3种组织来源的间充质干细胞体外成骨能力的比较

目的比较成人骨髓间充质干细胞(BMSCs)、人脐带间充质干细胞(UC-MSCs)和人胎盘间充质干细胞(P-MSCs)的成骨能力。方法用含10%胎牛血清的DMEM/Ham's F-12培养液培养3种MSCs,CCK8法检测增殖能力,流式细胞仪鉴定3种细胞。碱性磷酸酶(ALP)和茜素红染色观察细胞经成骨诱导后成骨分化蛋白-ALP的分泌和矿化钙结节的沉积。实时荧光定量PCR(RT-q PCR)法检测MSCs骨再生相关基因的表达。Western blot方法检测MSCs成骨再生相关基因的蛋白表达。结果 MSCs在第3天进入对数增殖期。3种细胞的表面标志物阳性率:CD44、CD90和CD105均高于98%。3种MSCs成骨诱导9 d时,3种MSCs的实验组均表达大量成骨分化蛋白-ALP,成骨诱导18 d时3种MSCs均呈现较好的矿化能力;3种MSCs成骨诱导9 d时,实验组RUNX2和ALP基因显著性高表达(P0.05),成骨诱导18 d时,实验组RUNX2和骨钙素(OCN)亦显著性高表达(P0.05);3种MSCs成骨诱导9 d时,实验组均检测到RUNX2和ALP的蛋白表达;成骨诱导18 d时,实验组细胞亦检测到RUNX2和OCN的蛋白表达。结论 UC-MSCs和P-MSCs具有良好的成骨分化能力,有望作为骨组织工程的种子细胞用于治疗骨缺损。     

动态力学信号对人骨髓基质细胞、骨膜细胞生长和成骨表达的作用

目的 探讨动态力学信号对体外分离培养的人骨髓基质细胞、骨膜细胞生长与分化特征的生物学效应。方法 使用Flexcell应力系统，将频率为1Hz、振幅为5％变形、正弦波状力学信号作用于体外分离培养的正常人骨髓基质细胞和骨膜细胞，在不同时间段检测其对细胞DNA、总蛋白合成、碱性磷酸酶（ALP）表达和骨钙素分泌量的影响。结果 动态力学刺激对人骨髓基质细胞、骨膜细胞蛋白与DNA合成无明显作用。接受力学刺激信号后骨膜细胞受维生素D3刺激后分泌骨钙素显著增加，而骨髓基质细胞则显著下降。结论 动态力学信号能够促进人骨膜细胞向成骨细胞分化，这可能是其对骨的生物学作用的机制之一。     

绿色荧光蛋白小鼠骨髓间充质干细胞的体外培养及成骨诱导

目的分离培养绿色荧光蛋白(GFP)小鼠骨髓间充质干细胞(MSCs)并成骨诱导分化。方法密度梯度离心法分离GFP小鼠MSCs，体外传代扩增，条件培养基成骨方向诱导，利用荧光显微镜、HE染色、细胞化学和免疫组织化学方法进行观察。结果MSCs在传代和诱导分化过程中稳定表达GFP，经成骨方向诱导10d后，出现大量碱性磷酸酶和骨钙素染色阳性细胞。结论成功分离了GFP小鼠MSCs，并体外向成骨方向诱导分化。     

碱性成纤维细胞生长因子基因转染对间充质干细胞生物学行为的调控

为探讨碱性成纤维细胞生长因子(bFGF)基因转染对间充质干细胞(MSCs)增殖、定向分化等生物学行为的调控作用，本文将体外具有促进MSCs增殖分化及毛细血管增殖等多重生物学效应的bFGF基因转入骨组织工程首选种子细胞——MSCs，通过免疫组化SABC法检测其瞬时与稳定表达，并检测转基因细胞增殖活力及碱性磷酸酶(ALP)与骨钙素(OC)合成情况。结果表明，bFGF基因能被转入MSCs并得到稳定表达，转基因细胞增殖与OC合成明显增强，ALP活性无明显变化。因此，转基因MSCs可使bFGF持续高效发挥作用，克服了使用外源性bFGF半衰期短，需反复大剂量给药的缺点；bFGF基因转染可促进MSCs增殖并调控其定向分化，使其获得良好的生物学活性；从而为把组织工程学与分子生物学有机结合，用分子组织工程学技术高质量地修复骨缺损奠定了良好的基础     

