Integration of hollow fiber membranes improves nutrient supply in three-dimensional tissue constructs

Sufficient nutrient and oxygen transport is a potent modulator of cell proliferation in in vitro tissue-engineered constructs. The lack of oxygen and culture medium can create a potentially lethal environment and limit cellular metabolic activity and growth. Diffusion through scaffold and multi-cellular tissue typically limits transport in vitro, leading to potential hypoxic regions and reduction in the viable tissue thickness. For the in vitro generation of clinically relevant tissue-engineered grafts, current nutrient diffusion limitations should be addressed. Major approaches to overcoming these include culture with bioreactors, scaffolds with artificial microvasculature, oxygen carriers and pre-vascularization of the engineered tissues. This study focuses on the development and utilization of a new perfusion culture system to provide adequate nutrient delivery to cells within large three-dimensional (3D) scaffolds. Perfusion of oxygenated culture medium through porous hollow fiber (HF) integrated within 3D free form fabricated (FFF) scaffolds is proposed. Mouse pre-myoblast (C2C12) cells cultured on scaffolds of poly(ethylene-oxide-terephthalate)-poly(butylene-terephthalate) block copolymer (300PEOT55PBT45) integrated with porous HF membranes of modified poly(ether-sulfone) (mPES, Gambro GmbH) is used as a model system. Various parameters such as fiber transport properties, fiber spacing within a scaffold and medium flow conditions are optimized. The results show that four HF membranes integrated with the scaffold significantly improve the cell density and cell distribution. This study provides a basis for the development of a new HF perfusion culture methodology to overcome the limitations of nutrient diffusion in the culture of large 3D tissue constructs.     

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.     

Hypoxia in static and dynamic 3D culture systems for tissue engineering of bone

Tissue engineering of sizeable cell-scaffold constructs is limited by gradients in tissue quality from the periphery toward the center. Because homogenous delivery of oxygen to three-dimensional (3D) cell cultures remains an unsolved challenge, we hypothesized that uneven oxygen supply may impede uniform cellular growth on scaffolds. In this study we challenged static and dynamic 3D culture systems designed for bone tissue engineering applications with a well-growing subclone of MC3T3-E1 preosteoblasts and continuously measured the oxygen concentrations in the center of cell-seeded scaffolds and in the surrounding medium. After as little as 5 days in static culture, central oxygen concentrations dropped to 0%. Subsequently, cells died in central regions of the scaffold but not in its periphery, where oxygen levels were approximately 4%. The use of perfusion bioreactors successfully prevented cell death, yet central oxygen concentrations did not rise above 4%. We conclude that 3D culture in vitro is associated with relevant oxygen gradients, which can be the cause of inhomogeneous tissue quality. Perfusion bioreactors prevent cell death but they do not entirely eliminate 3D culture-associated oxygen gradients. Therefore, we advise continuous oxygen monitoring of 3D culture systems to ensure tissue quality throughout engineered constructs.     

Effects of medium perfusion rate on cell-seeded three-dimensional bone constructs in vitro

Cellular activity at the center of tissue-engineered constructs in static culture is typically decreased relative to the construct periphery because of transport limitations. We have designed a tissue culture system that perfuses culture medium through three-dimensional (3D) porous cellular constructs to improve nutrient delivery and waste removal within the constructs. This study examined the effects of medium perfusion rate on cell viability, proliferation, and gene expression within cell-seeded 3D bone scaffolds. Human trabecular bone scaffolds were seeded with MC3T3-E1 osteoblast-like cells and perfused for 1 week at flow rates of 0.01, 0.1, 0.2, and 1.0 mL/min. Confocal microscopy after 1 week of culture indicated that a flow rate of 1.0 mL/min resulted in substantial cell death throughout the constructs whereas lowering the flow rate led to an increasing proportion of viable cells, particularly at the center of the constructs. DNA analysis showed increases in cell proliferation at a flow rate of 0.01 mL/min relative to 0.2 mL/min and static controls. Conversely, mRNA expressions of Runx2, osteocalcin, and alkaline phosphatase were upregulated at 0.2 mL/min compared with lower flow rates as quantified by real-time RT-PCR. These data suggest that medium perfusion may benefit the development of 3-D tissues in vitro by enhancing transport of nutrients and waste within the constructs and providing flow-mediated mechanical stimuli.     

