双相支架负载软骨细胞修复兔关节软骨缺损

目的研究自固化磷酸钙/纤维蛋白凝胶(CPC/FG)双相支架负载软骨细胞修复兔关节软骨缺损的可行性和有效性.方法将分离培养的第3代软骨细胞包埋在CPC/FG双相支架的FG中,体外培养1周后,将软骨细胞-支架复合体移植修复兔膝关节股骨髁的软骨缺损(φ4 mm,深3.5 mm,达软骨下骨质).然后对软骨缺损的修复情况进行大体、光镜和电镜观察.同时对移植后第12周的修复软骨进行胶原含量测定,并与正常的关节软骨细胞胶原含量进行比较.结果移植的软骨细胞能在双相支架上良好地生长,软骨缺损以透明软骨的形式被修复,而对照组为纤维组织修复.多孔自固化磷酸钙在软骨修复过程中能起软骨下骨的临时替代作用.胶原含量测定显示:移植术后12周的修复软骨胶原含量为(43.25±0.85)%;正常的关节软骨胶原含量为(55.69±0.76)%,两者差异有显著性(P<0.01).结论 CPC/FG双相支架负载软骨细胞能以透明软骨的形式修复兔关节软骨缺损.新环境中移植的软骨细胞生长的不适应和FG降解过快,可能是导致新生修复软骨与自身正常关节软骨胶原含量有差异的原因.     

软骨细胞—支架复合的修复兔耳廓软骨缺损

目的 在有免疫力的动物兔体内探索组织工程化软骨对软骨缺损的修复能力。方法 软骨细胞种于经不同物质修饰的聚羟基乙酸支架体外培养后修复兔耳廓软骨缺损，与对照组比较，并从大体及组织学进行评价。结果 软骨细胞种于经不同物质修饰的聚羟基乙酸支架体外培养后回植到兔耳廓软骨缺损部位，软骨缺损得到修复，但人骨交上为纤维组织。注射软骨细胞悬液、以聚羟基乙酸支架修复及空白对照组软骨缺损均未修复。结论 软骨细胞种于经不     

柱状分层的胶原-羟基磷灰石复合支架负载软骨细胞体外培养

目的 观察具有柱状分层结构的胶原-羟基磷灰石（hydroxyapatite，HA）复合支架对软骨细胞的吸附作用及对软骨细胞生物学性状的影响，评价其作为软骨组织工程支架的可行性及价值。方法 以胶原复合HA逐层制备胶原-HA复合支架，体外培养新西兰大白兔关节软骨细胞并扩增，吸附于多孔胶原-HA复合支架材料上进行三维立体培养3周，通过倒置相差显微镜、组织学、扫描电镜及免疫组织化学检测支架对软骨细胞的表型、增殖及功能的影响。结果 胶原-HA复合支架为柱状，支架表层为胶原。平均孔径为147μm，孔隙率89％；中层为多孔胶原-HA复合；底层为HA，平均孔径为85μm，孔隙率85％。胶原-HA复合支架亲水性好，软骨细胞吸附于支架表面24h后，增殖并逐渐顺孔隙迁徙至支架内部，在孔壁贴附良好，表型维持稳定，分泌细胞外基质，Ⅱ型胶原免疫组织化学染色阳性。结论 具有柱状分层结构的胶原-HA复合支架其细胞相容性良好，较胶原纤维具有更强的力学性能，有望成为一种比较理想的软骨组织工程支架材料。     

聚乙烯醇/羟基磷灰石复合水凝胶移植修复兔膝关节软骨缺损

目的：探讨聚乙烯醇／羟基磷灰石复合水凝胶移植修复兔膝关节软骨缺损的疗效及了解其组织相容性方法：采用溶胶凝胶法原位复合制备PVA／HA水凝胶，通过体外力学性能测试，植入到兔膝关节实验性关节软骨缺损中，对照组的缺损不作任何处理，术后4、8、12周取材作大体观察及组织学检查。结果：术后4周，实验组的缺损由PVMHA充填，材料与软骨下骨连接无间隙，术后12周，植入材料与软骨交界面有大量的软骨细胞增殖，未见软骨退变，植入材料与软骨下骨连接紧密，有骨样组织长入；对照组缺损主要由纤维肉芽组织修复。结沦：PVA／HA复合水凝胶可以作为良好的人工关节软骨替代材料，组织相容性良好。     

