背景:选择适合的生物支架与骨髓间充质干细胞作为新型的种子细胞复合能否构建出理想的组织工程化软骨?目的:观察将扩增的兔骨髓间充质干细胞接种于聚羟基乙酸构建人工软骨的可行性.设计:单一样本观察.单位:泸州医学院附属医院耳鼻咽喉头颈外科.材料:实验于2004-10/2005-10在泸州医学院完成.选用8只日本大耳兔,雌雄不拘,清洁级,二三个月龄,体质量1.5～2.0 kg,常温常湿环境饲养.聚羟基乙酸由美国Albany公司生产.方法:分离、获取、扩增实验兔骨髓间充质干细胞,将聚羟基乙酸剪成1 cm×1 cm×1 cm大小,并以多聚赖氨酸包被,通过将扩增的骨髓间充质干细胞接种于聚羟基乙酸上,按4 mL/cm3多点播散的方式均匀地接种在预湿的聚羟基乙酸支架上,体外诱导培养3周,作为实验组.将不加入骨髓间充质干细胞的聚羟基乙酸支架作为对照组.将实验组和对照组的标本分别植入4只自体兔腹腔内,进行体内培养6～12周后,分别取出实验组与对照组的标本,对其进行大体观察.同时以100 g/L中性甲醛固定,行5 μm切片,采用苏木精-伊红染色观察标本的组织形态学特点,进行阿新蓝染色以观察酸性粘多糖形成,进行甲苯胺蓝染色以观察异染性基质的形成,进行免疫组织化学染色检测Ⅱ型胶原蛋白的表达.主要观察指标:两组支架植入实验兔体内后6,12周时标本大体观察结果、不同染色观察结果及Ⅱ型胶原蛋白的表达.结果:纳入8只实验兔均进入结果分析.①支架植入实验兔体内后6及12周标本大体观察结果:支架植入兔腹腔6周后,实验组标本均仍被腹腔大网膜包裹,剥去大网膜后,有3个标本成淡黄色,光滑,质地中,外形与植入前基本一致,1个标本呈灰黑色,质地很软,标本不能成形.12周后,实验组标本的外形仍与植入前基本一致,呈灰白色,光滑,但质地明显较硬.而第6,12周的对照组的标本大部分均被吸收,以第12周的标本更明显,且均未见软骨样组织形成.②支架植入实验兔体内后6及12周不同染色观察结果及Ⅱ型胶原蛋白的表达:苏木精-伊红染色结果显示:6周时,有软骨陷窝样结构开始形成,聚羟基乙酸支架开始降解;12周时,复合物呈现软骨组织样的形态,有较明显的软骨陷窝样结构形成,细胞排列规则,成群存在于软骨陷窝样结构内,边缘的细胞小,中央的细胞大,聚羟基乙酸基本完全消失.阿新蓝染色结果:6周时,组织内有部分区域被染成淡蓝色;12周时组织的基质被广泛染成蓝紫色,说明有大量的酸性粘多糖形成.苯胺蓝染色结果:6周时,组织内可见蓝色异染性基质;12周时组织内呈现很强的蓝色异染.免疫组织化学染色结果:6周时,胞浆内可见棕黄色阳性颗粒,基质内有少量的Ⅱ型胶原蛋白表达;12周时,胞浆、基质内均有较强的阳性表达.Ⅱ型胶原蛋白的表达:6周时,胞浆内可见深棕黄色阳性颗粒,基质内有少量的Ⅱ型胶原mRNA表达;12周时,胞浆、基质内均有强的阳性表达.对照组标本基本上已被吸收,没有成形的组织工程化标本.结论:兔骨髓间充质干细胞在诱导剂作用下,经体外和体内培养后,可生成组织工程化类软骨.