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.     

基于丝素/胶原/羟基磷灰石仿生骨材料的多维结构及其性能

目的 体外构建丝素蛋白（silk fibroin,SF）、I型胶原(type I collagen,Col-I)和羟基磷灰石(hydroxyapatite, HA)共混体系制备二维复合膜和三维仿生支架,研究其理化性质和生物相容性,探讨其在组织工程支架材料中应用的可行性。方法 通过在细胞培养小室底部共混SF/Col-I/HA以及低温3D打印结合真空冷冻干燥法制备二维复合膜及三维支架。通过机械性能测试、电子显微镜和Micro-CT检测材料的理化性质,检测细胞的增殖评估其生物相容性。结果 通过共混和低温3D打印获得稳定的二维复合膜及三维多孔结构支架;力学性能具有较好的一致性,孔径、吸水率、孔隙率和弹性模量均符合构建组织工程骨的要求;支架为网格状的白色立方体,内部孔隙连通性较好; HA均匀分布在复合膜中,细胞黏附在复合膜上,呈扁平状;细胞分布在支架孔壁周围,呈梭形状,生长及增殖良好。结论 利用SF/Col-I/HA共混体系成功制备复合膜及三维支架,具有较好的孔连通性与孔结构,有利于细胞和组织的生长以及营养输送,其理化性能以及生物相容性符合骨组织工程生物材料的要求。     

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.     

The interactions of astrocytes and fibroblasts with defined pore structures in static and perfusion cultures

Open pores to maintain nutrient diffusion and waste removal after cell colonization are crucial for the successful application of constructs based on assembled membranes, in our case tubular scaffolds made of ?-polycaprolactone (PCL), for use in tissue engineering. Due to the complex three-dimensional structure and large size of such scaffolds needed for transplantable tissues, it is difficult to investigate the cell-pore interactions in situ. Therefore miniaturized bioreactors inside Petri dishes (30 mm in diameter), containing porous PCL or poly-dimethylsiloxane (PDMS) membranes, were developed to allow the interactions of different cells with defined pores to be investigated in situ during both static and perfusion cultures. Investigation of two different cell types (fibroblasts and cortical astrocytes) and how they interact with a range of pores (100-350 μm in diameter) for up to 50 days indicated that the cells either 'covered' or 'bridged' the pores. Three distinct behaviors were observed in the way cortical astrocytes interacted with pores, while fibroblasts were able to quickly bridge the pores based on consistent "joint efforts". Our studies demonstrate that the distinct pore sealing behaviors of both cell types were influenced by pore size, initial cell density and culture period, but not by medium perfusion within the range of shear forces investigated. These findings form important basic data about the usability of pores within scaffolds that could inform the design and fabrication of suitable scaffolds for various applications in tissue engineering.     

Finite element study of scaffold architecture design and culture conditions for tissue engineering

Tissue engineering scaffolds provide temporary mechanical support for tissue regeneration and transfer global mechanical load to mechanical stimuli to cells through its architecture. In this study the interactions between scaffold pore morphology, mechanical stimuli developed at the cell microscopic level, and culture conditions applied at the macroscopic scale are studied on two regular scaffold structures. Gyroid and hexagonal scaffolds of 55% and 70% porosity were modeled in a finite element analysis and were submitted to an inlet fluid flow or compressive strain. A mechanoregulation theory based on scaffold shear strain and fluid shear stress was applied for determining the influence of each structures on the mechanical stimuli on initial conditions. Results indicate that the distribution of shear stress induced by fluid perfusion is very dependent on pore distribution within the scaffold. Gyroid architectures provide a better accessibility of the fluid than hexagonal structures. Based on the mechanoregulation theory, the differentiation process in these structures was more sensitive to inlet fluid flow than axial strain of the scaffold. This study provides a computational approach to determine the mechanical stimuli at the cellular level when cells are cultured in a bioreactor and to relate mechanical stimuli with cell differentiation.     

