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。     

力学应变对体外培养动物细胞的影响

介绍了生物力学领域中细胞生长、增殖与分泌方面的实验研究进展.着重介绍了基底加载实验中不同环境对细胞分裂与增殖的作用;力学载荷对细胞黏附及生长的影响;力学应变对体外培养成骨细胞增殖与分泌的影响;流体环境对细胞生长增殖的作用.     

大鼠真皮间充质干细胞的体外长期培养及胶原海绵对其生长的影响

从新生大鼠真皮细胞中分离多能干细胞，体外长期传代培养，观察细胞形态和超微结构的变化。检测细胞增殖和分化能力的改变及胶原基质对细胞生长的影响。揭示体外长期传代培养对大鼠真皮间充质干细胞生物学性状的影响，结果表明；大鼠真皮组织中的间充质干细胞体外培养至180d，传至25代的细胞仍具有干细胞的特征；形态的均一，细胞核结构原始，细胞器不发达；体外增殖迅速，保持了向成骨细胞和软骨细胞分化的潜能，胶原基质成分可促进细胞的生长，而胶原海绵支架更利于细胞的三维生长。结论是体外长期传代培养后大鼠真皮间充质干细胞仍保持良好的干细胞特性，为真皮间充质干细胞的深入研究与应用提供了实验依据。     

骨组织工程中的应力与生长

力学环境是骨组织所处的重要微环境之一，应力(应变)可促进细胞增殖，引起细胞骨架重排及细胞形态的变化，加速细胞基质矿化，刺激细胞因子及骨代谢激素的分泌．从而调节骨代谢、促进骨组织的生长与重建。但体外培养时，应力(应变)水平和细胞反应程度间的关系及细胞间信号转导等的机制还不是十分清楚，而这些都是体外培养组织所必须解决的问题，因此，本综述了应力对骨组织及成骨细胞、软骨细胞的影响与机理，对今后研究应力对骨组织工程化培养的影响具有十分重的意义。     

力学刺激对3月龄骨质疏松大鼠成骨细胞增殖与合成功能的影响

探讨周期性双轴力学应变对成骨细胞增殖与分化合成功能的影响。将正常 3月龄雌性 SD大鼠和骨质疏松大鼠颅顶骨分离的成骨细胞分别在含 10 %胎牛血清的 F- 12培养液中培养 ,并接种在双轴力学应变装置中。当细胞生长至亚融合状态 (Subconfluence) ,给细胞施加力学刺激 ,频率为 1Hz,力学刺激分别为 4 0 0、10 0 0、4 0 0 0μ strain;作用时间分别为每天 30 m in,2、4、8h,共加载两天。以未受力学刺激的细胞为对照组 ,受力学刺激的细胞为实验组 ,并进行比较。采用流式细胞技术测定细胞增殖变化 ;采用同位素标记方法检测成骨细胞骨钙素、I型胶原 C端前肽 (PICP)和总蛋白的分泌量。结果表明 :1)在静态培养条件下 ,3ovx组与 3control组比较 ,其细胞功能活性无明显变化 ,但 3ovx组大鼠成骨细胞增殖活性明显增高 ,这与绝经后骨质疏松骨代谢的高转换率相一致。 2 )4 0 0、10 0 0 μstrain力学刺激可以促进 3control组成骨细胞 I型胶原、骨钙素和总蛋白的分泌量增加 ,促进成骨细胞的分化成熟 ;在 10 0 0μstrain力学刺激下 ,成骨细胞合成骨基质的能力增加最为明显。同时 ,在 4 0 0、10 0 0μstrain力学刺激的初期也可以促进成骨细胞的增殖 ,而促进成骨细胞的分化成熟的作用大于促进细胞增殖的作用。 3)在4 0 0 0 μ     

