Numerical fluid-dynamic optimization of microchannel-provided porous scaffolds for the co-culture of adherent and non-adherent cells

Computational fluid dynamic (CFD) techniques were used to optimize the microenvironment inside scaffolds for hematopoietic stem cell (HSC) culture in a perfusion bioreactor. These matrices are meant to be seeded with adherent bone marrow stromal cells and then co-cultivated with HSCs; the scaffold micro-architecture and the fluid-dynamic conditions have to be optimized to avoid non-adherent stem cells being dragged away while ensuring adequate nutrient supply. The insertion of longitudinal microchannels was tested as a tool to improve perfusion in a homogeneous porous scaffold. Models of microchannel-provided scaffolds, characterized by different values of geometric parameters concerning pores and channels, were built, and numerical fluid-dynamic and oxygen-transfer analyses were carried out. The results of the computations indicated that the microchannels created preferential paths for culture medium flow, causing low shear stresses and drag forces within the pores; meanwhile, they improved oxygen delivery by forcing its penetration into the scaffold bulk. In particular, an 85% porous, 3-mm-thick scaffold with 175-microm-diameter pores was considered; at a constant average drag force guaranteeing stem cell suspension inside this porous bulk, the addition of approximately 260-microm-diameter, 700-microm-spaced channels resulted in 34% higher oxygen partial pressure at the exit (approximately 135 vs 101 mmHg), maintaining a wall shear stress median value of approximately 0.14 mPa. The present work demonstrates the capacity of microchannel-provided scaffolds to ensure suitable conditions for HSC culture and shows that CFD methods are a valuable tool to retrieve significant clues for the design of the culture environment.     

Deformation simulation of cells seeded on a collagen-GAG scaffold in a flow perfusion bioreactor using a sequential 3D CFD-elastostatics model

Tissue-engineered bone shows promise in meeting the huge demand for bone grafts caused by up to 4 million bone replacement procedures per year, worldwide. State-of-the-art bone tissue engineering strategies use flow perfusion bioreactors to apply biophysical stimuli to cells seeded on scaffolds and to grow tissue suitable for implantation into the patient's body. The aim of this study was to quantify the deformation of cells seeded on a collagen-GAG scaffold which was perfused by culture medium inside a flow perfusion bioreactor. Using a μCT scan of an unseeded collagen-GAG scaffold, a sequential 3D CFD-deformation model was developed. The wall shear stress and the hydrostatic wall pressure acting on the cells were computed through the use of a CFD simulation and fed into a linear elastostatics model in order to calculate the deformation of the cells. The model used numerically seeded cells of two common morphologies where cells are either attached flatly on the scaffold wall or bridging two struts of the scaffold. Our study showed that the displacement of the cells is primarily determined by the cell morphology. Although cells of both attachment profiles were subjected to the same mechanical load, cells bridging two struts experienced a deformation up to 500 times higher than cells only attached to one strut. As the scaffold's pore size determines both the mechanical load and the type of attachment, the design of an optimal scaffold must take into account the interplay of these two features and requires a design process that optimizes both parameters at the same time.     

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.     

The influence of the scaffold design on the distribution of adhering cells after perfusion cell seeding

In natural tissues, the extracellular matrix composition, cell density and physiological properties are often non-homogeneous. Here we describe a model system, in which the distribution of cells throughout tissue engineering scaffolds after perfusion seeding can be influenced by the pore architecture of the scaffold. Two scaffold types, both with gyroid pore architectures, were designed and built by stereolithography: one with isotropic pore size (412 ± 13 μm) and porosity (62 ± 1%), and another with a gradient in pore size (250-500 μm) and porosity (35%-85%). Computational fluid flow modelling showed a uniform distribution of flow velocities and wall shear rates (15-24 s(-1)) for the isotropic architecture, and a gradient in the distribution of flow velocities and wall shear rates (12-38 s(-1)) for the other architecture. The distribution of cells throughout perfusion-seeded scaffolds was visualised by confocal microscopy. The highest densities of cells correlated with regions of the scaffolds where the pores were larger, and the fluid velocities and wall shear rates were the highest. Under the applied perfusion conditions, cell deposition is mainly determined by local wall shear stress, which, in turn, is strongly influenced by the architecture of the pore network of the scaffold.     

