软骨脱细胞基质的研制及其力学性质分析

目的 猪CACM的制作及其力学特征的检测。方法 切取猪软骨，采取TritonX－100、Teis-HCl液等4步法制取脱细胞的猪耳CACM。采用光镜、透射电镜、扫描电镜等对结构进行检测，同时对CACM进行力学检测。结果 肉眼观察，CACM与软骨除颜色略白外无差别。组织学HE染色，细胞隐窝已无细胞结构，阿尔新兰色呈淡染，阴性；透射电镜下CACM罗骨陷窝内已无细胞结构；细胞膜、核器均已溶解；扫描电镜下CACM基质纤维成分与正常软骨基质的胶原纤维在形态上无差异；力学试验，最大拉伸强度，最大拉伸比，猪耳软骨与其脱细胞基质比较及其弹性模量无统计学意义。结论 经TritonX-100为主的脱细胞处理方法可以脱去软骨的细胞成分，不改变软骨细胞基质的主要成分，不改变其原有力学性质。     

软骨脱细胞基质的研制及其力学性质分析

目的猪CACM的制作及其力学特征的检测.方法切取猪耳软骨,采取Triton X-100、 Tris-HCl液等4步法制取脱细胞的猪耳CACM.采用光镜、透射电镜、扫描电镜等对结构进行检测,同时对CACM进行力学检测.结果肉眼观察,CACM与软骨除颜色略白外无差别.组织学HE染色,细胞陷窝已无细胞结构,阿尔新兰染色呈淡染,阴性;透射电镜下CACM软骨陷窝内已无细胞结构,细胞膜、核器均已溶解;扫描电镜下CACM基质纤维成分与正常软骨基质的胶原纤维在形态上无差异;力学试验,最大拉伸强度,最大拉伸比,猪耳软骨与其脱细胞基质比较及其弹性模量无统计学意义.结论经Triton X-100为主的脱细胞处理方法可以脱去软骨的细胞成分,不改变软骨细胞基质的主要成分,不改变其原有力学性质.     

软骨脱细胞基质的生物力学性质研究

目的 研究猪耳CACM的力学特征.方法 切取猪耳软骨,采取Triton X-100液等4步法制取脱细胞的猪CACM.采用光镜、透射电镜分析其结构进行检测,同时对CACM进行力学检测.结果 肉眼观察,CACM与软骨除颜色略白外无差别.组织学HE染色,细胞陷窝无细胞结构;透射电镜下CACM软骨陷窝内无细胞结构,细胞膜、核器均已溶解;力学试验,最大拉伸强度,最大拉伸比,猪耳软骨与其脱细胞基质比较及其弹性模量无统计学意义.结论 经TritonX-100为主的脱细胞处理方法可以脱去软骨的细胞成分,不改变软骨细胞的原有力学性质.     

人关节软骨脱细胞基质的制备

目的制备人关节软骨脱细胞基质。方法切取人关节软骨,对之冷冻干燥后,采取化学去污剂TritonX100及DNA酶和RNA酶等试剂制备脱细胞的人关节软骨。做HE、番红0及软骨蛋白聚糖(aggrecan)免疫组化染色等方法进行检测。结果HE染色、番红0染色均显示细胞陷窝内己无细胞结构番红花0染色阳性;软骨蛋白聚糖免疫组化染色阳性。结论人关节软骨冻干后,经去污剂酶等处理方法可脱去软骨的细胞成分,并且保留了软骨细胞外基质,成功制备了人关节软骨脱细胞基质。     

软骨脱细胞基质复合脂肪源性干细胞构建软骨组织的实验研究

[目的] 探讨软骨脱细胞基质支架材料的制备,及其体外复合脂肪源性干细胞构建软骨组织的技术方法.[方法] 成年新西兰大白兔脂肪源性干细胞获得,培养,扩增.成年新西兰大白兔新鲜软骨,低温冻干12 h,后经曲拉通、DNA、RNA酶等处理制备成为软骨脱细胞基质支架材料,终浓度为2×107/L的脂肪干细胞种植于软骨脱细胞基质中于软骨细胞方向诱导培养基中培养2周,构建软骨组织.新鲜制备的软骨脱细胞基质及构建的软骨组织分别行组织学、免疫组织化学及透射电镜检测.[结果] 实验制备的软骨脱细胞基质支架材料内无细胞结构存在,仅残留空白软骨陷窝.具有合适的孔隙率和孔径大小;复合脂肪源性干细胞后细胞向材料内部迁移,粘附,生长良好.部分载体内细胞Ⅱ型胶原免疫组化染色阳性.[结论] 软骨脱细胞基质可作为支架材料应用于软骨组织工程,复合脂肪源性干细胞培养可成功构建软骨组织.     

