Nanofibrous Scaffold Containing Osteoblast-Derived Extracellular Matrix for the Proliferation of Bone Marrow Mesenchymal Stem Cells

Extracellular matrix( ECM) plays a prominent role in establishing and maintaining an appropriate microenvironment for tissue regeneration. The aims of this study were to construct a tissue engineered scaffold by reconstituting osteoblast cell-derived ECM( O-ECM) on the electrospun nanofibrous scaffold,and further to evaluate its subsequent application for promoting the proliferation of bone marrow mesenchymal stem cells( BMSCs). To engineer a biomimetic scaffold, calvarial osteoblasts and electrospun poly-llactic acid( PLLA) nanofibers were prepared and subjected to decellularize for O-ECM deposition. To evaluate and characterize the O-ECM/PLLA scaffold, the morphology was examined and several specific mark proteins of osteoblasts matrix were evaluated.Furthermore,the cell counting kit-8( CCK-8) assay was used to detect the proliferation of the BMSCs cultivated on the O-ECM/PLLA scaffold. The results indicated O-ECM/PLLA scaffold was loaded with Collagen I, Fibronectin, and Laminin, as the composition of the marrow ECM. After decellularization,O-ECM deposition was observed in O-ECM/PLLA scaffold. Moreover,the O-ECM/PLLA scaffold could significantly enhance the proliferation of BMSCs,suggesting better cytocompatibility compared to the other groups tested. Taken together,a biomimetic scaffold based on the joint use of O-ECM and PLLA biomaterials,which represents a promising approach to bone tissue engineering, facilitates the expansion of BMSCs in vitro.

TISSUE-ENGINEERED GRAFT CONSTRUCTED BY SELF-DERIVED BONE MARROW MONONUCLEAR CELLS AND HETEROGENEOUS ACELLULARIZED TISSUE MATRIX

Objective To create a method for constructing a tissue-engineered graft with self-derived bone marrow cells and heterogeneous acellular matrix.Methods The mononuclear cells were isolated from bone marrows drawn from piglets and cultured in different mediums including either vascular endothelial growth factor(VEGF)or platelet derived growth factor BB(PDGF-BB)to observe their expansion and differentiation.The aortas harvested from canines were processed by a multi-step decellularizing technique to erase.The bone marrow mononuclear cells cultured in the mediums without any growth factors were seeded to the acellular matrix.The cells-seeded grafts were incubated in vitro for 6 d and then implanted to the cells-donated piglets to substitute parts of their native pulmonary arteries.Results After 4 d culturing,the cells incubated in the medium including VEGF showed morphological feature of endothelial cells(ECs)and were positive to ECs-specific monoclonal antibodies of CD31,FLK-1,VE-Cadherin and vWF.The cells incubated in the medium including PDGF-BB showed morphological feature of smooth muscle cells(SMCs)and were positive to SMCs-specific monoclonal antibodies of α-SMA and Calponin.One hundred days after implantation of seeded grafts,the inner surfaces of explants were smooth without thrombosis,calcification and aneurysm.Under the microscopy,plenty of growing cells could be seen and elastic and collagen fibers were abundant.Conclusion Mesenchymal stem cells might exist in mononuclear cells isolated from bone marrow.They would differentiate into endothelial cells or smooth muscle cells in proper in vitro or in vivo environments.The bone marrow mononuclear cells might be a choice of seeding cells in constructing tissue-engineered graft.

Experimental Study on Allogenic Decalcified Bone Matrix as Carrier for Bone Tissue Engineering

The biocompatibility and osteogenic activity of allogenic decalcified bone matrix (DBM) used as a carrier for bone tissue engineering were studied. Following the method described by Urist, allogenic DBM was made. In vitro, DBM and bone marrow stromal cell (BMSC) from rabbits were co-cultured for 3-7 days and subjected to HE staining, and a series of histomorphological observations were performed under phase-contrast microscopy and scanning electron microscopy (SEM). In vivo the mixture of DBM/BMSC co-cultured for 3 days was planted into one side of muscules sacrospinalis of rabbits, and the DBM without BMSC was planted into other side as control. Specimens were collected at postoperative week 1, 2 and 4, and subjected to HE staining, and observed under SEM. The results showed during culture in vitro, the BMSCs adherent to the wall of DBM grew, proliferated and had secretive activity. The in vivo experiment revealed that BMSCs and undifferentiated mesenchymal cells in the perivascular region invaded gradually and proliferated together in DBM/BMSC group, and colony-forming units of chondrocytes were found. Osteoblasts, trabecular bone and medullary cavity appeared. The inflammatory reaction around muscles almost disappeared at the second weeks. In pure DBM group, the similar changes appeared from the surface of the DBM to center, and the volume of total regenerate bones was less than the DBM/BMSC group at the same time. The results indicated that the mixture of DBM and BMSC had good biocompatibility and ectopic induced osteogenic activity.

