选择性细胞滞留技术快速构建组织工程骨的实验研究
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摘要
背景和目的:因现代战争及和平时期交通、建筑等行业的高能量损伤、骨肿瘤切除、骨结核与感染等所致的骨缺损日益常见。作为治疗骨缺损金标准的自体骨移植,由于存在取骨量有限和取骨区并发症,已不能满足骨缺损治疗的需要。利用骨髓干细胞与生物材料结合构建的组织工程骨修复骨缺损的动物实验已获得较为肯定的效果,但其缺点在于MSCs来源少、体外培养扩增周期长、细胞对支架材料的粘附率不足、技术条件高,从而影响了其在骨缺损治疗中的应用。自体红骨髓经皮注射在临床治疗骨不连和填充骨缺损等方面已取得一定的成功,但应用直接注入的方法,局部干细胞容易流失,影响了疗效。SCR是骨髓干细胞富集技术之一,是通过基质材料适当的网孔结构和良好的表面粘附性能,使骨髓流经时选择性滞留利于成骨的干细胞及促成骨因子等成分,所构建的TEB即刻回植修复骨缺损,可获得与自体骨移植相接近的效果。但由于受到知识产权保护和入关限制的影响,该技术及相关产品至今难以在我国临床应用。为此,本实验主要目的包括:⑴制备一种新型的骨髓干细胞富集材料,并观察其体外细胞相容性和体内组织相容性;⑵探讨SCR技术快速构建的TEB的异位成骨效果;⑶探讨新型富集材料应用SCR技术处理后对骨髓细胞和促成骨生长因子的富集效果;⑷观察SCR技术快速构建的TEB对成骨标志物和成骨特异性转录激活因子Cbfα1表达的影响,探讨富集材料促成骨的可能机制。
方法:⑴采用多聚左旋赖氨酸修饰人脱钙骨基质制备一种新型的骨髓干细胞富集材料,用液体置换法、扫描电镜、三维视频显微镜、拉曼光谱、红外光谱和HE染色测定其密度、孔隙率、孔径等表面特征;⑵将富集材料和不同浓度的材料浸提液与hMSCs共培养,应用形态学观察、MTT法、流式细胞仪、免疫组织化学、溶血实验等评价材料的体外细胞相容性;⑶裸鼠分别予腹腔注射PLL-DBM的生理盐水浸提液、背部皮下注射PLL-DBM浸提液培养后的DOC细胞悬液、背部植入PLL-DBM材料后,采用全身急性毒性试验、致癌试验和皮下植入试验等评价材料的体内组织相容性。⑷采用SCR技术使PLL-DBM富集骨髓后快速构建TEB,分别将SCR技术构建的TEB(SCR组)、体外培养的MSCs复合DBM常规构建的TEB(TEB组)、注射骨髓复合DBM(骨髓组)、单纯DBM(DBM组)植入裸鼠皮下,4、8、12、16w时取材,应用X线摄片、CT扫描、扫描电镜和HE染色等方法,观察植骨处影像密度、植骨区组织学改变和成骨效果;⑸应用SCR技术处理富集材料,观察富集前后PLL-DBM对骨髓有核细胞、血小板及纤维母细胞集落形成单位的富集效果,ELISA法检测富集前后骨髓上清中TGF-β1和PDGF表达;⑹采用SCR技术富集骨髓后快速构建TEB,分别将SCR技术构建的TEB(SCR组)、MSCs复合DBM常规构建的TEB(TEB组)、注射骨髓复合DBM(骨髓组)、单纯DBM(DBM组)植入裸鼠皮下,应用免疫组化和RT-PCR方法,检测4、8、12、16w时组织块内成骨标志物I型胶原、整合素α2β1、骨钙素表达,及成骨特异性转录激活因子Cbfα1表达。
结果:⑴PLL在材料内外表面形成均匀的乳白色涂层。PLL-DBM密度为(0.27±0.02)g/ml,孔隙率为(73±11)%,孔径为(412.73±160.29)μm。孔与孔之间有约100μm左右小孔贯通,孔隙内部有大量孔隙相互连通,并在天然孔隙内形成更小、孔径较均匀的网孔结构。⑵将富集材料及其不同浓度的材料浸提液分别与hMSCs共培养,形态学观察示细胞生长良好;细胞毒性实验显示hMSCs均良好增殖,毒性0-1级;流式细胞仪检测证实hMSCs、DOC均无DNA倍体异常的细胞,其CD29、CD105呈阳性,CD34、CD45呈阴性,材料浸提液不影响hMSCs表面分子的表达,且可通过促其向增殖期转化而促进hMSCs增殖;成骨诱导后DOC的生长与成骨活性无异常,可形成钙结节、表达I型胶原,ALP活性未受影响。溶血试验测得溶血率2.617%,符合生物材料要求小于5%的标准。⑶全身急性毒性试验显示裸鼠体重正常增长;致癌试验显示裸鼠正常存活,8月内未见肿块形成,心、肝、脑、肺、肾、脾组织学观察未见肿瘤细胞;皮下植入试验显示取材后皮下组织紧裹材料表面,色泽无改变,材料周围为一层薄纤维组织包绕,组织学观察发现皮下及皮肤组织细胞结构正常,无淋巴细胞/巨噬细胞浸润,未见细胞溶解和坏死迹象。⑷随着植入时间的延长,各复合材料植入组植入物的密度逐渐增高,在各时间点与单纯DBM组比较均有显著性差异;SCR组植入物密度与TEB组类似,显著高于骨髓组和DBM组。扫描电镜和组织学观察发现,随时间延长,组织块内PLL-DBM材料逐渐降解吸收,支架材料内逐渐形成小血管和新骨。8w时DBM开始降解,支架材料内有大量细胞和少数小血管形成,植入TEB内可见部分新生骨形成;12w时DBM支架部分降解,组织块内有较多的小血管长入,新骨亦明显增加;16w时植入区可见较为坚硬的骨性组织,并有血管长入。TEB组扫描电镜和组织学改变与SCR组类似,优于骨髓组和DBM组。⑸PLL-DBM的富集效果随PLL浓度增大而逐渐增加。0.1%PLL修饰的富集材料对人骨髓NCs浓度富集倍数为3.18±0.31倍、粘附率达53%±12%,对血小板富集倍数为3.88±0.68倍、粘附率达34%±10%,对骨髓干细胞即CFU-F的浓度富集倍数为5.25±1.40倍、粘附率达73%±13%、选择率达1.41±0.34。0.1%PLL修饰的富集材料与更高浓度PLL制备的PLL-DBM比较无明显差异,说明PLL-DBM对NCs的粘附具有一定的饱和性。富集后TEB中促成骨生长因子TGF-β1的浓度富集倍数为42.327±4.561,PDGF的浓度富集倍数为9.618±1.251,富集技术可以显著提高TEB中TGF-β1和PDGF含量。⑹随时间延长,植入区成骨标志物I型胶原、整合素α2β1和骨钙素的表达均逐渐增高,16w时达高峰,SCR组和TEB组表达相似,显著高于骨髓组和DBM组;免疫组化和RT-PCR法显示成骨早期SCR组Cbfα1 mRNA和蛋白表达显著增加,随着骨组织的逐渐成熟,Cbfα1的表达逐渐下降,16w时表达明显减弱。SCR组Cbfα1的表达与TEB组类似,优于骨髓组和DBM组。
