骨组织工程化培养中成骨细胞力学响应的初步研究

由于骨具有支撑、保护、运动的功能,是承力器官,力学环境对骨组织的发生、发展有着十分重要的影响。活体研究表明:载荷可促进成骨细胞的增殖、分化和细胞外基质的分泌,及骨衬细胞的生物学活动;力学因素显著影响骨细胞生物学活动,包括骨细胞的凋亡;力学环境引起骨内细胞的协同反应,对骨组织变化起整合作用。由于骨组织力学环境如此重要,力学环境就有可能是工程化骨培养中的必要因素。由此,笔者尝试建立三维立体条件下成骨细胞力学响应的模型,研究骨内细胞的力学反应。模型包括支架材料、种子细胞和有力学作用的培养环境。支架材料除具有一般生物支架材料的要求外,还应与天然松质骨有相似的结构和力学性能,这里包括生物衍生松质骨、珊瑚支架等。种子细胞可采用乳鼠分离出的成骨细胞或成骨细胞系,接种在支架上进行培养。载荷直接施加在复合体上,复合体的表观应变被精确控制,可形成与骨活体内相似的力学环境,其中载荷可采用不同形状的波形,如正弦波、方波等。加载应变可达到0～10 000,频率0～100 Hz,大大包括了活体骨组织所受的力学环境。载荷形成细胞的力学环境是以支架材料的表观应变衡量,这正对应活体骨研究的力学指标。     

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     

生物衍生骨在骨组织工程研究中的应用

支架材料的选取是骨组织工程研究的关键 ,生物衍生骨具有较好的生物相容性和材料界面 ,三维立体孔隙 -网架合理 ,可塑性强 ,可降解 ,并具备一定的力学强度 ,兼备良好的骨传导及一定的骨诱导能力。可作为种子细胞的支架材料应用于骨组织工程研究。     

生物衍生骨在骨组织工程研究中的应用

支架材料的选取是骨组织工程研究的关键，生物衍生骨具有较好的生物相容性和材料界面，三维立体孔隙-网架合理，可塑性强，可降解，并具备一定的力学强度，兼备良好的骨传导及一定的骨诱导能力。可作为种子细胞的支架材料应用于骨组织工程研究。     

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。     

材料和孔隙率对可降解支架内骨形成的影响

目的研究可降解支架植入人体后的骨修复,不同材料和孔隙率对支架内骨形成的影响。方法根据骨折愈合自然反应机理,运用有限元方法,结合支架几何结构,搭建以材料降解曲线和骨重建控制方程为基础的计算耦合模型。通过这一平台,选择5种材料、4种孔隙率的支架代表性体积元进行计算模拟分析,并通过骨密度和支架最大应力反映这一动态过程。结果材料弹性模量对支架内骨组织生长的影响较大,材料弹性模量越小,骨形成量越大,但会对支架的力学性能造成较大影响。较高孔隙率的支架刚度小,能够更好地促进骨组织形成,但同时也会破坏支架的力学稳定性。结论根据不同年龄、性别和部位骨组织的性能需求,为可降解多孔骨支架的材料和孔隙率选择、结构设计以及临床应用提供个性化参考和计算依据。     

成骨细胞培养在骨组织工程和运动领域的研究进展

背景：成骨细胞在不同的培养环境、细胞因子刺激下增殖、分化及功能不同。 目的：总结成骨细胞体外培养技术与成骨细胞增殖速度的研究进展。 方法：检索1990/2011PubMed数据及万方数据库有关运动、成骨细胞培养和骨组织工程等方面的文献,英文检索词为“exercise, Osteoblast raise method”,中文检索词为“运动,成骨细胞,骨组织工程”。排除与研究目的无关和内容重复者。保留29篇文献做进一步分析。 结果与结论：骨组织工程构建中种子细胞的选择方式、支架材料的不同形式、细胞因子、培养环境、力学因素都对成骨细胞的培养有重要影响。构建骨组织工程中成骨细胞在不同的培养环境、细胞因子刺激下会有不同的增殖能力和分化结果。成骨细胞的增殖速度与旋转壁式生物反应器提供的低剪力环境、适宜的细胞因子、细胞支架的选择密切相关。 关键词：成骨细胞;培养;骨组织工程;运动;细胞增殖 doi:10.3969/j.issn.1673-8225.2012.11.033     

