Cell colonization in degradable 3D porous matrices

Cell colonization is an important in a wide variety of biological processes and applications including vascularization, wound healing, tissue engineering, stem cell differentiation, and biosensors. During colonization porous 3D structures are used to support and guide the ingrowth of cells into the matrix. In this review, we summarize our understanding of various factors affecting cell colonization in 3-dimensional environment. The structural, biological, and degradation properties of the matrix all play key roles during colonization. Further, specific scaffold properties such as porosity, pore size, fiber thickness, topography, and scaffold stiffness as well as important cell material interactions such as cell adhesion and mechanotransduction also influence colonization.     

Photoinitiated cross-linking of the biodegradable polyester poly(propylene fumarate). Part II. In vitro degradation

This study investigated the in vitro degradation of both solid PPF networks and porous PPF scaffolds formed by photoinitiated cross-linking of PPF polymer chains. Three formulations of scaffolds of differing porosity and pore size were constructed by varying porogen size and content. The effects of pore size and pore volume on scaffold mass, geometry, porosity, mechanical properties, and water absorption were then examined. Throughout the study, the solid networks and porous scaffolds exhibited continual mass loss and slight change in length. Porogen content appeared to have the greatest effect upon physical degradation. For example, scaffolds initially fabricated with 80 wt % porogen content lost approximately 30% of their initial PPF content after 32 weeks of degradation, whereas scaffolds fabricated with 70 wt % porogen content lost approximately 18% after 32 weeks of degradation. For all scaffold formulations, water absorption capacity, porosity, and compressive modulus were maintained at constant values following porogen leaching. These results indicate the potential of photo-cross-linked PPF scaffolds in tissue engineering applications which require maintenance of scaffold structure, strength, and porosity during the initial stages of degradation.     

Effects of pore size in 3-D fibrous matrix on human trophoblast tissue development

The effects of pore size in a 3-D polyethylene terephthalate (PET) nonwoven fibrous matrix on long-term tissue development of human trophoblast ED27 cells were studied. Thermal compression was used to modify the porosity and pore size of the PET matrix. The pore size distributions in PET matrices were quantified using a liquid extrusion method. Cell metabolic activities, estradiol production, and cell proliferation and differentiation were studied for ED27 cells cultured in the thermally compressed PET matrices with known pore structure characteristics. In general, metabolic activities and proliferation rate were higher initially for cultures grown in the low-porosity (LP) PET matrix (porosity of 0.849, average pore size of 30 microm in diameter) than those in the high-porosity (HP) matrix (porosity of 0.896, average pore size of 39 microm in diameter). However, 17beta-estradiol production and cell differentiation activity in the HP matrix surpassed those in the LP matrix after 12 days. The expression levels of cyclin B1 and p27kip1 in cells revealed progressively decreasing proliferation and increasing differentiation activities for cells grown in PET matrices. Also, difference in pore size controlled the cell spatial organization in the PET matrices and contributed to the tissue development in varying degrees of proliferation and differentiation. It was also found that cells grown on the 2-D surface behaved differently in cell cycle progression and did not show increased differentiation activities after growth had stopped and proliferation activities had lowered to a minimal level. The results from this study suggest that the 3-D cell organization guided by the tissue scaffold is important to tissue formation in vitro.     

甲壳素/海藻酸钠-纳米羟基磷灰石多孔复合支架材料的实验研究(英文)

