三维支架材料中软骨细胞生长过程的数值模拟

组织工程是临床上用于修复以及重建受损软骨的一项有广泛应用前景的方法。但是在支架材料内部物质传递仅仅依赖于扩散,这是支架材料中细胞生长的主要限制因素。利用氧扩散-反应基本原理建立了软骨细胞生长过程的数学模型,同时考虑了由于细胞生长引起的扩散系数下降以及空间抑制因素对细胞生长的影响。模拟结果与实验数据吻合良好,表明该模型对材料内部的细胞生长的分析具有较高的可靠性,可用于组织工程生物反应器以及三维多孔支架材料的优化设计。     

丝素蛋白在组织工程中的应用及进展

组织工程是生物支架材料、种子细胞和生物活性因子的有机组合,其中支架材料为种子细胞的黏附载体,为细胞的生长增殖及新陈代谢提供适宜的微环境,并最终被生物体逐渐降解而被再生组织替代。支架材料为周围组织提供机械支持,并引导再生组织按照预定结构和方向生长。同时,各种生物活性物质可以加入支架材料中,比如各种生长因子以及抗体等,扩大了支架材料的应用范围。丝素蛋白具有可控且缓慢的生物降解性,突出的机械性能,良好的生物相容性,支持多种细胞的黏附、生长和分化增殖,已经用于血管、骨、软骨及神经组织等方面的组织工程研究。     

丝素蛋白在电纺丝法构建组织工程支架中的应用进展

丝素蛋白是天然高分子纤维蛋白,具有良好的物理和机械力学性能及生物相容性,因而在组织工程领域有着广阔的应用前景。文中对丝素蛋白的化学组成、分子结构特点、提取方法以及利用静电纺丝技术在组织工程化支架构建中的应用作了概述。总结了丝素蛋白在用于组织工程材料上的性能和优势以及在人工血管、皮肤、骨组织等工程化支架方面的应用情况,探讨了丝素蛋白支架对细胞在其上生长、增殖和功能的影响,同时对丝素蛋白在组织工程化食道支架及其他再生医学上的应用前景进行了展望。     

组织工程生物支架材料

组织工程支架材料表面的微观和亚微观结构对细胞的黏附与生长有很重要的影响,纳米结构材料的应用为该结构展现了较广阔的前景。另外,组织工程支架材料的表面修饰及孔径调控对生物材料的改进有很重要的作用。介绍了生物材料的基本要求和分类,纳米结构材料在组织工程中的应用及生物材料表面修饰,以及以泡沫支架为例介绍材料孔径调控。     

微环境对细胞的影响以及仿生学在组织工程支架中的应用

微环境影响着细胞的增殖、迁移、分化以及细胞功能,细胞微环境影响细胞命运的因素包括细胞之间相互作用、细胞与细胞外基质相互作用、可溶性信号分子以及缺氧和营养对细胞的影响。组织工程支架的制备就是要利用仿生学原理最大程度模拟细胞微环境,从而应用于细胞行为研究以及临床治疗。全面了解细胞微环境对细胞的影响因素是制备组织工程支架的重要条件,而组织工程支架的研究也进一步推动了细胞微环境对细胞影响的认识。组织工程支架研究在组织工程研究中仍具有广阔前景,新的制备工艺也在组织工程支架研究中发挥着巨大推动作用。     

胶原蛋白-壳聚糖支架的构建及其与骨髓基质干细胞复合的实验研究

目的:构建一种组织工程神经支架,并观察体外培养的骨髓基质干细胞在其内部的生长情况,为后续种子细胞的移植提供阶段性实验数据.方法:以Ⅰ型胶原蛋白和壳聚糖为原料通过冷冻干燥技术制备神经支架,扫描电镜观察其内部结构,测量其孔径大小、孔隙率等指标.将体外培养的骨髓基质干细胞与Ⅰ型胶原蛋白-壳聚糖神经支架复合,共培养2天;扫描电镜观察细胞在支架内部的生长情况.结果:构建的神经支架均为圆柱状,内部为纵向平行排列的孔径均匀的微管样结构,细胞紧密贴附在支架微孔内壁上,细胞生长状况良好.结论:Ⅰ型胶原蛋白-壳聚糖支架具有良好的内部三维结构和生物相容性,可与细胞复合后用于修复周围神经缺损.     

