In vitro study of osteogenic differentiation of bone marrow stromal cells on heat-treated porcine trabecular bone blocks

This in vitro study investigated the potential of the heat-treated porcine trabecular bone block as a bone substitute for the treatment of bone defects or related diseases. Chemical, mechanical, and morphological studies of bone blocks were performed. The resultant properties were compared with the properties of currently available commercial products from bovine trabecular bones. The major component of the bone block was hydroxyapatite, and the ratio of Ca/P was 1.65-1.66. The average values of the compressive Young's modulus and the ultimate strength were 346.33 +/- 83.15 and 6.66 +/- 1.62 MPa, respectively. The pore size of the heat-treated bone blocks was approximately 300-500 microm. For the biological investigations, expanded bone marrow stromal cells (BMSCs) were isolated from the femurs of New Zealand White rabbits and were dynamically seeded into the heat-treated porcine bone block (10x10x5 mm3). Before the cells were seeded, the heat-treated porcine bone blocks were divided into two groups: collagen coated blocks (n=16) and uncoated blocks (n=16). Within each group, the blocks were again divided into two groups, depending on the culture method, i.e., static or rotating culture. Cells were cultured in the blocks for up to 6 weeks. Scanning electron microscopic examination after 4 weeks showed that the cell layers attached to the porcine bone block. Proliferation and osteogenic differentiation were analyzed by cell counting, an MTT assay, alkaline phosphatase activity, and total protein content. The deposition of extracellular substances and osteoid formation surrounded by osteoblast-like cuboidal cells were confirmed through histochemical staining and transmission electron microscopy. Based on the results of this study, we conclude that heat-treated porcine trabecular bone has great potential as a bone substitute and may even be superior to currently available commercial products.     

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.     

Incorporation of particulated bone implants in the facial skeleton.

The purpose of this study was to compare the regenerative response of autogenous cortical and cancellous bone chips and a natural particulate resorbable bone mineral (RBM) (Bio-Oss, Geistlich-Pharma, Wolhusen, Switzerland) in standardized bony defects relating paranasal sinuses to one another and to bone blocks. On 13 skeletally mature female goats four standardized critical-sized full thickness bone defects were made in the frontal bone overlying the frontal sinus. These defects were filled at random with cortical bone chips, cancellous bone chips, spongiosa granules of a RBM or left empty. Fluorochrome bone markers were injected subcutaneously 1 and 5 weeks after transplantation, and one week before the animals were killed. The animals were killed at 3, 6, 12 and 24 weeks after surgery. Autogenous cancellous bone chips is the material of choice for bridging a bony defect in the maxillofacial area where there is no need for mechanical strength. They heal in the same way as cancellous bone blocks do. Cortical bone chips are not reliable enough to be used as a solitary bone-grafting material under these conditions. A cortical block as a solitary implant gives better results. RBM granules as solitary implant in a critical-sized defect do not stimulate osteoconduction but give rise to an extensive osteoclastic activity stimulated by the mutual loose relation. A solid block of RBM is in a similar case more reliable.     

Cancellous bone repair using bovine trabecular bone matrix particulates.

At 5 and 15 weeks post-surgery, biomechanical and histological analyses of cancellous bone defects filled with the bovine trabecular bone matrix (BBM) and hydroxyapatite (Hap) particulates of dimensions 106-150 microm were investigated. It was observed that at 5 weeks post-surgery the stiffness properties of the BBM filled defects were significantly higher than those observed in the Hap filled defects (p < 0.01) but comparable to those recorded in intact cancellous bone from the same anatomical position. Histologically, no significant differences were observed in the percentage of new bone contact with the particles. The biomechanical properties of the Hap filled defects mirrored those in intact cancellous bone only at 15 weeks post-surgery. BBM particles thus appeared to accelerate the early healing of osteotomies. It is therefore suggested that particles of this bioceramic be the subject of intense research for more usage in both periodontal osseous defects and orthopaedic fractures.     