结论:⑴制备的PLL-DBM具有自体骨三维空间结构和促进细胞粘附的PLL,具有良好的细胞相容性、组织相容性和可降解性,能促进hMSCs的粘附与增殖,对DOC的成骨活性无影响,是一种较理想的骨髓干细胞富集材料。⑵SCR技术快速制备的TEB植入体内后能够成骨,并可以达到与体外培养的hMSCs复合制备的TEB同样的成骨效果。⑶PLL-DBM能明显增高骨髓有核细胞、血小板和CFU-F的浓度富集倍数和粘附率,显著提高TEB中TGF-β1和PDGF含量;推测SCR技术快速构建TEB后局部高浓度的骨髓干细胞和促成骨生长因子TGF-β1与PDGF,可能是SCR技术构建的TEB高成骨活性的可能机制之一。⑷SCR技术快速构建的TEB,通过在早期促进成骨特异性转录激活因子Cbfα1表达、中晚期上调成骨标志物I型胶原、整合素α2β1和骨钙素表达而促进成骨,可能是SCR技术构建的TEB高成骨活性的可能分子机制之一。
Background and objective: Bone defects induced by various kinds of causes are commonly seen in clinic. As a gold standard, autogenous bone transplantation can not satisfy the requirement for bone defects therapy because of limited bone mass and operation complications in donor site. Tissue engineered bone (TEB) constructed by bone marrow stem cells (BMSCs) and biomaterial in treating bone defects in animal studies had obtained certain effects, but the shortcomings were: BMSCs in little resource, long cycle of culture and amplification in vitro, low adhesion rate of cell to scaffold and high specification, thus limited the application in bone defect therapy. Autologous red bone marrow percutaneous injection had obtained some success in treating clinical bone nonunion and bone defect, but the method of direct injection, had the defect of partly stem cell running off. Selective cell retention (SCR) is a technique of bone marrow stem cell enrichment, the mechanism is seletive retenting stem cells and facilitating ossify factors when bone marrow pass through the adequate mesh and favourable surface adhesiveness of scaffolds. The TEB constructed by SCR were replanted immediately for treating bone defect, could obtained the same effect as autogenous bone transplantation. But due to intellectual property rights conservation and enter restriction, enrichment technique and correlated manufactures harded to applicate in our country. Thus, the objectives of our experiment are:⑴Preparation of a new kind of bone marrow stem cells enrichment scaffold, to observe its cellular compatibility in vitro and its histocompatibility in vivo;⑵To explore the enriching effect of new kind of bone marrow stem cells enrichment scaffold constructed by SCR;⑶To study the ectopia osteogenesis effects of tissue engineered bone fast constructed by SCR;⑷To observe the effect of tissue engineered bone fast constructed by SCR on ossify marker and ossify specific activating transcript factor Cbfα1, and explore the mechanism of enrichment scaffold on osteogenesis.
