生物反应器中的力学刺激促进组织工程软骨再生

BACKGROUND: Cartilage tissue engineering has been widely used to achieve cartilage regeneration in vitro and repair cartilage defects. Tissue-engineered cartilage mainly consists of chondrocytes, cartilage scaffold and in vitro environment. OBJECTIVE: To mimic the environment of articular cartilage development in vivo, in order to increase the bionic features of tissue-engineered cartilage scaffold and effectiveness of cartilage repair. METHODS: Knee joint chondrocytes were isolated from New Zealand white rabbits, 2 months old, and expanded in vitro. The chondrocytes at passage 2 were seeded onto a scaffold of articular cartilage extracellular matrix in the concentration of 1×106/L to prepare cell-scaffold composites. Cell-scaffold composites were cultivated in an Instron bioreactor with mechanical compression (1 Hz, 3 hours per day, 10% compression) as experimental group for 7, 14, 24, 28 days or cultured statically for 1 day as control group. RESULTS AND CONCLUSION: Morphological observations demonstrated that the thickness, elastic modulus and maximum load of the composite in the experimental group were significantly higher than those in the control group, which were positively related to time (P < 0.05). Histological staining showed the proliferation of chondrocytes, formation of cartilage lacuna and synthesis of proteoglycan in the experimental group through hematoxylin-eosin staining and safranin-O staining, which were increased gradually with mechanical stimulation time. These results were consistent with the findings of proteoglycan kit. Real-time quantitative PCR revealed that mRNA expressions of collagen type I and collagen type II were significantly higher in the experimental group than the control group (P < 0.05). The experimental group showed the highest mRNA expression of collagen type I and collagen type II at 21 and 28 days of mechanical stimulation, respectively (P < 0.05). With the mechanical stimulation of bioreactor, the cell-scaffold composite can produce more extracellular matrix, such as collagen and proteoglycan, strengthen the mechanical properties to be more coincident with the in vivo environment of cartilage development, and increase the bionic features. With the progress of tissue engineering, the clinical bioregeneration of damaged cartilage will be achieved.   中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

AZ31镁合金材料植入物在兔股骨髁内的降解：Micro-CT评价

BACKGROUND: Magnesium can be degraded voluntarily in vivo, so a second surgery is avoided. However, its alloys have not been widely used in the clinical orthopedics because there is a lack of accurate and reliable methods to assess its degradation in vivo. OBJECTIVE: To explore the degradation of micro-arc-oxidized AZ31 magnesium alloy in the femoral condyle of rabbits based on micro-CT images and relative data. METHODS: Forty micro-arc-oxidized AZ31 magnesium alloys were implanted into the right femoral condyle of 40 New Zealand rabbits. Then 10 right femoral condyles were removed at 5, 10, 15 and 20 weeks after surgery, respectively, to quantitatively analyze and evaluate the degradation of AZ31 magnesium alloys by micro-CT images and relative data. RESULTS AND CONCLUSION: The surface of AZ31 alloys was corroded progressively with dark color and distorted appearance at 5-20 weeks post implantation. Micro-CT images showed that in the first 5 weeks, the degradation was inactive, and at the 10th week, it turned active; at the 15th week, the corrosion pits were obviously increased in number, and the corrosion area and corrosion speed were enlarged and fastened, respectively. Up to the 20th week, the alloy surfaces were full of corrosion pits besides roughness and discontinuity. Relevant data analysis showed that the volume fraction of magnesium alloy was 98.6%, 97.1% and 86.4% at the 5th, 10th and 20th weeks after implantation, respectively, and it had a significant decrease from the 10th to 15th week and from the 15th to 20th week (P < 0.05). Within 15-20 weeks, the volume fraction of magnesium alloy was decreased by 6.5% that was the maximum volume reduction per unit cycle. With the progress of corrosion, the surface continuously became rough and vague, and its surface area was enlarged; the ratio of surface area to volume continuously increased, and there was a significant difference at 15 and 20 weeks (P < 0.05). Because of the increasing number of corrosion pits, the cross-sectional radius decreased, which was reflected by the trabecular thickness decreasing from 1.00 to 0.87 mm. From the view of the slope of curve, the trabecular thickness decreased most rapidly at 10-15 weeks. The mineral density of magnesium alloy continuously decreased from 649.302 to 356.445 mg/cm3 during the whole experiment period (P < 0.05). In addition, the micro-CT image density decreased from 679.710 to 644.947 mg/cm3, but there was no significant difference. To conclude, the degradation speed is peaked at 10-20 weeks after implantation, and the content of magnesium alloys decrease with degradation, but the magnesium density has no significant change. 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