去抗原异种松质骨复合骨髓基质干细胞修复骨缺损

目的研制理想的能够修复骨缺损的骨材料,为其在骨缺损修复领域中的临床应用提供实验依据。方法体外扩增诱导兔骨髓基质干细胞,将其与去抗原异种松质骨复合培养。造成兔桡骨中段10mm骨缺损模型,分复合细胞组、单纯材料组、空白对照组。通过大体观察、放射学检查、组织学检查观察骨缺损的修复情况。结果去抗原异种松质骨复合骨髓基质干细胞修复兔桡骨缺损,12周时缺损区完全被新骨代替,骨髓腔完全通畅,优于单纯材料组的修复效果,单纯材料组优于空白对照组。结论去抗原异种松质骨具有良好的成骨能力,有可能成为理想的骨支架材料。     

异种去抗原松质骨成骨效能的实验研究

 目的：研究异种去抗原松质骨与基因重组人骨形成蛋白2 (recombinant human bone morphogenetic protein 2, rhBMP-2)复合后的生物相容性和成骨效能。方法：28只新西兰大白兔28处颌骨缺损完全随机分为3组：异种去抗原松质骨/rhBMP-2组（A组）、异种去抗原松质骨组（B组）和空白对照组（C组）。A组和B组每组12处骨缺损,于4、8和12周分别进行大体观察、X线检查、HE染色和新生骨量测定;C组共4处骨缺损于12周时进行大体观察、X线检查和HE染色。结果：X线显示,随着时间的增长,2组的植入骨逐渐降解吸收,由自体骨替代,A组较B组显著;C组12周时无法完成愈合。骨密度测量显示,随着时间的增长,A和B组的骨密度值增加,2组之间存在显著差异（P<0.05）。HE染色可见A组的新生血管、纤维组织和新生骨均较B组多;植入骨降解速度快于B组;A组新生骨面积大于B组（P<0.05）。结论：异种去抗原松质骨是rhBMP-2的良好载体;异种去抗原松质骨/rhBMP-2复合材料能持续成骨,可加速骨缺损修复的速度,是一种较好的骨修复材料。     

骨髓间充质干细胞复合异体骨修复松质骨缺损

背景：有研究表明骨髓间充质干细胞及异体骨可促进骨缺损的修复,但骨髓间充质干细胞复合异体骨对于松质骨缺损的修复效果至今少有报道。 目的：观察骨髓间充质干细胞复合异体骨修复兔松质骨缺损效果。 方法：在新西兰大白兔双侧股骨外侧髁造成0.6 cm×1.2 cm 的松质骨缺损,一侧设为模型组,骨缺损处植入复合骨髓间充质干细胞的异体骨,另一侧设为对照组,单纯植入异体骨。 结果与结论：植入后4,8,12周,大体观察、X射线检查和苏木精-伊红染色观察结果显示,模型组在新骨成长方面,缺损区修复方面均优于对照组。植入后12周,模型组骨缺损区可见大量骨小梁形成及成熟的板层骨组织,骨缺损基本修复。对照组骨缺损区仅可见大量编织骨形成,骨缺损尚未得到有效修复。模型组Lane-Sandhu法X射线结合组织学观察评分高于对照组(P < 0.05)。生物力学检测结果显示,植入后12周,模型组股骨髁最大压力载荷、载荷/应变比值均高于对照组(P < 0.05),最大应变位移较对照组低(P < 0.05)。结果证实,骨髓间充质干细胞复合异体骨可有效修复兔股骨髁松质骨缺损,且修复效果明显优于单纯异体骨移植。     

Preformed grafts of porcine small intestine submucosa (SIS) for bridging segmental bone defects

Previous studies suggest that fresh, morselized porcine small intestine submucosa (SIS) may have promise in the treatment of large bone defects. This study evaluated the bone regenerative potential of preformed tubular SIS grafts, designed to provide a scaffold for regeneration of diaphyseal bone. Critical length segmental defects in the femurs of male rats were either left unfilled (n = 11) or filled with morselized cancellous bone (n = 12), or spanned with intramedullary tubes (n = 12) or periosteal sleeves (n = 12) fabricated from SIS. All of the animals were euthanized 12 weeks postoperatively. Healing was assessed with biweekly radiographs, routine histology, and mechanical testing. Copious new bone formed in the defects of all of the animals treated with cancellous bone; 10 of the 12 animals in that group had healed their defects. In contrast, no new bone was formed in the defects left unfilled or treated with SIS; only fibrous tissue was found. In both of the SIS-treated groups, the SIS persisted at twelve weeks. The cellular response to the SIS involved a mild mononuclear infiltrate in the loose or delaminated superficial layers of the tubes and sleeves, with few cells in the deeper layers. The results of this study cast doubt on the ability of SIS to support or stimulate growth of bone across a critical length segmental bone defect. Additional work will be required to determine whether our results reflect the protocols used to prepare and fabricate the SIS grafts used in the study or the inherent inability of SIS to support new bone growth.     