猪主动脉脱细胞基质的简化制备及生物学评价

目的应用酶法制备猪主动脉脱细胞基质，并评价其生物学性能。方法取新鲜屠宰的16根肉猪降主动脉，长10～12cm，应用0．1％胰蛋白酶和0．01％EDTA于37℃震荡条件下脱除细胞成分，HE和Masson染色行脱细胞基质的组织学观察，扫描电镜和透射电镜观察超微结构改变。应用单轴拉伸方法比较脱细胞前和脱细胞后48、96和120h时材料两断端厚度、极限抗张强度（ultimate tension stress，UTS）和断裂伸长率（strain of failure，SOF），绘制应力-应变曲线。取成年杂种犬3只，体重20～30kg，雌雄不限。将脱细胞前后片状主动脉基质埋植于犬脊柱两侧皮下，分别于埋植后1、3和6周取材，行HE染色观察，参照半定量的Wakitani评分法，对取材时的大体形态、浸润细胞种类和数量、新生血管等指标进行比较，观察宿主炎性细胞反应和脱细胞基质变化。将犬内皮细胞种植于猪脱细胞基质，HE染色和扫描电镜观察细胞相容性。结果HE染色和电镜检查显示在96h可将新鲜猪主动脉细胞成分脱除，Masson染色显示细胞外纤维成分保留完整。脱细胞前后的动脉基质厚度、UTS和SOF差异均无统计学意义（P〉0．05），但UTS显示降低趋势，SOF则呈现增加趋势，应力-应变曲线呈现力学强度降低和延展性增加的变化趋势。犬皮下埋植，各组脱细胞标本炎性细胞浸润明显减轻，6周时以成纤维细胞浸润为主，且基质中见新生毛细血管。半定量组织学评分，在大体观察、浸润细胞种类和数量方面与脱细胞前比较有统计学意义（P〈0．05）；新生血管数量比较，差异无统计学意义（P〉0．05）。HE染色和扫描电镜显示种植的内皮细胞可在基质表面形成单细胞层。结论应用0．1％胰蛋白酶和0．01％EDTA持续振荡96h制备的猪主动脉脱细胞基质，在生物力学、免疫原性和细胞相容性方面可满足血管组织工程的需要。     

利用脂肪干细胞构建软骨修复兔膝关节软骨缺损的初步研究

目的 利用兔同种异体软骨脱细胞基质支架和脂肪干细胞体外构建组织工程软骨,探讨其修复关节软骨损伤的可行性.方法 将新西兰大白兔的脂肪干细胞与软骨脱细胞基质支架复合,于软骨细胞方向诱导培养基中培养两周,构建组织工程软骨.兔24只随机分为A、B、C 3组, A组关节软骨缺损处置入经诱导的脂肪源干细胞复合软骨基质支架, B组缺损处只置入软骨基质支架, C组软骨缺损处不做任何处理.分别于术后第12周处死动物,修复处行大体、组织学、Ⅱ型胶原免疫组化染色和透射电镜检测.结果 A组软骨缺损处被类软骨组织填充,修复区表面光滑;Ⅱ型胶原免疫组化染色和甲苯胺蓝染色阳性;电镜下可见软骨陷窝内有细胞结构存在,且有大量均匀颗粒状细胞分泌基质成分存在,细胞周围大量胶原纤维.B组软骨缺损处为纤维组织状物填充,C组软骨缺损处无修复组织填充.结论 脂肪干细胞与软骨脱细胞基质复合并向软骨诱导后可良好地修复关节软骨缺损,具有替代正常软骨的潜力.     