肌腱组织工程支架与种子细胞研究进展

目的:综述肌腱组织工程支架材料、细胞来源、制备技术及体外构建的研究进展.方法:查阅近期肌腱组织工程研究的相关文献,对组织工程肌腱支架的材料来源、制备技术,复合细胞种类,体外构建力学刺激等进行分析、归纳.结果:肌腱组织工程支架材料有天然材料、人工合成材料及复合材料等;制备技术包括静电纺丝和编织法等;其中支架材料的表面修饰是组织工程化肌腱构建的重要环节.与肌腱材料进行复合的种子细胞有肌腱细胞、骨髓间充质干细胞及成纤维细胞等.结论:复合材料是近年肌腱组织工程支架材料研究的重点,静电纺丝技术是一种具有潜力的支架制备技术,支架材料的表面修饰可促进细胞在支架上的黏附及肌腱的形成,种子细胞的研究仍是肌腱组织工程发展的瓶颈,周期性张力的存在为组织工程化肌腱的形成创造了条件.     

基于组织工程研究的可降解支架材料选择策略

目前,器官或组织移植是治疗器官衰竭或大范围组织缺损唯一长期有效的方法,但存在供体短缺、免疫排斥等问题。组织工程技术作为一种潜在的替代治疗方法,支架材料的选择是其中具有决定意义的组成部分。组织工程支架材料按其来源可分为天然及其改性修饰材料、人工合成与复合支架材料3种。组织工程目的就是修复临床上的病损组织或器官,并达到较理想的结构和功能的恢复。因此组织工程支架也必须从基本性质上具有一定的仿生化结构及功能,即"活"支架,这样才能彻底代替病损组织或器官。通过多种支架材料的优化组合(即材料的复合),对材料进行表面改性、制备工艺优化及添加细胞因子缓释微球等技术,模拟病损器官组织的特性及周围环境,有望打开组织工程的新局面。理想的组织工程支架应当以临床需要为根本目的,依靠材料学、分子生物学、工程学等多学科间的交叉研究,取各家之长,优化配比组合,达到仿生的目的。本课题组前期工作已经将骨髓间充质干细胞体外诱导分化为胆管上皮样细胞,并设计出左旋聚乳酸/聚己内酯共聚物(PLCL)胆道支架,内部混有包含生长因子的纳米缓释微球,供细胞因子的远期释放,支架内表面涂有基质胶/胶原混合层,且胶内加入bFGF、EGF,提供诱导因子的早期释放。将诱导细胞与PLCL胆道支架复合,制备组织工程胆管。文中综述了现存各类支架材料的研究状况,简单介绍了制备工艺、表面修饰等影响支架性能的因素,力求探索组织工程支架材料的选择策略。     

成纤维细胞在壳聚糖/PHEA水凝胶膜上的粘附与生长行为

水凝胶是一类广泛溶涨于水 ,呈三维网状结构的聚合物具有很高的生物相容性 ,广泛地用于生物材料 ,如眼球的晶状体、人造脏器以及人造皮肤等。高含水量的水凝胶不利于细胞粘附 ,研究能使细胞粘附并生长的水凝胶是开发其在组织工程材料领域应用的关键 ,细胞易于粘附的水凝胶可用于细胞培养基材和组织工程移植支架材料。一般来说 ,由于细胞表面带有负电荷 ,带正电荷的基材表面 (如 ,多熔素 (Ｐｏｌｙｌ ｙｓｉｎｅ) )有利于细胞粘附 ,而带有酸性或中性基团的材料不利于细胞粘附[1 ] ,而且带高负电荷密度的基材会导致细胞新陈代谢的紊乱并抑制细…     

组织工程皮肤生物反应器的研究与设计

目的设计一套生物反应器,能针对不同支架材料———细胞复合物进行构建组织工程皮肤。方法根据皮肤的自身生长特点和不同支架材料-细胞复合物的特性,模拟皮肤的生长环境和力学环境,通过生物反应器解决组织工程皮肤构建中支架的装夹和气液界面问题。结果生物反应器由控制系统和生物反应器主体两部分构成,能提供对多种皮肤细胞复合物的动态培养。结论皮肤生物反应器能够满足不同组织工程皮肤产品的需要。能够形成气液界面和模拟生物力学的刺激。     

Analysis of cell growth kinetics and substrate diffusion in a polymer scaffold.