动态力学因素在组织工程化血管构建中的作用及其研究进展

动态培养环境是成功构建具有生理功能和力学性能的工程化组织的重要因素之一.越来越多的研究证实,接近生物体内生理环境的动态力学条件对细胞在体外支架上的黏附、增殖、分化以及细胞外基质的重建等一系列生长行为均有重要的作用,通过模拟和优化动态培养条件,可以促进细胞的生长和细胞外基质的重建,从而提高组织工程化血管的性能.本文对近年来在组织工程化血管动态培养环境方面的主要研究进展做了综合评述,重点讨论了不同力学环境对血管细胞生长和细胞外基质重建的影响,以及生物反应器在组织工程化血管构建中的作用,并通过组织工程化血管的生物学性能分析,讨论了动态力学培养环境对血管组织的形成及其力学性能的作用.     

微振动在骨髓间充质干细胞成骨向分化中的作用机制研究进展

微振动是指系统相对平衡位置幅度很小的周期性偏离,而高频率、低振幅的微振动(low-magnitude high-frequency vibration, LMHFV)对骨骼系统细胞的作用力与肌肉运动时对骨骼产生的力学刺激相似。骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)作为力学敏感细胞,存在于骨髓基质,具有多向分化潜能。在体外适当机械刺激下,BMSCs增殖、分化等生物学特性发生功能性变化,对力学刺激做出适应性应答。LMHFV可促进BMSCs向成骨细胞分化,探明其机制有助于将微振动应用于骨质疏松、骨折、成骨不全症、肥胖症等疾病的治疗以及正畸牙移动的加速等方面。综述微振动对BMSCs成骨向分化的影响以及可能的作用机制,为研究微振动刺激下BMSCs的力学生物学改变提供思路。     

骨组织工程中的应力与生长

力学环境是骨组织所处的重要微环境之一 ,应力 (应变 )可促进细胞增殖 ,引起细胞骨架重排及细胞形态的变化 ,加速细胞基质矿化 ,刺激细胞因子及骨代谢激素的分泌 ,从而调节骨代谢、促进骨组织的生长与重建。但体外培养时 ,应力(应变 )水平和细胞反应程度间的关系及细胞间信号转导等的机制还不是十分清楚 ,而这些都是体外培养组织所必须解决的问题 ,因此 ,本文综述了应力对骨组织及成骨细胞、软骨细胞的影响与机理 ,对今后研究应力对骨组织工程化培养的影响具有十分重的意义     

人骨髓间质干细胞体外扩增和向成骨细胞分化的实验研究

骨髓间质干细胞（mesenchymal stem cell,MSCs）是一类存在于骨髓中的具有多向分化潜能的干细胞，在体外不仅可分化为间质类细胞，而且可以分化为非间质细胞。本研究探讨人骨髓间质干细胞的体外分离纯化、培养扩增和向成骨细胞诱导分化的条件。利用密度为1．073g/ml的Percoll分离骨髓的单个核细胞，以含10％胎牛血清的低糖DMEM培养基培养与扩增MSCs，细胞纯度可达95％左右。取第二代和第三代的MSCs，以含有不同浓度的抗坏血酸、β－磷酸甘油、地塞米松的诱导培养基向成骨细胞诱导，诱导向的细胞呈现典型的成骨细胞样改变，免疫组化技术显示其I型胶原染色阳性，ALP染色阳性，表明MSCs在体外具有向成骨细胞分化的潜力。     

影响骨髓间充质干细胞成骨分化的力学信号传导机制研究进展

骨髓间充质干细胞(bone marrow stem cell,BMSC)是一种具有多向分化潜能的成体干细胞,适量的力学刺激能促进BMSC向成骨细胞分化。近几年来,一些学者利用力学刺激作用于体外培养的BMSC,促进其向成骨细胞分化,并且对其诱导分化的机制进行了大量研究。尽管这一机制目前尚不十分清楚,但是已有的研究表明,多条信号通路参与了该力学信号传导。本文就国内外近几年来关于力学信号影响BMSC成骨分化的信号转导机制研究进展做一综述。     