骨骼肌无细胞基质的制备及其生物相容性研究

目的细胞外基质是脂肪组织工程材料的研究热点之一。通过探讨骨骼肌无细胞基质的制作方法及生物相容性,为其在脂肪组织工程中的应用奠定基础。方法取健康成年小香猪新鲜骨骼肌组织,横切成厚2～3 mm的组织块,采用低渗-去垢剂法脱细胞处理。处理后采用HE染色、Masson三色染色、免疫组织化学染色及扫描电镜检测骨骼肌无细胞基质是否有细胞成分残留,并观察其基本结构;应用MTT法检测骨骼肌无细胞基质细胞毒性。取乳腺癌患者自愿捐赠脂肪组织,分离培养人脂肪干细胞(human adipose-derived stem cells,hADSCs),从形态学、流式细胞学和成脂、成骨分化能力方面进行鉴定。将骨骼肌无细胞基质与第3代hADSCs共培养,于培养后第1、3、5、7天通过细胞活性检测材料上细胞黏附、扩散和增殖情况,了解其与细胞之间的相互作用。结果 HE、Masson、免疫组织化学染色及扫描电镜观察显示骨骼肌无细胞基质肌纤维去除完全,无细胞核残留,基质结构保留完整;大量连接成网状的胶原纤维呈多孔隙样结构,规则排列。MTT检测示骨骼肌无细胞基质细胞毒性为1级,细胞相容性好。细胞活性检测示hADSCs在骨骼肌无细胞基质上能很好地伸展,且能与周围基质黏附,进入基质内部并相互交织。结论经脱细胞处理的骨骼肌无细胞基质具有良好生物相容性,可能作为脂肪组织工程的支架材料。     

不同浓度曲拉通X-100对制备颌下腺脱细胞基质的影响

目的探讨制备颌下腺脱细胞基质生物衍生支架材料过程中曲拉通X-100的最佳浓度。方法用3组不同浓度的曲拉通X-100试剂对30个颌下腺腺体进行脱细胞处理，并通过光镜、透射电镜及扫描电镜观察。结果0．5％和1％曲拉通X-100组脱细胞后，光镜下见大部分细胞轮廓存在，呈拥挤状态。透射电镜下见细胞膜破损，细胞器破碎，形成多个散在的碎块。扫描电镜下见细胞膜被破坏，表面呈丰窝状改变。而3．0％曲拉通X-100组脱细胞后，光镜下见颌下腺细胞成分完全消失，胶原纤维呈网状排列。透射电镜下见细胞器及细胞核消失，只见残留的细胞轮廓。扫描电镜下见细胞完全消失，只残留空虚的陷窝，基质胶原纤维粗细不等，相互交错呈网状结构。结论在制备颌下腺脱细胞基质时曲拉通X-100试剂的最佳浓度是3．0％。     

脱细胞血管基质和间充质干细胞构建组织工程血管

目的 探讨利用异种脱细胞血管基质和间充质干细胞体外构建小口径血管移植物的方法.方法 采用去垢剂和胰蛋白酶去除猪髂动脉血管壁的细胞成分,对脱细胞基质进行组织学、力学检测及孔隙率评估.分离培养犬骨髓问充质干细胞,种植到脱细胞基质上,并进一步在搏动性生物反应器内培养,采用HE染色和扫描电镜对构建的组织工程血管进行检测.结果 脱细胞处理后,猪髂动脉的细胞成分完全去除,细胞外基质保存完好,力学强度轻度下降;脱细胞基质的孔隙率为94.9%.间充质干细胞能够种植到脱细胞基质上,在剪切力的作用下细胞基本融合,高度伸长并且其排列与流体的方向一致.结论 小口径血管移植物可以通过将间充质干细胞种植到异种脱细胞血管基质并在搏动性生物反应器内培养的方法进行构建.     