Methods:⑴Demineralized bone matrix decorated with poly-L-lysine (PLL-DBM) as a new kind of bone marrow stem cells enrichment scaffold, its surface character such as density, porosity and pore size were determined by liquid displacement method, scanning electron microscope, three dimension video microscope, raman spectroscopy, infrared spectrum and HE staining.⑵New enrichment scaffold and different dense of scaffold leaching liquor were co-cultured with hMSCs, cellular compatibility in vitro were evaluated by morphologic observation, MTT assay, FCM, hemolysis experiment and immunohistochemistry.⑶Nude mouse were accepted intraperitoneal injection of saline leaching liquor extracted from PLL-DBM, back subcutaneous injection of DOC cell suspension after cultured with PLL-DBM leaching liquor, back implantation of PLL-DBM scaffold respectively, histocompatibility in vivo were evaluated by acute toxicity test, carcinogenesis test and subcutaneous implantation test.⑷TEB were constructed quickly after bone marrow enrichment by SCR. PLL-DBM combinated with bone marrow by SCR (SCR group), deminerilized bone matrix ( DBM ) combinated with MSCs (TEB group), DBM combinated with bone marrow (bone marrow group) and DBM (DBM group) were implantated into subcutaneous area of nude mouse, drow the materials at 4, 8, 12, 16 weeks. Image density, histological changes of bone graft area and ossify effects were determined by X ray, CT scanning, scanning electron microscope and HE staining.⑸New enrichment scaffold were handled by SCR, the enriching effects of PLL-DBM to bone marrow nucleated cells (NCs) ,platelets (PLTs) and fibroblast colony forming unit ( CFU-F) were observed before and after enrichment, contents of TGF-β1 and PDGF in bone marrow supernatant were detected by ELISA method before and after enrichment.⑹TEB were constructed fast after bone marrow enrichment by SCR, PLL-DBM combinated with SCR (SCR group), DBM combinated with MSCs (TEB group), DBM combinated with bone marrow (bone marrow group) and DBM (DBM group) were implantated into subcutaneous area of nude mouse, Collagen type I, integrinα2β1, osteocalcin and Cbfα1 mRNA and protein expression at 4、8、12、16 weeks were studied by immunohistochemistry and RT-PCR method.