Role of Dynamic Loading on Early Stage of Bone Fracture Healing

After fracture, mesenchymal stem cells (MSCs) and growth factors migrate into the fracture callus to exert their biological actions. Previous studies have indicated that dynamic loading induced tissue deformation and interstitial fluid flow could produce a biomechanical environment which significantly affects the healing outcomes. However, the fundamental relationship between the various loading regimes and different healing outcomes has not still been fully understood. In this study, we present an integrated computational model to investigate the effect of dynamic loading on early stage of bone fracture healing. The model takes into account cell and growth factor transport under dynamic loading, and mechanical stimuli mediated MSC differentiation and tissue production. The developed model was firstly validated by the available experimental data, and then implemented to identify the loading regimes that produce the optimal healing outcomes. Our results demonstrated that dynamic loading enhances MSC and growth factor transport in a spatially dependent manner. For example, compared to free diffusion, dynamic loading could significantly increase MSCs concentration in endosteal zone; and chondrogenic growth factors in both cortical and periosteal zones in callus. Furthermore, there could be an optimal dynamic loading regime (e.g. 10% strain at 1 Hz) which could potentially significant enhance endochondral ossification.     

Molecular Variations Related to the Regional Differences in Periosteal Growth at the Mandibular Ramus

Periosteal growth at human mandibular ramus is characterized by bone apposition at the posterior border and resorption at the anterior border. Molecular control of this regional variation is unclear. This study examined the expression of several molecules involved in bone apposition/resorption at these regions in vivo and in vitro. By using growing pigs as a model, the periosteal growth was assessed at the mandibular ramus by vital staining and histological observations. In parallel, periosteal tissues were harvested and pulverized for RNA and protein extraction. Periosteal cells were also isolated, expanded in osteogenic media, and subjected to a single dose of dynamic tensile strain (0, 5, or 10% magnitude at 0.5 Hz) to examine their responses to mechanical loading. Real‐time RT‐PCR and Western blot analyses were used to examine mRNA and protein expression from periosteal tissues and cultured cells. Histological observation confirmed an anterior‐resorption/posterior‐apposition pattern in the pig mandibular ramus. Both in vivo tissue and in vitro cells demonstrated greater mRNA expression of receptor activator of NF‐κB ligand (RANKL)/osteoprotegerin (OPG) ratio and bone morphogenetic protein 2 (BMP2) at the anterior region, while OPG expression at the anterior region was lower than the posterior region. In response to the application of a single dose of dynamic tensile strain, cultured periosteal cells appeared to change the expression profile of osteogenic markers but not that of RANKL/OPG and BMP2. These findings suggest that the unique regional variation of periosteal activity at the mandibular ramus is regulated by a differential expression of RANKL/OPG ratio (likely through differential induction of OPG) and BMP2. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Dynamic mechanical stimulations induce anisotropy and improve the tensile properties of engineered tissues produced without exogenous scaffolding

Mechanical strength and the production of extracellular matrix (ECM) are essential characteristics for engineered tissues designed to repair and replace connective tissues that are subject to stress and strain. In this study, dynamic mechanical stimulation (DMS) was investigated as a method to improve the mechanical properties of engineered tissues produced without the use of an exogenous scaffold, referred to as the self-assembly approach. This method, based exclusively on the use of human cells without any exogenous scaffolding, allows for the production of a tissue sheet comprised of cells and ECM components synthesized by dermal fibroblasts in vitro. A bioreactor chamber was designed to apply cyclic strain to engineered tissues in order to determine if dynamic culture had an impact on their mechanical properties and ECM organization. Fibroblasts were cultured in the presence of ascorbic acid for 35 days to promote ECM production and allow the formation of a tissue sheet. This sheet was grown on a custom-built anchoring system allowing for easy manipulation and fixation of the tissue in the bioreactor. Following the 35 day period, tissues were maintained for 3 days in static culture (SC), or subjected either to a static mechanical stimulation of 10% strain, or a dynamic DMS with a duty cycle of 10% uniaxial cyclic strain at 1Hz. ECM was characterized by histology, immunofluorescence labeling and Western blotting. Both static and dynamic mechanical stimulation induced the alignment of assessed cytoskeletal proteins and ECM components parallel to the axis of applied strain and increased the ECM content of the tissues compared to SC. Measurement of the tensile mechanical properties revealed that mechanical stimulation significantly increases both the ultimate tensile strength and tensile modulus of the engineered tissues when compared to the non-stimulated control. Moreover, we demonstrated that cyclic strain significantly increases these parameters when compared to a static-loading stimulation and that mechanical stimulation contributes to the establishment of anisotropy in the structural and mechanical properties of self-assembled tissue sheets.     