Effect of scaffold architecture and pore size on smooth muscle cell growth

Tissue engineering has the potential to replace damaged tissues and organs. Diffusion limitation of cell growth in three-dimensional (3D) scaffolds is a significant constraint in most tissue engineering applications. This study describes a scaffold architecture that improves mass transfer. Scaffolds with three different geometries of villi architecture (0.5, 1, 0.5; 0.5, 1, 1; 1, 1, 1 mm; villus diameter, height, intervillus spacing, respectively) were fabricated by indirect 3D printing technique. The ability of these scaffolds to support smooth muscle cell growth was investigated in vitro. Smooth muscle cells attached to the scaffolds uniformly after 1 day of culture, and the cell density in the scaffold with small villi feature (0.5 mm) was significantly higher as compared to that for the scaffold with large villi features (1 mm) after 14 days of culture. To evaluate the effect of scaffold pore size on cell growth, scaffolds with three different pore size ranges (50-100, 100-150, and 150-200 microm) were fabricated by the solvent casting and particulate leaching technique. Scaffold pore size did not significantly affect cell growth after 14 days of culture. Optimization in the architectural design of scaffolds provides an alternative method to improve diffusion limitation in the 3D constructs.     

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.     

Influence of the porosity of starch-based fiber mesh scaffolds on the proliferation and osteogenic differentiation of bone marrow stromal cells cultured in a flow perfusion bioreactor

This study investigates the influence of the porosity of fiber mesh scaffolds obtained from a blend of starch and poly(epsilon-caprolactone) on the proliferation and osteogenic differentiation of marrow stromal cells cultured under static and flow perfusion conditions. For this purpose, biodegradable scaffolds were fabricated by a fiber bonding method into mesh structures with two different porosities-- 50 and 75%. These scaffolds were then seeded with marrow stromal cells harvested from Wistar rats and cultured in a flow perfusion bioreactor or in 6-well plates for up to 15 days. Scaffolds of 75% porosity demonstrated significantly enhanced cell proliferation under both static and flow perfusion culture conditions. The expression of alkaline phosphatase activity was higher in flow cultures, but only for cells cultured onto the higher porosity scaffolds. Calcium deposition patterns were similar for both scaffolds, showing a significant enhancement of calcium deposition on cellscaffold constructs cultured under flow perfusion, as compared to static cultures. Calcium deposition was higher in scaffolds of 75% porosity, but this difference was not statistically significant. Observation by scanning electron microscopy showed the formation of pore-like structures within the extracellular matrix deposited on the higher porosity scaffolds. Fourier transformed infrared spectroscopy with attenuated total reflectance and thin-film X-ray diffraction analysis of the cell-scaffold constructs after 15 days of culture in a flow perfusion bioreactor revealed the presence of a mineralized matrix similar to bone. These findings indicate that starch-based scaffolds, in conjunction with fluid flow bioreactor culture, minimize diffusion constraints and provide mechanical stimulation to the marrow stromal cells, leading to enhancement of differentiation toward development of bone-like mineralized tissue. These results also demonstrate that the scaffold structure, namely, the porosity, influences the sequential development of osteoblastic cells and, in combination with the culture conditions, may affect the functionality of tissues formed in vitro.     

Quantitative analysis of three-dimensional fluid flow in rotating bioreactors for tissue engineering

Tissue engineering has emerged as a viable alternative to the problem of organ and tissue shortage. Our laboratory has developed matrices for bone tissue engineering based on sintered spherical particles and, using bioreactor technology, has demonstrated the ability to produce highly mineralized matrices in vitro. In this study, porous microcapsule scaffolds were developed for bone tissue engineering in the high aspect ratio vessel rotating bioreactor. The motion of individual microcapsules as well as scaffolds in the bioreactor were studied by numerical simulation and in situ imaging analysis. Results show that spherical microcapsules with density less than the surrounding fluid exhibited two motions: (1) a periodic circular orbit with tangential speed equal to the free fall speed of the particle, and (2) an inward radial migration of the circular orbit toward the center of the bioreactor vessel. Lighter-than-water scaffolds were fabricated by sintering poly(lactic-co-glycolic acid) hollow microcarriers with diameter from 500 to 860 microm into a fixed three-dimensional geometry with approximately 30% pore volume and 180 to 190 microm median pore size. Scaffolds were fabricated with aggregate densities ranging from 0.65 g/mL and 0.99 g/mL by appropriate combinations of hollow and solid microcarriers within the scaffold. Scaffold velocity in the bioreactor for the above range of densities was accurately predicted by numerical simulation and ranged from 100 mm/s to 3 mm/s. Maximum shear stress estimation due to media flow over the exterior of the scaffold ranged from 0.3 N/m(2) to 0.006 N/m(2). Internal perfusion velocity through scaffolds also was calculated and ranged from 13 mm/s to 0.2 mm/s. Estimates of maximum interior shear stress ranged from 0.03 to 0.0007 N/m(2). These analytical methods provide an excellent vehicle for the study of bone tissue synthesis in three-dimensional culture with fluid flow.     