胶原/羟基磷灰石骨软骨一体化支架的生物学特性研究

[目的]对体外条件下胶原/羟基磷灰石一体化复合材料的生物学特性进行评价,探讨其作为骨软骨组织工程支架的可行性。[方法]以胶原和羟基磷灰石为原料,研制出由胶原逐层过渡到羟基磷灰石为主的一体化支架材料,其软骨层主要成分为胶原,骨层主要成分为羟基磷灰石。通过扫描电镜观察材料内部结构及孔径大小,液体位移法测定材料的孔隙率。将材料与兔骨髓基质细胞复合培养,MTT法测定细胞的生长曲线,扫描电镜观察细胞在材料上的生长粘附情况。[结果]胶原/羟基磷灰石一体化复合支架具有疏松多孔结构,其中软骨层孔径大小(90±15)μm,骨层孔径大小(120±20)μm,孔隙率(75±5.0)%。细胞在材料上生长良好。[结论]胶原/羟基磷灰石一体化复合材料具有适宜的孔隙结构和良好的生物相容性,可用作骨软骨组织工程支架材料。     

聚乙烯醇/羟基磷灰石复合水凝胶移植修复兔膝关节软骨缺损

目的:探讨聚乙烯醇/羟基磷灰石复合水凝胶移植修复兔膝关节软骨缺损的疗效及了解其组织相容性.方法:采用溶胶凝胶法原位复合制备PVA/HA水凝胶,通过体外力学性能测试,植入到兔膝关节实验性关节软骨缺损中,对照组的缺损不作任何处理,术后4、8、12周取材作大体观察及组织学检查.结果:术后4周,实验组的缺损由PVA/HA充填,材料与软骨下骨连接无间隙,术后12周,植入材料与软骨交界面有大量的软骨细胞增殖,未见软骨退变,植入材料与软骨下骨连接紧密,有骨样组织长入;对照组缺损主要由纤维肉芽组织修复.结论:PVA/HA复合水凝胶可以作为良好的人工关节软骨替代材料,组织相容性良好.     

壳聚糖/羟基磷灰石支架修复骨软骨缺损的实验研究

[目的] 探讨双层壳聚糖(chitosan CS)/羟基磷灰石复合支架(hydroxyapatite HA)修复兔骨软骨缺损的可行性.[方法] 采用冻干法和烧结法制作双层壳聚糖(CS)/羟基磷灰石(HA)复合支架,以骨髓间充质干细胞为种子细胞,运用纤维蛋白胶种植技术,以双层壳聚糖(CS)/羟基磷灰石(HA)复合支架为载体,修复骨软骨缺损,实验分3组,A组:BMSc 支架,B组:单纯支架,C组:未处理.将修复材料植入骨软骨缺损模型,分别于6、12周取材,进行大体观察,组织学检测,改良Wakitani法评分,经统计学处理,比较各组修复效果差异(P＜0.05).[结果] (1)CS/HA支架CS层孔隙率为76%± 5.01%,孔径为200～400 μm,平均为300 μm左右,孔相通性好,HA层孔隙率为72%± 4.23%,孔径为200～500 μm,平均为350 μm左右,孔相通性好,结合部结合好;(2)P2骨髓间充质干细胞较纯,扫描电镜观察MSCs附着在复合支架上.大体观察和组织学检测显示, A组基本修复软骨缺损,骨缺损有骨小梁长入.B、C组骨软骨缺损修复不良,组织学检测以纤维性组织或无新生组织形成,软骨及骨缺损均明显存在,改良Wakitani评分显示A组在6周、12周2个时间点的各项评分结果,均优于B、C组,且差异有统计学意义(P＜0.05).[结论] 双层壳聚糖(CS)/羟基磷灰石(HA)复合支架可作为骨软骨组织工程支架,结合BMSc可修复软骨与骨的缺损,重建关节的解剖结构和功能.     