墨鱼骨转化羟基磷灰石制备及细胞相容性

背景：从自然界中寻找骨移植替代材料来充填骨缺损是目前骨组织工程支架是研究的热点。目的：探索墨鱼骨转化羟基磷灰石三维支架及其作为骨组织工程支架材料的可行性。方法：以墨鱼骨为原料,与磷酸氢二铵在特定条件下发生水热反应,利用X射线衍射、傅里叶变换红外光谱、X射线光电子能谱法和扫描电镜分别对水热反应产物进行表征。结果与结论：墨鱼骨水热反应产物为羟基磷灰石。墨鱼骨转化羟基磷灰石保持了良好的文石相结构,将其与MG63细胞体外培养并考察其细胞相容性,证实墨鱼骨转化羟基磷灰石材料对细胞的生长无明显影响且无明显毒性,不影响细胞的正常增殖,可作为新型骨组织工程细胞支架材料。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

骨组织工程用聚磷酸钙的结构和性能的研究

多孔聚磷酸钙（Calcium polyphosphate，CPP）生物陶瓷是一种新型的骨组织工程支架材料，国外有研究表明聚磷酸钙作为骨组织工程支架材料具有良好的生物相容性及可降解性。通过对原料煅烧过程的研究，探索了聚合度的计算方法，制备出不同聚合度的材料。通过对材料烧结温度的试验，制得了不同晶型的聚磷酸钙多孔材料。在Tris—HCl缓冲液中进行的体外降解实验表明，CPP多孔支架材料是可控降解的，不同聚合度和晶型的支架材料力学强度和降解性能不同。随着聚合度的增加，材料的力学强度增大，降解速率变小；非晶态的CPP17d就可以完全降解，7晶型的CPP25d可完全降解，β和α晶型的CPP降解缓慢，30d分别降解了大约12％和5％。因此聚磷酸钙是一种很有前途的骨组织工程支架材料，凭借其独特的无机聚合物结构及降解性能，有望实现可控速率的降解。     

冠脉可降解支架介入的血管力学特性数值模拟与实验研究

目的探究可降解支架在动态降解过程中,血管应力变化对内皮功能恢复以及血管再狭窄抑制作用的影响。方法拟合超弹性血管本构关系的材料参数,通过数值模拟计算支架介入前以及动态降解过程中血管内膜应力分布;采用体外培养实验,设置硅胶腔体拉伸率分别为0%、5%、10%、15%,模拟不同降解阶段的力学环境,探究不同拉伸率下对内皮细胞生长状态的影响。结果支架完全降解后,血管内膜周向应力、应变恢复到0.137 MPa、5.5%,与支架介入前生理参数(0.122 MPa、4.8%)接近;体外实验表明,在0.1 MPa周向应力、5%应变条件下,内皮细胞成活率最高,能实现全部黏附生长。结论支架随降解进程的发生,内膜周向应力、应变恢复到接近生理参数范围,促进内皮细胞的生长,完整内皮功能的维持有效抑制了血管再狭窄进程。结果可为研究冠脉介入治疗血管再狭窄问题提供理论依据和实验平台。     

Using dihydropyridine-release strategies to enhance load effects in engineered human bone constructs

We report on the development of a novel biodegradable scaffold capable of enhancing mechanical signals for tissue-engineering applications. It has been shown that mechanotransduction enhances bone formation in vitro and in vivo; in tissue-engineering applications, this phenomenon is exploited through the use of mechanical bioreactors to generate bone tissue. The dihydropyridine agonist Bay K8644 (Bay) acts to increase the opening time of mechanosensitive voltage-operated calcium channels (VOCCs), specifi- cally L-type VOCCs, which are known to play a fundamental role in the early mediation of mechanotransduction. We have produced porous 3-dimensional, Bay-encapsulated biodegradable poly(L-lactide) acid scaffolds using a solvent-casting and salt-leaching technique. The effects of the released Bay on osteoid production and mineralization in human bone cell-seeded constructs following incubation in a perfusion-compression bioreactor in vitro was investigated using Western blotting techniques and a calcium assay protocol developed in our lab. Our newly developed scaffolds act by slowly releasing the calcium channel agonist Bay K8644 as observed using ultraviolet spectroscopy, maintaining the open state of mechanosensitive VOCCs responding to load, which augments the load signal at sites of strain across the scaffold. Our results demonstrate that, in the presence of physiological loading regimes in vitro, release of Bay enhances collagen I protein production and osteoid calcification more than non-Bay control constructs do. Osteopontin and alpha2delta1 VOCC subunit protein levels were also higher as a result of perfusion-compression conditioning. These results indicate that Bay-encapsulated scaffolds can be used in the presence of load to enhance the production of load-bearing engineered tissue.     