Effect of flow perfusion on the osteogenic differentiation of bone marrow stromal cells cultured on starch-based three-dimensional scaffolds

This study aims to investigate the effect of culturing conditions (static and flow perfusion) on the proliferation and osteogenic differentiation of rat bone marrow stromal cells seeded on two novel scaffolds exhibiting distinct porous structures. Specifically, scaffolds based on SEVA-C (a blend of starch with ethylene vinyl alcohol) and SPCL (a blend of starch with polycaprolactone) were examined in static and flow perfusion culture. SEVA-C scaffolds were formed using an extrusion process, whereas SPCL scaffolds were obtained by a fiber bonding process. For this purpose, these scaffolds were seeded with marrow stromal cells harvested from femoras and tibias of Wistar rats and cultured in a flow perfusion bioreactor and in 6-well plates for 3, 7, and 15 days. The proliferation and alkaline phosphatase activity patterns were similar for both types of scaffolds and for both culture conditions. However, calcium content analysis revealed a significant enhancement of calcium deposition on both scaffold types cultured under flow perfusion. This observation was confirmed by Von Kossa-stained sections and tetracycline fluorescence. Histological analysis and confocal images of the cultured scaffolds showed a much better distribution of cells within the SPCL scaffolds than the SEVA-C scaffolds, which had limited pore interconnectivity, under flow perfusion conditions. In the scaffolds cultured under static conditions, only a surface layer of cells was observed. These results suggest that flow perfusion culture enhances the osteogenic differentiation of marrow stromal cells and improves their distribution in three-dimensional, starch-based scaffolds. They also indicate that scaffold architecture and especially pore interconnectivity affect the homogeneity of the formed tissue.     

Resorbable polymeric scaffolds for bone tissue engineering: the influence of their microstructure on the growth of human osteoblast-like MG 63 cells

Degradable three-dimensional porous scaffolds applicable as cell carriers for bone tissue engineering were developed by an innovative solvent casting/particulate leaching technique from poly(L-lactide-co-glycolide) (PLG). Three types of PLG scaffolds were prepared, and these had the same high porosity (83%) but increasing diameter of the pores (180-200 microm, 250-320 microm, and 400-600 microm) and increasing pore interconnectivity. The colonization of the scaffolds with human osteoblast-like MG 63 cells was then studied in vitro in a conventional static cell culture system. The number of cells growing on the scaffolds on days 1 and 7 after seeding was highest in the material with the largest pore diameter, but on day 15, the differences among the scaffolds disappeared. Confocal microscopy revealed that on day 1 after seeding, the cells penetrated to a depth of 490 +/- 100 microm, 720 +/- 170 microm, and 720 +/- 120 microm into the scaffolds of small, medium, and large pore size, respectively. Incorporation of bromodeoxyuridine into newly synthesized DNA and the concentration of vinculin, beta-actin, osteopontin, and osteocalcin in cells on the scaffolds of all pore sizes were similar to the values obtained on standard tissue culture polystyrene, which indicated good biocompatibility of the scaffolds. These results suggest that all scaffolds could serve as good carriers for bone cells, although the quickest colonization with cells was found in the scaffolds with the largest pore diameter from 400 to 600 microm.     

