带血供肌瓣复合BMP微球修复兔桡骨缺损的实验研究

目的：探讨以带血供肌瓣(VMF)复合骨形态发生蛋白微球(BMPMs)修复兔桡骨缺损的效果。方法：将壳聚糖与海藻酸钠制成复合微球，包裹骨形态发生蛋白，以带血供肌瓣复合BMP微球植入骨缺损处。通过大体观察、X线检查及组织学观察了解其修复效果。结果：带血供肌瓣复合BMP微球植入兔桡骨缺损处，在8周时缺损修复，组织学检查显示成熟骨组织形成，效果明显优于带血供肌瓣复合单纯BMP组及空白微球组。结论：壳聚糖海藻酸钠微球可作为骨形态发生蛋白的良好载体，以带血供肌瓣复合BMP微球可以修复免桡骨15mm的骨缺损.     

脂肪干细胞与小肠黏膜下层构建仿生骨膜的体内成骨☆

背景：脂肪干细胞与小肠黏膜下层两者具有良好的组织相容性,但将二者复合构建仿生骨膜修复骨缺损的效果如何尚需进一步验证。 目的：探讨以小肠黏膜下层为支架材料复合成骨诱导的脂肪干细胞构建仿生骨膜,体内成骨的可行性。 方法：取兔腹股沟区的脂肪分离培养脂肪干细胞经成骨诱导后,与猪小肠黏膜下层复合体外培养3周,构建仿生骨膜。将仿生骨膜埋置于裸鼠皮下,并以小肠黏膜下层复合未成骨诱导的脂肪干细胞的复合材料置于裸鼠皮下做对照。 结果与结论：兔脂肪干细胞经成骨诱导后与小肠黏膜下层体外复合培养,见两者黏附良好,细胞分泌大量的细胞外基质。植入4,8,12周组织学和透射电镜观察见有大量骨组织形成,未成骨诱导材料复合物组内无成骨细胞。仿生骨膜植入体内8周免疫组织化学测试骨钙蛋白、骨桥蛋白呈阳性反应。提示仿生骨膜植入裸鼠体内后可形成具有良好血运的新生骨组织。     

掺锶聚磷酸钙的细胞相容性

背景:研究显示,以可降解生物陶瓷聚磷酸钙为基材,加入微量锶元素,通过调控材料结构如孔径、孔隙率等性能,可制备出有足够力学强度并可控降解的掺锶聚磷酸钙。目的:观察成骨细胞与掺锶聚磷酸钙的细胞相容性。方法:将兔成骨细胞与不同含锶质量分数(0～100%)的掺锶聚磷酸钙体外复合培养,进行形态学和功能测定。MTT法检测掺锶聚磷酸钙中成骨细胞的活力,并测定成骨细胞碱性磷酸酶活性。结果与结论:在倒置显微镜、扫描电子显微镜和激光共聚焦显微镜下观察到成骨细胞在掺锶聚磷酸钙的表面和孔隙内大量黏附、增殖和生长,并且分泌细胞外基质及细胞表面的大量微绒毛,显示成骨细胞在掺锶聚磷酸钙材料上生长形态良好;同时通过MTT和碱性磷酸酶测定表明细胞功能良好,其中含锶1%掺锶聚磷酸钙细胞增殖活性及碱性磷酸酶活性最高,对成骨细胞无毒性,具有良好的细胞相容性。     

PDLLA/PLA-PEG-PLA新型人工骨修复恒河猴颅骨缺损实验研究

目的　评估PDLLA/PLA-PEG-PLA在骨组织工程中的应用前景。 方法　在4只恒河猴双侧顶骨24个10 mm颅骨去骨膜全层骨缺损中分别植入4种筛选出的支架/成骨细胞复合体,术后6、　12周采用X线、组织学及扫描电镜观察评价骨缺损修复情况。 结果　大体观察,都未引起明显的全身反应,局部切口愈合良好。X线、组织学及扫描电镜均证实人工骨在术后6周、12周成骨能力明显高于空白对照组,也高于单纯注射成骨细胞对照组,而改性的PDLLA/PLA-PEG-PLA、PDLLA/β-TCP及　PDLLA/BG成骨能力优于单纯PDLLA,X线评分证实PDLLA/PLA-PEG-PLA成骨能力最强,12周时成骨量达缺损区70%。 结论　4种筛选的支架与成骨细胞复合构建组织工程人工骨具有较强的体内成骨效能,PDLLA/PLA-PEG-PLA在骨组织工程中有很好的应用前景。     