Results:⑴Steady ivory converage were formed at exterior and interior surface by PLL. The density of PLL-DBM is (0.27±0.02) g/ml, the porosity is (73±11)%, the pore size is (412.73±160.29)μm. there are many micropore about 100μm size among pores, a great quantity of pore cross-connected each other in the interior pore, and formed smaller, more steady mesh structure in the natural pore.⑵New enrichment scaffold and different density scaffold leaching liquor were co-cultured with hMSCs, morphologic observation showed that MSCs growth well, cytotoxicity showed that MSCs proliferate well and toxity range from 0 to 1 grade. FCM showed that hMSCs and DOC had no abnormal DNA ploid, the marker of CD29 and CD105 are positive, CD34 and CD45 are negative, scaffold leaching liquor had no affect on hMSCs surface molecule, and could promote its proliferation by transform it into multiplication period. After ossify induction, DOC could form calcium nodule and express Collagen type I, and the ALP activity had not been affected. Hemolysis experiment showed that hemolysis rate was 2.617% which in accordance with the standard of biomaterial that the rate must less than 5%.⑶In acute toxicity test, nude mouse growth well; in carcinogenesis test, nude mouse survival better, tumour had not been seen after 8 months, tumour cells had not been in the organization such as heart, liver, brain, lung, kidney and spleen. In subcutaneous implantation test, histological observation showed that subcutaneous tissue tighted the scaffold surface with a thin fiber tissue around it, lymphocyte/macrophage infiltration, cytolysis and necrosis had not been seen.⑷The density of each composite implantation increase gradually followed the time, and had significant difference at each time point when compared with single DBM; the density in SCR group was similar to that in TEB group, but was superior to in bone marrow group and DBM group. Scanning electron microscope and HE staining showed that, PLL-DBM scaffold degradated gradually as time passed, bone trabecula and small vessels formed gradually into the scaffold. DBM started to degradation at 8 weeks, a lot of cells and a few small vessels formed, and part of newly formed bone generated into the scaffold. DBM partly degraded at 12 weeks, smaller vessels entered scaffold and new bone formation was found; rigid bone tissue were seen at 16 weeks, and medullary tissue and vessels were seen in some region of implantation site. Scanning electron microscope and HE staining in TEB group were similar to SCR group, but superior to bone marrow group and DBM group.⑸The enriching effects of PLL-DBM increased gradually with the PLL dnsity. The concentration enrichment multiple rate of enrichment scaffold decorated with 0.1% PLL of bone marrow NCs was 3.18±0.31, the adhesion rate was 53%±12%; The concentration enrichment multiple rate of platelet was 3.88±0.68, the adhesion rate was 34%±10%, The concentration enrichment multiple rate of CFU-F was 5.25±1.40, the adhesion rate was 73%±13%, the selective rate was 1.41±0.34. Compared with high density PLL, 0.1% PLL-DBM had the same effect, showed that PLL-DBM had saturability to NCs adhesion. concentration enrichment multiple rate of TGF-β1 was 42.327±4.561 and concentration enrichment multiple rate of PDGF was 9.618±1.251 in the enrichment scaffold, and enrichment technique could increase the content of TGF-β1 and PDGF significantly.⑹Expression of Collagen type I, integrinα2β1, osteocalcin mRNA and protein were increased gradually followed the time, and reached the peak at 16 weeks, the expression in SCR group was similar to that in TEB group, superior to bone marrow group and DBM group. Immunohistochemistry and RT-PCR showed that, Cbfα1 mRNA and protein expression increased significantly at ossify earlier period, but decreased gradually followed the bone maturation. The expression of Cbfα1in SCR group was similar to in TEB group, superior to in bone marrow group and DBM group.