重组人骨形态发生蛋白-7对NIH3T3细胞生物学行为的影响

目的:探讨重组人骨形态发生蛋白-7(rhBMP-7)对NIH3T3增殖和骨向分化的影响。方法:向培养的成纤维细胞NIH3T3中加入不同浓度的rhBMP-7,观察NIH3T3增殖、碱性磷酸酶(ALP)活性和骨钙素(OCN)含量的变化。结果:rh-BMP-7在一定浓度范围内可以明显促进NIH3T3细胞增殖、提高ALP活性与OCN水平。结论:rhBMP-7可以刺激NIH3T3细胞增殖,诱导NIH3T3细胞向成骨细胞表型分化。     

动态力学应变对人骨膜细胞的生物学作用

探讨动态力学应变对体外分离培养的人骨膜细胞的生物学效应。使用Flexercell应力系统，将频率为1Hz、振幅为5％变形、正弦波状力学应变作用于体外培养的人骨膜细胞，在不同时间段检测细胞总蛋白含量、DNA合成量、ALP活性及骨钙素分泌量，并与空白对照组相比较，观察动态力学应变对人骨膜细胞生长、分化指标的影响。结果表明：所施加的动态力学应变对骨膜细胞的生长增殖无明显影响，但显著促进骨膜细胞向成骨细胞的分化。动态力学应变能够促进人骨膜细胞向成骨细胞分化，这可能是其对骨的生物学作用的机制之一。     

#br# rhTGF-β1对SD大鼠MSCs骨向分化的影响及机制研究

 目的：通过观察重组人转化生长因子 β1(rhTGF-β1)对大鼠骨髓间充质干细胞(MSCs)增殖和骨向分化能力的影响,以及对骨形态发生蛋白2(BMP-2)、Smad4及核心结合因子α1(Cbfa1)的作用,阐释其对MSCs骨向分化影响以及可能的作用机制。方法：用全骨髓贴壁法分离、纯化SD大鼠MSCs;用MTT法检测0、5、10、20、40、80和100 μg/L rhTGF-β1对MSCs增殖活性的影响;以碱性磷酸酶(ALP)活性及ALP染色阳性率确定rhTGF-β1的最佳促MSCs骨向分化浓度,并以该浓度对MSCs骨向分化进行干预。按是否添加经典成骨诱导液将实验分为：正常组、经典组、rhTGF-β1组和rhTGF-β1+经典组。通过检测ALP、I型胶原、骨钙素表达和钙化结节的数目,评价各组骨向分化能力;通过检测BMP-2、Smad4和Cbfa1 mRNA的表达,评价各组促MSCs骨向分化的可能作用机制。结果：rhTGF-β1最佳促MSCs增殖浓度为10 μg/L,最佳促MSCs骨向分化浓度为5 μg/L。经典组、rhTGF-β1组和rhTGF-β1+经典组均能促进MSCs骨向分化,刺激BMP-2分泌,并上调Smad4和Cbfa1 mRNA的表达,且rhTGF-β1对MSCs成骨分化的早期、中期效果好,而rhTGF-β1+经典组对MSCs成骨分化的晚期效果更为明显。结论:经典组、rhTGF-β1组和rhTGF-β1+经典组均有促MSCs骨向分化的作用,其机制可能是促进BMP-2的分泌,通过TGF-β超家族/Smads信号通路调控骨向分化。     

重组人骨形成蛋白-2对细胞成骨分化的作用

目的 进一步探讨rhBMP-2的促细胞成骨分化作用，以期找到合适的成骨分化标志作为rhBMP-2的定量活性测定指标。方法 首先表达制备rhBMP-2，用小鼠股部肌袋包埋法进行诱骨活性实验，然后检测rhBMP-2作用后的骨髓基质细胞（MSC）、NIH3T3和C2C12等3种细胞的碱性磷酸酶（ALP）和骨钙素（OC）、细胞总蛋白合成量以及细胞增殖的变化。结果 rhBMP-2具有良好的诱导骨形成的活性，可增加3种细胞的OC含量和蛋白合成量，对MSC的ALP活性变化影响明显，且可促进MSC的增殖，抑制NIH3T3细胞的生长。结论 rhBMP-2具有促进上述细胞向成骨细胞分化的作用；在一定剂量范围内，rhBMP-2的作用与细胞骨钙素合成量的增加呈线性正相关，故定量测定OC的含量基本可反映rhBMP-2的活性。     