核素骨显像评价组织工程骨修复骨缺损效果的价值

目的 应用放射性核素骨显像评价组织工程骨修复兔股骨髁骨缺损的效果。方法 取人白兔15只，抽取骨髓，行骨髓间充质干细胞分离、培养、骨向诱导。双侧股骨髁制作0．6×1．2cm的骨缺损，将诱导的成骨细胞复合珊瑚羟基磷灰石植入左侧，右侧单纯植入羟基磷灰石为对照组。术后4间、8周和12周分别行静态核素骨显像评价骨缺损的修复能力。结果表明术后4、8、12周实验组ROI计数（单位像素）均较对照组有显著性增高（P〈0．001）。实验组ROI计数随时间的延长呈明显的上升趋势，但术后8周始增长放缓；对照组ROI也有卜升趋势，但术后8周始增长加快，均在12周达到峰值。结论 骨髓间充质干细胞诱导后复合珊瑚羟基磷灰石可有效的修复股骨髁松质骨缺损。放射性核素骨显像在骨修复过程中具有动态评价血管化和骨生长的作用。     

异种松质骨支架体内移植免疫的实验研究

观察异种松质骨支架植入后宿主动态免疫应答,为进一步改进材料和临床应用提供实验依据。采用物理化学方法对异种(猪)松质骨进行处理得到猪松质骨支架,对猪松质骨进行蛋白提取,考马斯亮蓝法测定蛋白浓度,用提取的蛋白作抗原包被ELISA条板备用。将异种(猪)松质骨支架植入兔背部近头侧皮下,分别于移植后1周、2周、4周、8周、12周五个时间点作支架周围组织的组织学检查(HE)及外周血清抗体检测(ELISA)。结果:异种(猪)松质骨及松质骨支架的蛋白质浓度分别为(1.242±0.26)mg/ml和(0.024±0.004)mg/ml。异种(猪)松质骨蛋白抗原液经聚丙烯酰胺凝胶(SDS-PAGE)电泳分离后可清楚显示十条不同相对分子质量大小的蛋白条带(从15000到180000),异种(猪)松质骨支架蛋白抗原液经SDS-PAGE电泳分离后10000 Mr以上的蛋白条带消失,说明松质骨支架不含有效激发免疫反应的抗原蛋白成分。外周血中特异抗体水平在移植术后1周达最高,与处理前有明显差异(P<0.05),2~4周时逐渐降低,8~12周时维持在较低水平,与移植前无显著差异(P>0.05)。植术后1周,植入物周围组织光学显微镜下可见大量炎症细胞浸润,以淋巴细胞和单核细胞为主,2周以后炎症细胞浸润逐渐减轻,4周时未见明显炎性细胞浸润,术后12周,组织内炎性细胞罕见。异种(猪)松质骨支架不含有效激发免疫反应的抗原蛋白成分,植入后表现为一过性轻度的炎性细胞反应。     

Incorporation of three types of bone block implants in the facial skeleton

The regenerative response on autogenous cancellous and cortical bone grafts, and on a commercial available xenogenous resorbable bone mineral (RBM) (Bio-Oss, Geistlich-Pharma, Wolhusen, Switzerland) was compared in standardized bony defects related to a paranasal sinus. On 15 skeletally mature goats four critical sized full thickness bone defects were trephined in the frontal bone. These defects were filled at random with a cortical bone plug, a cancellous bone plug, a plug of spongious RBM cut into shape or left empty. Fluorochrome bone markers were injected subcutaneously 1 and 5 weeks after transplantation, and one week before the animals were killed. The animals were killed at 3, 6, 12 and 24 weeks after surgery. Histological evaluation showed that autogenous bone grafts were all accepted and incorporated in a similar way as in calvarial defects. RBM was only osteoconductive. New bone was formed at the margins of the defects, and only little of the RBM was incorporated. Most of the RBM was gradually resorbed by multinucleated osteoclast-like cells.     