Temporal evolution of skeletal regenerated tissue: what can mechanical investigation add to biological?

The objective here was to experimentally characterize the temporal evolution of the structural and mechanical properties of large volume immature regenerated tissues. We studied these evolving tissues from their genesis in controlled mechanical conditions. We developed an animal model based on the periosteal properties leading to unloaded regenerated skeletal tissue. To characterize the temporal evolution of mechanical properties, we carried out indentation tests coupled with macroscopic examinations and histological studies. This combined methodology yielded a range of information on osteogenesis at different scales: macroscopic by simple observation, mesoscopic by indentation test and microscopic by histological study. Results allowed us to identify different periods, providing a link between biological changes and material property evolution in bone tissue regeneration. The regenerated tissue evolves from a viscous, homogeneous, soft material to a heterogeneous stiffer material endowed with a lower viscosity. From a biological point of view, cell organization progresses from a proliferated cell clot to a mature structure closer to that of the bone. During the first 7 days, mechanical and biological results revealed the same evolution: first, the regenerated tissue grew, then, differentiated into an osteochondral tissue and finally calcification began. While our biological results confirm those of other studies, our mechanical results provide the first experimental mechanical characterization by reduced Young’s modulus of such tissue.     

Cell mechanics of alveolar epithelial cells (AECs) and macrophages (AMs)

Cell mechanics provides an integrated view of many biological phenomena which are intimately related to cell structure and function. Because breathing constitutes a sustained motion synonymous with life, pulmonary cells are normally designed to support permanent cyclic stretch without breaking, while receiving mechanical cues from their environment. The authors study the mechanical responses of alveolar cells, namely epithelial cells and macrophages, exposed to well-controlled mechanical stress in order to understand pulmonary cell response and function. They discuss the principle, advantages and limits of a cytoskeleton-specific micromanipulation technique, magnetic bead twisting cytometry, potentially applicable in vivo. They also compare the pertinence of various models (e.g., rheological; power law) used to extract cell mechanical properties and discuss cell stress/strain hardening properties and cell dynamic response in relation to the structural tensegrity model. Overall, alveolar cells provide a pertinent model to study the biological processes governing cellular response to controlled stress or strain.     

Effect of Strain Magnitude on the Tissue Properties of Engineered Cardiovascular Constructs

Mechanical loading is a powerful regulator of tissue properties in engineered cardiovascular tissues. To ultimately regulate the biochemical processes, it is essential to quantify the effect of mechanical loading on the properties of engineered cardiovascular constructs. In this study the Flexercell FX-4000T (Flexcell Int. Corp., USA) straining system was modified to simultaneously apply various strain magnitudes to individual samples during one experiment. In addition, porous polyglycolic acid (PGA) scaffolds, coated with poly-4-hydroxybutyrate (P4HB), were partially embedded in a silicone layer to allow long-term uniaxial cyclic mechanical straining of cardiovascular engineered constructs. The constructs were subjected to two different strain magnitudes and showed differences in biochemical properties, mechanical properties and organization of the microstructure compared to the unstrained constructs. The results suggest that when the tissues are exposed to prolonged mechanical stimulation, the production of collagen with a higher fraction of crosslinks is induced. However, straining with a large strain magnitude resulted in a negative effect on the mechanical properties of the tissue. In addition, dynamic straining induced a different alignment of cells and collagen in the superficial layers compared to the deeper layers of the construct. The presented model system can be used to systematically optimize culture protocols for engineered cardiovascular tissues.     