Engineering three-dimensional bone tissue in vitro using biodegradable scaffolds: investigating initial cell-seeding density and culture period

New three-dimensional (3D) scaffolds for bone tissue engineering have been developed throughout which bone cells grow, differentiate, and produce mineralized matrix. In this study, the percentage of cells anchoring to our polymer scaffolds as a function of initial cell seeding density was established; we then investigated bone tissue formation throughout our scaffolds as a function of initial cell seeding density and time in culture. Initial cell seeding densities ranging from 0.5 to 10 x 10(6) cells/cm(3) were seeded onto 3D scaffolds. After 1 h in culture, we determined that 25% of initial seeded cells had adhered to the scaffolds in static culture conditions. The cell-seeded scaffolds remained in culture for 3 and 6 weeks, to investigate the effect of initial cell seeding density on bone tissue formation in vitro. Further cultures using 1 x 10(6) cells/cm(3) were maintained for 1 h and 1, 2, 4, and 6 weeks to study bone tissue formation as a function of culture period. After 3 and 6 weeks in culture, scaffolds seeded with 1 x 10(6) cells/cm(3) showed similar tissue formation as those seeded with higher initial cell seeding densities. When initial cell seeding densities of 1 x 10(6) cells/cm(3) were used, osteocalcin immunolabeling indicative of osteoblast differentiation was seen throughout the scaffolds after only 2 weeks of culture. Von Kossa and tetracycline labeling, indicative of mineralization, occurred after 3 weeks. These results demonstrated that differentiated bone tissue was formed throughout 3D scaffolds after 2 weeks in culture using an optimized initial cell density, whereas mineralization of the tissue only occurred after 3 weeks. Furthermore, after 6 weeks in culture, newly formed bone tissue had replaced degrading polymer.     

In vitro localization of bone growth factors in constructs of biodegradable scaffolds seeded with marrow stromal cells and cultured in a flow perfusion bioreactor

Tissue engineering strategies aim at controlling the behavior of individual cells to stimulate tissue formation. This control is achieved by mimicking signals that manage natural tissue development or repair. Flow perfusion bioreactors that create culture environments with minimal diffusion constraints and provide cells with mechanical stimulation may closely resemble in vivo conditions for bone formation. Therefore, these culturing systems, in conjunction with an appropriate scaffold and cell type, may provide significant insight towards the development of in vitro tissue engineering models leading to improved strategies for the construction of bone tissue substitutes. The objective of this study was to investigate the in vitro localization of several bone growth factors that are usually associated with bone formation in vivo by culturing rat bone marrow stromal cells seeded onto starch-based biodegradable fiber meshes in a flow perfusion bioreactor. The localization of several bone-related growth factors-namely, transforming growth factor-beta1, platelet-derived growth factor- A, fibroblast growth factor-2, vascular endothelial growth factor, and bone morphogenetic protein- 2-was determined at two different time points in scaffolds cultured under perfusion conditions at two different flow rates using an immunohistochemistry technique. The results show the presence of regions positively stained for all the growth factors considered, except platelet-derived growth factor-A. Furthermore, the images obtained from the positively stained sections suggest an increase in the immunohistochemically stained area at the higher flow rate and culture time. These observations demonstrate that flow perfusion augments the functionality of scaffold/cell constructs grown in vitro as it combines both biological and mechanical factors to enhance cell differentiation and cell organization within the construct. This study also shows that flow perfusion bioreactor culture of marrow stromal cells, combined with the use of appropriate biodegradable fiber meshes, may constitute a useful model to study bone formation and assess bone tissue engineering strategies in vitro.     

Porous alginate/poly(ε-caprolactone) scaffolds: preparation, characterization and in vitro biological activity

In bone tissue engineering, scaffolds with controlled porosity are required to allow cell ingrowth, nutrient diffusion and sufficient formation of vascular networks. The physical properties of synthetic scaffolds are known to be dependent on the biomaterial type and its processing technique. In this study, we demonstrate that the separation phase technique is a useful method to process poly(ε-caprolactone) (PCL) into a desired shape and size. Moreover, using poly(ethylene glycol), sucrose, fructose and Ca2+ alginate as porogen agents, we obtained PCL scaffolds with three-dimensional porous structures characterized by different pore size and geometry. Scanning electron microscopy and porosity analysis indicated that PCL scaffolds prepared with Ca2+ alginate threads resemble the porosity and the homogeneous pore size distribution of native bone. In parallel, MicroCT analysis confirmed the presence of interconnected void spaces suitable to guarantee a biological environment for cellular growth, as demonstrated by a biocompatibility test with MC3T3-E1 murine preosteoblastic cells. In particular, scaffolds prepared with Ca2+ alginate threads increased adhesion and proliferation of MC3T3-E1 cells under basal culture conditions, and upon stimulation with a specific differentiation culture medium they enhanced the early and later differentiated cell functions, including alkaline phosphatase activity and mineralized extracellular matrix production. These results suggest that PCL scaffolds, obtained by separation phase technique and prepared with alginate threads, could be considered as candidates for bone tissue engineering applications, possessing the required physical and biological properties.     