Regeneration of bicortical defects in the iliac crest of estrogen-deficient sheep, using new biodegradable polyurethane bone graft substitutes

Porous scaffolds for cancellous bone graft substitutes were prepared from new experimental biodegradable aliphatic polyurethane elastomers with varying hydrophilicity. The ratios of the hydrophilic-to-hydrophobic content in the polymers were 30-70, 50-50, and 70-30%, respectively. The hydrophilic component consisted of poly(ethylene oxide) diol and the hydrophobic component of poly(epsilon-caprolactone) diol. To promote the materials' biological performance, the calcium complexing moiety, the polysaccharide, and vitamin D(3) were incorporated into the polymer chain upon synthesis. The scaffolds had an interconnected porous structure with an average pore size in the range of 300-2,000 microm and pore-to-volume ratios of (85 +/- 5)%. The bone substitutes were implanted (press-fit) in biocortical 10 x 10 mm(2) defects created in the tuber coxae of 21 skeletally mature Warhill ewes, which were ovariectomized 12 months prior to implantation. At the time of euthanasia at 18 and 25 months, all the defects in the ilium implanted with polyurethane bone substitutes had healed with new bone. The extent of bone healing depended on the chemical composition of the polymer from which the implant was made, although for the same material there were animal-related differences in healing. The structure of the newly formed cancellous bone was radiographically and histologically similar to the native bone. The implants from polymers with the incorporated calcium-complexing additive were the most effective promoters of bone healing, followed by those with vitamin D(3) and polysaccharide-containing polymer. There was no bone healing in the control defects.     

The performance of a bone-derived scaffold material in the repair of critical bone defects in a rhesus monkey model

The efficacy and safety of a material derived from human bones in the repair of critical segmental bone defects are evaluated in a rhesus monkey model. Frozen human bones were chemically and physically processed into a partially demineralized and deproteinized material in blocks. The complete tissue-engineered (TE) bone was constructed of the material preseeded with allogeneic bone marrow mesenchymal stem cells (MSCs). The material alone and the TE bone were, respectively, implanted to bridge 2.5cm-long critical defects in right and left radii of 15 monkeys. At weeks 1, 2, 3, 6 and 12 post-implantation, the grafts were collected from three animals and assessed for the local expression of osteogenic markers, histological and roentgenographic features, and immune reactions. It was shown that defects were well repaired with both treatments whereas the bone defects in 2 additional untreated animals remained the same size after 12 weeks. In radii implanted with the TE bones, the repair processes were approximately 3 weeks faster and new bones were formed in a multipoint way. There was neither observable toxic effect nor overt immune rejection in any animals. Taken together, these observations suggest that the TE bone blocks composited of the allogeneic or xenogeneic bone-derived scaffold and allogeneic MSCs may provide an ideal method for repairing large segmental bone defects.     

聚左旋乳酸/壳聚糖纳米纤维三维多孔支架复合骨髓间充质干细胞修复兔骨缺损**△

背景：骨髓间充质干细胞具有向多种间质细胞谱系分化的能力,且支架材料的性能对骨缺损的修复有重要影响。 目的：观察聚左旋乳酸/壳聚糖纳米纤维三维多孔支架复合骨髓间充质干细胞治疗骨缺损。 方法：对骨缺损模型兔分别采用空白植入、髂后上棘自体松质骨移植、聚左旋乳酸/壳聚糖纳米纤维多孔支架移植和复合了骨髓间充质干细胞的聚左旋乳酸/壳聚糖纳米纤维多孔支架移植修复缺损部位。 结果与结论：至移植12周,移植复合了骨髓间充质干细胞的聚左旋乳酸/壳聚糖纳米纤维多孔支架的实验兔的缺损处有骨组织生成,支架材料降解,已完成缺损修复,其修复情况接近松质骨组;髂后上棘自体松质骨移植的实验兔的缺损修复完好,新形成的骨组织较规则;只植入聚左旋乳酸/壳聚糖纳米纤维多孔支架的实验兔有少量骨组织形成,材料部分降解;空白植入的实验兔缺损处无新生骨组织生成,主要由纤维结缔组织填充。说明新型的生物支架材料聚左旋乳酸/壳聚糖纳米纤维三维多孔支架与来源于新西兰大白兔的骨髓间充质干细胞复合培养后,植入同种异体兔股骨髁缺损处,使骨缺损的修复速度加快,表现为较好的体内诱导成骨的作用。     