Hydrophobicity as a design criterion for polymer scaffolds in bone tissue engineering

Porous polymeric scaffolds play a key role in most tissue-engineering strategies. A series of non-degrading porous scaffolds was prepared, based on bulk-copolymerisation of 1-vinyl-2-pyrrolidinone (NVP) and n-butyl methacrylate (BMA), followed by a particulate-leaching step to generate porosity. Biocompatibility of these scaffolds was evaluated in vitro and in vivo. Furthermore, the scaffold materials were studied using the so-called demineralised bone matrix (DBM) as an evaluation system in vivo. The DBM, which is essentially a part of a rat femoral bone after processing with mineral acid, provides a suitable environment for ectopic bone formation, provided that the cavity of the DBM is filled with bone marrow prior to subcutaneous implantation in the thoracic region of rats. Various scaffold materials, differing with respect to composition and, hence, hydrophilicity, were introduced into the centre of DBMs. The ends were closed with rat bone marrow, and ectopic bone formation was monitored after 4, 6, and 8 weeks, both through X-ray microradiography and histology. The 50:50 scaffold particles were found to readily accommodate formation of bone tissue within their pores, whereas this was much less the case for the more hydrophilic 70:30 counterpart scaffolds. New healthy bone tissue was encountered inside the pores of the 50:50 scaffold material, not only at the periphery of the constructs but also in the center. Active osteoblast cells were found at the bone-biomaterial interfaces. These data indicate that the hydrophobicity of the biomaterial is, most likely, an important design criterion for polymeric scaffolds which should promote the healing of bone defects. Furthermore, it is argued that stable, non-degrading porous biomaterials, like those used in this study, provide an important tool to expand our comprehension of the role of biomaterials in scaffold-based tissue engineering approaches.     

Ex Vivo bone formation in bovine trabecular bone cultured in a dynamic 3D bioreactor is enhanced by compressive mechanical strain

Our aim was to test cell and trabecular responses to mechanical loading in vitro in a tissue bone explant culture model. We used a new three-dimensional culture model, the ZetOS system, which provides the ability to exert cyclic compression on cancellous bone cylinders (bovine sternum) cultured in forced flow circumfusion chambers, and allows to assess mechanical parameters of the cultivated samples. We evaluated bone cellular parameters through osteocyte viability test, gene and protein expression, and histomorphometric bone formation rate, in nonloaded versus loaded samples. The microarchitecture of bone cores was appraised by in vivo micro-CT imaging. After 3 weeks, the samples receiving daily cyclic compression exhibited increased osteoblast differentiation and activity associated with thicker, more plate-like-shaped trabeculae and higher Young's modulus and ultimate force as compared to unloaded samples. Osteoclast activity was not affected by mechanical strain, although it was responsive to drug treatments (retinoic acid and bisphosphonate) during the first 2 weeks of culture. Thus, in the ZetOS apparatus, we reproduce in vitro the osteogenic effects of mechanical strain known in vivo, making this system a unique and an essential laboratory aid for ex vivo testing of lamellar bone remodeling.     

一种体外贴壁细胞应变加载装置的研制

目的开发一套新型的应变加载装置,用于贴壁细胞力学生物学研究。方法该装置基于基底形变加载技术,采用可控制编程器驱动步进器,引起硅橡胶小室变形,实现多单元大应变的细胞加载;研制该装置,检测机械性能;建立硅橡胶小室的三维模型,利用有限元技术对硅橡胶小室进行仿真,分析该小室的应变场均匀性问题;采用该装置对骨髓间充质干细胞(bone marrow stromal cells,BMSCs)加载5%机械应变,频率0.5 Hz,2 h/d,持续5 d,并在倒置显微镜下观察细胞形态的变化。结果所研制的适用于体外细胞加载装置可对3组细胞加载基底实现最大至50%机械单向应变;在10%应变范围内,硅橡胶小室底部的均匀应变场面积占比保持在50%以上,保证了细胞受力均匀; BMSCs形态发生明显变化,排列方向趋于垂直主应变加载方向。结论该装置运行可靠,应变范围宽,频率可调,操作方便,可同时对多组细胞培养基底进行应变加载,为细胞力学生物学研究提供了便利条件。     

