改良法制备异种脱蛋白骨复合重组人骨形态发生蛋白支架修复长骨大段骨缺损

目的：异种脱蛋白骨因其来源广泛和独特的生物学特性作为组织工程骨支架材料在修复长骨大段骨缺损上有较好的应用前景。 目的：采用改良法制备异种脱蛋白骨复合重组人骨形态发生蛋白支架材料,评价其修复大动物大段长骨缺损的成骨能力。 方法：山羊24只,随机分为单纯异种脱蛋白骨组、自体骨组及异种脱蛋白骨+重组人骨形态发生蛋白组,在每只山羊右侧胫骨中下段截除胫骨总长度20%形成节段性骨缺损,按分组情况植入不同材料,采用半环槽式外固定器固定。术后4~24周每隔4周进行X射线检查,术后24周取新生骨进行双能X射线、组织学、生物力学检测骨缺损修复效果。 结果与结论：①制得的异种脱蛋白骨为三维多孔结构,其孔隙率为(78.5±6.45)%,孔径大小为(472.5±7.02) μm。②各组骨缺损表现为时间依从性的骨修复,生物力学试验抗压缩、三点抗弯曲和抗扭转试验均为自体骨组> 异种脱蛋白骨+重组人骨形态发生蛋白组>单纯异种脱蛋白骨组。③自体骨组、异种脱蛋白骨+重组人骨形态发生蛋白组与正常组比较,山羊胫骨骨密度及骨矿含量、抗压缩压强及极限压强、抗弯曲载荷及极限载荷、抗扭转扭矩及极限扭矩平均值差异均无显著性意义。结果提示改良法制备的异种脱蛋白骨复合重组人骨形态发生蛋白修复山羊胫骨大段缺损成骨能力与自体骨相当,可以作为组织工程支架材料。 关键词：异种脱蛋白骨;重组人骨形态发生蛋白;骨缺损;组织工程;支架材料 doi:10.3969/j.issn.1673-8225.2010.16.043     

Biomechanical researches on tissue engineering bone constructed by deproteinated bone

Objective： To study biomechanical changes of newly formed bones 24 weeks after repairing large defects of long bones of goats using heterogeneous deproteinated bone （DPB） prepared by modified methods as an engineering scaffold. Methods： According to a fully randomized design, 18 goats were evenly divided into three groups： normal bone control group （Group A）, autologous bone group （Group B） and experimental group （Group C）. Each goat in Groups B and C were subjected to the periosteum and bone defect at middle-lower part of the right tibia （20% of the whole tibia in length）, followed by autologous bone or DPB plus autolognus MSCs ＋ rhBMP2 implantation, respectively and semi- ring slot fixation; while goats in Group A did not perform osteotomy. At 24 weeks after surgery, biomechanical tests were carried out on the tibias. Results： At 24 weeks after surgery, the results of anticompression test on tibias in three groups were recorded by a functional recorder presented as linear pressure-deformation curve. The shapes of the curves and their change tendency were similar among three groups. The ultimate pressure values were 10.74 MPa±1.23 MPa, 10. 11 MPa±1.35 MPa and 10.22 MPa±1.32 MPa and fracture compression rates were 26.82%±0.87%, 27.17%±0.75% and 28.22%±1.12% in Groups A, B and C, respectively. Comparisons of anti-compression ultimate pressures and fracture compression rates among three groups demonstrated no significant difference （PAB=0.415, PBC=0.494）. Three-point antibend test on tibias was recorded as load-deformation curves, and the shapes of the curves and their change tendency were similar among three groups. The ultimate pressure values of the anti-bend test were 481.52 N±12.45 N, 478.34 N±14.68 N and 475.62 N±13.41 N and the fracture bend rates were 2.62 mm±0.12 mm, 2.61 mm±0.15 mm and 2.81 mm±0.13 mm in Groups A, B and C, respectively. There was no significant difference between groups （PAB=0.7, PBc=0.448）. The ultimate anti-torsion torque values were 6.55 N.mi-0.25 N.m, 6.34 N＇m~0.18 N＇m and 6.42 N＇m~0.21 N＇m and fracture torsion rates were 29.51°±1.64°, 28.88±1.46° and 28.81°±1.33° in Groups A, B and C, respectively. There was no significant difference between groups （PAB=0.123, PBc=0.346）. Conclusions： The biomechanical characteristics of newly formed bones from heterogeneous DPB for repairing large segmental long bone defect are comparable to those of normal bones and autologous bones. DPB has the potential for clinical usage as bone graft material.     