揹景:選擇適閤的生物支架與骨髓間充質榦細胞作為新型的種子細胞複閤能否構建齣理想的組織工程化軟骨?目的:觀察將擴增的兔骨髓間充質榦細胞接種于聚羥基乙痠構建人工軟骨的可行性.設計:單一樣本觀察.單位:瀘州醫學院附屬醫院耳鼻嚥喉頭頸外科.材料:實驗于2004-10/2005-10在瀘州醫學院完成.選用8隻日本大耳兔,雌雄不拘,清潔級,二三箇月齡,體質量1.5～2.0 kg,常溫常濕環境飼養.聚羥基乙痠由美國Albany公司生產.方法:分離、穫取、擴增實驗兔骨髓間充質榦細胞,將聚羥基乙痠剪成1 cm×1 cm×1 cm大小,併以多聚賴氨痠包被,通過將擴增的骨髓間充質榦細胞接種于聚羥基乙痠上,按4 mL/cm3多點播散的方式均勻地接種在預濕的聚羥基乙痠支架上,體外誘導培養3週,作為實驗組.將不加入骨髓間充質榦細胞的聚羥基乙痠支架作為對照組.將實驗組和對照組的標本分彆植入4隻自體兔腹腔內,進行體內培養6～12週後,分彆取齣實驗組與對照組的標本,對其進行大體觀察.同時以100 g/L中性甲醛固定,行5 μm切片,採用囌木精-伊紅染色觀察標本的組織形態學特點,進行阿新藍染色以觀察痠性粘多糖形成,進行甲苯胺藍染色以觀察異染性基質的形成,進行免疫組織化學染色檢測Ⅱ型膠原蛋白的錶達.主要觀察指標:兩組支架植入實驗兔體內後6,12週時標本大體觀察結果、不同染色觀察結果及Ⅱ型膠原蛋白的錶達.結果:納入8隻實驗兔均進入結果分析.①支架植入實驗兔體內後6及12週標本大體觀察結果:支架植入兔腹腔6週後,實驗組標本均仍被腹腔大網膜包裹,剝去大網膜後,有3箇標本成淡黃色,光滑,質地中,外形與植入前基本一緻,1箇標本呈灰黑色,質地很軟,標本不能成形.12週後,實驗組標本的外形仍與植入前基本一緻,呈灰白色,光滑,但質地明顯較硬.而第6,12週的對照組的標本大部分均被吸收,以第12週的標本更明顯,且均未見軟骨樣組織形成.②支架植入實驗兔體內後6及12週不同染色觀察結果及Ⅱ型膠原蛋白的錶達:囌木精-伊紅染色結果顯示:6週時,有軟骨陷窩樣結構開始形成,聚羥基乙痠支架開始降解;12週時,複閤物呈現軟骨組織樣的形態,有較明顯的軟骨陷窩樣結構形成,細胞排列規則,成群存在于軟骨陷窩樣結構內,邊緣的細胞小,中央的細胞大,聚羥基乙痠基本完全消失.阿新藍染色結果:6週時,組織內有部分區域被染成淡藍色;12週時組織的基質被廣汎染成藍紫色,說明有大量的痠性粘多糖形成.苯胺藍染色結果:6週時,組織內可見藍色異染性基質;12週時組織內呈現很彊的藍色異染.免疫組織化學染色結果:6週時,胞漿內可見棕黃色暘性顆粒,基質內有少量的Ⅱ型膠原蛋白錶達;12週時,胞漿、基質內均有較彊的暘性錶達.Ⅱ型膠原蛋白的錶達:6週時,胞漿內可見深棕黃色暘性顆粒,基質內有少量的Ⅱ型膠原mRNA錶達;12週時,胞漿、基質內均有彊的暘性錶達.對照組標本基本上已被吸收,沒有成形的組織工程化標本.結論:兔骨髓間充質榦細胞在誘導劑作用下,經體外和體內培養後,可生成組織工程化類軟骨.