The cultivation of cartilage cells (chondrocytes) in polymer scaffolds leads to implants that may potentially be used to repair damaged joint cartilage or for reconstructive surgery. For this technique to be medically applicable, the physical parameters that govern cell growth in a polymer scaffold must be understood. This understanding of cell behavior under in vitro conditions, where diffusion is the primary mode of transport of nutrients, may aid in the scale-up of the cartilage generation process. A mathematical model of chondrocyte generation and nutrient consumption is developed here to analyze the behavior of cell growth in a biodegradable polymer matrix for a series of different thickness polymers. Recent literature has implied that the diffusion of nutrients is a major factor that limits cell growth (Freed et al., 1994). In the present paper, a mathematical model is developed to directly relate the effects of increasing cell mass in the polymer matrix on the transport of nutrients. Reaction and diffusion of nutrients in the cell-polymer system are described using the fundamental species continuity equations and the volume averaging method. The volume averaging method is utilized to derive a single averaged nutrient continuity equation that includes the effective transport properties. This approach allows for the derivation of effective diffusion and rate coefficients as functions of the cell volume fraction. The cell volume fraction as a function of time is determined by solution of a material balance on cell mass. Growth functions including the Moser, a modified Contois, and an nth-order heterogeneous growth kinetic model are evaluated through a parameter analysis, and the results are compared to experimental data found in the literature. The results indicate that cellular functions in conjunction with mass transfer processes can account partially for the general trends in the cell growth behavior for various thickness polymers. The Contois growth function appeared to describe the data more accurately in terms of the lag period at early times and the long time limits. However, all kinetic growth functions required variations in the kinetic parameters to fully describe the effects of polymer thickness. This result implies that restricted diffusion of nutrients is not the sole factor limiting cell growth when the thickness of the polymer is changed. Therefore, further experimental data and model improvements are needed to accurately describe the cell growth process.     

Analysis of cell growth and diffusion in a scaffold for cartilage tissue engineering

Developments in tissue engineering over the past decade have offered promising future for the repair and reconstruction of damaged tissues. To regenerate three dimensional and weight-bearing implants, advances in biomaterials and manufacturing technologies prompted cell cultivations with natural or artificial scaffolds, in which cells are allowed to proliferate, migrate, and differentiate in vitro. In this article, we develop a mathematical model for cell growth in a porous scaffold. By treating the cell-scaffold construct as a porous medium, a continuum model is set up based on basic principles of mass conservation. In addition to cell growth kinetics, we incorporate cell diffusion in the model to describe the effects of cell random walks. Computational results are compared to experimental data found in the literature. With this model, we are able to investigate cell motility, heterogeneous cell distributions, and non-uniform seeding for tissue engineering applications. Results show that random walks tend to enhance uniform cell spreads in space, which in turn increases the probabilities for cells to acquire nutrients; therefore random walks are likely to be a positive contribution to the overall cell growth on scaffolds.     

A mathematical model for bone tissue regeneration inside a specific type of scaffold

Bone tissue regeneration using scaffolds is receiving an increasing interest in orthopedic surgery and tissue engineering applications. In this study, we present the geometrical characterization of a specific family of scaffolds based on a face cubic centered (FCC) arrangement of empty pores leading to analytical formulae of porosity and specific surface. The effective behavior of those scaffolds, in terms of mechanical properties and permeability, is evaluated through the asymptotic homogenization theory applied to a representative volume element identified with the unit cell FCC. Bone growth into the scaffold is estimated by means of a phenomenological model that considers a macroscopic effective stress as the mechanical stimulus that regulates bone formation. Cell migration within the scaffold is modeled as a diffusion process based on Fick's law which allows us to estimate the cell invasion into the scaffold microstructure. The proposed model considers that bone growth velocity is proportional to the concentration of cells and regulated by the mechanical stimulus. This model allows us to explore what happens within the scaffold, the surrounding bone and their interaction. The mathematical model has been numerically implemented and qualitatively compared with previous experimental results found in the literature for a scaffold implanted in the femoral condyle of a rabbit. Specifically, the model predicts around 19 and 23% of bone regeneration for non-grafted and grafted scaffolds, respectively, both with an initial porosity of 76%.     