目的:制备性能优良的复合支架一直是骨组织工程学研究的重点和难点。比较分析甲壳素对复合支架材料的孔隙率、含水量、降解率及生物力学特性的影响。方法:将甲壳素溶液与海藻酸钠溶液充分混合,然后将一定质量的羟基磷灰石加入混合液。根据甲壳素溶液在混合液中的质量分数不同分为两组:sca1(0%chitin)、sca2(50%chitin)。扫描电镜下观察材料的表面结构以及检测材料的孔径。测量并计算出复合支架材料的孔隙率、降解率、含水量以及生物力学性能。结果:两组支架材料均表现为多孔隙结构,平均孔径大小分别为:121.2±12.6μm、213.3±27.3μm。孔隙率分别为:(90.53±1.62)%、(87.73±1.22)%,统计学分析显示,两组材料孔隙率的差异比较有统计学意义(P0.05)。两组支架材料第6周的降解率分别(:59.12±1.93)%、(22.91±0.953)%,统计学分析显示,两组材料降解率的差异比较有统计学意义(P0.05)。两组含水量分别为:(95.52±1.17)%、(90.42±0.85)%,统计学分析显示,两组材料含水量的差异比较有统计学意义(P0.05)。第二组生物力学特性显著提高。结论:从本实验的实验数据可以看出,甲壳素可以增大材料的孔径,提高材料的降解稳定性,提高材料的生物力学强度。因此,甲壳素在骨组织工程领域具有重要的研究价值,同时为今后的进一步实验提供一定的实验依据。关键词:甲壳素;海藻酸钠;纳米羟基磷灰石;复合支架材料;组织工程     

Systematic investigation of porogen size and content on scaffold morphometric parameters and properties

A systematic investigation of tissue engineering scaffolds prepared by salt leaching of a photopolymerized dimethacrylate was performed to determine how the scaffold structure (porosity, pore size, etc.) can be controlled and also to determine how the scaffold structure and the mechanical properties are related. Two series of scaffolds were prepared with (1) the same polymer-to-salt ratio but different salt sizes (ranging from average size of 100 to 390 microm) and (2) the same salt size but different polymer-to-salt ratios (ranging from salt mass of 70 to 90%). These scaffolds were examined to determine how the fabrication parameters affected the scaffold morphometric parameters and corresponding mechanical properties. Combined techniques of X-ray microcomputed tomography (microCT), mercury porosimetry, and gravimetric analysis were used to determine the scaffold parameters, such as porosity, pore size, and strut thickness and their size distributions, and pore interconnectivity. Scaffolds with porosities ranging from 57% to 92% (by volume) with interconnected structures could be fabricated using the current technique. The porosity and strut thickness were subsequently related to the mechanical response of the scaffolds, both of which contribute to the compression modulus of the scaffold. The current study shows that the structure and properties of the scaffold could be tailored by the size and the amount of porogen used in the fabrication of the scaffold.     

制备大孔径静电纺丝组织工程支架的研究进展

研究表明静电纺丝可以制备出模拟细胞外基质的三维结构,其中限制静电纺丝纤维支架应用的问题之一就是纤维排列紧密导致支架的孔径较小,从而阻碍了细胞的浸入,组织中血管化的形成以及支架与宿主细胞的融合。为了增大支架的孔径,提高孔隙率,许多研究者提出了相应的策略。本文综述了多种制备大孔径静电纺丝纤维支架的方法,主要包括不同接收装置控制电场分布、盐粒子/聚合物析出法、水浴接收、低温静电纺丝以及激光/紫外烧蚀法等,以上的方法都能够有效的增大静电纺丝三维支架的孔径,进而提高了细胞的浸润性、营养物质的传输以及废物的排出,为静电纺丝纤维支架在组织工程中的应用奠定了基础。     

Theoretical analysis of insulin-dependent glucose uptake heterogeneity in 3D bioreactor cell culture

Three-dimensional (3D) cell cultures in bioreactors are becoming relevant as models for biological and physiological in vitro studies. In such systems, mathematical models can assist the experiment design that links the macroscopic properties to single-cell responses. We investigated the relationship between biochemical stimuli and cell response within a 3D cell culture in scaffold with heterogeneous porosity. Specifically, we studied the effect of insulin on the local glucose metabolism as a function of 3D pore size distribution. The multiscale mathematical model combines the mass transport within a 3D scaffold and a signaling pathways model. It considers the scaffold heterogeneity, and it describes spatiotemporal concentration of metabolites, biochemical stimuli, and cell density. The signaling model was integrated into this model, linking the local insulin concentration at cell membrane to the glucose uptake rate through glucose transporter type 4 (GLUT4) translocation from the cytosol to the cell membrane. The integrated model determines the cell response heterogeneities in a single channel, hence the biological response distribution in a 3D system. It also provides macroscopic outcomes to evaluate the feasibility of an experimental measurement of the system response. From our analysis, it became apparent that the flow rate is the most important operative variable, and that an optimum value ensures a fast and detectable cell response. This model on insulin-dependent glucose consumption rate offers insight into the cell metabolism physiology, which is a fundamental requirement for the study metabolic disorder such as Type 2 diabetes mellitus, in which the physiological insulin-dependent glucose metabolism is impaired.     