Conclusion:⑴PLL-DBM possess three dimensional space as autogenous bone and PLL for cell adhesion, and have favourable cellular compatibility, histocompatibility and biodegradability, can promote the adhesion and proliferation of hMSCs, but not affect the ossify activity of DOC, is an ideal enrichment scaffold for bone marrow stem cells.⑵TEB constructed fast by SCR have favourable bone formation, and the effect of ossify is similar to MSCs combined with DBM.⑶Enrichment scaffold can increase the concentration enrichment multiple rate and the adhesion rate of bone marrow NCs, platelet and CFU-F significantly, and increase the contents of TGF-β1 and PDGF in TEB, it is said that the local high concentration of bone marrow stem cells and facilitate ossify growth factor TGF-β1 and PDGF, maybe the one of the mechanism of high ossify activity of TEB constructed fast by SCR.⑷Increased Cbfα1 expression in earlier period of osteogenesis and increased expression of Collagen type I, integrinα2β1 and osteocalcin in middle-advanced stage maybe the one of the molecule mechanism of high ossify activity of TEB constructed fast by SCR.
方法:⑴采用多聚左旋赖氨酸修饰人脱钙骨基质制备一种新型的骨髓干细胞富集材料,用液体置换法、扫描电镜、三维视频显微镜、拉曼光谱、红外光谱和HE染色测定其密度、孔隙率、孔径等表面特征;⑵将富集材料和不同浓度的材料浸提液与hMSCs共培养,应用形态学观察、MTT法、流式细胞仪、免疫组织化学、溶血实验等评价材料的体外细胞相容性;⑶裸鼠分别予腹腔注射PLL-DBM的生理盐水浸提液、背部皮下注射PLL-DBM浸提液培养后的DOC细胞悬液、背部植入PLL-DBM材料后,采用全身急性毒性试验、致癌试验和皮下植入试验等评价材料的体内组织相容性。⑷采用SCR技术使PLL-DBM富集骨髓后快速构建TEB,分别将SCR技术构建的TEB(SCR组)、体外培养的MSCs复合DBM常规构建的TEB(TEB组)、注射骨髓复合DBM(骨髓组)、单纯DBM(DBM组)植入裸鼠皮下,4、8、12、16w时取材,应用X线摄片、CT扫描、扫描电镜和HE染色等方法,观察植骨处影像密度、植骨区组织学改变和成骨效果;⑸应用SCR技术处理富集材料,观察富集前后PLL-DBM对骨髓有核细胞、血小板及纤维母细胞集落形成单位的富集效果,ELISA法检测富集前后骨髓上清中TGF-β1和PDGF表达;⑹采用SCR技术富集骨髓后快速构建TEB,分别将SCR技术构建的TEB(SCR组)、MSCs复合DBM常规构建的TEB(TEB组)、注射骨髓复合DBM(骨髓组)、单纯DBM(DBM组)植入裸鼠皮下,应用免疫组化和RT-PCR方法,检测4、8、12、16w时组织块内成骨标志物I型胶原、整合素α2β1、骨钙素表达,及成骨特异性转录激活因子Cbfα1表达。
结果:⑴PLL在材料内外表面形成均匀的乳白色涂层。PLL-DBM密度为(0.27±0.02)g/ml,孔隙率为(73±11)%,孔径为(412.73±160.29)μm。孔与孔之间有约100μm左右小孔贯通,孔隙内部有大量孔隙相互连通,并在天然孔隙内形成更小、孔径较均匀的网孔结构。⑵将富集材料及其不同浓度的材料浸提液分别与hMSCs共培养,形态学观察示细胞生长良好;细胞毒性实验显示hMSCs均良好增殖,毒性0-1级;流式细胞仪检测证实hMSCs、DOC均无DNA倍体异常的细胞,其CD29、CD105呈阳性,CD34、CD45呈阴性,材料浸提液不影响hMSCs表面分子的表达,且可通过促其向增殖期转化而促进hMSCs增殖;成骨诱导后DOC的生长与成骨活性无异常,可形成钙结节、表达I型胶原,ALP活性未受影响。溶血试验测得溶血率2.617%,符合生物材料要求小于5%的标准。⑶全身急性毒性试验显示裸鼠体重正常增长;致癌试验显示裸鼠正常存活,8月内未见肿块形成,心、肝、脑、肺、肾、脾组织学观察未见肿瘤细胞;皮下植入试验显示取材后皮下组织紧裹材料表面,色泽无改变,材料周围为一层薄纤维组织包绕,组织学观察发现皮下及皮肤组织细胞结构正常,无淋巴细胞/巨噬细胞浸润,未见细胞溶解和坏死迹象。⑷随着植入时间的延长,各复合材料植入组植入物的密度逐渐增高,在各时间点与单纯DBM组比较均有显著性差异;SCR组植入物密度与TEB组类似,显著高于骨髓组和DBM组。扫描电镜和组织学观察发现,随时间延长,组织块内PLL-DBM材料逐渐降解吸收,支架材料内逐渐形成小血管和新骨。8w时DBM开始降解,支架材料内有大量细胞和少数小血管形成,植入TEB内可见部分新生骨形成;12w时DBM支架部分降解,组织块内有较多的小血管长入,新骨亦明显增加;16w时植入区可见较为坚硬的骨性组织,并有血管长入。TEB组扫描电镜和组织学改变与SCR组类似,优于骨髓组和DBM组。⑸PLL-DBM的富集效果随PLL浓度增大而逐渐增加。0.1%PLL修饰的富集材料对人骨髓NCs浓度富集倍数为3.18±0.31倍、粘附率达53%±12%,对血小板富集倍数为3.88±0.68倍、粘附率达34%±10%,对骨髓干细胞即CFU-F的浓度富集倍数为5.25±1.40倍、粘附率达73%±13%、选择率达1.41±0.34。0.1%PLL修饰的富集材料与更高浓度PLL制备的PLL-DBM比较无明显差异,说明PLL-DBM对NCs的粘附具有一定的饱和性。富集后TEB中促成骨生长因子TGF-β1的浓度富集倍数为42.327±4.561,PDGF的浓度富集倍数为9.618±1.251,富集技术可以显著提高TEB中TGF-β1和PDGF含量。⑹随时间延长,植入区成骨标志物I型胶原、整合素α2β1和骨钙素的表达均逐渐增高,16w时达高峰,SCR组和TEB组表达相似,显著高于骨髓组和DBM组;免疫组化和RT-PCR法显示成骨早期SCR组Cbfα1 mRNA和蛋白表达显著增加,随着骨组织的逐渐成熟,Cbfα1的表达逐渐下降,16w时表达明显减弱。SCR组Cbfα1的表达与TEB组类似,优于骨髓组和DBM组。