Mathematical model and numerical simulation of the cell growth in scaffolds

A scaffold is a three-dimensional matrix that provides a structural base to fill tissue lesion and provides cells with a suitable environment for proliferation and differentiation. Cell-seeded scaffolds can be implanted immediately or be cultured in vitro for a period of time before implantation. To obtain uniform cell growth throughout the entire volume of the scaffolds, an optimal strategy on cell seeding into scaffolds is important. We propose an efficient and accurate numerical scheme for a mathematical model to predict the growth and distribution of cells in scaffolds. The proposed numerical algorithm is a hybrid method which uses both finite difference approximations and analytic closed-form solutions. The effects of each parameter in the mathematical model are numerically investigated. Moreover, we propose an optimization algorithm which finds the best set of model parameters that minimize a discrete l ₂ error between numerical and experimental data. Using the mathematical model and its efficient and accurate numerical simulations, we could interpret experimental results and identify dominating mechanisms.     

Evaluation of RGD- or EGF-immobilized chitosan scaffolds for chondrogenic activity

Chitosan scaffolds were prepared by freeze-drying method and modified with Arg-Gly-Asp (RGD) sequence of fibronectin or epidermal growth factor (EGF) by covalent immobilization. The results obtained from FTIR-ATR, fluorescence visualization and quantitative measurements showed that biosignal molecules, RGD and EGF, were successfully immobilized on chitosan scaffolds. ATDC5 murine chondrogenic cells were seeded on both type of scaffolds, chitosan-RGD and chitosan-EGF, and cultured for 28 days in stationary conditions. According to the results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) test, considerable increase in cell proliferation was only detected on chitosan-EGF scaffolds. Biochemical analysis of the chondrocyte seeded scaffolds showed that glycosaminoglycan (GAG) and deoxyribonucleic acid (DNA) content of the scaffolds increases with time. In conclusion, EGF-modified chitosan scaffolds (containing 1.83 microg EGF/3 mg dry scaffold) have been proposed to promote chondrogenesis and to have potential for reticular cartilage regeneration.     

Adult mesenchymal stem cells for bone and cartilage engineering: effect of scaffold materials

Bone marrow is a useful cell source for skeletal tissue engineering approaches. In vitro differentiation of marrow mesenchymal stem cells (MSCs) to chondrocytes or osteoblasts can be induced by the addition of specific growth factors to the medium. The present study evaluated the behaviour of human MSCs cultured on various scaffolds to determine whether their differentiation can be induced by cell-matrix interactions. MSCs from bone marrow collected from the acetabulum during hip arthroplasty procedures were isolated by cell sorting, expanded and characterised by a flow cytometry system. Cells were grown on three different scaffolds (type I collagen, type I + II collagen and type I collagen + hydroxyapatite membranes) and analysed by histochemistry, immunohistochemistry and spectrophotometry (cell proliferation, alkaline phosphatase activity) at 15 and 30 days. Widely variable cell adhesion and proliferation was observed on the three scaffolds. MSCs grown on type I+II collagen differentiated to cells expressing chondrocyte markers, while those grown on type I collagen + hydroxyapatite differentiated into osteoblast-like cells. The study highlighted that human MSCs grown on different scaffold matrices may display different behaviours in terms of cell proliferation and phenotype expression without growth factor supplementation.     