骨化三醇通过PI3K/AKT促进BMP9诱导的间充质干细胞成骨分化作用

目的研究骨化三醇(calcitriol)对骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)诱导的间充质干细胞(mesenchymal stem cells,MSCs)成骨分化作用的影响。方法实验分为4组:对照组、calcitriol组、BMP9组、calcitriol联合BMP9组。通过PNPP法检测各组碱性磷酸酶(alkaline phosphatase,ALP)活性;通过RT-PCR和Western blotting方法检测成骨分化标记物骨钙蛋白(osteocalcin,OCN)和骨桥蛋白(osteopontin,OPN)表达变化,同时检测AKT和β-catenin磷酸化水平以及和ALP活性水平;茜素红染色检测矿化结节形成。此外,用原子力显微镜测试MSCs成骨分化过程中细胞形态及细胞弹性模量改变。结果 calcitriol单独作用对MSCs成骨分化过程无明显作用,但是calcitriol可以增强BMP9诱导MSCs的ALP、OCN、OPN表达和矿化结节形成。同时,calcitriol和BMP9作用均不影响细胞弹性模量数值。BMP9和calcitriol联合作用可以增强AKT和β-catenin磷酸化水平,而PI3K抑制剂应用以后可以抑制这种磷酸化变化,并抑制联合作用后的ALP活性。calcitriol作用以后不影响BMP9诱导的BMP/Smad信号通路。结论 calcitriol通过激活PI3K/AKT信号通路从而协同BMP9促进MSCs成骨分化。研究不同调控因子对MSCs成骨分化的作用及机制对于骨质疏松等疾病的治疗和骨组织工程的发展有一定意义。     

成骨诱导:兔骨髓间充质干细胞的形态和功能特征

目的 探讨体外成骨诱导的兔骨髓间充质干细胞(MSCs)的形态和功能特征，为骨组织工程中种子细胞的研究提供依据。方法 选用生长状态良好的传l代的兔MSCs，在体外进行成骨活性诱导，并进行形态学观察和碱性磷酸酶、骨钙紊等功能性指标的检测。结果 体外成骨诱导的兔骨髓间充质干细胞从诱导的第2周即开始表达成骨细胞的活性，到第4周趋于成熟，此过程中性态和功能均具有一定的阶段性。结论 经过体外成骨诱导的兔MSCs表现出典型的成骨细胞阶段性形态特征和功能特征，可以作为骨组织工程的种子细胞。     

Flow Perfusion Culture of Marrow Stromal Cells Seeded on Porous Biphasic Calcium Phosphate Ceramics

Calcium phosphate ceramics have been widely used for filling bone defects to aid in the regeneration of new bone tissue. Addition of osteogenic cells to porous ceramic scaffolds may accelerate the bone repair process. This study demonstrates the feasibility of culturing marrow stromal cells (MSCs) on porous biphasic calcium phosphate ceramic scaffolds in a flow perfusion bioreactor. The flow of medium through the scaffold porosity benefits cell differentiation by enhancing nutrient transport to the scaffold interior and by providing mechanical stimulation to cells in the form of fluid shear. Primary rat MSCs were seeded onto porous ceramic (60% hydroxyapatite, 40% β-tricalcium phosphate) scaffolds, cultured for up to 16 days in static or flow perfusion conditions, and assessed for osteoblastic differentiation. Cells were distributed throughout the entire scaffold by 16 days of flow perfusion culture whereas they were located only along the scaffold perimeter in static culture. At all culture times, flow perfused constructs demonstrated greater osteoblastic differentiation than statically cultured constructs as evidenced by alkaline phosphatase activity, osteopontin secretion into the culture medium, and histological evaluation. These results demonstrate the feasibility and benefit of culturing cell/ceramic constructs in a flow perfusion bioreactor for bone tissue engineering applications.     