羊膜细胞外基质与膀胱细胞外基质材料修复大鼠子宫内膜损伤

背景:大量研究已证实,羊膜细胞外基质与膀胱细胞外基质材料均可作为干细胞载体用于子宫内膜损伤的治疗,但是有关两种材料的比较研究相对少见。目的:对比羊膜细胞外基质与膀胱细胞外基质材料作为干细胞载体治疗子宫内膜损伤的差异。方法:采用全骨髓贴壁法分离纯化SD大鼠骨髓间充质干细胞。分别制备SD大鼠羊膜细胞外基质与膀胱细胞外基质材料,然后将骨髓间充质干细胞分别接种于两种材料表面,检测细胞增殖与黏附情况。将40只SD大鼠随机分为4组(n=10),除假手术组外,子宫内膜损伤组、羊膜细胞外基质组、膀胱细胞外基质组均通过机械干预的方式建立子宫内膜损伤模型,分别将羊膜细胞外基质/骨髓间充质干细胞复合物、膀胱基质细胞外基质/骨髓间充质干细胞复合物移植至羊膜细胞外基质组、膀胱细胞外基质组大鼠损伤内膜部位,移植后14,28 d取材检测,苏木精-伊红染色观察大鼠子宫内膜组织形态,酶联免疫吸附法分析子宫内膜组织中碱性成纤维细胞生长因子、胰岛素样生长因子1与血管内皮生长因子水平,免疫组化染色分析子宫内膜组织中波形蛋白与CD34表达。结果与结论:(1)两种细胞外基质材料均有利骨髓间充质干细胞的增殖,相较于膀胱细胞外基质材料,羊膜细胞外基质材料可促进骨髓间充质干细胞的黏附;(2)相较于假手术组,子宫内膜损伤组碱性成纤维细胞生长因子、胰岛素样生长因子1与血管内皮生长因子水平降低(P <0.01),子宫内膜组织形态发育不良,内膜厚度与腺体数量减少,波形蛋白与CD34阳性表达减少(P <0.01);相较于子宫内膜损伤组,羊膜细胞外基质组、膀胱细胞外基质组碱性成纤维细胞生长因子、胰岛素样生长因子1与血管内皮生长因子水平均升高(P <0.05或P <0.01),子宫内膜组织形态明显改善,内膜厚度与腺体数量增加,波形蛋白与CD34阳性表达增加(P <0.05或P <0.01),并且羊膜细胞外基质组的改善作用优于膀胱细胞外基质组(P <0.05);(3)结果表明,相较于膀胱细胞外基质材料,羊膜细胞外基质材料作为骨髓间充质干细胞的载体可进一步促进损伤子宫内膜的修复。

Differentiation of mesenchymal stem cells into neuronal cells on fetal bovine acellular dermal matrix as a tissue engineered nerve scaffold

The purpose of this study was to assess fetal bovine acellular dermal matrix as a scaffold for supporting the differentiation of bone marrow mesenchymal stem cells into neural cells fol-lowing induction with neural differentiation medium. We performed long-term, continuous observation of cell morphology, growth, differentiation, and neuronal development using several microscopy techniques in conjunction with immunohistochemistry. We examined speciifc neu-ronal proteins and Nissl bodies involved in the differentiation process in order to determine the neuronal differentiation of bone marrow mesenchymal stem cells. The results show that bone marrow mesenchymal stem cells that differentiate on fetal bovine acellular dermal matrix display neuronal morphology with unipolar and bi/multipolar neurite elongations that express neuro-nal-speciifc proteins, includingβIII tubulin. The bone marrow mesenchymal stem cells grown on fetal bovine acellular dermal matrix and induced for long periods of time with neural differen-tiation medium differentiated into a multilayered neural network-like structure with long nerve ifbers that was composed of several parallel microifbers and neuronal cells, forming a complete neural circuit with dendrite-dendrite to axon-dendrite to dendrite-axon synapses. In addition, growth cones with filopodia were observed using scanning electron microscopy. Paraffin sec-tioning showed differentiated bone marrow mesenchymal stem cells with the typical features of neuronal phenotype, such as a large, round nucleus and a cytoplasm full of Nissl bodies. The data suggest that the biological scaffold fetal bovine acellular dermal matrix is capable of supporting human bone marrow mesenchymal stem cell differentiation into functional neurons and the subsequent formation of tissue engineered nerve.

