2 for callus volume (p = .016), bone mineral density ratio (p = .03), bone mineral content ratio (p = .04), torsional strength (p = .005), and torsional stiffness (p = .01). For all end points, the outcome of group 3 was lower than that of group 4. In the histology, there was direct contact between newly formed bone and remnants of the particles. There were no signs of inflammatory reactions. Although a stimulatory effect of bone marrow was seen, the combination of resorbable calcium phosphate particles with bone marrow does not provide an alternative for autologous bone grafting.     

骨修复替代材料修复骨缺损的选择与应用

背景：在骨缺损治疗方面,近几年集中在骨修复替代材料领域出现了一系列重大科研突破,催生了很多全新的骨修复替代材料。目前临床上可供选择的骨修复替代材料种类繁多,各种材料的特性不同,各有优势和缺点。如何正确选择与应用是创伤骨科医师面临的实际问题,值得讨论和推敲。 目的：通过对自体骨、同种异体骨和人工骨材料在应用中的相关文献分析,评价3种骨修复替代材料的生物性能,有利于骨科医师更好的理解几种骨修复替代材料的理化、生物性能,进一步提高诊断和治疗水平。 方法：人工骨材料在骨缺损修复中起到不可取代的作用,并且也获得良好的临床效果,但近期临床效果并非是与传统材料的最终比较结果。人工骨材料安全性和稳定性的验证尚缺乏足够的实验依据,确切疗效尚须长期的临床观察。寻找具有良好生物特性的人工骨材料抑制是骨科的研究热点。分别对自体骨、同种异体骨、人工骨材料3种骨修复替代材料治疗骨缺损的理化、生物性能,安全性综合比较进行实验数据分析。 结果与结论：①在骨缺损修复过程中,从修复质量、免疫排斥和疾病传播等多方面来衡量,自体骨都是最佳的选择,成为骨移植的金标准。但来源有限且取骨区可能产生并发症,取骨又造成第二术区的创伤,因而它的临床应用受到了很大的限制。②同种异体骨的来源比自体骨多,但与宿主间的免疫排斥反应,并有感染疾病如肝炎病毒的可能。③人工骨材料成型迅速,在计算机辅助设计和辅助制作的帮助下,能更好的合成与缺损骨形态接近的骨修复替代材料,人工骨材料的骨诱导性和仿生物性骨结构方面还不能令人满意。④现有的骨修复替代材料均存在各自不同某些缺陷,不能完全满足临床质量的需要。未来能代替天然骨骼,解决骨缺损修复的材料应当是优势的组合,是骨修复替代材料的发展趋势。     

Increased new bone formation with a surface magnesium-incorporated deproteinized porcine bone substitute in rabbit calvarial defects

This study investigated the effects of magnesium ion (Mg) incorporation into the surface of deproteinized porcine cancellous bone in the bone healing of rabbit calvarial defects with the expectation of utilizing the integrin-ligand binding enhancement effect of Mg, and compared its bone healing capacity with that of untreated porcine cancellous bone and deproteinized bovine bone (Bio-Oss). Hydrothermal treatment was performed to produce Mg-incorporated porcine bone using an alkaline Mg-containing solution. The surface morphology and chemical composition of the samples were investigated using scanning electron microscopy, energy-dispersive X-ray spectrometry, and X-ray photoelectron spectroscopy. Defects 7 mm in diameter were created in the calvaria of 14 adult male New Zealand White rabbits and were filled with (1) untreated porcine bone (PB), (2) Bio-Oss, and (3) Mg-containing porcine bone (MG). The percentage of newly formed bone (NB%) was evaluated histomorphometrically at 2 and 4 weeks after implantation. Hydrothermal treatment resulted in a Mg-containing surface in porcine bone covered with nanostructures ~100 nm in size. The MG group supported better new bone formation compared with the other groups. Osteoconductive new bone formation was observed in the central defect area in the MG group at an early healing time-point. Histomorphometric analysis revealed significantly greater NB% in the MG group when compared with the untreated PB and Bio-Oss groups at 4 weeks (p < 0.05). The Mg-incorporated porcine bone with surface nanostructures achieved rapid new bone formation in the osseous defects of rabbit calvaria compared with untreated xenografts of porcine and bovine origin.     

Effect of porosity on the osteointegration and bone ingrowth of a weight-bearing nickel-titanium bone graft substitute

Porous nickel-titanium (NiTi) alloy is a promising new material for a bone graft substitute with good strength properties and an elastic modulus closer to that of bone than any other metallic material. The purpose of this study was to evaluate the effect of porosity on the osteointegration of NiTi implants in rat bone. The porosities (average void volume) and the mean pore size (MPS) were 66.1% and 259+/-30 microm (group 1, n=14), 59.2% and 272+/-17 microm (group 2, n=4) and 46.6% and 505+/-136 microm (group 3, n=15), respectively. The implants were implanted in the distal femoral metaphysis of the rats for 30 weeks. The proportional bone-implant contact was best in group 1 (51%) without a significant difference compared to group 3 (39%). Group 2 had lower contact values (29%) than group 1 (p=0.038). Fibrotic tissue within the porous implant was found more often in group 1 than in group 3 (p=0.021), in which 12 samples out of 15 showed no signs of fibrosis. In conclusion, porosity of 66.1% (MPS 259+/-30 microm) showed best bone contact (51%) of the porosities tested here. However, the porosity of 46.6% (MPS 505+/-136 microm) with bone contact of 39% was not significantly inferior in this respect and showed lower incidence of fibrosis within the porous implant.     

回盲肠间置幽门替代术动物模型的初步探讨

目的探讨回盲肠间置幽门替代术应用于动物胃肠道重建的可行性.方法将21头成年健康小型猪分为3组：假手术对照组（正常组）、B-I式胃大部切除组和远端胃大部切除带蒂回盲肠间置幽门替代手术组（回盲肠间置组）.术后观察对照动物的生存情况.结果手术后4个月,回盲肠间置组几乎未见呕吐、厌食及食量下降等现象;营养、精神状态及体力与正常组无甚差别;体重与术前无明显变化.而上述现象在B-I式组可见,且精神体力较差,体重较术前降低明显（P〈0.05）.结论回盲肠间置替代幽门重建消化道的动物模型可以借鉴.