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.     

Three-dimensional macroporous calcium phosphate bioceramics with nested chitosan sponges for load-bearing bone implants

Three-dimensional macroporous calcium phosphate bioceramics embedded with porous chitosan sponges were synthesized to produce composite scaffolds with high mechanical strength and a large surface/volume ratio for load-bearing bone repairing and substitutes. The macroporous calcium phosphate bioceramics with pore diameters of 300 microm to 600 microm were developed using a porogen burnout technique, and the chitosan sponges were formed inside the pores of the bioceramics by first introducing chiosan solution into the pores followed by a freeze-drying process. Our scanning electron microscopy results showed that the pore size of chitosan sponges formed inside the macroporous structure of bioceramics was approximately 100 microm, a structure favorable for bone tissue in-growth. The compressive modulus and yield stress of the composite scaffolds were both greatly improved in comparison with that of HA/beta-TCP scaffolds. The simulated body fluid (SBF) and cell culture experiments were conducted to assess the bioactivity and biocompatibility of the scaffolds. In the SBF tests, a layer of randomly oriented needle-like apatite crystals formed on the scaffold surface after sample immersion in SBF, which suggested that the composite material has good bioactivity. The cell culture experiments showed that MG63 osteoblast cells attached to the composite scaffolds, proliferated on the scaffold surface, and migrated onto the pore walls, indicating good cell biocompatibility of the scaffold. The cell differentiation on the composite scaffolds was evaluated by alkaline phosphatase (ALP) assay. Compared with the control in tissue culture dishes, the cells had almost the same ALP activity on the composite scaffolds during the first 11 days of culture.     

Influence of freezing rate on pore structure in freeze-dried collagen-GAG scaffolds

The cellular structure of collagen-glycosaminoglycan (CG) scaffolds used in tissue engineering must be designed to meet a number of constraints with respect to biocompatibility, degradability, pore size, pore structure, and specific surface area. The conventional freeze-drying process for fabricating CG scaffolds creates variable cooling rates throughout the scaffold during freezing, producing a heterogeneous matrix pore structure with a large variation in average pore diameter at different locations throughout the scaffold. In this study, the scaffold synthesis process was modified to produce more homogeneous freezing by controlling of the rate of freezing during fabrication and obtaining more uniform contact between the pan containing the CG suspension and the freezing shelf through the use of smaller, less warped pans. The modified fabrication technique has allowed production of CG scaffolds with a more homogeneous structure characterized by less variation in mean pore size throughout the scaffold (mean: 95.9 microm, CV: 0.128) compared to the original scaffold (mean: 132.4 microm, CV: 0.185). The pores produced using the new technique appear to be more equiaxed, compared with those in scaffolds produced using the original technique.     

通过骨支架的数字化建模分析其力学性能与内部流场分布

目的研究不同骨支架结构的力学性能与内部流场分布,为模型结构的优劣提供直观上的比较和评判,为骨支架结构设计提供有效的指导方法。方法利用Pro/Engineer和MIMICS等软件重建自然结构骨支架、编织状骨支架和球形孔骨支架,并通过有限元方法分析3种支架的有效弹性模量、应力分布和三维灌注培养下支架内部流场分布。结果采用相同材料设计得到的自然结构骨支架表现出更小的有效弹性模量;当3种支架受到相同压力时,自然结构骨支架内部应力峰值更小且应力分布更均匀;初始流速和流体黏度相同时,自然结构骨支架表现出更小的内部流速、壁面剪切应力和壁面压力。结论自然结构骨支架模型具有相对较好的生物力学性能,在3种骨支架中最适合用于骨组织工程中骨支架结构选型。     

Cell distribution in a scaffold with random architectures under the influence of fluid dynamics