纳米羟基磷灰石复合基因转染生长因子修复牙槽骨缺损

背景：研究发现成骨细胞可促进牙槽骨的形成,而血小板衍生生长因子A因子转染成骨细胞对于牙槽骨形成的作用尚不清楚。 目的：将基因转染的血小板衍生生长因子A重组质粒与纳米羟基磷灰石复合移植到骨缺损处,观察其对骨缺损修复的影响。 方法：将24只新西兰大白兔随机分成实验组与对照组,制作双侧下颌下缘10 mm×6 mm×4 mm骨质缺损,实验组植入血小板衍生生长因子A转染成骨细胞与纳米羟基磷灰石的复合材料,对照组单纯植入纳米羟基磷灰石。术后4,8,12周取材行大体标本、锥形束CT、组织学观察及扫描电镜观察。 结果与结论：术后不同时间点,实验组缺损处新骨生成,成骨细胞、骨小梁、骨陷窝及新生血管修复情况,以及材料与牙槽骨连接处的骨结合均明显优于对照组(P < 0.05)。表明血小板衍生生长因子A转染成骨细胞与纳米羟基磷灰石复合生成的新型材料,有较好的生物相容性,可加速骨组织再生,促进骨组织缺损修复。     

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。     

兔骨髓间充质干细胞自体皮下移植的成骨研究

目的探讨以兔骨髓间充质干细胞(BMSCs)为种子细胞,10%珊瑚羟基磷灰石(CHA)为支架材料,构建组织工程骨的可行性。方法取兔骨髓中的BMSCs,成骨细胞诱导,以5×107/ml的细胞密度接种于10%CHA,在联合培养1、3、7天,扫描电镜检查。联合培养7天的10%CHA+BMSCs细胞复合物,埋植入自体皮下,以单纯10%CHA为对照(n=4),分别观察6、12周取出做大体、组织学观察。结果 BMSCs与10%CHA联合培养1、3、7天,扫描电镜显示细胞能在10%CHA很好地黏附生长。皮下回植6周,10%CHA/BMSCs细胞复合物组可见大量骨组织形成,单纯10%CHA组未见骨组织生成;12周时,细胞组骨组织较6周时明显增多,单纯10%CHA组未见骨组织生成。     

掺锶冻干骨体内植入的生物相容性

背景：同种异体冻干骨是良好的骨移植材料,但在制备过程,其天然活性成分有所丢失或部分失活,成骨活性下降。努力改善冻干骨成骨活性,希望研制出一种能克服上述缺点的新型材料运用于临床。 目的：观察掺锶冻干骨植入大鼠体内的生物学活性。 方法：按照标准的冻干骨加工程序制备大鼠股骨髁冻干骨材料,采用氯化锶溶液浸泡方法制备掺锶冻干骨。将27只健康成年SD大鼠随机分为3组,每组9只。SD大鼠钝性分离肌肉,造成股部肌袋,双侧均行手术。植入掺锶冻干骨作为实验组,同法植入未掺锶的普通冻干骨作为对照组。空白对照组双侧均不植入任何材料。分别于植入后4,8,12周处死各组动物3只,行大体观察、组织学观察,并依据炎性浸润及组织纤维化程度进行组织学评分。 结果与结论：采用氯化锶溶液浸泡方法制作掺锶冻干骨,锶相对钙元素掺入摩尔浓度为4.2%时在骨质内层锶元素浓度分布比较均匀。实验组和对照组植入材料周围均可见炎性细胞、肉芽组织和血管生成,并随着时间推移,炎性浸润程度明显降低,而周围纤维组织包绕增加。空白对照组炎性浸润程度轻,少量组织纤维化。实验组和对照组与空白对照组相比,炎性浸润程度及组织纤维化程度组织学评分差异有显著性意义(P < 0.05)。说明通过温和的锶-钙离子交换方法可以在冻干骨中有效的掺入锶元素,获得一种掺锶冻干骨材料;该掺锶冻干骨具有良好的生物组织相容性,与普通冻干骨比较无明显差异。     