NUCLEAR MATRIX PROTEIN IN LEUKEMIA CELLS

Objective: To compare the composition of nuclear matrix proteins (NMP) between leukemia cells and normal bone marrow cells. Methods: NMP was isolated by high-salt extraction and identified in acute and chronic myelogenous leukemia cells as well as in the blast phase of chronic leukemia. On SDS-PAGE, NMPs with molecular myelogenous ferment from what were seen in normal bone marrow cells were present in both acute and chronic myelogenous leukemia. Conclusion: Marked changes of NMP, not only in contents but also in compositions, exist in leukemic cells compared with normal bone marrow cells. NMP may serve as a target of chemotherapeutic drug against leukemia.

Dynamic culture of a thermosensitive collagen hydrogel as an extracellular matrix improves the construction of tissue-engineered peripheral nerve

Tissue engineering technologies offer new treatment strategies for the repair of peripheral nerve injury, hut cell loss between seeding and adhesion to the scaffold remains inevitable. A thermosensitive collagen hydrogel was used as an extracellular matrix in this study and combined with bone marrow mesenchymal stem cells to construct tissue-engineered peripheral nerve composites in vitro. Dynamic culture was performed at an oscillating frequency of 0.5 Hz and 35° swing angle above and below the horizontal plane. The results demonstrated that bone marrow mesenchymal stem cells formed membrane-like structures around the poly-L-lactic acid scaffolds and exhibited regular alignment on the composite surface. Collagen was used to fill in the pores, and seeded cells adhered onto the poly-L-lactic acid fibers. The DNA content of the bone marrow mesenchymal stem cells was higher in the composites constructed with a thermosensitive collagen hydrogel compared with that in collagen I scaffold controls. The cellular DNA content was also higher in the thermosensitive collagen hydrogel composites constructed with the thermosensitive collagen hydrogel in dynamic culture than that in static culture. These results indicate that tissue-engineered composites formed with thermosensitive collagen hydrogel in dynamic culture can maintain larger numbers of seeded cells by avoiding cell loss during the initial adhe-sion stage. Moreover, seeded cells were distributed throughout the material.

转化生长因子β亚家族调控骨关节炎中的作用

背景:骨关节炎作为中国最常见的老年慢性退行性疾病之一,因其复杂的发病机制及细胞分子交流途径,目前尚未有行之有效的方法去延缓骨关节炎的进展。而转化生长因子β在早期关节的形成、骨和软骨的发育以及关节重塑各阶段发挥重要作用,是维持与调节关节稳态的关键因子之一。目的:综述近年来国内外关于转化生长因子β亚家族在骨关节炎的发生发展中所起到的调控作用,分析其在骨关节炎不同阶段所产生的影响,探究转化生长因子β在临床治疗骨关节炎上的应用前景,以期能为临床治疗方案提供参考。方法:应用计算机检索中国知网和PubMed数据库收录的相关文献,中文检索词为“骨关节炎,转化生长因子,信号通路,骨重塑,软骨退变,血管生成,治疗”,英文检索词为“Osteoarthritis,Transforming Growth Factor,Signaling Pathway,Bone Remodeling,Cartilage Degeneration,Angiogenesis,Treatment”,最终纳入57篇文献进行综述分析。结果与结论:(1)目前,关于骨关节炎复杂的发病机制尚未有统一定论,大量研究表明骨关节炎与细胞因子和信号通路关系密切,以转化生长因子β超家族作为其发病机制和治疗突破口的相关研究也是当前的热点。(2)转化生长因子β在早期关节软骨形成与稳态维持上起到关键作用,并对于软骨损伤修复有促进作用;而在关节成型后,转化生长因子β的保护作用会减弱甚至造成破坏效应,其双重调节作用也是目前转化为临床治疗手段的重点,需后续研究明确适用范围以制定标准。(3)高水平活性转化生长因子β在机械应力的介导下参与骨细胞、成骨细胞与破骨细胞的调控,并干预随后骨微观结构的重塑,特异性抑制剂可作为治疗疾病的靶向药物,但其作用的安全性与有效性仍需临床进一步完善。(4)血管增生可能在转化生长因子β介导软骨退变及软骨下骨重塑中提供潜在的串扰途径,异常的交流途径会进一步破坏骨软骨单元微环境的稳态从而加速骨关节炎中关键的病理学进展。(5)关于转化生长因子β在骨关节炎中的研究已经较为全面,临床应用前景广泛,目前已有相关药物处于临床试验阶段,但如何控制对其他组织的潜在影响和精准控制靶向递送等关键问题亟需解决,随着研究的深入,未来有望在延缓骨关节炎治疗方式上作出新的突破。