Preparation and physicochemical properties of scaffold materials of heterogeneous deproteinized bone

Objective:To prepare and observe the physicochemical properties of scaffold materials of heterogeneous deproteinized tissue-engineered bone. Methods: Deproteinized bone was made through a series of physicochemical treatments in pig ribs and analyzed with histological observation, scanning electron microscopy, infrared spectrum, X-ray diffraction and energy dispersive analysis, Kjeldahl determination and mechanics analysis. Results: Interstitial collagen fiber was positive and mucin was negative in deproteinized bone, but, both were positive in fresh bone. Deproteinized bone maintained natural pore network. Its pore size was 472.51μm±7.02μm and the porosity was 78.15%±6.45%. The results of infrared spectrum showed that collagen was present in deproteinized bone. Both fresh and deproteinized bone had curve of hydroxyapatite. The Ca/P ratios were 1.71±0. 95 and 1. 68±0. 76 ( P > 0. 05 ), and the protein contents were 26.6%±2.23% and 19.1%±2.14% (P < 0.05) in fresh and deproteinized bone, respectively. There was no significant difference of destruction load under compression and maximal destruction load between fresh and deproteinized bone (P > 0. 05). The elastic modulus was higher in deproteinized bone than that in fresh bone (P < 0.05). Conclusions:Physicochemical properties and mechanic strength of deproteinized tissue-engineered bone meet the demands of ideal scaffold materials. But, its immunogenicity should be observed through further experiments for its clinical applications.     

手术治疗Pipkin＇s骨折38例临床分析

Pipkin’s骨折是指髋关节后脱位合并股骨头、股骨颈或髋臼骨折，多由强大暴力所致，伤情复杂，常合并其他严重损伤。手术时机、手术入路及内固定方法的选择等问题争论目前尚无标准可寻。我院自2000—01—2009—01采用早期切开复位内固定Pipkin’s骨折38例，其中30例获随访，按美国矫形外科学院评定标准评定效果良好。现报告如下。     

Study of biological safety of scaffold material with heterologous deproteinized bone

Objective: To evaluate the biological safety of manufactured heterologous deproteinized bone and to provide an experimental basis for clinical applications. Methods: Deproteinized bone (10 mm) and leaching liquor were made from pig ribs with a series of physical and chemical methods, then were evaluated through acute and subacute toxicity test, hemolysis test, pyrogen test, intracutaneous test, intramuscular implantation test and cytotoxity test. Results: No obvious toxicity, hemolysis, pyrogenic characteristics, skin irritation, inflammatory reaction after intramuscular implantation and cytotoxity were observed. Conclusions: The heterologous deproteinized bone has good biological safety and meets all the demands of scaffold material for tissue engineering.     

一期后路单侧病灶清除植骨融合内固定治疗胸椎结核的临床疗效*

目的探讨一期后路经单侧入路病灶清除植骨融合内固定治疗胸椎结核的安全性及有效性。方法回顾性分析我科收治的12例单节段胸椎结核患者,男4例,女8例,平均年龄31.1岁。合并脊髓功能障碍者10例,其中Frankle B级2例,C级6例,D级2例,术前给予2周以上的抗结核化疗。记录手术时间、出血量、卧床时间、术前、术后的ODI评分、VAS评分、神经功能改善率、并发症等。结果所有病例均得到随访,平均随访时间16.6月,神经功能改善率100%,平均手术时间280.8分钟,平均失血量1037.5 m L,平均卧床时间7.4天。术前、术后神经功能、ODI评分、VAS评分相比差异具有统计学意义(0.05)。术后3个月内ESR及CRP恢复至正常水平,治愈率为100%。随访时间内无复发病例,无脊髓功能损伤,无严重并发症发生。结论该术式对于单节段的胸椎结核能满意地清除病灶,创伤小,能最大限度保留脊柱正常结构,是一种有效,安全的术式。     