软骨脱细胞基质支架材料的软骨组织工程实验研究

目的应用软骨脱细胞基质作为支架材料，按照组织工程的原理再生软骨，为修复软骨缺损探索新的途径。方法取新西兰大耳白兔1只，体重2．4kg，按改良Courtman法对兔耳软骨行脱细胞处理后用于实验。选用纯种6月龄新西兰大耳白兔18只，雌雄不限，体重2．4～2．6kg。每只耳形成2处1cm×1cm软骨缺损，按修复方式不同随机分为3组，每组24处缺损。A组，软骨脱细胞基质加软骨膜；B组，软骨脱细胞基质；C组软骨膜，作为对照。术后每日观察兔耳修复区的大体变化，并分别于4、12周每组处死3只动物，于修复区切取标本，行HE染色、藏红花红一奥尔新蓝染色、II型胶原基因探针原位杂交实验。结果术后4周内A、B组大体形态无明显改变；术后12周可见修复区轻度增厚，触摸时质地较正常软骨稍硬。C组术后2周，2只2处修复区形成痂皮，术后5周痂皮脱落形成穿孔。术后4周，A、B组HE染色可见软骨脱细胞基质周边有轻度的炎性细胞浸润，以淋巴细胞为主，未形成包囊，藏红花红．奥尔新蓝染色均阴性；C组软骨缺损处的软骨膜塌陷，无细胞增殖现象；各组修复区均未见II型胶原基因探针原位杂交显色。术后12周，A组HE染色示部分软骨脱细胞基质内有新生细胞，细胞排列不规律，ECM嗜碱性增强，藏红花红-奥尔新蓝染色呈阳性，II型胶原基因原位杂交显色示新生细胞内均有大片棕黄色阳性染色区域；B组HE染色示软骨脱细胞基质无吸收现象，周边无包囊，炎性细胞消失，藏红花红一奥尔新蓝染色呈阴性，未见II型胶原基因原位杂交显色；C组HE染色示近软骨断端处可见部分形成新生组织，藏红花红一奥尔新蓝染色呈阳性，II型胶原基因原位杂交显色可见棕黄色阳性染色细胞。结论脱细胞软骨可诱导软骨膜细胞向其中生长而重建软骨，软骨膜和脱细胞软骨复合移植是软骨组织工程的另一种选择。     

Staining of intracellular granules in fresh epiphyseal cartilage by cationic dyes

Hand-cut sections of fresh epiphyseal cartilage from young rats were stained at pH 4.5 in 0.01% solutions of various cationic dyes of the thiazine, oxazine, azine, triphenylmethane, acridine, and phthallocyanin classes. The intracellular β-and γ-metachromatic granules, previously demonstrated in fresh tissues with toluidine blue, were also demonstrated well with azure A, methylene blue, and brilliant cresyl blue. The granules were also demonstrated, but not as well, by thionin, neutral red, safranin O, toluylene blue, and acridine orange. Under the conditions of staining, the reserve zone matrix and the lower hypertrophic (calcifying) zone matrix stained, whereas the proliferative and upper hypertrophic zone matrix did not stain. Gallocyanin, crystal violet, basic fuchsin, azocarmine B, gallamine blue, and alcian blue either did not stain, or gave a different pattern of staining from that described above. It is suggested that the pK and molecular weight of the dyes are important, but not necessarily the only factors in determining the staining of the granules. The results indicate that there is a change in the metachromatic material (presumably proteinpolysaccharide) in both the matrix and cells of epiphyseal cartilage, which appears to be related to calcification.     

人耳软骨移植抗原性及脱细胞处理对其的影响

目的 研究人耳软骨的移植抗原性及脱细胞处理对其的影响。方法 用低渗的TritonX—100为主的方法脱去人耳软骨细胞，然后通过抗人主要组织相容性复合体—I(MHC—I)免疫组织化学染色，与单个核细胞共同培养来研究人耳软骨的移植抗原性及脱细胞处理对其的影响。结果 软骨细胞脑浆抗人MHC—I为阳性，抗原的分布以细胞核周边的细胞质为主。细胞外基质抗人MHC—I为阴性。而经过脱细胞处理后的人耳软骨则呈抗人MHC—I阴性。单个核细胞与软骨共同培养时则显示出较高DNA合成能力，与对照组比较有统计学意义(P＜0．05)。单个核细胞与脱细胞处理的软骨共同培养后的DNA合成能力较低，与单纯的单个核细胞培养展示出新的DNA合成能力，但无显著差异(P＞0．05)。单个核细胞与正常人耳软骨共同培养的过程中，可见移向软骨的单个核细胞随时间增加而增加。相反，单个核细胞与脱细胞处理的软骨共同培养后，却少见单个核细胞移向脱细胞处理的软骨(P＜0．01)。结论 软骨具有移植抗原性；以低渗的TritonX—100为主的脱细胞方法可以有效的去除移植物的抗原性，从而避免同种异体组织移植排斥反应的发生。     