In vitro cartilage production using an extracellular matrix-derived scaffold and bone marrow-derived mesenchymal stem cells

Background Cartilage repair is a challenging research area because of the limited healing capacity of adult articular cartilage.We had previously developed a natural,human cartilage extracellular matrix （ECM）-derived scaffold for in vivo cartilage tissue engineering in nude mice.However,before these scaffolds can be used in clinical applications in vivo,the in vitro effects should be further explored.Methods We produced cartilage in vitro using a natural cartilage ECM-derived scaffold.The scaffolds were fabricated by combining a decellularization procedure with a freeze-drying technique and were characterized by scanning electron microscopy （SEM）,micro-computed tomography （micro-CT）,histological staining,cytotoxicity assay,biochemical and biomechanical analysis.After being chondrogenically induced,the induction results of BMSCs were analyzed by histology and Immunohisto-chemistry.The attachment and viability assessment of the cells on scaffolds were analyzed using SEM and LIVE/DEAD staining.Cell-scaffold constructs cultured in vitro for 1 week and 3 weeks were analyzed using histological and immunohistochemical methods.Results SEM and micro-CT revealed a 3-D interconnected porous structure.The majority of the cartilage ECM was found in the scaffold following the removal of cellular debris,and stained positive for safranin O and collagen Ⅱ.Viability staining indicated no cytotoxic effects of the scaffold.Biochemical analysis showed that collagen content was （708.2±44.7）μg/mg,with GAG （254.7±25.9） μg/mg.Mechanical testing showed the compression moduli （E） were （1.226±0.288） and （0.052±0.007） MPa in dry and wet conditions,respectively.Isolated canine bone marrow-derived stem cells （BMSCs） were induced down a chondrogenic pathway,labeled with PKH26,and seeded onto the scaffold.Immunofluorescent staining of the cell-scaffold constructs indicated that chondrocyte-like cells were derived from seeded BMSCs and excreted ECM.The cell-scaffold constructs contained pink,smooth and translucent cartilage-like tissue after 3 weeks of culture.We observed evenly distributed cartilage ECM proteoglycans and collagen type Ⅱ around seeded BMSCs on the surface and inside the pores throughout the scaffold.Conclusion This study stuggests that a cartilage ECM scaffold holds much promise for in vitro cartilage tissue engineering.

红景天苷对骨髓抑制贫血小鼠骨髓基质金属蛋白酶的影响

本研究用免疫组织化学方法和酶谱电泳法观察了红景天苷对骨髓抑制贫血小鼠骨髓基质金属蛋白酶-2和-9(MMP-2、MMP-9)的影响,并探讨其在造血调控中的可能作用。免疫组化结果显示,各组小鼠骨髓细胞中均检测到MMP-2和MMP-9表达。与对照组相比,模型组及高、中、低剂量红景天苷组骨髓细胞中MMP-2、MMP-9蛋白表达明显增强。在造模后第4d,中剂量红景天苷组中MMP-2和MMP-9表达最强。在8d,分别是低剂量和中剂量红景天苷组中MMP-2和MMP-9表达最强。酶谱电泳结果显示,在对照组骨髓中可检测到66kD(MMP-2的酶原形式,proMMP-2)、62kD(MMP-2的活性形式,MMP-2)、86kD(MMP-9活性形式,MMP-9)和94kD(MMP-9的酶原形式,proMMP-9)4条明胶酶的活性带,其中MMP-9的活性最强。造模后骨髓造血微环境中明胶酶的活性明显降低,不同剂量的红景天苷均能明显促进proMMP-9和MMP-9的活性增强,使proMMP-2的活性减弱。结果表明,红景天苷可能通过促进骨髓细胞中基质金属蛋白酶表达增强、骨髓造血微环境中基质金属蛋白酶活性升高,进而导致ECM中或基质细胞膜上细胞因子的释放、骨髓微血管损伤的修复以及HSCs增殖、迁移和分化能力的增加来促进骨髓造血功能的恢复。