关节镜下手术治疗膝半月板损伤478例流行病学调查分析

目的调查分析近期关节镜下手术治疗膝半月板损伤病例的临床流行病学特点及其变化趋势。方法回顾性调查西南医院关节外科中心2001年1月~2005年3月期间关节镜下手术治疗膝半月板损伤的全部病例,统计分析其性别比例、年龄分布、病因分布、术前诊断准确率、发病至手术时间、损伤部位分布、术后住院天数、手术方式频次及合并症发生率。结果关节镜下手术治疗膝半月板损伤者478例,其性别比(男:女)为0.83∶1,年龄4~83岁,平均(40.88±16.96)岁;其病因分布:无外伤病史者(50.42%)>其他外伤者(43.31%)>运动损伤(6.28%);术前诊断准确度为92.15%,发病至手术平均时间(28.96±60.37)个月,右膝半月板损伤的发生率(52.60%)与左膝半月板损伤(47.40%)相近(P>0.05),外侧半月板损伤的发生率(65.56%)高于内侧半月板损伤(34.44%,P<0.05),外侧盘状半月板损伤的病因分布为:无外伤病史者(57.00%)>其他外伤者(39.00%)>运动损伤者(4.00%);手术方式频次为:部分切除术(46.72%)≈修整术(46.23%)>全部切除术(6.12%)>缝合术(0.92%);术后平均住院天数(6.40±6.27)d;合并症发生率为:滑膜炎(42.97%)≈关节软骨损伤(42.37%)>滑膜皱襞综合征(7.43%)≈前交叉韧带损伤(7.23%)。结论目前关节镜下手术治疗膝半月板损伤病例的性别比、年龄分布、病因分布、术前诊断准确率、发病至手术时间、术后住院天数、手术方式及并发症,较其开放手术治疗时期有一定变化,损伤部位分布则无明显改变;外侧盘状半月板损伤的病因分布具有其自身特点。     

异种脱蛋白骨的生物相容性研究

目的 评价制备的异种脱蛋白骨植入体内的生物相容性,为临床应用提供实验依据.方法 对猪肋骨进行一系列理化处理后,制成10 mm长脱蛋白骨及浸提液.然后采用标准的毒理学实验方法进行急性、亚急性毒理实验、溶血实验、热源实验、皮内注射实验、肌肉埋植实验及细胞毒性实验.结果 异种脱蛋白骨的毒性程度为无毒,溶血率＜5%,无热源性,对皮肤无刺激,肌肉内埋植后无明显炎症反应,对细胞无明显毒性作用,细胞的毒性均为0级.结论 异种脱蛋白骨具有良好的生物相容性,可满足作为组织工程骨支架材料生物相容性的要求.     

半环槽外固定器建立山羊胫骨缺损模型

目的 探索半环槽外固定器建立山羊胫骨段缺损模型.方法 山羊9只分3组,制备胫骨中下段20%的骨膜和骨缺损,半环槽外固定器外固定,术后第5、10、15周分别处死1组动物.用放射学、组织学方法评价骨缺损修复情况.结果 术后山羊胫骨段缺损处固定稳定,5、10、15周骨缺损处未见骨修复,15周时见骨断端硬化,放射学和组织学检查未见骨形成.结论 应用半环槽外固定器建立组织工程用大段骨缺损动物模型效果较好,山羊胫骨20%的骨膜和骨缺损不能自行修复.     

SD大鼠脂肪干细胞的体外分离培养及实验研究

目的探索从脂肪组织中分离、培养SD大鼠脂肪干细胞(Adipose tissue-derivedstromal cells,ADSCs)的方法,同时观察其生物学特性。方法取SD大鼠腹股沟区皮下脂肪组织,0.1%Ⅰ-型胶原酶消化分离、培养ADSCs,流式细胞术测定CD44、CD45和CD49d抗原的表达,MTT比色法测定细胞生长活力,诱导培养基向脂肪细胞定向分化,Oil Red O染色鉴定。结果分离出的细胞CD44表达阳性,CD49d表达弱阳性和CD45表达阴性,其生长曲线呈倒"S"形,成脂诱导培养基定向诱导分化,经Oil Red O染色呈红色。结论本次实验所分离出来的SD大鼠ADSCs在体外具有生长稳定,增殖较快并能诱导分化的特点。