人骨形成蛋白7修饰骨髓间充质干细胞复合仿生型支架材料的体内成骨研究

目的 应用携带人骨形成蛋白7(hBMP7)基因的兔骨髓间充质干细胞(BMSC)与仿生型生物玻璃-胶原-透明质酸-磷脂酰丝氨酸(BG-COL-HYA-PS)支架材料复合培养,植入兔桡骨缺损模型中观察其在体内成骨的能力.方法 携带hBMP7基因或增强型绿色荧光蛋白(EGFP)基因的2型重组腺相关病毒(rAAV2)载体在体外分别转染兔BMSC,再将转染后和未转染的兔BMSC分别与BG-COL-HYA-PS支架材料复合培养7 d后植入3组兔桡骨缺损模型,每组6只兔.各组在分别术后8周、12周通过大体标本观察、影像学、组织学等方法观察骨缺损的修复情况.以正常兔桡骨为对照组(n=3),术后12周比较各组骨缺损修复组织生物力学差异.结果 rAAV2-hBMP7转染的兔BMSC与BG-COL-HYA-PS复合支架材料有良好的生物相容性,植入兔桡骨缺损模型内表现出明确的成骨能力和骨修复能力,而形成的新骨最大压力载荷值低于正常桡骨组织[(188.46±12.24)N比(203.25±19.29)N,P＜0.05].结论 用hBMP7修饰BMSC复合仿生型BG-COL-HYA-PS支架材料构建的组织工程骨具有较强的骨修复能力,但形成的新生骨组织与正常骨组织比较仍然有早期生物力学方面的不足.     

三种骨修复材料植入兔股骨内的组织病理学研究

将三种骨修复生物材料分别植入兔股骨内，在植入后７天，１４天，３０天，６０天，９０天，１８０天，３６０天进行了组织病理学研究，三种骨修复生物材料植入早期组织学观察基本相同，植入３６０天组织学观察有所不同，羟基磷灰石（ＨＡ）已有部分吸收，骨组织及骨髓已长入生物材料内部；氧化锆主物陶瓷（Ｚｒ）可见材料周围形成大量成熟骨组织，表面光滑，生物材料表面未见明显吸收现象，生物活性玻璃陶瓷（ＢＧ）可见生物材料周围     

In vivo cancellous bone remodeling on a strontium-containing hydroxyapatite (sr-HA) bioactive cement

The purpose of this study was to investigate the in vivo bone response to the strontium-containing hydroxyapatite (Sr-HA) bioactive bone cement injected into the cancellous bone. Sr-HA cement was injected into the iliac crest of rabbits for 1, 3, and 6 months. Active bone formation and remodeling were observed after 1 month. Newly formed bone was observed to grow onto the bone cement after 3 months. Thick osteoid layer with osteoblasts formed along the bone and guided over the bone cement surface reflected the stimulating effect of Sr-HA. From scanning electron microscopy (SEM) and energy-dispersive X-ray (EDX) analysis, high calcium and phosphorus levels were detected at the interface with a thick layer of 70 microm in width, and fusion of Sr-HA with the bone was observed. Blood vessels were found developing in remodeling sites. The affinity of bone on Sr-HA cement was increased from 73.55 +/- 3.50% after 3 months up to 85.15 +/- 2.74% after 6 months (p < 0.01). In contrast to Sr-HA cement, poly(methyl methacrylate) (PMMA) bone cement was neither osteoconductive nor bioresorbable. Results show that the Sr-HA cement is biocompatible and osteoconductive, which is suitable for use in treating osteoporotic vertebral fractures.     

Material-dependent bone induction by calcium phosphate ceramics: a 2.5-year study in dog

Bone induction by different calcium phosphate biomaterials has been reported previously. With regard to (1) whether the induced bone would disappear with time due to the absence of mechanical stresses and (2) whether this heterotopically formed bone would give rise to uncontrolled growth, a long-time investigation of porous hydroxyapatite ceramic (HA), porous biphasic calcium phosphate ceramic (TCP/HA, BCP), porous alpha-tricalcium phosphate ceramic (alpha-TCP) and porous beta-tricalcium phosphate ceramic (beta-TCP) was performed in dorsal muscles of dog, for 2.5 years. Histological observation, backscattered scanning electron microscopy observation and histomorphometric analysis were made on thin un-decalcified sections of retrieved samples. Normal compact bone with bone marrow was found in all HA implants (n = 4) and in all BCP implants (n = 4), 48 +/- 4% pore area was filled with bone in HA implants and 41 +/- 2% in BCP implants. Bone-like tissue, which was a mineralised bone matrix with osteocytes but lacked osteoblasts and bone marrow, was found in all beta-TCP implants (n = 4) and in one of the four alpha-TCP implants. Both normal bone and bone-like tissues were confined inside the pores of the implants. The results show that calcium phosphate ceramics are osteoinductive in muscles of dogs. Although the quality and quantity varied among different ceramics, the induced bone in both HA and BCP ceramics did neither disappear nor grow uncontrollably during the period as long as 2.5 years.     