丝素蛋白/羟基磷灰石组织工程化骨修复兔桡骨节段性骨缺损

目的 探讨丝素蛋白/羟基磷灰石(SF/HA)组织工程化骨的成骨作用,以期为临床治疗骨缺损提供新的人工骨材料.方法 将SF/HA与成骨诱导的兔骨髓基质干细胞(BMSCs)复合,构建组织工程化骨.取54只兔于左侧桡骨中上段制备15 mm节段性骨缺损.实验分3组(A、B组各24只,C组6只):A组:植入SF/HA组织工程化骨,B组:单纯植入SF/HA;C组:骨缺损区不植入任何材料.于术后4、8、12及16周摄X线片,并于16周行螺旋CT扫描重建,观察骨缺损修复及骨塑形情况,参照Lane-Sandhu X线评分标准对各组骨缺损的骨修复程度评分.骨痂标本行 HE染色组织学观察,按照Lane-Sandhu组织学评分法比较12周和16周时各组的骨修复情况. 结果 术后16周,X线片示A组髓腔通畅,新骨塑形好,骨皮质连续;B组缺损区有缩小,两断端不连接;C组缺损区无明显骨痂生长.16周时螺旋CT扫描重建显示:A组骨塑形明显,骨缺损完全修复;B组有部分皮质骨形成,缺损区不能完全修复;C组骨缺损基本无修复.每组术后4、8、12、16周不同时间点的放射学评分差异均有统计学意义(P＜0.05).术后12、16周时3组间Lane-Sandhu组织学评分差异均有统计学意义(P＜0.05). 结论 SF/HA组织工程化骨具有良好的节段性骨缺损修复能力,但SF/HA本身缺乏骨诱导作用,单独修复节段性骨缺损作用有限.     

自体骨髓间充质干细胞复合壳聚糖/羟基磷灰石支架修复兔膝骨软骨缺损

目的 探讨骨髓间充质干细胞(bone mesenehymal stem cells,BMSCs)复合壳聚糖(chitosan,CS)/羟基磷灰石(hydmxyapatite,HA)支架修复兔膝关节局部骨软骨缺损.方法 选健康日本大耳白兔36只,2～3月龄,体重1.7～2.0 kg,每只抽取自体骨髓4～6ml,体外分离培养BMSCs后以2×107/ml密度植于CS/HA支架上体外培养10 h,制成BMSCs-CS/HA支架复合物.将36只实验动物手术制成右膝股骨外侧髁负重区骨缺损模型后,随机分成A、B、C 3组,每组12只.A组植入BMSCs-CS/HA复合物,B组植入单纯CS/HA支架;C组不作任何植入,为空白对照组.分别于术后6周、12周各处死6只动物,取材后进行大体、组织学观察6根据改良Wakitani评分标准进行评分,评估软骨组织的修复情况,并行成组设计方差分析.结果 A组术后6周即可重建关节软骨缺损;修复软骨在观察期内逐渐变厚,软骨下骨有少量骨修复;术后12周透明软骨样修复,表面光整,与周围软骨色泽相近,软骨下骨有部分修复.而B组和C组12周时缺损区仍为纤维软骨样纤维组织修复,色泽浅黄.术后6、12周各组组织学半定量评分显示:股骨髁负重区修复A组评分明显优于B、C组(F=27.26,P＜0.05).结论 自体BMSCs复合CS/HA支架在体内环境下可形成透明软骨修复兔膝关节负重区骨软骨缺损.     

羟基磷灰石修复骨缺损17例

我科从 1993年～ 2 0 0 0年 ,与四川大学生物工程研究所合作 ,应用羟基磷灰石 (hydroxyapatite,HA)修复骨缺损 17例 ,临床效果满意 ,现报告如下。1　临床资料1.1　一般资料本组 17例 ,男 8例 ,女 9例。年龄 2 6～ 44岁 ,平均 35岁。骨肿瘤 12例 ,其中骨巨细胞瘤 9例 ,骨纤维瘤 3例 ;肿瘤位于股骨粗隆间 5例 ,股骨下段 2例 ,股骨头 1例 ,胫骨上段2例 ,肱骨上段 2例。修复骨肿瘤切除术后骨缺损最大10 cm× 4cm ,最小 4cm× 3 cm。颅脑术后颅骨缺损 5例 ,颅骨缺损最大 6 cm× 4cm,最小 4cm× 4cm。1.2　 HA规格本组应用的 HA 规格有两种 :一…     