Three-dimensional,bioactive, biodegradable,polymer-bioactive glass composite scaffolds with improved mechanical properties support collagen synthesis and mineralization of human osteoblast-like cells in vitro

In the past decade, tissue engineering-based bone grafting has emerged as a viable alternative to biological and synthetic grafts. The biomaterial component is a critical determinant of the ultimate success of the tissue-engineered graft. Because no single existing material possesses all the necessary properties required in an ideal bone graft, our approach has been to develop a three dimensional (3-D), porous composite of polylactide-co-glycolide (PLAGA) and 45S5 bioactive glass (BG) that is biodegradable, bioactive, and suitable as a scaffold for bone tissue engineering (PLAGA-BG composite). The objectives of this study were to examine the mechanical properties of a PLAGA-BG matrix, to evaluate the response of human osteoblast-like cells to the PLAGA-BG composite, and to evaluate the ability of the composite to form a surface calcium phosphate layer in vitro. Structural and mechanical properties of PLAGA-BG were measured, and the formation of a surface calcium phosphate layer was evaluated by surface analysis methods. The growth and differentiation of human osteoblast-like cells on PLAGA-BG were also examined. A hypothesis was that the combination of PLAGA with BG would result in a biocompatible and bioactive composite, capable of supporting osteoblast adhesion, growth and differentiation, with mechanical properties superior to PLAGA alone. The addition of bioactive glass granules to the PLAGA matrix resulted in a structure with higher compressive modulus than PLAGA alone. Moreover, the PLAGA-BA composite was found to be a bioactive material, as it formed surface calcium phosphate deposits in a simulated body fluid (SBF), and in the presence of cells and serum proteins. The composite supported osteoblast-like morphology, stained positively for alkaline phosphatase, and supported higher levels of Type I collagen synthesis than tissue culture polystyrene controls. We have successfully developed a degradable, porous, polymer bioactive glass composite possessing improved mechanical properties and osteointegrative potential compared to degradable polymers of poly(lactic acid-glycolic acid) alone. Future work will focus on the optimization of the composite scaffold for bone tissue-engineering applications and the evaluation of the 3-D composite in an in vivo model.     

Direct compression as an appropriately mechanical environment in bone tissue reconstruction in vitro

Determining how to apply an appropriately mechanical environment which can improve the quality and function of bone-like construct in vitro is a required problem to be solved for the current development of bone tissue engineering. A specific mechanical force may be a key determinant of tissue development in vitro in bone tissue engineering. From the standpoint of bionics, the mechanical environments applied on bone tissue engineering should work in three aspects: providing adequately mechanical stimuli to the cells seeded in 3-D scaffold; ensuring the efficient mass-transport of the nutrients and waste products of the cells; promoting the development of functionally extracellular matrix in 3-D scaffold. After the analysis of several differently mechanical environments comparing with that in vivo, the directly dynamical compression environment, instead of hydrostatic pressure or microgravity or direct perfusion, can recreate the in vivo mechanisms of mechanosensation, mechanotransduction and mass-transport during engineered bone-like tissue culturing process in vitro. Therefore, it is hypothesized that the directly dynamic compression will be a specific mechanical environment to bone tissue reconstruction in vitro.     

Biomechanical considerations of animal models used in tissue engineering of bone

Tissue engineering combines the aspects of cell biology, engineering, material science, and surgery to generate new functional tissue, and provides an important approach to the repair of segmental defects and in restoring biomechanical function. The development of tissue-engineering strategies into clinical therapeutic protocols requires extensive, preclinical experimentation in appropriate animal models. The ultimate success of any treatment strategy must be established in these animal models before clinical application. It is clear that the demands of the biological and mechanical environment in the clinical repair of critical size defects with tissue-engineered materials is significantly different from those existing in experimental animals. The major considerations facing any tissue-engineering testing logic include the choice of the defect, the animal, the age of the animal, the anatomic site, the size of the lesion, and most importantly, the micro-mechanical environment. With respect to biomechanical considerations when selecting animals for tissue- engineering of bone, it is evident that no common criteria have been reported. While in smaller animals due to size constraint only structural properties of whole bones can be measured, in larger animals and humans both material properties and structural properties are of interest. Based on reported results, comparison between the tissue-engineered bone across species may be of importance in establishing better model selection criteria. It has already been found that the deformation of long bones is fairly constant across species, and that stress levels during gait are dependent on the weight of the animal and the material properties of the bone tissue. Future research should therefore be geared towards developing better biomechanical testing systems and then finding the right animal model for the existing equipment.     