Evaluation of the expression of collagen type I in porous calcium phosphate ceramics implanted in an extra-osseous site

The aim of the present study is to demonstrate the newly formed tissue in calcium phosphate (Ca/P) ceramics after extra-osseous implantation by histological and immunohistochemistry (IHC) methods. Synthesis porous Ca/P ceramics without adding any growth factor and living cell were implanted in the dorsal muscle of dogs for 1 and 2 months. Undecalcified and decalcified sections were stained by hematoxylin and eosin (H&E), and IHC, respectively. The histological results showed the beginning of osteogenesis and angiogenesis after being implanted for 1 month and the obvious new bone formation after being implanted for 2 months. IHC were conducted via the avidin-biotin peroxidase complex (ABC) method and the primary antibody was collagen type I. IHC results indicated that collagen type I was expressed within osteoblast-like cells and newly formed bone-like tissue in Ca/P ceramics after 1 month, and in the mineralized matrix of newly formed bone and osteoblasts, some osteocytes and some lacunae after 2 months. No cartilage and chondrocytes were observed in the histological and IHC-stained sections. Evidence of intramembranous osteogenesis was confirmed.     

A comparison of bone formation in biphasic calcium phosphate (BCP) and hydroxyapatite (HA) implanted in muscle and bone of dogs at different time periods

Physicochemical modification could implement synthetic materials into osteoinductive materials, which induce bone formation in nonosseous tissues. We hereby studied the relevance between the osteogenic capacities of osteoinductive materials in nonosseous tissues and in osseous sites. Biphasic calcium phosphate ceramic (BCP) and hydroxyapatite ceramic (HA) were implanted in femoral muscles and femoral cortical bone of dogs for 7, 14, 21, 30, 45, 60, 90, 180, and 360 days, respectively. Two dogs were used in each time point. In each dog, four cylinders (phi5x6 mm) per material were implanted in femoral muscles and 2 cylinders (phi5x6 mm) per material in femoral cortical bone. The harvested samples were processed for both histological and histomorphometric analyses. Bone was observed in BCP implanted in femoral muscles since day 30, while in HA since day 45. Quantitatively, more bone was formed in BCP than in HA at each time point after day 30 (p<0.05). The earlier and more bone formed in BCP than in HA suggests BCP a higher osteoinductive potential than HA in muscle. In femoral cortical bone defects, a bridge of bone in the defect with BCP was observed at day 21, while with HA at day 30. At days 14, 21, and 30, significantly more bone was formed in BCP than in HA (p<0.05). The results herein show that osteogenic capacities of osteoinductive materials in nonosseous tissues and osseous sites are correlated: the higher the osteoinductive potential of the material, the faster the bone repair.     

Enhanced bone ingrowth into hydroxyapatite with interconnected pores by Electrical Polarization

Hydroxyapatite (HA) ceramics are used as implants to repair damaged/removed bone, and negative or positive electrical polarization enhances osteoblast and decreases osteoclast activity, respectively, in vivo. We compared the ability of electrically polarized and non-polarized HA with interconnected pores (IPHA) implants to promote bone growth. Polarized or non-treated IPHAs were implanted into the right or left femoral condyle of rabbits (N = 10 in each group), and we performed histological examination, including enzymatic staining for osteoblasts and osteoclasts, 3 and 6 weeks after implantation. We observed improved bone ingrowth and increased osteoblast activity in polarized implants with complete bone penetration into polarized implants occurring as early as 3 weeks after surgery. In contrast, non-polarized implants were not fully ossified at 6 weeks after surgery. Furthermore, positively charged implant regions had decreased osteoclast activity compared to negatively charged or uncharged regions. We propose two different models to explain these observations.     

多孔碳酸钙陶瓷与干细胞源性成骨细胞的相容性

背景：国内外的研究证实普通碳酸钙陶瓷作为骨替代材料时具有细胞支架作用。 目的：观察多孔碳酸钙陶瓷与成骨细胞的相容性,及作为骨组织工程支架的可能性。 方法：SD大鼠骨髓基质干细胞经矿化诱导培养、扩增并检测证实其已具成骨细胞表型后,分别与多孔碳酸钙陶瓷支架、普通羟基磷灰石陶瓷支架体外复合培养。 结果与结论：骨髓基质干细胞经体外诱导形成成骨细胞,钙结节、Ⅰ型胶原和碱性磷酸酶免疫染色结果阳性。多孔碳酸钙陶瓷支架材料与羟基磷灰石陶瓷材料皆有细胞附着生长,但多孔碳酸钙陶瓷支架材料细胞的黏附能力、增殖活力及成骨活性均强于羟基磷灰石陶瓷材料。提示多孔碳酸钙陶瓷支架材料与SD大鼠骨髓基质干细胞源性成骨细胞有良好相容性。     

Early weight bearing of porous HA/TCP (60/40) ceramics in vivo: a longitudinal study in a segmental bone defect model of rabbit