载IL-2磁性纳米粒的制备及其联合体外磁场在肿瘤组织中的靶向富集作用

目的:以Fe3O4磁性纳米粒(Fe3O4magnetic nanoparticle,Fe3O4-MNP)和聚乳酸-聚乙醇酸共聚物(poly lactide-co-glycolide,PLGA)为材料,制备载IL-2磁性纳米颗粒(IL-2-loaded magnetic nanoparticle,IL-2-Fe3O4-PLGA),观察其在肿瘤免疫治疗中的靶向富集作用。方法:采用复乳法制备IL-2-Fe3O4-PLGA,激光粒度分析仪和扫描电镜观察其大小和形态。ELISA检测IL-2-Fe3O4-PLGA的包封率、体外释药特性;MTT法检测其释放IL-2的生物学活性。构建S180肉瘤细胞小鼠肿瘤模型,研究其瘤体内注射联合体外磁场对S180细胞移植瘤生长的影响,普鲁士蓝染色观察IL-2-Fe3O4-PLGA在肿瘤组织以及肝脏和肾脏组织中的富集和分布。结果:成功制备了IL-2-Fe3O4-PLGA,IL-2-Fe3O4-PLGA呈圆球形,平均粒径为(697±0.51)nm,包封率为(83.76±1.24)%。IL-2-Fe3O4-PLGA突释期释放的IL-2质量浓度为100 ng/ml,第15 d后质量浓度为180 ng/ml,并且保持原有生物学活性的85%～55%。在肿瘤组织中,IL-2-Fe3O4-PLGA联合体外磁场组可见大量的普鲁士蓝染色阳性IL-2-Fe3O4-PLGA颗粒沉积,IL-2-Fe3O4-PLGA组只有少量的IL-2-Fe3O4-PLGA阳性颗粒沉积;两组肝脏中均可偶见少量阳性颗粒沉积,而肾脏几乎未见IL-2-Fe3O4-PLGA颗粒沉积。结论:载IL-2磁性纳米颗粒IL-2-Fe3O4-PLGA具有缓释IL-2的功能,联合体外磁场可有效富集于肿瘤组织中。     

无菌性松动假体周围界膜组织中PDGF-B和BMP-7受体的分布及其临床意义

目的 通过对比研究PDGF－B和BMP－7受体在无菌性松动假体周围界膜组织细胞中的分布,探讨骨－假体界膜组织的分化趋势、假体松动的生物学原因并提出防治因界膜形成而导致假体松动的相应措施。方法 以预聚合SABC法,采用PDGF－B受体（PDGFR－β）和BMP－7受体（ActR-I)的多克隆抗体分别对10例无菌性松动假体周围溶骨区和非溶骨区界膜组织行免疫组织化学染色。检测每例每种阳性各100个的相对灰度值,在不同部位的组织和细胞间进行比较。结果 界膜组织中PDGFR－β在不同细胞上的分布没有特异性,但溶骨区界膜中巨噬细胞的成纤维细胞该受体的表达量明显高于非溶骨区（P＜0．01）,并以成纤维细胞表达量较高（P＜0．05）,ActR-I主要分布于无磨损颗粒非溶骨区界中层丰富的间充质细胞或成纤维细胞上,存在磨损颗粒的界膜中细胞也可有表达。结论 溶骨和成骨因子受体在界膜组织细胞上均有分布。磨损颗粒等因素致PCGF－B表达增加可能促进界面骨溶解和纤维增生,从而造成假体的不稳定或松动。而在界面施加成骨因子则可能是在一定程度上削弱人工关节无菌性松动生物学因素的一种尝试。     

骨骺软骨细胞的培养和保存方法

目的建立大量生产骨骺软骨细胞的培养方法和保存方法,为组织工程骺软骨的培养提供种子细胞。方法2周龄兔胫骨上端骨骺软骨经机械剪切和化学消化后,接种、培养及液氮冻存。通过显微镜观察其生物学表现,并通过蕃红花“O”染色和Ⅱ型胶原免疫组化染色对其生物学特征进行鉴定。结果在体外观察到了骨骺软骨细胞从静止、增殖到肥大这个近似体内的生长过程,经多次传代后增殖能力降低,经过冻存的细胞生物学特征不变。蕃红花“O”和Ⅱ型胶原免疫组化染色阳性。结论我们成功地建立了骨骺软骨细胞的培养和冻存方法,并证明培养出的细胞是骨骺软骨细胞。     

人脂肪干细胞复合脱细胞软骨基质支架在生物反应器中构建组织工程软骨

目的:观察人脂肪干细胞复合脱细胞软骨基质支架在生物反应器中初步构建组织工程软骨的可行性。方法:实验于2005-04/2006-05在解放军总医院骨科研究所完成。脂肪组织和关节软骨均来自膝关节置换术中切除的组织,并经患者知情同意。关节软骨冻干后经粉碎机粉碎,过筛,选取25～38μm大小的软骨微粒。在样品中先加入2.5g/L胰蛋白酶,37℃消化24h,再加入1%Triton X-100震荡72h。将软骨微粒和蒸馏水按1∶3的比例混合后滴加在模板中,置入冷冻干燥机冻干后行紫外线交联。紫外线照射8h完成。最后经25kGy 60Co辐照灭菌完成支架制备。取膝关节置换术中切除的髌下脂肪垫,酶消法获得脂肪干细胞,扩增后复合于脱细胞软骨基质制成圆柱状三维支架上(细胞密度5×1010L-1),置于生物反应器中进行诱导培养,同时设静态培养组作为对照,3周后观测大体形态和组织学形态变化,同时进行组织化学(包括番红花O,阿利新蓝染色)和Ⅱ型胶原免疫组织化学分析。结果:生物反应器组诱导培养3周苏木精-伊红染色显示支架结构消失,只有中心区域残存少量支架结构;静态培养组支架结构尚存在,有少量基质分泌。番红花O染色显示生物反应器组细胞外有大量蛋白聚糖沉积,阿利新蓝染色表明有软骨特异性蛋白多糖的聚集;而静态培养组只有部分区域染色且淡于生物反应器组。Ⅰ型胶原免疫组化的结果显示,在生物反应器组细胞能够合成大量软骨细胞特异性胶原成分,而静态培养组呈弱阳性。结论:生物反应器培养明显促进了脂肪干细胞的增殖与软骨分化,是体外构建组织工程软骨的良好方法。     