배경:선택괄합적생물지가여골수간충질간세포작위신형적충자세포복합능부구건출이상적조직공정화연골?목적:관찰장확증적토골수간충질간세포접충우취간기을산구건인공연골적가행성.설계:단일양본관찰.단위:로주의학원부속의원이비인후두경외과.재료:실험우2004-10/2005-10재로주의학원완성.선용8지일본대이토,자웅불구,청길급,이삼개월령,체질량1.5～2.0 kg,상온상습배경사양.취간기을산유미국Albany공사생산.방법:분리、획취、확증실험토골수간충질간세포,장취간기을산전성1 cm×1 cm×1 cm대소,병이다취뢰안산포피,통과장확증적골수간충질간세포접충우취간기을산상,안4 mL/cm3다점파산적방식균균지접충재예습적취간기을산지가상,체외유도배양3주,작위실험조.장불가입골수간충질간세포적취간기을산지가작위대조조.장실험조화대조조적표본분별식입4지자체토복강내,진행체내배양6～12주후,분별취출실험조여대조조적표본,대기진행대체관찰.동시이100 g/L중성갑철고정,행5 μm절편,채용소목정-이홍염색관찰표본적조직형태학특점,진행아신람염색이관찰산성점다당형성,진행갑분알람염색이관찰이염성기질적형성,진행면역조직화학염색검측Ⅱ형효원단백적표체.주요관찰지표:량조지가식입실험토체내후6,12주시표본대체관찰결과、불동염색관찰결과급Ⅱ형효원단백적표체.결과:납입8지실험토균진입결과분석.①지가식입실험토체내후6급12주표본대체관찰결과:지가식입토복강6주후,실험조표본균잉피복강대망막포과,박거대망막후,유3개표본성담황색,광활,질지중,외형여식입전기본일치,1개표본정회흑색,질지흔연,표본불능성형.12주후,실험조표본적외형잉여식입전기본일치,정회백색,광활,단질지명현교경.이제6,12주적대조조적표본대부분균피흡수,이제12주적표본경명현,차균미견연골양조직형성.②지가식입실험토체내후6급12주불동염색관찰결과급Ⅱ형효원단백적표체:소목정-이홍염색결과현시:6주시,유연골함와양결구개시형성,취간기을산지가개시강해;12주시,복합물정현연골조직양적형태,유교명현적연골함와양결구형성,세포배렬규칙,성군존재우연골함와양결구내,변연적세포소,중앙적세포대,취간기을산기본완전소실.아신람염색결과:6주시,조직내유부분구역피염성담람색;12주시조직적기질피엄범염성람자색,설명유대량적산성점다당형성.분알람염색결과:6주시,조직내가견람색이염성기질;12주시조직내정현흔강적람색이염.면역조직화학염색결과:6주시,포장내가견종황색양성과립,기질내유소량적Ⅱ형효원단백표체;12주시,포장、기질내균유교강적양성표체.Ⅱ형효원단백적표체:6주시,포장내가견심종황색양성과립,기질내유소량적Ⅱ형효원mRNA표체;12주시,포장、기질내균유강적양성표체.대조조표본기본상이피흡수,몰유성형적조직공정화표본.결론:토골수간충질간세포재유도제작용하,경체외화체내배양후,가생성조직공정화류연골.