A comparative study of shear stresses in collagen-glycosaminoglycan and calcium phosphate scaffolds in bone tissue-engineering bioreactors

The increasing demand for bone grafts, combined with their limited availability and potential risks, has led to much new research in bone tissue engineering. Current strategies of bone tissue engineering commonly use cell-seeded scaffolds and flow perfusion bioreactors to stimulate the cells to produce bone tissue suitable for implantation into the patient's body. The aim of this study was to quantify and compare the wall shear stresses in two bone tissue engineering scaffold types (collagen-glycosaminoglycan (CG) and calcium phosphate) exposed to fluid flow in a perfusion bioreactor. Based on micro-computed tomography images, three-dimensional numerical computational fluid dynamics (CFD) models of the two scaffold types were developed to calculate the wall shear stresses within the scaffolds. For a given flow rate (normalized according to the cross-sectional area of the scaffolds), shear stress was 2.8 times as high in the CG as in the calcium-phosphate scaffold. This is due to the differences in scaffold geometry, particularly the pore size (CG pore size approximately 96 microm, calcium phosphate pore size approximately 350 microm). The numerically obtained results were compared with those from an analytical method that researchers use widely experimentalists to determine perfusion flow rates in bioreactors. Our CFD simulations revealed that the cells in both scaffold types were exposed to a wide range of wall shear stresses throughout the scaffolds and that the analytical method predicted shear stresses 12% to 21% greater than those predicted using the CFD method. This study demonstrated that the wall shear stresses in calcium phosphate scaffolds (745.2 mPa) are approximately 40 times as high as in CG scaffolds (19.4 mPa) when flow rates are applied that have been experimentally used to stimulate the release of prostaglandin E(2). These findings indicate the importance of using accurate computational models to estimate shear stress and determine experimental conditions in perfusion bioreactors for tissue engineering.     

振荡流动对组织工程用灌注式生物 反应器中剪切力分布影响

目的 考察振荡流动以及三维支架孔径和孔隙率对生物反应器内流速和剪切力分布的影响,并根据理论计算结果为脱细胞骨三维支架和灌注式生物反应器制备提出优化方法。方法 针对实验室前期制备的骨组织工程用脱细胞骨三维支架和灌注式生物反应器,将脱细胞骨三维支架简化为各向同性的多孔介质,对生物反应器内的流速和剪切力分布进行理论建模。结果 振荡流作用时,多孔支架材料内速度和达西剪切力呈现一致的变化规律,不同半径处流速和达西剪切力差异减小,有利于在骨组织工程中对种子细胞进行均匀三维培养。提高入口灌流速度可提高平均达西剪切力;增加多孔支架孔径或孔隙率对支架内流速峰值影响不大,但会显著降低平均达西剪切力;提高入口振荡流动振荡频率可降低支架内流速最大峰值,显著减小不同半径处流速的差异。结论 适宜的振荡流易产生利于骨组织工程干细胞所需剪切力,研究结果有望为优化骨组织工程中种子细胞的三维培养方法提供理论指导。     

Flow perfusion culture of human fetal bone cells in large beta-tricalcium phosphate scaffold with controlled architecture

One unsolved problem in bone tissue engineering is how to enable the survival and proliferation of osteoblastic cells in large scaffolds. In this work, large beta-tricalcium phosphate scaffolds with tightly controlled channel architectures were fabricated and a custom-designed perfusion bioreactor was developed. Human fetal bone cells in third passage were seeded onto the scaffolds and cultured in static or flow perfusion conditions for up to 16 days. Compared with nonperfused constructs, flow perfused constructs demonstrated improved cells proliferation and differentiation according to cell viability, glucose consumption, alkaline phosphatase activity, and osteopontin. Moreover, after 16 days of perfusion culture, a homogenous layer composed of cells and mineralized matrix throughout the whole scaffold was observed by scanning electron microscopy and histological study. In contrast, cells were located only along the scaffold perimeter in static culture. These results demonstrated the feasibility and benefit of perfusion culture in conjunction with well-defined three-dimensional environment for large bone graft construction. Porous scaffold with controlled architecture can be a potential tool to evaluate the effects of scaffold specific geometry on fluid flow configuration and cell behavior under perfusion culture.     