Fluid dynamic environment and scaffold architectures have an important influence on cell growth and distribution inside the scaffold. A porous cylindrical scaffold with a central channel is seeded with the sheep mesenchymal stem cells (MSCs) in this study. Then the cell seeded scaffold is continuously perfused with alpha-MEM medium by a peristaltic pump for 7, 14, and 28 days. Histological study shows that the cell proliferation rates are different throughout the whole scaffolds. The different cell coverage is shown in various positions of the scaffold. A computational fluid dynamics (CFD) modeling is used to simulate the flow conditions within perfused cell-seeded scaffolds to give insight into the mechanisms of these cell growth phenomena. Relating the simulation results to perfusion experiments, the even fluid velocity (approximately 0.26-0.64 mm/s) and shear stress (approximately 0.0029-0.027 Pa) are found to correspond to increased cell proliferation within the cell-scaffold constructs. This method exhibits novel capabilities to compare results obtained for different perfusion rates or different scaffold microarchitectures and may allow specific fluid velocities and shear stresses to be determined that optimize the perfusion flow rate, porous scaffold architecture, and distribution of in vitro tissue growth.     

In vitro and in vivo characteristics of PCL scaffolds with pore size gradient fabricated by a centrifugation method

Polycaprolactone (PCL) cylindrical scaffolds with gradually increasing pore size along the longitudinal direction were fabricated by a novel centrifugation method to investigate pore size effect on cell and tissue interactions. The scaffold was fabricated by the centrifugation of a cylindrical mold containing fibril-like PCL and the following fibril bonding by heat treatment. The scaffold showed gradually increasing pore size (from approximately 88 to approximately 405 microm) and porosity (from approximately 80% to approximately 94%) along the cylindrical axis by applying the centrifugal speed, 3000 rpm. The scaffold sections were examined for their in vitro cell interactions using different kinds of cells (chondrocytes, osteoblasts, and fibroblasts) and in vivo tissue interactions using a rabbit model (skull bone defects) in terms of scaffold pore sizes. It was observed that different kinds of cells and bone tissue were shown to have different pore size ranges in the scaffold for effective cell growth and tissue regeneration. The scaffold section with 380-405 microm pore size showed better cell growth for chondrocytes and osteoblasts, while the scaffold section with 186-200 microm pore size was better for fibroblasts growth. Also the scaffold section with 290-310 microm pore size showed faster new bone formation than those of other pore sizes. The pore size gradient scaffolds fabricated by the centrifugation method can be a good tool for the systematic studies of the interactions between cells or tissues and scaffolds with different pore size.     

振荡流动对组织工程用灌注式生物 反应器中剪切力分布影响

目的 考察振荡流动以及三维支架孔径和孔隙率对生物反应器内流速和剪切力分布的影响,并根据理论计算结果为脱细胞骨三维支架和灌注式生物反应器制备提出优化方法。方法 针对实验室前期制备的骨组织工程用脱细胞骨三维支架和灌注式生物反应器,将脱细胞骨三维支架简化为各向同性的多孔介质,对生物反应器内的流速和剪切力分布进行理论建模。结果 振荡流作用时,多孔支架材料内速度和达西剪切力呈现一致的变化规律,不同半径处流速和达西剪切力差异减小,有利于在骨组织工程中对种子细胞进行均匀三维培养。提高入口灌流速度可提高平均达西剪切力;增加多孔支架孔径或孔隙率对支架内流速峰值影响不大,但会显著降低平均达西剪切力;提高入口振荡流动振荡频率可降低支架内流速最大峰值,显著减小不同半径处流速的差异。结论 适宜的振荡流易产生利于骨组织工程干细胞所需剪切力,研究结果有望为优化骨组织工程中种子细胞的三维培养方法提供理论指导。     