新型脱细胞软骨基质三维多孔支架的制备

目的 探讨脱细胞软骨基质三维多孔支架的制备方法以及将其应用于关节软骨组织工程的可行性. 方法 取天然人软骨粉碎后,采用梯度离心法取100 nm～5μm软骨微丝,脱细胞处理后制备为质量体积比为3%的悬液,采用冷冻冻干法制备脱细胞软骨基质三维多孔支架.254nm紫外线和碳化二亚胺/N-羟基琥珀酰亚胺对支架进行交联.冷冻冻干后,对支架材料进行组织学及扫描电镜观察,测定支架孔径和孔隙率、吸水率,并采用MTT法分析支架浸提液毒性.分离培养犬BMSCs,用TGF-β1成软骨诱导后种植至支架,倒置显微镜、电镜观察细胞在支架上的生长、分化情况. 结果 组织学观察显示,三维多孔支架中无软骨细胞碎片残留,甲苯胺蓝染色、番红O染色、Ⅱ型胶原免疫组织化学染色均呈阳性.扫描电镜显示支架内孔洞相互连通,孔径为(155±34)μm,孔隙率为91.3%±2.0%,吸水率为2 451%±155%.MTT法显示不同浓度支架浸提液与对照DMEM培养液吸光度值比较,差异无统计学意义(P＞0.05),支架无细胞毒性.倒置显微镜观察,细胞在支架上黏附良好;扫描电镜下细胞在支架上均匀分布,细胞呈圆形或椭圆形,并有基质分泌. 结论 制备的脱细胞软骨基质三维多孔支架去细胞彻底,保留了软骨ECM主要成分,无毒,具备合适的孔径和孔隙率,生物相容性良好,是软骨组织工程良好的支架载体.     

A Prospective Randomised Study of Laser Reshaping of Cartilage In Vivo

Laser reshaping using low laser energy levels was performed on the cartilage of ten porcine ears. The ears were examined up to 4 months after laser reshaping and the stability of the reshaping was assessed by photography and casts obtained from alginate impressions of the ears. The cartilage was also studied histologically in three animals at 3 weeks, 8 weeks, and 4 months using haematoxylin and eosin (H&E), periodic-acid–Schiff (PAS) and alcian blue stains as well as Mib-1 antibody stain to detect cells in cycle. These ears were compared with the other control or non-irradiated ear. All the treated ears initially demonstrated a controlled alteration in their shape. Four were exposed to a pulse energy of 2 J/cm² at 20 Hz with a spot diameter of 1.1 mm for 7 s and retained their shape for up to 14 days. However, the three exposed to the same levels of energy for 9 s showed tissue necrosis whereas the three which were treated for less than 4 s returned to their original shape after 24 h. Histological examination revealed that there was a dramatic eccentric proliferation of perichondrium away from the centre of irradiation with a noticeable absence of any inflammation. In all laser-irradiated areas there was a loss of staining with PAS and a dramatic increase in staining with alcian blue within the cartilage matrix. Viable chondrocytes were absent from the irradiated cartilage. Staining with a Mib-1 antibody that detects cells in cycle showed positive staining in a small fraction of proliferated perichondrial cells where new cartilage had been produced. Paper received 16 August 2000; accepted after revision 16 March 2001.     

Reduced NO accumulation in arthrotic cartilage by exposure to methylene blue

Nitric oxide (NO) appears to be a final common inflammation mediator of cartilage degradation. Halting the pathological formation of excessive NO, by suppressing the inducible NO synthase (iNOS) activity, may help to preserve cartilage integrity. We used fresh ex-vivo human articular cartilage explants from normal and arthrotic joints for assessment of NO levels, as determined by its nitrite degradation products and nitric oxide synthase expression. We measured matrix proteoglycan content, assessed by image analysis of alcian blue staining, and proteoglycan synthesis, assessed by sulfate incorporation into proteoglycans. The effect of methylene blue, a nitric oxide synthase inhibitor, on matrix preservation was evaluated. Cartilage discs in vitro, derived from normal appearing joints, secreted about one tenth as much NO compared to discs derived from arthrotic cartilage. Cartilage explants showed a time-dependent reduction in the amount of aggrecan within the cartilaginous matrix. Addition of methylene blue to the growth medium lowered nitric oxide accumulation and prevented matrix degradation in the cultured cartilage discs. The cartilage matrix preservation effect was mediated through downregulation of all three isoforms of NOS, i.e., the neuronal NOS, endothelial NOS and inducible NOS and upregulation of TGF beta receptor in the chondrocytes. Our findings indicate that inhibition of NOS activity preserves cartilage matrix in vitro.     