BACKGROUND: Whether choosing a suitable biological scaffold compounding with mesenchymal stem cells (MSCs) can construct an ideal tissue-engineering cartilage or not should be researched further.OBJ ECTIVE: To investigate the feasibility of constructing artificial cartilage by using amplified rabbit MSCs which were inoculated on poly-glycolic acid (PGA).DESIGN: Single sample observation.SETTING: Department of Otolaryngology-Head & Neck Surgery, Affiliated Hospital of Sichuan Luzhou Medical College.MATERIALS: The experiment was carried out in the Luzhou Medical College from October 2004 to October 2005. A total of 8 Japanese large ear rabbits, of both genders, clean grade, aged from 2 to 3 months, weighing 1.5-2.0 kg, were fed in normal temperature and humidity. Poly-glycolic acid was provided by Albany Company, USA.METHODS: Rabbit MSCs were separated, obtained and amplified. In addition, poly-glycolic acid was sheared into pieces with the size of 1 cm × 1 cm × 1 cm and embedded with poly-L-lysine. Amplified MSCs were inoculated on the surface of poly-glycolic acid, and then, they were averagely grown on pre-wet scaffold of poly-glycolic acid according to 4 mL/cm3multi-points spreading style and cultured in vitro for 3 weeks. After the operations mentioned above, samples were regarded as the experimental group. Scaffolds of poly-glycolic acid without MSCs were considered as the control group.Samples in both experimental group and control group were transplanted into abdominal cavity of 4 rabbits, respectively,cultured in vivo for 6-12 weeks, taken out and observed generally. Meanwhile, the samples were fixed with 100 g/L neutrality formaldehyde, cut into sections with the thickness of 5 μm, stained with haematine-eosin (HE) and observed their histomorphological characteristics. Moreover, the samples were stained with alcian blue for observation of glycosaminoglycans formation, with toluidine blue for observation of metachromasia matrix formation, and with immunohistochemical staining for detection of type- Ⅱ collagen expression.MAIN OUTCOME MEASURES: Generally observational results of two kinds ofscaffolds at 6 and 12 weeks after transplanting into experimental rabbits, various staining results and expression of type- Ⅱ collagen.RESULTS: A total of 8 experimental rabbits were involved in the final analysis. ① Generally observational results of two kinds of scaffolds at 6 and 12 weeks after transplanting into experimental rabbits: At 6 weeks after transplanting scaffold into abdominal cavity of rabbits, samples in the experimental group were still coated with greater omentum of abdominal cavity. After clearing greater omentum, three samples were light yellow, smooth and moderate quality; meanwhile, their appearances were coincidence with those before transplantation. However, one sample was gray black and soft quality;meanwhile, it was not able to take shape. Twelve weeks later, appearances of samples in the experimental group were still coincidence with those before transplantation. They were gray white, smooth but hard quality. However, samples in the control group were mostly absorbed at 6 and 12 weeks after transplantation, especially, samples were remarkably absorbed at 12 weeks after transplantation. Any tissue like cartilage did not form in both two durations. ② Various staining results and expression of type- Ⅱ collagen at 6 and 12 weeks after transplanting scaffolds into experimental rabbits: Results of HE staining showed that, at 6 weeks after transplantation, structure of cartilage lacuna-like was started form, and PGA scaffold began to be degraded. In contrast, at 12 weeks postoperatively, some cartilage-like tissue were observed in compound, cartilage lacuna-like structure formed obviously; cells arrayed regularly and geminately existed In cartilage lacuna-like atructure; the smaller cell sizes observed at borders and the bigger ones observed at the center;PGA degraded completely. Results of alcian blue staining showed that, at 6 weeks after transplantation, a partial of regions in tissue were light blue; meanwhile, at 12 weeks after transplantation, matrixes in tissue were mostly blue. This suggested that a lot of glycosaminoglycans were formed. Results of toluidine blue staining suggested that, at 6 weeks after transplantation, blue metachromasia matrixes were observed in tissue; meanwhile, at 12 weeks after transplantation, blue metachromasia matrixes were stronger and stronger in tissue. Results of immunohistochemical staining indicated that, at 6 weeks after transplantation, buffy positive granules were observed in plasma and a few of type-Ⅱ collagen expressed in matrix; meanwhile, at 12 weeks after transplantation, powerfully positive expressions were observed in both plasma and matrix. Expression of type- Ⅱ collage showed that, at 6 weeks after transplantation, dark buffy positive granules were observed in plasma and a few of type- Ⅱ collagen mRNA expressed in matrix; meanwhile, at 12 weeks after transplantation, powerfully positive expressions were observed in both plasma and matrix. Samples in the control group were completely absorbed and any tissue-engineering samples did not form.CONCLUSION: Affecting by osteogenic inducer, rabbit MSCs can generate tissue-engineering cartilage after culture in vitro and in vivo.