同种异体骨复合自体红骨髓移植治疗关节周围骨折

背景：关节周围骨折复位后常出现骨缺损,需进行植骨填充骨缺损以早期支撑关节面以防止关节面塌陷及移位。同种异体骨是治疗骨缺损的移植材料,但成骨能力差。自体红骨髓有成骨能力,但同种异体骨复合自体红骨髓移植治疗关节周围骨折的临床效果有待评定。 目的：采用锁定板固定、同种异体骨复合自体红骨髓移植治疗关节周围骨折的临床效果。 方法：纳入河北医科大学第三医院骨伤科治疗关节周围骨折患者43例。采用切开解剖复位关节面、将红骨髓与同种异体骨颗粒复合体植于骨缺损处,植骨完成后常规解剖锁定板内固定。胫骨平台骨折采用内侧、外侧或双侧锁定板固定。桡骨远端骨折采用背侧或掌侧锁定板固定,胫骨远端骨折采用胫骨远端内侧或外侧板锁定内固定。 结果与结论：患者43例共随访12个月至6年,平均4.3年。X射线片及CT复查结果显示,43例患者达骨性愈合,塌陷骨折复位良好。其中新鲜骨折愈合时间2-6个月,平均4个月;陈旧骨折愈合时间3-7个月,平均5.5个月。植骨后43例患者无明显免疫排斥反应,2例患者切口渗液较多,经换药2周愈合。切口感染患者1例,经引流换药4周伤口愈合,随访4年1个月至今感染未复发。根据Mankin和Komender标准评定,同种骨移植满意患者40例,占93%;不满意患者3例,占7%。结果证实,在锁定板支撑固定下,异体松质骨与自体红骨髓复合体移植治疗周围关节骨折可以起到近期支撑作用,防止关节面塌陷及骨折移位,并为关节周围骨折骨缺损提供骨重建材料,远期可以达到骨折愈合的目的。     

异种去蛋白松质骨复合骨髓间充质干细胞修复骨缺损的组织学变化

背景：目前应用各种人造支架复合细胞修复骨缺损研究很多,但是各种人造支架都没有骨的天然结构,所以修复效果不够理想。 目的：将大鼠骨髓间充质干细胞接种到异种去蛋白松质骨上,移植修复大鼠股骨节段性缺损,以体内修复效果来评价复合体应用前景。 方法：分离培养大鼠骨髓间充质干细胞并进行扩增,用BrdU体外进行标记。同时制备牛去蛋白松质骨,在体外与标记后的细胞复合。制备大鼠双侧股骨中段5 mm缺损模型,实验分成3组,缺损处分别移植骨髓间充质干细胞/去蛋白松质骨复合体、单纯去蛋白松质骨及单纯骨髓间充质干细胞。 结果与结论：BrdU免疫染色结果显示,在各组细胞均呈阳性表达,但随着移植时间的延长而减弱。X射线放射学评分及苏木精-伊红染色组织学评分结果显示,各时间段复合体组成骨效果均好于其他组。复合体组Ⅰ型胶原蛋白表达随着时间的延长有明显增强,强于其他组。提示骨髓间充质干细胞复合异种去蛋白松质骨的成骨能力明显强于单纯的支架修复能力,单纯的骨髓间充质干细胞虽然有成骨能力,但不能修复节段性骨缺损。     

Augmentation of screw fixation with injectable calcium sulfate bone cement in ovariectomized rats

The objective of this study was to determine the effect of augmenting screw fixation with an injectable calcium sulfate cement (CSC) in the osteoporotic bone of ovariectomized rats. The influence of the calcium sulfate (CS) on bone remodeling and screw anchorage in osteoporotic cancellous bone was systematically investigated using histomorphometric and biomechanical analyses. The femoral condyles of 55 Sprague-Dawley ovariectomized rats were implanted with screw augmented with CS, while the contralateral limb received a nonaugmented screw. At time intervals of 2, 4, 8, 12, and 16 weeks, 11 rats were euthanized. Six pair-matched samples were used for histological analysis, while five pair-matched samples were preserved for biomechanical testing. Histomorphometric data showed that CS augmented screws activated cancellous bone formation, evidenced by a statistically higher (p < 0.05) percentage of osteoid surface at 2, 4, and 8 weeks and a higher rate of bone mineral apposition at 12 weeks compared with nonaugmented screws. The amount of the bone-screw contact at 2, 8, and 12 weeks and of bone ingrowth on the threads at 4 and 8 weeks was greater in the CS group than in the nonaugmented group (p < 0.05), although these parameters increased concomitantly with time for both groups. The CS was resorbed completely at 8 weeks without stimulating fibrous encapsulation on the screw surface. Also, the cement significantly increased the screw pull-out force and the energy to failure at 2, 4, 8, and 12 weeks after implantation, when compared with the control group (p < 0.05). These results imply that augmentation of screw fixation with CS may have the potential to decrease the risk of implant failure in osteoporotic bone.     