Study of osteoblastic cells in a microfluidic environment

Bone tissue engineering consists of culturing osteoblastic cells onto synthetic three-dimensional (3D) porous scaffolds. The organization of bone cells into 3D scaffolds is crucial for ex vivo tissue formation. Diffusional rates of nutrients could be greatly improved by perfusing media through the 3D microporous scaffolds. However, bone cells cultured in vitro are responsive to a variety of different mechanical signals including fluid flow and shear stresses. In this work, we attempt to study osteoblastic cells behaviour cultured within microdevices allowing continuous and homogenous feeding of cells. We have fabricated polydimethylsiloxane PDMS microdevices with a 3D microstructured channel network. Mouse calvarial osteoblastic cells MC3T3-E1 were seeded at 2x10(6)cells/ml and cultured into the microdevices under flow rates of 0, 5, 35 microl/min. Cells attached and proliferated well in the designed microdevices. Cell viability was found around 85% up to 1 to 2 weeks for shear stress value under 5 mPa. The alkaline phosphatase (ALP) activity was enhanced 3- and 7.5-fold inside the microdevices under static and dynamic flow of 5 microl/min as compared to flat static cultures in PDMS coated Petri dishes. Therefore, osteoblastic cells could be successfully cultured inside the microdevices under dynamic conditions and their ALP activity was enhanced. These results are promising for bone cell growth and differentiation as well as future tissue regeneration using larger 3D microfluidic microdevices.     

Design of a Modular Bioreactor to Incorporate Both Perfusion Flow and Hydrostatic Compression for Tissue Engineering Applications

Physiological models have demonstrated that cells undergo a cyclic regimen of hydrostatic compression and fluid shear stress within the lacunar-canalicular porosity of bone. A new modular bioreactor was designed to incorporate both perfusion fluid flow and hydrostatic compression in an effort to more accurately simulate the mechanical loading and stress found in natural bone in vivo. The bioreactor design incorporated custom and off-the-shelf components to produce levels of mechanical stimuli relevant to the physiologic range, including hydrostatic compression exceeding 300 kPa and perfusion shear stress of 0.7 dyne/cm². Preliminary findings indicated that the novel system facilitated the viable growth of cells on discrete tissue engineering scaffolds. The bioreactor has established an experimental platform for ongoing investigation of the interactive effect of perfusion fluid flow and hydrostatic compression on multiple cell types.     

循环灌注接种方法在人脐带间充质干细胞骨组织工程中的应用

背景：课题前期设计了一套模块式三维灌注生物反应器系统,并初步将其应用于大鼠骨髓间充质干细胞接种于三维非纺型聚对苯二甲酸乙二醇酯(polyethylene terephthalate,PET)纤维片状载体的骨组织工程研究中。 目的：应用自制的循环灌注接种系统将传代人脐带间充质干细胞接种至三维无纺布PET片状载体,并与传统的静态接种方法接种的载体进行比较。 方法：培养传代人脐带间充质干细胞,流式细胞仪检测细胞表面标记,将细胞用循环灌注方法(高速率组和低速率组)和静态接种方法接种至三维聚PET无纺布片状载体。 结果与结论：传代细胞形态稳定、活力好,高标达 CD90,CD105,不表达CD14,CD45。循环灌注方法组的接种效率,细胞密度,增殖能力均优于静态接种组。循环灌注高速率组乳酸脱氢酶偏高,并且细胞的延迟期长于静态接种。循环灌注组的碱性磷酸酶活性高于静态接种组。结果表明,循环灌注接种方法更适合间充质干细胞骨组织工程的应用,进一步应用仍需对工程参数进行优化。     