A novel exogenous concentration-gradient collagen scaffold augments full-thickness articular cartilage repair

OBJECTIVES: A collagen scaffold has been long used in order to enhance the regeneration of articular cartilage. In the present study, we investigate the effectiveness of a concentration-gradient (CG) collagen that is designed to recruit efficiently the mesenchymal stem cells (MSCs) to the central region of the full-thickness cartilage defects via haptotaxis. METHODS: The present study used Cellmatrix((R)) (0.3% type I collagen; Nitta gelatin, Osaka, Japan) as the collagen material. We prepared 33%CG collagen gel and 50%CG collagen gel. No gradient collagen gel served as negative control. Full-thickness cartilage defects were created at the patella groove of the rabbit knee, to which the three different collagen gels were transplanted. Bromodeoxyuridine (BrdU) positive, proliferating cells were enumerated and localized, whereas the histological grading score for cartilage regeneration was counted. The expression of type I and type II collagens was evaluated by immunohistochemistry. We also confirmed that the MSCs migrate toward the collagen substrate of higher concentration in a stringently in vitro haptotactic manner. RESULTS: Enumeration of the BrdU-positive cells demonstrated that 33%CG collagen gel recruited a significantly larger number of proliferating cells to the central region of the cartilage defect. The histological grading score for the regenerated cartilage treated with 33%CG collagen gel was superior to the other groups. CONCLUSIONS: CG collagen scaffold recruits effectively the MSCs to the center of full-thickness cartilage defect and enhances regeneration of the full-thickness cartilage defect.     

胶原凝胶包埋软骨细胞复合聚磷酸钙纤维/左旋聚乳酸支架异体移植修复兔关节软骨缺损

目的探讨胶原凝胶包埋软骨细胞复合聚磷酸钙纤维／左旋聚乳酸(CPPf／PLLA)支架异体移植修复兔关节软骨缺损的有效性和可行性。方法将胶原凝胶包埋的软骨细胞接种CPPf/PLLA支架构建的复合物体外培养3周，行倒置显微镜和扫描电镜观察，并将复合物异体移植入兔关节软骨缺损，术后4、8、12周取材，从大体、组织学和Ⅱ型胶原免疫组织化学对再生软骨组织进行评价。结果复合物体外培养3周，细胞被大量基质包裹，在支架内分布均匀；新形成的组织为透明软骨样组织、表面光滑且与周围组织整合良好、基质内有Ⅱ型胶原分布。结论胶原凝胶包埋软骨细胞接种CPPf／PLLA支架的方法能提高细胞一支架复合物构建质量，胶原凝胶复合CPPf／PLLA支架可作为软骨细胞载体修复关节软骨缺损。     

Repair of osteochondral defects in articular weightbearing areas in the rabbit's knee

Summary Repair of osteochondral defects in articular weightbearing areas presents its own particular problems because of the low potential of hyaline cartilage for regeneration.Our first group of experiments on the knee of the rabbit confirms that the new regenerated cartilage comes from bone marrow which degenerates before developing into true hyaline cartilage. The second group of experiments shows that autologous grafts from the non-weightbearing articular area suitable for the repair of defects in weightbearing areas. In an third group, autologous mensical fibro-cartilage was used as a graft for the repair of osteochondral defects.

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Résumé La réparation des pertes de substance ostéo-cartilagineuses au niveau des zones d'appui pose des problèmes particuliers en raison du faible pouvoir de régénération du cartilage hyalin.Notre premier groupe d'expériences sur le genou du lapin montre que le cartilage régénéré provient de la moelle osseuse qui dégénère avant de se transformer en véritable cartilage hyalin. Le deuxième groupe d'expériences démontre que des autogreffes, prélevées sur la partie non portante du cartilage articulaire, sont susceptibles de combler les pertes de substance en zone d'appui. Dans un troisième groupe on a utilisé comme greffe le fibro-cartilage ménsial autologue.