Osteogenic differentiation of dura mater stem cells cultured in vitro on three-dimensional porous scaffolds of poly(epsilon-caprolactone) fabricated via co-extrusion and gas foaming

A novel scaffold fabrication method utilizing both polymer blend extrusion and gas foaming techniques to control pore size distribution is presented. Seventy-five per cent of all pores produced using polymer blend extrusion alone were less than 50mum. Introducing a gas technique provided better control of pore size distribution, expanding the range from 0-50 to 0-350mum. Varying sintering time, annealing temperature and foaming pressure also helped to reduce the percentage of pore sizes below 50mum. Scaffolds chosen for in vitro cellular studies had a pore size distribution of 0-300mum, average pore size 66+/-17mum, 0.54+/-0.02% porosity and 98% interconnectivity, measured by micro-computed tomography (microCT) analysis. The ability of the scaffolds to support osteogenic differentiation for subsequent cranial defect repair was evaluated by static and dynamic (0.035+/-0.006ms(-1) terminal velocity) cultivation with dura mater stem cells (DSCs). In vitro studies showed minimal increases in proliferation over 28 days in culture in osteogenic media. Alkaline phosphatase expression remained constant throughout the study. Moderate increases in matrix deposition, as assessed by histochemical staining and microCT analysis, occurred at later time points, days 21 and 28. Although constructs cultured dynamically showed greater mineralization than static conditions, these trends were not significant. It remains unclear whether bioreactor culture of DSCs is advantageous for bone tissue engineering applications. However, these studies show that polycaprolactone (PCL) scaffolds alone, without the addition of other co-polymers or ceramics, support long-term attachment and mineralization of DSCs throughout the entire porous scaffold.     

Optimally porous and biomechanically compatible scaffolds for large-area bone regeneration

Large-area or critical-sized bone defects pose a serious challenge in orthopedic surgery, as all current treatment options present with shortcomings. Bone tissue engineering offers a more promising alternative treatment strategy. However, this approach requires mechanically stable scaffolds that support homogenous bone formation throughout the scaffold thickness. Despite advances in scaffold fabrication, current scaffold-based techniques are unable to support uniform, three-dimensional bone regeneration, and are limited to only the scaffold surface in vitro and in vivo. This is mainly because of inadequate scaffold pore sizes (<200?μm) and accessible pore volume, and the associated limited oxygen diffusion and vascular invasion. In this study, we have adopted a method combining microsphere-sintering and porogen-leaching techniques to fabricate scaffolds with an increased accessible pore volume. Of the scaffolds developed, moderately porous poly(85 lactide-co-15 glycolide) (PLGA) microsphere scaffolds were selected as most advantageous, since they retain mechanical strength in the range of human cancellous bone and display a significantly higher accessible pore volume, which is attributed to an increased percentage of larger pores (i.e., size range 200-600?μm). Unlike control scaffolds with a limited pore size and an accessible pore volume, moderately porous scaffolds displayed increased oxygen diffusion, pre-osteoblast cell infiltration, proliferation, and survival throughout the entire scaffold. Furthermore, moderately porous PLGA microsphere scaffolds displayed enhanced and homogenous mineralization in vitro. Since these newly designed moderately porous scaffolds are weight bearing, are fully osteoconductive, and have the ability to support vascularization, they may serve as effective scaffolds for large-area bone defect repair/regeneration. In addition, this study demonstrates the ability to modulate scaffold porosity and, in turn, to develop oxygen tension-controlled matrices that are effective for large-area bone regeneration.