Simulation of cell differentiation in fracture healing: mechanically loaded composite scaffolds in a novel bioreactor system

Cell differentiation during bone healing following a fracture is influenced by various biological and mechanical factors. We introduce a method for the examination of cell and tissue differentiation simulating a fracture gap in vitro. A closed bioreactor system allows the imitation of the biological, mechanical, and biochemical conditions in vitro. The initial hematoma formed in a fracture is simulated with a mixed construct composed of lyophilized cancellous bone and a fibrin matrix in a sandwich configuration. The construct may be loaded with osteoprogenitor cells. Exemplarily, constructs were loaded with rabbit periosteal cells and cultivated under mechanical loading with 7 kPa at 0.05 Hz for up to two weeks. During the observation period, cell morphology and correlating protein synthesis changed under mechanical stimulation. Cell differentiation differed between the various regions of the constructs. The periosteal cells were arranged perpendicularly to the mechanical loading and differentiated to osteoblastic forms with rising collagen type I synthesis, constant alkaline phosphatase activity, and initiation of the calcification of the extracellular matrix. The observed pattern of cell and tissue differentiation was similar to the one seen in the early phase of bone healing. In conclusion, the presented method allows simulation of cell and tissue differentiation during the early phase of fracture healing. It could serve as an in vitro model for the examination of mechanical and pharmacological influences during the early phase of bone healing on a cellular level.     

Investigation of osteogenic activity of primary rabbit periosteal cells stimulated by multi-axial tensile strain

Periosteum–derived cells was indicated to respond to mechanical force and have stem cell potential capable of differentiating into multiple tissue. Investigation of osteogenic activity under mechanical stimulation is important to understand the therapeutic conditions of fracture healing. In this work, a cell culture platform was developed for respectively providing isotropic and anisotropic axial strain. Primary rabbit periosteal cells were isolated and cultured in the chamber. Multi-axial tensile strain was received and osteogenic activity was investigated by mRNA expressions of CBFA1 and OPN. The highest mRNA expression was found in moderate strain (5-8%) under anisotropic axial strain. These results provided important foundation for further in vivo studies and development of tailor-made stretching rehabilitation equipment.     

Cyclic biaxial strain affects U937 macrophage-like morphology and enzymatic activities

As monocytes migrate to the site of a foreign body and differentiate into mature monocyte-derived macrophages (MDMs), the cells undergo a morphological transformation that involves mechanical stimulation via membrane stretch. Because the site of many cardiovascular implant devices includes substrates that are also undergoing mechanical change, it is of interest to assess the effect of such dynamic conditions on cellular-biomaterial responses. This study investigated the influence of cyclic (0.25 Hz) biaxial strain (maximum 10% amplitude) on human U937 macrophage-like cells cultured on a flexible siloxane membrane. Cell attachment was unaffected by the strain but total protein levels were significantly higher in stimulated cells. Intracellular esterase and released acid phosphatase activities were elevated by dynamic loading in addition to a strain-induced increase of monocyte-specific esterase protein as demonstrated by immunoblotting analysis. The morphology of static cells changed with cyclic strain from a round cell shape to an irregular, spread phenotype with a progressive reorganization of filamentous actin. The focal adhesion protein vinculin showed distinct reorganization in structure going from a well-defined arrangement in static cells to a diffuse staining pattern in mechano-stimulated cells. This study has demonstrated that U937 cells respond to cyclic deformation with an augmentation of select enzymatic activities that have been identified as being important in polymer biodegradation processes, as well as morphological changes, which may be characteristic of mechanical stress-induced cell activation.