Engineering porous scaffolds using gas-based techniques

Scaffolds are used in tissue engineering as a matrix for the seeding and attachment of human cells. The creation of porosity in three-dimensional (3D) structures of scaffolds plays a critical role in cell proliferation, migration, and differentiation into the specific tissue while secreting extracellular matrix components. These pores are used to transfer nutrients and oxygen and remove wastes produced from the cells. The lack of oxygen and nutrient supply impedes the cell migration more than 500μm from the surface. The physical properties of scaffolds such as porosity and pore interconnectivity can improve mass transfer and have a great impact on the cell adhesion and penetration into the scaffolds to form a new tissue. Various techniques such as electrospinning, freeze-drying, and solvent casting/salt leaching have been used to create porosity in scaffolds. The major issues in these methods include lack of 3D structure, control on pore size, and pore interconnectivity. In this review, we provide a brief overview of gas-based techniques that have been developed for creating porosity in scaffolds.     

Preparation and biological properties of a novel composite scaffold of nano-hydroxyapatite/chitosan/carboxymethyl cellulose for bone tissue engineering

In this study, we report the physico-chemical and biological properties of a novel biodegradable composite scaffold made of nano-hydroxyapatite and natural derived polymers of chitosan and carboxymethyl cellulose, namely, n-HA/CS/CMC, which was prepared by freeze-drying method. The physico-chemical properties of n-HA/CS/CMC scaffold were tested by infrared absorption spectra (IR), transmission electron microscope(TEM), scanning electron microscope(SEM), universal material testing machine and phosphate buffer solution (PBS) soaking experiment. Besides, the biological properties were evaluated by MG63 cells and Mesenchymal stem cells (MSCs) culture experiment in vitro and a short period implantation study in vivo. The results show that the composite scaffold is mainly formed through the ionic crossing-linking of the two polyions between CS and CMC, and n-HA is incorporated into the polyelectrolyte matrix of CS-CMC without agglomeration, which endows the scaffold with good physico-chemical properties such as highly interconnected porous structure, high compressive strength and good structural stability and degradation. More important, the results of cells attached, proliferated on the scaffold indicate that the scaffold is non-toxic and has good cell biocompatibility, and the results of implantation experiment in vivo further confirm that the scaffold has good tissue biocompatibility. All the above results suggest that the novel degradable n-HA/CS/CMC composite scaffold has a great potential to be used as bone tissue engineering material.     

Prediction of the micro-fluid dynamic environment imposed to three-dimensional engineered cell systems in bioreactors

Bioreactors allowing culture medium perfusion overcome diffusion limitations associated with static culturing and provide flow-mediated mechanical stimuli. The hydrodynamic stress imposed to cells will depend not only on the culture medium flow rate, but also on the scaffold three-dimensional (3D) micro-architecture. We developed a CFD model of the flow of culture medium through a 3D scaffold of homogeneous geometry, with the aim of predicting the shear stress acting on cells as a function of parameters that can be controlled during the scaffold fabrication process, such as the scaffold porosity and the pore size, and during the cell culture, such as the medium flow rate and the diameter of the perfused scaffold section. We built three groups of models corresponding to three pore sizes: 50, 100 and 150 microm. Each group was made of four models corresponding to 59%, 65%, 77%, and 89% porosity. A commercial finite-element code was used to set up and solve the problem and to analyze the results. The mode value of shear stress varied between 2 and 5 mPa, and was obtained for a circular scaffold of 15.5 mm diameter, perfused by a flow rate of 0.5 ml/min. The simulations showed that the pore size is a variable strongly influencing the predicted shear stress level, whereas the porosity is a variable strongly affecting the statistical distribution of the shear stresses, but not their magnitude. Our results provide a basis for the completion of more exhaustive quantitative studies to further assess the relationship between perfusion, at known micro-fluid dynamic conditions, and tissue growth in vitro.     