The effect of pore size on cell adhesion in collagen-GAG scaffolds

The biological activity of scaffolds used in tissue engineering applications hypothetically depends on the density of available ligands, scaffold sites at which specific cell binding occurs. Ligand density is characterized by the composition of the scaffold, which defines the surface density of ligands, and by the specific surface area of the scaffold, which defines the total surface of the structure exposed to the cells. It has been previously shown that collagen-glycosaminoglycan (CG) scaffolds used for studies of skin regeneration were inactive when the mean pore size was either lower than 20 microm or higher than 120 microm (Proc. Natl. Acad. Sci., USA 86(3) (1989) 933). To study the relationship between cell attachment and viability in scaffolds and the scaffold structure, CG scaffolds with a constant composition and solid volume fraction (0.005), but with four different pore sizes corresponding to four levels of specific surface area were manufactured using a lyophilization technique. MC3T3-E1 mouse clonal osteogenic cells were seeded onto the four scaffold types and maintained in culture. At the experimental end point (24 or 48 h), the remaining viable cells were counted to determine the percent cell attachment. A significant difference in viable cell attachment was observed in scaffolds with different mean pore sizes after 24 and 48 h; however, there was no significant change in cell attachment between 24 and 48 h for any group. The fraction of viable cells attached to the CG scaffold decreased with increasing mean pore size, increasing linearly (R2 = 0.95, 0.91 at 24 and 48 h, respectively) with the specific surface area of the scaffold. The strong correlation between the scaffold specific surface area and cell attachment indicates that cell attachment and viability are primarily influenced by scaffold specific surface area over this range (95.9-150.5 microm) of pore sizes for MC3T3 cells.     

Accurately shaped tooth bud cell-derived mineralized tissue formation on silk scaffolds

Based on the successful use of silk scaffolds in bone tissue engineering, we examined their utility for mineralized dental tissue engineering. Four types of hexafluoroisopropanol (HFIP) silk scaffolds-(250 and 550 microm diameter pores, with or without arginine-glycine-aspartic acid (RGD) peptide) were seeded with cultured 4-day postnatal rat tooth bud cells and grown in the rat omentum for 20 weeks. Analyses of harvested implants revealed the formation of bioengineered mineralized tissue that was most robust in 550 microm pore RGD-containing scaffolds and least robust in 250 microm pore sized scaffolds without RGD. The size and shape of the silk scaffold pores appeared to guide mineralized tissue formation, as revealed using polarized light imaging of collagen fiber alignment along the scaffold surfaces. This study is the first to characterize bioengineered tissues generated from tooth bud cells seeded onto silk scaffolds and indicates that silk scaffolds may be useful in forming mineralized osteodentin of specified sizes and shapes.     

Synthetic scaffold morphology controls human dermal connective tissue formation

Engineering tissues in bioreactors is often hampered by disproportionate tissue formation at the surface of scaffolds. This hinders nutrient flow and retards cell proliferation and tissue formation inside the scaffold. The objective of this study was to optimize scaffold morphology to prevent this from happening and to determine the optimal scaffold geometric values for connective tissue engineering. After comparing lyophilized crosslinked collagen, compression molded/salt leached PEGT/PBT copolymer and collagen-PEGT/PBT hybrid scaffolds, the PEGT/PBT scaffold was selected for optimization. Geometric parameters were determined using SEM, microcomputed tomography, and flow permeability measurements. Fibroblast were seeded and cultured under dynamic flow conditions for 2 weeks. Cell numbers were determined using CyQuant DNA assay, and tissue distribution was visualized in H&E- and Sirius Red-stained sections. Scaffolds 0.5 and 1.5 mm thick showed bridged connected tissue from top-to-bottom, whereas 4-mm-thick scaffolds only revealed tissue ingrowth until a maximum depth of 0.6-0.8 mm. Rapid prototyped scaffold were used to assess the maximal void space (pore size) that still could be filled with tissue. Tissue bridging between fibers was only found at fiber distances < or =401 +/- 60 microm, whereas filling of void spaces in 3D-deposited scaffolds only occurred at distances < or =273 +/- 55 microm. PEGT/PBT scaffolds having similar optimal porosities, but different average interconnected pore sizes of 142 +/- 50, 160 +/- 56 to 191 +/- 69 microm showed comparable seeding efficiencies at day 1, but after 2 weeks the total cell numbers were significantly higher in the scaffolds with intermediate and high interconnectivity. However, only scaffolds with an intermediate interconnectivity revealed homogenous tissue formation throughout the scaffold with complete filling of all pores. In conclusion, significant amount of connective tissue was formed within 14 days using a dynamic culture process that filled all void spaces of a PEGT/PBT scaffolds with the following geometric parameters: thickness 1.5-1.6 mm, pore size range 90-360 microm, and average interconnecting pore size of 160 +/- 56 microm.     