针灸对化疗小鼠骨髓基质细胞支持造血功能的影响

目的:了解针灸对化疗后骨髓基质细胞支持造血能力的影响。方法:将40只小鼠分为对照组、针刺组、艾灸组和正常组,每组10只。除正常组外,其余3组均给予一次性腹腔注射环磷酰胺150 mg/kg。然后,针刺组每d针刺“足理”、“三阴交”,留针5min;艾灸组每d无瘢痕灸“丈椎”、“膈俞”3次,其余2组仅陪同固定。5 d后,行外周血白细胞计数,取骨髓进行体外培养,前14 d用不含集落刺激因子的培养体系Ⅰ,再用含正常小鼠骨髓细胞的培养体系Ⅱ继续培养7 d后计数产生的造血细胞集落。结果:外周血白细胞总数艾灸组高于对照组(P>0.05),低于正常组(P<0.05),但与针刺组比较,差异无统计学意义(P>0.05)。集落数量:艾灸组大于对照组(P<0.05),但与正常组及针刺组比较,差异无统计学意义(P>0.05)。结论:针灸可以提高化疗后骨髓基质细胞支持造血的能力。

松质骨基质-骨髓基质成骨细胞复合物的异位成骨作用

目的：观察松质骨基质－ 骨髓基质成骨细胞复合移植皮下成骨作用，以探索松质骨基质作为骨组织工程支架材料的可行性。方法：新生兔骨髓细胞体外培养、诱导后，接种于藻酸盐／松质骨基质中，形成松质骨基质／藻酸盐／骨髓基质成骨细胞复合物，并植入裸鼠背部皮下，对照组单纯植入松质骨基质。植入４、８ 周后行 Ｘ线摄片和组织学检查，观察骨形成情况。结果：松质骨基质／骨髓基质成骨细胞复合物皮下成骨效果明显优于松质骨基质。前者兼有纤维成骨和软骨成骨，以纤维成骨为主；后者仅见少量软骨成骨。结论：松质骨基质可以作为一种良好的骨组织工程支架材料。

多聚赖氨酸修饰的山羊脱钙骨富集骨髓干细胞的性能评价

目的研究经多聚左旋赖氨酸修饰的山羊脱钙骨基质(demineralized bone matrix decorated with poly-L-lysine,PLL-DBM)的性能,为选择性细胞滞留技术(selective cell retention,SCR)提供一种对骨髓干细胞黏附性能良好的富集基质材料。方法将取材于山羊股骨近端的松质骨去皮质理化处理制成脱钙骨基质(demineralized bonem atrix,DBM),用多聚左旋赖氨酸(poly-L-lysine,PLL)对其进行修饰。扫描电镜分析其显微结构;氨基酸成分分析检测PLL与DBM的复合情况;通过纤维母细胞集落形成单位(CFU-F)计数,检测山羊PLL-DBM对骨髓干细胞的富集效果;在山羊横突间融合模型中,通过X线片及组织学评价其成骨能力。结果PLL-DBM具有天然网状孔隙结构系统,空隙内修饰有PLL形成的蜘蛛网样结构;PLL与DBM能较好地复合;山羊PLL-DBM对骨髓干细胞富集效果明显优于空白DBM(P<0.01)。PLL-DBM的成骨能力与自体髂骨相当。结论山羊PLL-DBM对骨髓干细胞有较好的富集效果,成骨能力强,是一种理想的富集基质材料。