The impact of substrate stiffness and mechanical loading on fibroblast-induced scaffold remodeling

Fibroblasts as many other cells are known to form, contract, and remodel the extracellular matrix (ECM). The presented study aims to gain an insight into how mechanical boundary conditions affect the production of ECM components, their remodeling, and the feedback of the altered mechanical cell environment on these processes. The influence of cyclic mechanical loading (f=1?Hz, 10% axial compression) and scaffold stiffness (E=1.2 and 8.5?kPa) on the mechanical properties of fibroblast-seeded scaffold constructs were investigated in an in vitro approach over 14 days of culture. To do so, a newly developed bioreactor system was employed. While mechanical loading resulted in a clear upregulation of procollagen-I and fibronectin production, scaffold stiffness showed to primarily influence matrix metalloproteinase-1 (MMP-1) secretion and cell-induced scaffold contraction. Higher stiffness of the collagen scaffolds resulted in an up to twofold higher production of collagen-degrading MMP-1. The changes of mechanical parameters like Young's modulus, maximum compression force, and elastic portion of compression force over time suggest that from initially distinct mechanical starting conditions (scaffold stiffness), the construct's mechanical properties converge over time. As a consequence of mechanical loading a shift toward higher construct stiffness was observed. The results suggest that scaffold stiffness has only a temporary effect on cell behavior, while the impact of mechanical loading is preserved over time. Thus, it is concluded that the mechanical environment of the cell after remodeling is depending on mechanical loading rather than on initial scaffold stiffness.     

A three-dimensional osteochondral composite scaffold for articular cartilage repair

There is a recognized and urgent need for improved treatment of articular cartilage defects. Tissue engineering of cartilage using a cell-scaffold approach has demonstrated potential to offer an alternative and effective method for treating articular defects. We have developed a unique, heterogeneous, osteochondral scaffold using the TheriForm three-dimensional printing process. The material composition, porosity, macroarchitecture, and mechanical properties varied throughout the scaffold structure. The upper, cartilage region was 90% porous and composed of D,L-PLGA/L-PLA, with macroscopic staggered channels to facilitate homogenous cell seeding. The lower, cloverleaf-shaped bone portion was 55% porous and consisted of a L-PLGA/TCP composite, designed to maximize bone ingrowth while maintaining critical mechanical properties. The transition region between these two sections contained a gradient of materials and porosity to prevent delamination. Chondrocytes preferentially attached to the cartilage portion of the device, and biochemical and histological analyses showed that cartilage formed during a 6-week in vitro culture period. The tensile strength of the bone region was similar in magnitude to fresh cancellous human bone, suggesting that these scaffolds have desirable mechanical properties for in vivo applications, including full joint replacement.     

A three-dimensional nonlinear finite element analysis of the mechanical behavior of tissue engineered intervertebral discs under complex loads

The use of tissue-engineering method holds great promise for treating degenerative disc disease [Gan JC, Ducheyne P, Vresilovic E, Shapiro IM. J Biomed Mater Res 2000; 51(4): 596-604]. This concept typically implies that nucleus pulposus (NP) cells are seeded on a scaffold, while the NP tissue is regenerated. Such hybrid implant is inserted into the host intervertebral disc. Because the success of a tissue engineering approach depends on maintenance or restoration of the mechanical function of the intervertebral disc, it is useful to study the initial mechanical performance of the disc after implantation of the hybrid. A three-dimensional finite element model (FEM) of the L2-L3 disc-vertebrae unit has been analyzed. The model took into account the material nonlinearities and it imposed different and complex loading conditions. In this study, we validated the model by comparison of its predictions with several sets of experimental data; we determined the optimal Young's modulus as well as the failure strength for the tissue-engineered scaffold under different loading conditions; and we analyzed the effects of implanted scaffold on the mechanical behavior of the intervertebral disc. The results of this study suggest that a well-designed tissue-engineered scaffold preferably has a modulus in the range of 5-10 MPa and a compressive strength exceeding 1.67 MPa. Implanted scaffolds with such properties can then achieve the goal of restoring the disc height and distributing stress under different loading conditions.