结论:⑴制备的PLL-DBM具有自体骨三维空间结构和促进细胞粘附的PLL,具有良好的细胞相容性、组织相容性和可降解性,能促进hMSCs的粘附与增殖,对DOC的成骨活性无影响,是一种较理想的骨髓干细胞富集材料。⑵SCR技术快速制备的TEB植入体内后能够成骨,并可以达到与体外培养的hMSCs复合制备的TEB同样的成骨效果。⑶PLL-DBM能明显增高骨髓有核细胞、血小板和CFU-F的浓度富集倍数和粘附率,显著提高TEB中TGF-β1和PDGF含量;推测SCR技术快速构建TEB后局部高浓度的骨髓干细胞和促成骨生长因子TGF-β1与PDGF,可能是SCR技术构建的TEB高成骨活性的可能机制之一。⑷SCR技术快速构建的TEB,通过在早期促进成骨特异性转录激活因子Cbfα1表达、中晚期上调成骨标志物I型胶原、整合素α2β1和骨钙素表达而促进成骨,可能是SCR技术构建的TEB高成骨活性的可能分子机制之一。
Background and objective: Bone defects induced by various kinds of causes are commonly seen in clinic. As a gold standard, autogenous bone transplantation can not satisfy the requirement for bone defects therapy because of limited bone mass and operation complications in donor site. Tissue engineered bone (TEB) constructed by bone marrow stem cells (BMSCs) and biomaterial in treating bone defects in animal studies had obtained certain effects, but the shortcomings were: BMSCs in little resource, long cycle of culture and amplification in vitro, low adhesion rate of cell to scaffold and high specification, thus limited the application in bone defect therapy. Autologous red bone marrow percutaneous injection had obtained some success in treating clinical bone nonunion and bone defect, but the method of direct injection, had the defect of partly stem cell running off. Selective cell retention (SCR) is a technique of bone marrow stem cell enrichment, the mechanism is seletive retenting stem cells and facilitating ossify factors when bone marrow pass through the adequate mesh and favourable surface adhesiveness of scaffolds. The TEB constructed by SCR were replanted immediately for treating bone defect, could obtained the same effect as autogenous bone transplantation. But due to intellectual property rights conservation and enter restriction, enrichment technique and correlated manufactures harded to applicate in our country. Thus, the objectives of our experiment are:⑴Preparation of a new kind of bone marrow stem cells enrichment scaffold, to observe its cellular compatibility in vitro and its histocompatibility in vivo;⑵To explore the enriching effect of new kind of bone marrow stem cells enrichment scaffold constructed by SCR;⑶To study the ectopia osteogenesis effects of tissue engineered bone fast constructed by SCR;⑷To observe the effect of tissue engineered bone fast constructed by SCR on ossify marker and ossify specific activating transcript factor Cbfα1, and explore the mechanism of enrichment scaffold on osteogenesis.