骨髓基质干细胞与不同支架材料复合异位成骨能力的实验研究

目的:研究骨髓基质干细胞(bone marrow stromal cells,BMSCs)与不同支架材料复合后的异位成骨能力。方法:将体外诱导培养的成年犬骨髓基质干细胞,以1×106/cm2的密度接种到冻干骨基质(fdDBM)、磷酸三钙(TCP)、孔径分别为200μm及500μm的珊瑚羟磷灰石(CH200、CH500)等支架材料上,分别于体外培养的第4￣7天进行扫描电镜及石蜡切片HE染色,了解细胞在不同支架上的黏附及生长情况,并于1周后将大小为10mm×5mm×2mm的上述细胞支架复合物(fdDBM、TCP、CH200、CH500)分别植入裸鼠(n=8)背部皮下,每只裸鼠均植入4个实验组及1组无细胞的fdDBM空白支架对照组。9周后,取材行石蜡切片及HE染色,以IMAGER-PROPLUS软件测量每组的新生骨小梁量,并采用SAS软件包行单因素方差分析。结果:犬BMSCs与4种支架材料均黏附良好,TCP、CH200及CH5003组各样本均能观察到新骨形成,fdDBM及fdDBM空白对照组分别有75%、25%的样本有新骨形成;其中,TCP组的TBV值(28.2%±2.86%)显著高于CH200组(24.1%±4.12%)及CH500组(18.1%±4.66%)(P<0.01)。结论:BMSCs与4种材料的相容性良好。TCP与BMSCs复合后异位成骨效果显著,是很有希望的骨组织工程支架材料。

69例恶性淋巴瘤骨髓侵犯后骨髓象与血象分析

目的 了解恶性淋巴瘤骨髓侵犯后骨髓与外周血白细胞之间的关系。方法 对69例恶性淋巴瘤具有骨髓侵犯的骨髓细胞分类及外周血白细胞检验结果作回顾性分析。结果 69例恶性淋巴瘤骨髓侵犯者外周血白细胞计数异常33例,分类计数异常35例,骨髓增生正常者19例,增生异常者50例。结论 当恶性淋巴瘤发展到骨髓侵犯或淋巴瘤细胞白血病时,外周血白细胞计数和分类计数与骨髓象之间没有绝对的对应关系,但如明确诊断为恶性淋巴瘤时则应把骨髓涂片细胞形态学列为常规检查,如其外周血白细胞形态出现异常,则应高度怀疑有淋巴瘤细胞白血病。

脱钙骨基质支架构建组织工程骨的实验研究

目的 以同种异体骨脱钙骨基质(demineralizedbonematrix ,DBM )为支架材料,复合体外诱导培养的人骨髓间充质干细胞(humanmesenchymalstemcells ,hMSCs) ,构建组织工程骨并通过裸鼠皮下异位成骨实验及裸鼠皮下致瘤性实验验证其成骨效果及安全性。方法 贴壁法培养hMSCs ,体外诱导培养扩增,以1 85 7×10 6/ml的密度与DBM复合构建组织工程骨,体外培养,并在扫描电镜下观察细胞与材料复合情况,进行组织学观察。同时进行了裸鼠皮下异位成骨实验。结果 细胞与支架复合后3d ,细胞与DBM表面及孔隙可实现良好复合;复合后5d ,扫描电镜观察到细胞基质分泌旺盛,充满支架孔隙。裸鼠皮下成骨实验证明其成骨效果良好。结论 以本实验中使用的细胞培养方法扩增、诱导分化的种子细胞生物安全性好,自制的DBM具有良好的生物相容性,可为种子细胞生长提供较好的三维空间,按照标准化工艺流程制备的组织工程骨安全、有效。

异种骨基质明胶复合自体红骨髓治疗骨缺损的实验结果观察

目的观察异种骨基质明胶(bonematrixgelatin,BMG)复合自体红骨髓(redbonemarrow,RBM)治疗骨缺损的疗效,为临床应用提供证据。方法制备猪BMG,将之与自体红骨髓复合移植兔桡骨中段1cm骨缺损,通过影像学和组织形态学等检测,观察其成骨效果,并与单纯BMG移植及自体骨移植进行比较。结果BMG/RBM表现出较强的骨形成能力及骨传导作用,16周基本修复骨缺损,骨髓腔再通,其修复能力明显优于BMG组;与自体骨相近。骨修复过程为膜内成骨和软骨内成骨。结论使用BMG/RBM治疗骨缺损,可明显促进骨形成,加快骨修复,具有良好的诱导骨生成和骨传导作用。