Flow perfusion culture of marrow stromal osteoblasts in titanium fiber mesh

The objective of this study was to evaluate the effect of two cell culture techniques, static and flow perfusion, on the osteogenic expression of rat bone marrow cells seeded into titanium fiber mesh for a period up to 16 days. A cell suspension of rat bone marrow stromal osteoblasts (5 x 10(5) cells/300 microL) was seeded into the mesh material. Thereafter, the constructs were cultured under static conditions or in a flow perfusion system for 4, 8, and 16 days. To evaluate cellular proliferation and differentiation, constructs were examined for DNA, calcium content, and alkaline phosphatase activity. Samples were also examined with scanning electron microscopy (SEM) and plastic-embedded histological sections. Results showed an increase in DNA from day 4 to day 8 for the flow perfusion system. At day 8, a significant enhancement in DNA content was observed for flow perfusion culture compared with static culture conditions, but similar cell numbers were found for each culture system at 16 days. Calcium measurements showed a large increase in calcium content of the meshes subjected to flow perfusion at day 16. The SEM examination revealed that the 16-day samples subjected to flow perfusion culture were completely covered with layers of cells and mineralized matrix. In addition, this matrix extended deep into the scaffolds. In contrast, meshes cultured under static conditions had only a thin sheet of matrix present on the upper surface of the meshes. Evaluation of the light microscopy sections confirmed the SEM observations. On the basis of our results, we conclude that a flow perfusion system can enhance the early proliferation, differentiation, and mineralized matrix production of bone marrow stromal osteoblasts seeded in titanium fiber mesh.     

Mechano-active tissue engineering of vascular smooth muscle using pulsatile perfusion bioreactors and elastic PLCL scaffolds

Blood vessels are subjected in vivo to mechanical forces in a form of radial distention, encompassing cyclic mechanical strain due to the pulsatile nature of blood flow. Vascular smooth muscle (VSM) tissues engineered in vitro with a conventional tissue engineering technique may not be functional, because vascular smooth muscle cells (VSMCs) cultured in vitro typically revert from a contractile phenotype to a synthetic phenotype. In this study, we hypothesized that pulsatile strain and shear stress stimulate VSM tissue development and induce VSMCs to retain the differentiated phenotype in VSM engineering in vitro. To test the hypothesis, rabbit aortic smooth muscle cells (SMCs) were seeded onto rubber-like elastic, three-dimensional PLCL [poly(lactide-co-caprolactone), 50:50] scaffolds and subjected to pulsatile strain and shear stress by culturing them in pulsatile perfusion bioreactors for up to 8 weeks. As control experiments, VSMCs were cultured on PLCL scaffolds statically. The pulsatile strain and shear stress enhanced the VSMCs proliferation and collagen production. In addition, a significant cell alignment in a direction radial to the distending direction was observed in VSM tissues exposed to radial distention, which is similar to that of native VSM tissues in vivo, whereas VSMs in VSM tissues engineered in the static condition randomly aligned. Importantly, the expression of SM alpha-actin, a differentiated phenotype of SMCs, was upregulated by 2.5-fold in VSM tissues engineered under the mechano-active condition, compared to VSM tissues engineered in the static condition. This study demonstrates that tissue engineering of VSM tissues in vitro by using pulsatile perfusion bioreactors and elastic PLCL scaffolds leads to the enhancement of tissue development and the retention of differentiated cell phenotype.     

Deformation simulation of cells seeded on a collagen-GAG scaffold in a flow perfusion bioreactor using a sequential 3D CFD-elastostatics model

Tissue-engineered bone shows promise in meeting the huge demand for bone grafts caused by up to 4 million bone replacement procedures per year, worldwide. State-of-the-art bone tissue engineering strategies use flow perfusion bioreactors to apply biophysical stimuli to cells seeded on scaffolds and to grow tissue suitable for implantation into the patient's body. The aim of this study was to quantify the deformation of cells seeded on a collagen-GAG scaffold which was perfused by culture medium inside a flow perfusion bioreactor. Using a μCT scan of an unseeded collagen-GAG scaffold, a sequential 3D CFD-deformation model was developed. The wall shear stress and the hydrostatic wall pressure acting on the cells were computed through the use of a CFD simulation and fed into a linear elastostatics model in order to calculate the deformation of the cells. The model used numerically seeded cells of two common morphologies where cells are either attached flatly on the scaffold wall or bridging two struts of the scaffold. Our study showed that the displacement of the cells is primarily determined by the cell morphology. Although cells of both attachment profiles were subjected to the same mechanical load, cells bridging two struts experienced a deformation up to 500 times higher than cells only attached to one strut. As the scaffold's pore size determines both the mechanical load and the type of attachment, the design of an optimal scaffold must take into account the interplay of these two features and requires a design process that optimizes both parameters at the same time.