Reduced NO accumulation in arthrotic cartilage by exposure to methylene blue

Nitric oxide (NO) appears to be a final common inflammation mediator of cartilage degradation. Halting the pathological formation of excessive NO, by suppressing the inducible NO synthase (iNOS) activity, may help to preserve cartilage integrity. We used fresh ex-vivo human articular cartilage explants from normal and arthrotic joints for assessment of NO levels, as determined by its nitrite degradation products and nitric oxide synthase expression. We measured matrix proteoglycan content, assessed by image analysis of alcian blue staining, and proteoglycan synthesis, assessed by sulfate incorporation into proteoglycans. The effect of methylene blue, a nitric oxide synthase inhibitor, on matrix preservation was evaluated. Cartilage discs in vitro, derived from normal appearing joints, secreted about one tenth as much NO compared to discs derived from arthrotic cartilage. Cartilage explants showed a time-dependent reduction in the amount of aggrecan within the cartilaginous matrix. Addition of methylene blue to the growth medium lowered nitric oxide accumulation and prevented matrix degradation in the cultured cartilage discs. The cartilage matrix preservation effect was mediated through downregulation of all three isoforms of NOS, i.e., the neuronal NOS, endothelial NOS and inducible NOS and upregulation of TGF beta receptor in the chondrocytes. Our findings indicate that inhibition of NOS activity preserves cartilage matrix in vitro.     

Reduced NO accumulation in arthrotic cartilage by exposure to methylene blue

Nitric oxide (NO) appears to be a final common inflammation mediator of cartilage degradation. Halting the pathological formation of excessive NO, by suppressing the inducible NO synthase (iNOS) activity, may help to preserve cartilage integrity. We used fresh ex-vivo human articular cartilage explants from normal and arthrotic joints for assessment of NO levels, as determined by its nitrite degradation products and nitric oxide synthase expression. We measured matrix proteoglycan content, assessed by image analysis of alcian blue staining, and proteoglycan synthesis, assessed by sulfate incorporation into proteoglycans. The effect of methylene blue, a nitric oxide synthase inhibitor, on matrix preservation was evaluated. Cartilage discs in vitro, derived from normal appearing joints, secreted about one tenth as much NO compared to discs derived from arthrotic cartilage. Cartilage explants showed a time-dependent reduction in the amount of aggrecan within the cartilaginous matrix. Addition of methylene blue to the growth medium lowered nitric oxide accumulation and prevented matrix degradation in the cultured cartilage discs. The cartilage matrix preservation effect was mediated through downregulation of all three isoforms of NOS, i.e., the neuronal NOS, endothelial NOS and inducible NOS and upregulation of TGF beta receptor in the chondrocytes. Our findings indicate that inhibition of NOS activity preserves cartilage matrix in vitro.     

去细胞猪主动脉瓣叶的获取和内皮细胞的种植

目的　探讨猪主动脉瓣叶去细胞后作为组织工程心脏瓣膜支架的可行性。　方法　经胰酶 - EDTA、表面活性剂和核酸酶处理 ,去除猪主动脉瓣叶的细胞成分 ,测定瓣叶去细胞前、后的生物力学特性 ,并在其表面种植新生牛主动脉内皮细胞 (BAECs) ;分别行大鼠皮下包埋实验。　结果　猪主动脉瓣叶中的细胞成分能完全去除 ,获得完整无细胞的纤维网状支架 ,断裂强度和断裂伸长率无明显变化 ;种植的 BAECs在去细胞瓣叶表面可形成一层连续的细胞层 ,其分泌前列环素 (PGI2 )的能力同直接种植在 2 4孔板中的比较 ,差异无统计学意义 (P>0 .0 5 )。　结论　猪主动脉瓣去细胞后获得的纤维支架可以用来构建组织工程瓣膜 ,适宜于血管内皮细胞的生长。