PRP-fibrinogen gel-like chondrocyte carrier stabilized by TXA-preliminary study

Three dimensional scaffolds are becoming increasingly popular in the treatment of cartilage defects. Platelet-rich plasma (PRP) and fibrinogen can be used potentially as a three dimensional cell delivery vehicle. PRP is a fraction of plasma containing high levels of growth factors such as PDGF, IGF-I and TGF-I, which stimulate chondrocyte to synthesize extracellular matrix. The aim of this study was to prepare grafts based on fibrinogen, and PRP with fibrinogen as a chondrocyte carrier. Another goal was to estimate tranexamic acid as an antifibrynolytic agent in chondrocyte grafts and in monolayer culture for about 3 weeks. 450 ml blood was collected to produce fibrinogen and PRP from a Regional Blood Center voluntary donor. To prepare gel grafts, chondrocytes were mixed with PRP and, fibrinogen and then with thrombin in calcium chloride. Different doses of tranexamic acid or aprotinin were used to stabilize the constructs. Grafts were cultivated for 4 weeks in vitro to evaluate and compare their disintegration. Grafts were stable for the entire observation period and revealed no shrinkage. During graft storage, cells appeared to be viable, and cell migration from the graft to the culture plate was observed. Chondrocyte graft preparation based on PRP and fibrinogen is a promising method. PRP-fibrinogen carrier in combination with cells constitutes highly plastic and adhesive grafts. Tranexamic acid can be used as an anti-fibrinolytic agent in chondrocyte graft preparation instead of aprotinin.     

Improvement of PHBV Scaffolds with Bioglass for Cartilage Tissue Engineering

Polymer scaffold systems consisting of poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) have proven to be possible matrices for the three-dimensional growth of chondrocyte cultures. However, the engineered cartilage grown on these PHBV scaffolds is currently unsatisfactory for clinical applications due to PHBV’s poor hydrophilicity, resulting in inadequate thickness and poor biomechanical properties of the engineered cartilage. It has been reported that the incorporation of Bioglass (BG) into PHBV can improve the hydrophilicity of the composites. In this study, we compared the effects of PHBV scaffolds and PHBV/BG composite scaffolds on the properties of engineered cartilage in vivo. Rabbit articular chondrocytes were seeded into PHBV scaffolds and PHBV/BG scaffolds. Short-term in vitro culture followed by long-term in vivo transplantation was performed to evaluate the difference in cartilage regeneration between the cartilage layers grown on PHBV and PHBV/BG scaffolds. The results show that the incorporation of BG into PHBV efficiently improved both the hydrophilicity of the composites and the percentage of adhered cells and promoted cell migration into the inner part the constructs. With prolonged incubation time in vivo, the chondrocyte-scaffold constructs in the PHBV/BG group formed thicker cartilage-like tissue with better biomechanical properties and a higher cartilage matrix content than the constructs in the PHBV/BG group. These results indicate that PHBV/BG scaffolds can be used to prepare better engineered cartilage than pure PHBV.     

Melatonin delivery from PCL scaffold enhances glycosaminoglycans deposition in human chondrocytes – Bioactive scaffold model for cartilage regeneration

The therapeutic potential of an engineered cartilage construct can be enhanced by sustained delivery of chondrogenic drug like melatonin from 3D porous scaffolds embedded with melatonin loaded bovine serum albumin nanoparticles (MNP). In this study, MNP was synthesized and loaded into polycaprolactone (PCL) scaffolds. 12 % (w/v) and 10 % (w/v) PCL scaffolds were fabricated with different concentrations of MNP. X- ray diffraction and Raman analysis of MNP and scaffolds revealed amorphization of melatonin which is highly desired in drug delivery applications. Additionally, Fourier Transform Infrared spectroscopic analysis confirmed the drug to be chemically inert to fabrication process. Field emission scanning electron microscopic analysis suggested highly interlinked porous scaffold (diameter 50 μm – 300 μm) and MNP diameters in the range of 110−200 nm. Importantly, UV spectrophotometric analysis showed that all groups of scaffolds showed sustained release for 21 days, wherein MNP concentration had an influence on release behaviour of melatonin from scaffolds. Drug release kinetics studied using mathematical models revealed, diffusion and dissolution mechanism of release. Furthermore, in vitro evaluation of MNP loaded scaffolds with Human chondrocytes for 21 days increased glycosaminoglycans deposition significantly. In brief, sustained release of melatonin from polycaprolactone scaffolds increased the therapeutic potential of the engineered construct.