Mesenchymal stem cell cultivation in electrospun scaffolds: mechanistic modeling for tissue engineering

Tissue engineering is a multidisciplinary field of research in which the cells, biomaterials, and processes can be optimized to develop a tissue substitute. Three-dimensional (3D) architectural features from electrospun scaffolds, such as porosity, tortuosity, fiber diameter, pore size, and interconnectivity have a great impact on cell behavior. Regarding tissue development in vitro, culture conditions such as pH, osmolality, temperature, nutrient, and metabolite concentrations dictate cell viability inside the constructs. The effect of different electrospun scaffold properties, bioreactor designs, mesenchymal stem cell culture parameters, and seeding techniques on cell behavior can be studied individually or combined with phenomenological modeling techniques. This work reviews the main culture and scaffold factors that affect tissue development in vitro regarding the culture of cells inside 3D matrices. The mathematical modeling of the relationship between these factors and cell behavior inside 3D constructs has also been critically reviewed, focusing on mesenchymal stem cell culture in electrospun scaffolds.     

Physical limits of cell migration: Control by ECM space and nuclear deformation and tuning by proteolysis and traction force

Cell migration through 3D tissue depends on a physicochemical balance between cell deformability and physical tissue constraints. Migration rates are further governed by the capacity to degrade ECM by proteolytic enzymes, particularly matrix metalloproteinases (MMPs), and integrin- and actomyosin-mediated mechanocoupling. Yet, how these parameters cooperate when space is confined remains unclear. Using MMP-degradable collagen lattices or nondegradable substrates of varying porosity, we quantitatively identify the limits of cell migration by physical arrest. MMP-independent migration declined as linear function of pore size and with deformation of the nucleus, with arrest reached at 10% of the nuclear cross section (tumor cells, 7 µm²; T cells, 4 µm²; neutrophils, 2 µm²). Residual migration under space restriction strongly depended upon MMP-dependent ECM cleavage by enlarging matrix pore diameters, and integrin- and actomyosin-dependent force generation, which jointly propelled the nucleus. The limits of interstitial cell migration thus depend upon scaffold porosity and deformation of the nucleus, with pericellular collagenolysis and mechanocoupling as modulators.     

Microarchitecture of three-dimensional scaffolds influences cell migration behavior via junction interactions

Cell migration plays a critical role in a wide variety of physiological and pathological phenomena as well as in scaffold-based tissue engineering. Cell migration behavior is known to be governed by biochemical stimuli and cellular interactions. Biophysical processes associated with interactions between the cell and its surrounding extracellular matrix may also play a significant role in regulating migration. Although biophysical properties of two-dimensional substrates have been shown to significantly influence cell migration, elucidating factors governing migration in a three-dimensional environment is a relatively new avenue of research. Here, we investigate the effect of the three-dimensional microstructure, specifically the pore size and Young's modulus, of collagen-glycosaminoglycan scaffolds on the migratory behavior of individual mouse fibroblasts. We observe that the fibroblast migration, characterized by motile fraction as well as locomotion speed, decreases as scaffold pore size increases across a range from 90 to 150 μm. Directly testing the effects of varying strut Young's modulus on cell motility showed a biphasic relationship between cell speed and strut modulus and also indicated that mechanical factors were not responsible for the observed effect of scaffold pore size on cell motility. Instead, in-depth analysis of cell locomotion paths revealed that the distribution of junction points between scaffold struts strongly modulates motility. Strut junction interactions affect local directional persistence as well as cell speed at and away from the junctions, providing a new biophysical mechanism for the governance of cell motility by the extracellular microstructure.     