Methods:⑴Demineralized bone matrix decorated with poly-L-lysine (PLL-DBM) as a new kind of bone marrow stem cells enrichment scaffold, its surface character such as density, porosity and pore size were determined by liquid displacement method, scanning electron microscope, three dimension video microscope, raman spectroscopy, infrared spectrum and HE staining.⑵New enrichment scaffold and different dense of scaffold leaching liquor were co-cultured with hMSCs, cellular compatibility in vitro were evaluated by morphologic observation, MTT assay, FCM, hemolysis experiment and immunohistochemistry.⑶Nude mouse were accepted intraperitoneal injection of saline leaching liquor extracted from PLL-DBM, back subcutaneous injection of DOC cell suspension after cultured with PLL-DBM leaching liquor, back implantation of PLL-DBM scaffold respectively, histocompatibility in vivo were evaluated by acute toxicity test, carcinogenesis test and subcutaneous implantation test.⑷TEB were constructed quickly after bone marrow enrichment by SCR. PLL-DBM combinated with bone marrow by SCR (SCR group), deminerilized bone matrix ( DBM ) combinated with MSCs (TEB group), DBM combinated with bone marrow (bone marrow group) and DBM (DBM group) were implantated into subcutaneous area of nude mouse, drow the materials at 4, 8, 12, 16 weeks. Image density, histological changes of bone graft area and ossify effects were determined by X ray, CT scanning, scanning electron microscope and HE staining.⑸New enrichment scaffold were handled by SCR, the enriching effects of PLL-DBM to bone marrow nucleated cells (NCs) ,platelets (PLTs) and fibroblast colony forming unit ( CFU-F) were observed before and after enrichment, contents of TGF-β1 and PDGF in bone marrow supernatant were detected by ELISA method before and after enrichment.⑹TEB were constructed fast after bone marrow enrichment by SCR, PLL-DBM combinated with SCR (SCR group), DBM combinated with MSCs (TEB group), DBM combinated with bone marrow (bone marrow group) and DBM (DBM group) were implantated into subcutaneous area of nude mouse, Collagen type I, integrinα2β1, osteocalcin and Cbfα1 mRNA and protein expression at 4、8、12、16 weeks were studied by immunohistochemistry and RT-PCR method.