异体骨基质联合重组人骨形成蛋白2与红骨髓修复兔骨缺损

目的 :采用NaOH消蚀脱细胞制备异体骨基质 (BMM ) ,观察其与重组人骨形成蛋白 2 (rhBMP) 2 及自体红骨髓 (ARM )联合移植的成骨效果 ,为该材料应用提供实验依据。方法 :用 6%NaOH消蚀兔皮质骨。将支架、rhBMP2及ARM组合分组 ,移植修复兔桡骨节段性骨缺损 ,定期X线、组织学及生物力学检测 ,观察成骨效果。结果 :( 1 )支架几乎未见细胞成分 ,骨基质 胶原纤维及骨盐结构无改变 ,无明显排异反应。 ( 2 )BMM/rhBMP2 /ARM共同移植 ,修复效果优于其它组 ,应用ARM组有早期修复现象。结论 :采用NaOH消蚀法制作的骨基质支架 ,可用于骨组织工程 ;ARM促进早期成骨 ,与rhBMP2 联合应用 ,可有效修复骨缺损。

壳聚糖温敏凝胶负载釉基质蛋白对骨髓基质细胞的作用

目的:探讨在壳聚糖温敏凝胶支架材料中釉基质蛋白(enamel matrix proteins,EMPs)对骨髓基质细胞(bone marrow stromal cells,BMSCs)增殖和碱性磷酸酶(alkaline phosphate,ALP)活性的影响。方法:合成壳聚糖温敏凝胶并加入适当浓度的EMPs,通过考马斯亮蓝试剂盒检测其对EMPs的缓释作用。用全骨髓培养法获得大鼠BMSCs,含10%胎牛血清的DMEM培养液进行原代培养。含EMPs浓度分别为0、50、100、150μg/mLDMEM培养液培养第3代大鼠BMSCs,MTT法测定各组细胞的增殖活性。MTT法及ALP试剂盒检测大鼠BMSCs在负载100μg/mLEMPs及不负载EMPs的壳聚糖温敏凝胶支架材料中的增殖情况及ALP的活性。采用SPSS11.0软件包对实验数据进行单因素方差分析及两样本t检验。结果:EMPs可在壳聚糖温敏凝胶中持续释放3周以上。DMEM培养液中,50μg/mLEMPs组从实验的第3天开始,显著促进大鼠BMSCs的增殖(P<0.01)。壳聚糖温敏凝胶负载100μg/mLEMPs后,于实验的第3天和第5天明显促进大鼠BMSCs的增殖(P<0.05)并且在实验的第7天(P<0.05)和第9天(P<0.01)显著促进大鼠BMSCsALP水平的提高。结论:壳聚糖温敏凝胶对EMPs具有缓释性,负载EMPs后,可以促进大鼠BMSCs增殖,提高ALP的活性。

多发性骨髓瘤患者MMP-9、MMP-2表达的研究

背景与目的:近年来的研究发现基质金属蛋白酶(MMPs)与骨重建、骨质重吸收和肿瘤浸润转移等过程有关。我们检测了多发性骨髓瘤(MM)患者的MMP-9和MMP-2水平,以探讨其与MM发病的关系和临床意义。方法:用酶联免疫吸附试验(ELISA)检测了30例MM患者单个核细胞培养液的MMP-9水平、24例MM患者骨髓基质细胞(BMSCs)培养液的MMP-2水平和12例对照组MMP-9、MMP-2水平。结果:19/30例进展期MM组的MMP-9表达水平显著高于11/30稳定期MM组和12例对照组(P<0.05);14/24例进展期MM组BMSCs的MMP-2表达水平显著高于10/24例稳定期MM组和对照组(P<0.05),稳定期MM组和对照组的MMP-9、MMP-2水平无显著差别(P>0.05);12例IgG型MM患者的MMP-9和血免疫球蛋白(M蛋白)水平呈正相关(r=0.87,P=0)。rIL-6(终浓度100ng/m l)、rIL-1β(终浓度10ng/m l)并不能显著改变BMSCs分泌的MMP-2,但9例进展期MM组BMSCs与人骨髓瘤细胞系U266细胞(终浓度2×105/mL)混合培养48小时后,MMP-2水平明显增高,显著高于上述进展期和稳定期MM患者和对照组的BMCSs(均为P<0.05)。结论:MMP-9、MMP-2水平升高与MM的病情进展有关;MMP-9可反映MM患者的肿瘤负荷,可能与MM的肿瘤浸润病理过程有关;而这些过程需骨髓瘤细胞和BMSCs相互作用而完成。