Development of biodegradable scaffolds based on magnetically guided assembly of magnetic sugar particles

Biodegradable scaffolds with controlled pore layout and porosity have great significance in tissue engineering for cell penetration, tissue ingrowth, vascularization, and nutrient delivery. Porogen leaching has been commonly used to control pore size, pore structure and porosity in the scaffold. In this paper we focus on the use/development of two magnetically guided porogen assembly methods using magnetic sugar particles (MSPs) for scaffold fabrication. First, a patterning device is utilized to align MSPs following designed templates. Then a magnetic sheet film is fabricated by mixing poly(vinyl alcohol, PVA) and NdFeB powder for steering the MSPs. After poly(l-lactide-co-?-caprolactone) (PLCL) casting and removal of the sugar template, a scaffold with spherical pores is obtained. The surface and the inner structure of the scaffolds are evaluated using light and electron micrographs showing their interconnection of pores, pore wall morphology and porosity. Single layer scaffolds with the size of 8mm in width and 10mm in length were constructed with controllable pore diameters in the ranges of 105-150 μm, 250-300 μm and 425-500 μm.     

Geometry Design Optimization of Functionally Graded Scaffolds for Bone Tissue Engineering: A Mechanobiological Approach

Functionally Graded Scaffolds (FGSs) are porous biomaterials where porosity changes in space with a specific gradient. In spite of their wide use in bone tissue engineering, possible models that relate the scaffold gradient to the mechanical and biological requirements for the regeneration of the bony tissue are currently missing. In this study we attempt to bridge the gap by developing a mechanobiology-based optimization algorithm aimed to determine the optimal graded porosity distribution in FGSs. The algorithm combines the parametric finite element model of a FGS, a computational mechano-regulation model and a numerical optimization routine. For assigned boundary and loading conditions, the algorithm builds iteratively different scaffold geometry configurations with different porosity distributions until the best microstructure geometry is reached, i.e. the geometry that allows the amount of bone formation to be maximized. We tested different porosity distribution laws, loading conditions and scaffold Young’s modulus values. For each combination of these variables, the explicit equation of the porosity distribution law–i.e the law that describes the pore dimensions in function of the spatial coordinates–was determined that allows the highest amounts of bone to be generated. The results show that the loading conditions affect significantly the optimal porosity distribution. For a pure compression loading, it was found that the pore dimensions are almost constant throughout the entire scaffold and using a FGS allows the formation of amounts of bone slightly larger than those obtainable with a homogeneous porosity scaffold. For a pure shear loading, instead, FGSs allow to significantly increase the bone formation compared to a homogeneous porosity scaffolds. Although experimental data is still necessary to properly relate the mechanical/biological environment to the scaffold microstructure, this model represents an important step towards optimizing geometry of functionally graded scaffolds based on mechanobiological criteria.     

担载b-FGF的PLGA微球复合明胶支架的体外释放研究

目的:研究担载碱性成纤维细胞生长因子(b-FGF)微球复合明胶支架的外形特征、孔径、孔隙率及体外释放动力学,以期构建具有缓释功能、高孔隙率的担载细胞因子的新型复合明胶支架。方法:本文利用冷冻相分离法和S/O/W法先将b-FGF水溶液包裹于PLGA微球中,然后埋置于明胶溶液中制备为多孔复合明胶支架。分别对微球的形态和复合明胶支架的基本形态、孔径、孔隙率进行表征,通过Elisa法测定b-FGF在复合明胶支架中的体外释放行为。结果:制备成形态良好的三维复合明胶支架,其孔隙率为82.90%±1.45%,孔径范围为150~300μm,复合明胶支架中b-FGF在体外缓慢释放20余天。结论:担载蛋白微球复合明胶支架不仅满足组织工程支架的要求,还能有效缓释细胞因子,为细胞和组织生长提供良好的微环境,为进一步应用于组织工程领域提供了可能。     