Bioactivation of an anorganic bone matrix by P-15 peptide for the promotion of early bone formation

This animal experiment compared the regenerative processes within defined bony defects of the porcine skull after delivery of routinely utilized bone graft materials: anorganic bone matrix (ABM) and an identical ABM carrying the cell binding peptide P-15. Particulated autogenous bone was used as a control group. The chosen porcine model guaranteed the transferability of the obtained results to clinical practice. A total observation period of 6 months was defined. The bone samples were examined microradiographically and histologically at 8 specific times. Sufficient osseointegration and osseoconduction could be demonstrated for both anorganic bone minerals. However, in the selected model significantly higher mineralization rates (p = 0.0286) were found in the microradiographic image at 12 weeks after application of the bioactive form. The histological examination confirmed this accelerating effect on bone formation starting at day 3. At the end of the study after 6 months, the mineralization values had equalized in both study groups. For the first time, the material was demonstrated to be suitable as a bone substitute material for the treatment of larger bony defects in a large animal model. The P-15 sequence accelerated the process of bone formation on the surface of the anorganic bone matrix as early as 3 days but was not traced over the whole term of the study.     

超临界二氧化碳萃取的猪松质骨

背景:近年来国外使用超临界流体进行生物材料处理的研究较多,但是超临界流体应用于骨组织清洗的研究少见,国内相关报道更少。目的:评估超临界二氧化碳萃取技术处理猪股骨松质骨的有效性及对骨生物学性能的影响。方法:分别制备超临界二氧化碳萃取前(对照组)及萃取后猪股骨骨块(实验组),检测两组骨密度、微观结构、最大抗压强度、弹性模量、骨组织组成成分、胶原含量与组织学分析。将骨髓间充质干细胞分别接种于两组骨块上,培养1 d后,扫描电镜观察材料骨小梁微孔结构及骨材料-细胞复合物中细胞黏附及生长情况。将两组骨块分别植入SD大鼠皮下,术后1,2,4周组织学观察皮下包埋炎症反应。动物实验方案已经解放军总医院伦理委员会批准。结果与结论:①两组材料孔径大小、骨密度、最大抗压强度、弹性模量、胶原含量比较差异均无显著性意义(P>0.05);②扫描电镜显示,对照组材料孔隙连通欠佳,有软组织残留;实验组材料孔隙相互连通、结构完整;③傅里叶红外光谱及X射线衍射分析显示,两组骨组织材料具有相似的吸收峰及衍射峰,热重分析显示超临界二氧化碳萃取可减少骨组织水分含量;④苏木精-伊红染色显示实验组骨无软组织残留,骨陷凹内细胞残留明显减少,对照组有软组织及细胞残留;天狼星红及改良Masson染色显示实验组骨胶原结构完整,胞质成分减少,对照组胞质成分残留明显;⑤扫描电镜显示,对照组骨无明显细胞黏附,实验组骨可见明显细胞黏附生长;⑥实验组骨组织植入皮下的血管周围炎症反应明显轻于对照组骨组织;⑦结果表明,超临界二氧化碳萃取技术是一种有效、环保的骨组织处理技术,在不影响其胶原结构及含量、力学性能的基础上,能够有效去除猪股骨松质骨细胞及软组织,保留骨孔隙结构完整,增加细胞黏附及生长,有效减低炎性排斥反应。     

Hierarchically biomimetic bone scaffold materials: nano-HA/collagen/PLA composite

A bone scaffold material (nano-HA/ collagen/PLA composite) was developed by biomimetic synthesis. It shows some features of natural bone both in main composition and hierarchical microstructure. Nano-hydroxyapatite and collagen assembled into mineralized fibril. The three-dimensional porous scaffold materials mimic the microstructure of cancellous bone. Cell culture and animal model tests showed that the composite material is bioactive. The osteoblasts were separated from the neonatal rat calvaria. Osteoblasts adhered, spread, and proliferated throughout the pores of the scaffold material within a week. A 15-mm segmental defect model in the radius of the rabbit was used to evaluate the bone-remodeling ability of the composite. Combined with 0.5 mg rhBMP-2, the material block was implanted into the defect. The segmental defect was integrated 12 weeks after surgery, and the implanted composite was partially substituted by new bone tissue. This scaffold composite has promise for the clinical repair of large bony defects according to the principles of bone tissue engineering.