肿瘤疫苗与白介素-12联合免疫治疗小鼠肉瘤的研究

目的:探讨混合热休克蛋白(HSP)/肽与白介素-12(IL-12)和环磷酰胺(CTX)联合治疗小鼠肉瘤的作用及机制。方法:以S-180肉瘤为模型,将瘤组织剪碎,裂解,离心,取上清经Sephacryl S-200层析,截取相对分子质量(50～200)×103段,经SDS-PAGE电泳及Western-blot法鉴定。用提取的HSP/肽免疫小鼠后,再用低剂量CTX及IL-12治疗(强化疫苗),观察无瘤生存率、抑瘤生长率、抑制转移率和存活期。用流式细胞仪,ELISPOT及乳酸脱氢酶等方法检测免疫功能的变化。结果:HSP疫苗与IL-12和CTX联合治疗小鼠S-180肉瘤,80.0%肿瘤完全消退,且100.0%抑制了肺部转移,达长期生存,而对照组全部在40d内死亡。检测表明HSP/肽疫苗形成了免疫预存,从而使IL-12和CTX发挥了抗瘤作用。结论:HSP/肽与IL-12和CTX联合治疗诱发了机体细胞免疫功能,产生强大的抗瘤作用,可能具有临床应用前景。     

TJ骨粘固剂的研制及临床应用

骨粘固剂俗称骨水泥,是由甲基丙烯酸甲醋均聚物(PMMA)或共聚物(如本研究采用的甲基丙烯酸/苯乙烯共聚物)与甲基丙烯酸甲酯单体(MMA)所组成的室温自凝塑料,主要用作人工关节的固定材料及骨缺损充填材料。1975年以来中国人民解放军总医院与天津市合成材料研究所医用高分子组协作,采用国产原料,制成TJ骨粘固剂,进行了动物实验及临床应用研究,现综合报告如下:(＊:TJ是代号,由天津合成材料研究所与解放军总医院两个汉语拼音的第一个字母组成)     

低浓度唑来膦酸对成骨细胞和破骨细胞影响的体外实验

目的：观察低浓度(10-6 mol/L)唑来膦酸(zoledronate acid,ZA)对体外大鼠破骨细胞及成骨细胞的影响。方法体外分别培养大鼠来源的成骨细胞和破骨细胞,将两种细胞各分为两组：空白对照组及低浓度(10-6 mol/L)ZA组。应用抗酒石酸酸性磷酸酶染色、图像分析计算骨吸收陷窝面积,检测破骨细胞形态及骨吸收情况。碱性磷酸酶(alkaline phosphatase, ALP)染色、四甲基偶氮唑盐比色法了解成骨细胞的形态及增殖情况。结果培养1周后破骨细胞具有典型的形态特征,并在骨片上形成了吸收陷窝;ZA组与对照组相比,破骨细胞数量及生成吸收陷窝的数目和面积减少,差异有统计学意义(P＜0.05)。成骨细胞有典型的梭形、ALP染色阳性特征,培养至第7天ZA组成骨细胞光吸收值(3.37±0.11)高于对照组(2.87±0.12),差异有统计学意义(P＜0.05)。结论低浓度(10-6 mol/L)的ZA能够抑制破骨细胞的增殖和活性,促进成骨细胞的增殖,选择恰当给药方式和剂量能够在抑制破骨的同时促进成骨。     

人工全髋关节置换术后股骨应力变化的有限元分析

目的：建立全髋关节置换术后股骨的有限元模型，探讨应力变化的特点、规律，为临床应用提供参考。方法：用有限元计算法对不同设计的人工全部关节假体在骨内应力分布规律进行分析。结果：①正常股骨压应力主要分布在股骨内侧面，有托式无骨水泥型人工假体压应力主要分布在人工假体柄与股骨内侧皮质的交界面，有骨水泥型人工假体主要分布在骨水泥与假体交界面上。②有托式无骨水泥型在股骨内侧皮质应力比有骨水泥型小。③假体长度增加，假体末端应力略有减少。结论：人工关节置换术后较正常股骨应力发生改变，骨水泥对应有缓冲作用，增加假体长度应力减少不显著。