Vinculin, cadherin mechanotransduction and homeostasis of cell–cell junctions

Cell adhesion junctions characteristically arise from the cooperative integration of adhesion receptors, cell signalling pathways and the cytoskeleton. This is exemplified by cell–cell interactions mediated by classical cadherin adhesion receptors. These junctions are sites where cadherin adhesion systems functionally couple to the dynamic actin cytoskeleton, a process that entails physical interactions with many actin regulators and regulation by cell signalling pathways. Such integration implies a potential role for molecules that may stand at the interface between adhesion, signalling and the cytoskeleton. One such candidate is the cortical scaffolding protein, vinculin, which is a component of both cell–cell and cell–matrix adhesions. While its contribution to integrin-based adhesions has been extensively studied, less is known about how vinculin contributes to cell–cell adhesions. A major recent advance has come with the realisation that cadherin adhesions are active mechanical structures, where cadherin serves as part of a mechanotransduction pathway by which junctions sense and elicit cellular responses to mechanical stimuli. Vinculin has emerged as an important element in cadherin mechanotransduction, a perspective that illuminates its role in cell–cell interactions. We now review its role as a cortical scaffold and its role in cadherin mechanotransduction.     

A multiscale investigation of mechanical properties of bio-inspired scaffolds

Abstract

Finding a structural design which allows the scaffold to have a high porosity and large pore size while retaining high strength is essential. Here, a bio-inspired scaffold is designed based on the observed geometrical pattern of the apatite atomic crystal structure, and mechanical properties are compared with other common scaffold geometries. The bio-inspired scaffold design is proven superior using a multiscale computational approach, which combines density functional theory and finite element analysis to predict the stress reaction and substitution effects on the scaffolds. This study provides insight into better scaffold design using bio-inspired structures and the effect of substitutions.     

Porosity and pore size regulate the degradation product profile of polylactide

Porosity and pore size regulated the degradation rate and the release of low molar mass degradation products from porous polylactide (PLA) scaffolds. PLA scaffolds with porosities above 90% and different pore size ranges were subjected to hydrolytic degradation and compared to their solid analog. The solid film degraded fastest and the degradation rate of the porous structures decreased with decreasing pore size. Degradation products were detected earlier from the solid films compared to the porous structures as a result of the additional migration path within the porous structures. An intermediate degradation rate profile was observed when the pore size range was broadened. The morphology of the scaffolds changed during hydrolysis where the larger pore size scaffolds showed sharp pore edges and cavities on the scaffold surface. In the scaffolds with smaller pores, the pore size decreased during degradation and a solid surface was formed on the top of the scaffold. Porosity and pore size, thus, influenced the degradation and the release of degradation products that should be taken into consideration when designing porous scaffolds for tissue engineering.     

Hybrid chitosan–ß‐glycerol phosphate–gelatin nano‐/micro fibrous scaffolds with suitable mechanical and biological properties for tissue engineering

Scaffold‐based tissue engineering is considered as a promising approach in the regenerative medicine. Graft instability of collagen, by causing poor mechanical properties and rapid degradation, and their hard handling remains major challenges to be addressed. In this research, a composite structured nano‐/microfibrous scaffold, made from a mixture of chitosan–ß‐glycerol phosphate–gelatin (chitosan–GP–gelatin) using a standard electrospinning set‐up was developed. Gelatin–acid acetic and chitosan ß‐glycerol phosphate–HCL solutions were prepared at ratios of 30/70, 50/50, 70/30 (w/w) and their mechanical and biological properties were engineered. Furthermore, the pore structure of the fabricated nanofibrous scaffolds was investigated and predicted using a theoretical model. Higher gelatin concentrations in the polymer blend resulted in significant increase in mean pore size and its distribution. Interaction between the scaffold and the contained cells was also monitored and compared in the test and control groups. Scaffolds with higher chitosan concentrations showed higher rate of cell attachment with better proliferation property, compared with gelatin‐only scaffolds. The fabricated scaffolds, unlike many other natural polymers, also exhibit non‐toxic and biodegradable properties in the grafted tissues. In conclusion, the data clearly showed that the fabricated biomaterial is a biologically compatible scaffold with potential to serve as a proper platform for retaining the cultured cells for further application in cell‐based tissue engineering, especially in wound healing practices. These results suggested the potential of using mesoporous composite chitosan–GP–gelatin fibrous scaffolds for engineering three‐dimensional tissues with different inherent cell characteristics. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 163–175, 2016.