Results:⑴Steady ivory converage were formed at exterior and interior surface by PLL. The density of PLL-DBM is (0.27±0.02) g/ml, the porosity is (73±11)%, the pore size is (412.73±160.29)μm. there are many micropore about 100μm size among pores, a great quantity of pore cross-connected each other in the interior pore, and formed smaller, more steady mesh structure in the natural pore.⑵New enrichment scaffold and different density scaffold leaching liquor were co-cultured with hMSCs, morphologic observation showed that MSCs growth well, cytotoxicity showed that MSCs proliferate well and toxity range from 0 to 1 grade. FCM showed that hMSCs and DOC had no abnormal DNA ploid, the marker of CD29 and CD105 are positive, CD34 and CD45 are negative, scaffold leaching liquor had no affect on hMSCs surface molecule, and could promote its proliferation by transform it into multiplication period. After ossify induction, DOC could form calcium nodule and express Collagen type I, and the ALP activity had not been affected. Hemolysis experiment showed that hemolysis rate was 2.617% which in accordance with the standard of biomaterial that the rate must less than 5%.⑶In acute toxicity test, nude mouse growth well; in carcinogenesis test, nude mouse survival better, tumour had not been seen after 8 months, tumour cells had not been in the organization such as heart, liver, brain, lung, kidney and spleen. In subcutaneous implantation test, histological observation showed that subcutaneous tissue tighted the scaffold surface with a thin fiber tissue around it, lymphocyte/macrophage infiltration, cytolysis and necrosis had not been seen.⑷The density of each composite implantation increase gradually followed the time, and had significant difference at each time point when compared with single DBM; the density in SCR group was similar to that in TEB group, but was superior to in bone marrow group and DBM group. Scanning electron microscope and HE staining showed that, PLL-DBM scaffold degradated gradually as time passed, bone trabecula and small vessels formed gradually into the scaffold. DBM started to degradation at 8 weeks, a lot of cells and a few small vessels formed, and part of newly formed bone generated into the scaffold. DBM partly degraded at 12 weeks, smaller vessels entered scaffold and new bone formation was found; rigid bone tissue were seen at 16 weeks, and medullary tissue and vessels were seen in some region of implantation site. Scanning electron microscope and HE staining in TEB group were similar to SCR group, but superior to bone marrow group and DBM group.⑸The enriching effects of PLL-DBM increased gradually with the PLL dnsity. The concentration enrichment multiple rate of enrichment scaffold decorated with 0.1% PLL of bone marrow NCs was 3.18±0.31, the adhesion rate was 53%±12%; The concentration enrichment multiple rate of platelet was 3.88±0.68, the adhesion rate was 34%±10%, The concentration enrichment multiple rate of CFU-F was 5.25±1.40, the adhesion rate was 73%±13%, the selective rate was 1.41±0.34. Compared with high density PLL, 0.1% PLL-DBM had the same effect, showed that PLL-DBM had saturability to NCs adhesion. concentration enrichment multiple rate of TGF-β1 was 42.327±4.561 and concentration enrichment multiple rate of PDGF was 9.618±1.251 in the enrichment scaffold, and enrichment technique could increase the content of TGF-β1 and PDGF significantly.⑹Expression of Collagen type I, integrinα2β1, osteocalcin mRNA and protein were increased gradually followed the time, and reached the peak at 16 weeks, the expression in SCR group was similar to that in TEB group, superior to bone marrow group and DBM group. Immunohistochemistry and RT-PCR showed that, Cbfα1 mRNA and protein expression increased significantly at ossify earlier period, but decreased gradually followed the bone maturation. The expression of Cbfα1in SCR group was similar to in TEB group, superior to in bone marrow group and DBM group.
Conclusion:⑴PLL-DBM possess three dimensional space as autogenous bone and PLL for cell adhesion, and have favourable cellular compatibility, histocompatibility and biodegradability, can promote the adhesion and proliferation of hMSCs, but not affect the ossify activity of DOC, is an ideal enrichment scaffold for bone marrow stem cells.⑵TEB constructed fast by SCR have favourable bone formation, and the effect of ossify is similar to MSCs combined with DBM.⑶Enrichment scaffold can increase the concentration enrichment multiple rate and the adhesion rate of bone marrow NCs, platelet and CFU-F significantly, and increase the contents of TGF-β1 and PDGF in TEB, it is said that the local high concentration of bone marrow stem cells and facilitate ossify growth factor TGF-β1 and PDGF, maybe the one of the mechanism of high ossify activity of TEB constructed fast by SCR.⑷Increased Cbfα1 expression in earlier period of osteogenesis and increased expression of Collagen type I, integrinα2β1 and osteocalcin in middle-advanced stage maybe the one of the molecule mechanism of high ossify activity of TEB constructed fast by SCR.
引文
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