     

组织工程骨-软骨复合体修复犬股骨头负重区大面积骨软骨缺损的实验研究

目的 探讨采用犬股骨头负重区骨和天然软骨制备的骨-软骨双层支架复合成软骨诱导的骨髓间质干细胞(bone marrow mesenchymal stem cells,BMSCs)修复犬股骨头负重区大面积骨软骨缺损的疗效.方法 利用软骨细胞外基质作为软骨支架部分,犬股骨头负重区骨柱经脱细胞处理后作为骨支架部分,采用相分离技术制备骨-软骨双层支架.将成软骨诱导的BMSCs种植到双层支架上体外构建组织工程骨-软骨复合体,并以此修复犬股骨头负重区大面积骨软骨缺损(直径11 mm,高10 mm),第3、6个月时分别取材,行大体、X线片、组织学、Micro-CT和生物力学等检测.结果 X线片及大体观察:3个月时可见股骨头负重区出现轻度塌陷;6个月时出现严重塌陷,呈重度骨关节炎改变.组织学观察:第3、6个月时软骨缺损部分均以纤维组织或纤维软骨充填,周围软骨退变,骨缺损部分不同程度塌陷,与宿主骨质结合紧密.第3、6个月时骨软骨缺损的骨体积分数均低于正常股骨头,差异有统计学意义.6个月时重建软骨下骨的刚度明显低于正常股骨头,差异有统计学意义.结论 结构性骨-软骨双层支架复合成软骨诱导的BMSCs修复犬股骨头负重区骨软骨缺损效果不佳,易导致股骨头塌陷.

Abstract：

Objective To investigate the effects of the novel scaffold on repairing large,high-loadbearing osteochondral defects of femoral head in a canine model.Methods The biphasic scaffolds were fabricated using cartilage extracellular matrix (ECM)-derived scaffold (cartilage layer) and acellular bone matrix (bone layer) by phase separation technique.Articular high-load-bearing osteochondral defects with a diameter of 11-mm and the depth of 10-mm were created in femoral heads.The defects were treated with constructs of a biphasic scaffold seeded with chondrogenically induced bone marrow-derived mesenehymal stem cells (BMSCs).The outcomes were evaluated for gross morphology,histological,biomechanical and micro-CT analysis at the third and sixth month after implantation.Results The gross and X-ray results showed femoral head slightly collapsed at the third month and severely collapse at the sixth month.Histological analysis showed cartilage defects were repaired with fibrous tissue or fibrocartilage with severe osteoarthritis and the varied degrees of the collapse of femoral heads were presented.Micro-CT showed that the values of bone volume fraction in defect area were always lower than those of the normal area in the femoral heads.Biomechanical analysis showed rigidity of the subchondral bone in defect area was significantly lower than that in normal area in the femoral heads at the sixth month.Conclusion The ECM-derived,integrated biphasic scaffold seeded with chondrogenically induced BMSCs could not successfully repair the large high-load-bearing osteochondral defects of the femoral head.     

Transplantation of tissue-engineered osteochondral plug using cultured chondrocytes and interconnected porous calcium hydroxyapatite ceramic cylindrical plugs to treat osteochondral defects in a rabbit model

Abstract:  The purpose of this study was to evaluate the macroscopic and histological results of transplanting a tissue-engineered composite plug made of tissue-engineered cartilage and interconnected porous calcium hydroxyapatite ceramics (IP-CHA) with a very high porosity of 94.9% to treat osteochondral defects. Twelve 12-week-old male Japanese white rabbits were used. Fresh articular cartilage slices were taken, and isolated chondrocytes (2 × 10⁶ cells) were embedded in atelocollagen gel. They were seeded on the top of IP-CHA plugs and cultured for 2 weeks. These tissue-engineered composite plugs were transplanted into the osteochondral defects in the patellar grooves (the experimental group). In the control group, the defects were treated with composite plugs without chondroytes. Twelve weeks after transplantation in the experimental group, the defects were repaired with cartilage-like tissue with good subchondral bone formation histologically. Histological scores in the experimental group were significantly better than those in the control group. This study clearly showed the defects that had been treated with tissue-engineered composite plugs.     