Porous interconnected hydroxyapatite (HA) and HA/tricalcium phosphate (TCP) (60/40) ceramics are promising materials for hard tissue repair. However, the mechanical properties of these materials have not been accurately determined under weight-bearing conditions. In this study, newly developed HA and HA/TCP (60/40) ceramics were used with intramedullary fixation in segmental bone defects of rabbits. Early radiological, histological, densitometric and biomechanical changes were evaluated. The mean radiological grade of healing and bonding to bone was higher in HA/TCP (60/40) ceramics than that of pure HA ceramics but the difference was not statistically significant. The densities of both implanted ceramics improved with time, supported by the histological evaluation of bone matrix ingrowth into ceramic pores, whereas the densities at the bone–ceramic interface decreased gradually. Flexural resonant frequencies and three-point bending strength increased, revealing an increase in mechanical stability during this early critical time interval where implant and/or bone–implant interface failures occur frequently. It can be concluded that both HA and HA/TCP (60/40) ceramics have a limited application in the treatment of load-bearing segmental bone defects but did not fail at the early stages of implantation.     

在不同动物肌肉中磷酸钙陶瓷表面类骨磷灰石的形成研究

磷酸钙陶瓷材料植入动物体内后其表面类骨磷灰石层的形成对骨的形成有非常重要的作用，并被认为是骨诱导发生的先决条件。我们将相同大小的孔壁有微孔的多孔材料和致密磷酸钙陶瓷材料植入猪，狗、兔和鼠的背肌或腿肌内，研究陶瓷表面类骨磷灰石的形成，以了解类骨磷灰石层的形成与骨诱导的联系。结果表明：磷酸钙陶瓷材料植入动物的肌肉内14d后，狗，兔和鼠体内的多孔材料孔隙内表面（包括陶瓷表面较深孔隙）有一层类骨磷灰石层形成；植入猪体内的多孔材料内外表面都形成了一层类骨磷灰石，致密材料在几种动物体内都未观察到类骨磷灰石层形成，类骨磷灰石层形成的快慢次序与动物组织学观察到的在不同动物的肌内骨诱导性高低的次序不一致。证实了类骨磷灰石层的形成的确是骨诱导的先决条件。但还有其它因素影响骨诱导的发生。     

Ectopic osteogenesis with biphasic ceramics of hydroxyapatite and tricalcium phosphate in rabbits.

Porous calcium phosphate ceramics consisting of hydroxyapatite (HA) and tricalcium phosphate (TCP) with different HA to TCP ratio were implanted intramuscularly in rabbits for six months in order to carry out a comparative study on osteogenic activity of the ceramics. Bone formation was detected only in HT73 (HA to TCP ratio, 7-3) specimens. Other implants, HT28 (2-8) and HT010 (0-10), could not induce bone. After a six-month period of implantation, HT28 and HT010 implants showed obvious degradation of the implants changing their shape and size macro and microscopically. Microscopically, they showed aggregates of fine particles and appearance of multinucleated cells. However, HT73 implants was less degraded and could maintain their original structure macro and microscopically. This study showed that HT73 ceramics can induce bone in rabbit muscle tissue and it is considered that maintenance of porous structure, that is, degradation rate of the materials may be one of the affecting factors in ceramic-induced osteogenesis.     

A preliminary study on osteoinduction of two kinds of calcium phosphate ceramics.

With respect to the effect of material factors on calcium phosphate biomaterial-induced osteogenesis, the osteoinductive property of two kinds of porous hydroxyapatite ceramics, which were made by different producers, was investigated in dorsal muscles of dogs. One hydroxyapatite ceramic (S-HA), macroporous implants with rough pore walls containing abundant micropores, was made by Sichuan Union University (Chengdu, China); the other hydroxyapatite ceramic (J-HA), porous implants with smooth macropore walls composed of regularly aligned crystal grains, was provided by Mitsubishi Ceramic Int. (Japan). Different tissue response was detected histologically and microradiographically after the ceramic samples had been implanted in dorsal muscles of dogs for 3 and 6 months. Bone formation was found in S-HA at 3 months, which increased at 6 months. In contrast, no bone formation was detected in J-HA at both 3 and 6 months. These results indicate that with the special architecture, calcium phosphate ceramic can induce bone formation in soft tissue. As both materials were very similar in their chemical and crystallographic structures, but varied in their microstructures, the latter seem to be an important factor affecting the osteoinductive capacity of calcium phosphate ceramics. These data suggest that, by controlling the preparation of calcium phosphate ceramic, bone substitutes with intrinsic osteoinductive property can be developed from calcium phosphates.