Cell-based treatment of osteochondral defects in the rabbit knee with natural and synthetic matrices: cellular seeding determines the outcome

Introduction: Matrix-associated transplantation of cartilage constructs is an appealing method in cartilage repair. Three different matrices seeded with allogenic chondrocytes were compared in an osteochondral defect model in the rabbit. An investigation was conducted to identify the best matrix for cell-based treatment of osteochondral defects in the rabbit knee joint. Materials and methods: Osteochondral defects (diameter 3 mm) were created in the trochlea and the femoral condyles of 33 New Zealand White rabbits, which were then treated with bioartificial cartilage constructs. The cartilage constructs were created in vitro using three different resorbable carrier materials (two fleece matrices: one of PLLA, and one composite of polydioxanon/polyglactin, as well as one consisting of lyophilized dura) cultured with isolated allogenic chondrocytes. The defects were evaluated macroscopically, by histological and immunhistological techniques, and by scanning electron microscopy after 6 weeks, 6 months, and 12 months. The chondrocyte-seeded constructs were compared to defects treated with carrier material alone as well as to untreated control defects. Results: There was a significant improvement in defect repair quality in the transport materials, which were cultured with chondrocytes prior to implantation (P<0.0005). No significant differences were observed between the three carrier matrices, and no significant differences were seen between the unseeded matrices and the untreated control defects. Conclusion: There is no difference in the outcome between the three tested matrices in the treatment of osteochondral defects in the rabbit knee. The results of this in vitro experiment are promising and with refinement may lead to useful clinical therapies.     

动物源性骨软骨支架修复兔膝关节复合缺损

目的探讨软骨细胞-动物源性骨软骨支架复合体修复兔膝关节骨软骨复合缺损的可行性和影响因素。方法将改良贴壁离心法获取的骨髓间充质干细胞（bone marrowm esenchymal stem cells,BMSCs）/诱导分化的软骨细胞共培养后与经深低温冷冻、脱脂、脱钙、真空冷冻干燥和辐照消毒的动物源性骨软骨支架复合,构建共培养细胞＋软骨-骨一体化复合支架。27只新西兰大白兔随机分为实验组（A组）、对照组（B组）和空白组（C组）,每组9只。于兔股骨髁间窝处钻一深6mm的骨软骨复合缺损,A组植入共培养细胞＋骨软骨复合支架,B组植入骨软骨复合支架,C组不植入任何支架材料和细胞,分别于术后4周、8周和12周取材,行大体观察、苏木精—伊红染色和甲苯胺蓝染色,并对各标本的软骨切片进行组织学评分。结果随着时间的延长,A组大体观察见复合缺损区已完全修复,局部无凹陷,新生组织和周围组织融合;B组新生组织仍不能完全填充缺损;C组缺损区仍明显。苏木精—伊红染色和甲苯胺蓝染色见A组软骨缺损区由新生的透明软骨样组织修复,细胞呈柱状排列,极性好,软骨陷窝明显,骨缺损区由骨样组织修复,新生软骨和软骨下骨以及宿主骨界面耦合良好;B组新生软骨细胞无软骨陷窝,排列混乱,各界面藕合欠理想;C组可见陈旧性肉芽组织生长并突出于缺损区表面。甲苯胺蓝染色阳性率和组织学评分结果表明,A组与B、C两组之间的差异具有统计学意义（P〈0.05）。结论 BMSCs/诱导分化的软骨细胞共培养细胞复合动物源性骨软骨支架对兔膝关节软骨和软骨下骨的复合缺损具有修复作用。     

Pulsing direct current-induced repair of articular cartilage in rabbit osteochondral defects

Osteochondral defects in the distal femoral condyles of rabbits exposed to a pulsing direct current exhibits an enhanced quality of repair. The signal, with a peak value of 2 microA repeating at 100 Hz, imposed an electric field in the tissue of 20-60 mV/cm2. Maximum efficacy was seen with a shorter period of exposure (40 vs. 160 h) initiated 48 h after surgery for 4 h/day. Repair tissue originated primarily from metaplasia of subchondral elements although hyperplasia of pre-existing chondrocytes at the margins of the defect could be detected. Defects in treated joints contained Safranin O staining material that was histologically similar to a disorganized hyaline cartilage. Central areas of the defects in control animals contained Safranin O-negative material that generally extruded over the surface as a pannus. The edges of nontreated defects also had characteristics of cartilaginous healing, stressing the importance of using serial sectioning techniques in this model of cartilage repair.