Optimising bioactive glass scaffolds for bone tissue engineering

A 3D scaffold has been developed that has the potential to fulfil the criteria for an ideal scaffold for bone tissue engineering. Sol-gel derived bioactive glasses of the 70S30C (70 mol% SiO2, 30 mol% CaO) composition have been foamed to produce 3D bioactive scaffolds with hierarchical interconnected pore morphologies similar to trabecular bone. The scaffolds consist of a hierarchical pore network with macropores in excess of 500 microm connected by pore windows with diameters in excess of 100 microm, which is thought to be the minimum pore diameter required for tissue ingrowth and vasularisation in the human body. The scaffolds also have textural porosity in the mesopore range (10-20 nm). The scaffolds were sintered at 600, 700, 800 and 1000 degrees C. As sintering temperature was increased to 800 degrees C the compressive strength increased from 0.34 to 2.26 MPa due to a thickening of the pore walls and a reduction in the textural porosity. The compressive strength is in the range of that of trabecular bone (2-12 MPa). Importantly, the modal interconnected pore diameter (98 microm) was still suitable for tissue engineering applications and bioactivity is maintained. Bioactive glass foam scaffolds sintered at 800 degrees C for 2 h fulfill the criteria for an ideal scaffold for tissue engineering applications.     

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.     

Tissue engineered bone: measurement of nutrient transport in three-dimensional matrices

The classic paradigm for in vitro tissue engineering of bone involves the isolation and culture of donor osteoblasts or osteoprogenitor cells within three-dimensional (3D) scaffold biomaterials under conditions that support tissue growth and mineralized osteoid formation. Our studies focus on the development and utilization of new dynamic culture technologies to provide adequate nutrient flux within 3D scaffolds to support ongoing tissue formation. In this study, we have developed a basic one-dimensional (1D) model to characterize the efficiency of passive nutrient diffusion and transport flux to bone cells within 3D scaffolds under static and dynamic culture conditions. Internal fluid perfusion within modeled scaffolds increased rapidly with increasing pore volume and pore diameter to a maximum of approximately 1% of external fluid flow. In contrast, internal perfusion decreased significantly with increasing pore channel tortuosity. Calculations of associated nutrient flux indicate that static 3D culture and some inappropriately designed dynamic culture environments lead to regions of insufficient nutrient concentration to maintain cell viability, and can result in steep nutrient concentration gradients within the modeled constructs. These quantitative studies provide a basis for development of new dynamic culture methodologies to overcome the limitations of passive nutrient diffusion in 3D cell-scaffold composite systems proposed for in vitro tissue engineering of bone.     

Fabrication and characterization of poly(DL-lactic-co-glycolic acid)/zirconia-hybridized amorphous calcium phosphate composites

Several minerals, such as hydroxyapatite and beta-tricalcium phosphate, have been incorporated into bioresorbable polyester bone scaffolds to increase the osteoconductivity both in vitro and in vivo. More soluble forms of calcium phosphate that release calcium and phosphate ions have been postulated as factors that increase osteoblast differentiation and mineralization. Recently, a zirconia-hybridized pyrophosphate-stabilized amorphous calcium phosphate (Zr-ACP) has been synthesized allowing controlled release of calcium and phosphate ions. When incorporated into bioresorbable scaffolds, Zr-ACP has the potential to induce osteoconductivity. In this study, 80-90% (w/v) porous poly(DL-lactic-co-glycolic acid) (PLGA) scaffolds were formed by thermal phase separation from dioxane while incorporating Zr-ACP. Scanning electron microscopy revealed a highly porous structure with a pore size ranging from a few microm to about 100 microm, smaller than we had hoped for. Zr-ACP particles were evenly dispersed in the composite structure and incorporated into the pore walls. The amorphous structure of the Zr-ACP was maintained during composite fabrication, as found by X-ray diffraction. Composite scaffolds had larger compressive yield strengths and moduli compared to pure polymer scaffolds. These initial efforts demonstrate that PLGA/Zr-ACP composites can be formed in ways that ultimately serve as promising bone scaffolds in tissue engineering.     

Oscillatory perfusion seeding and culturing of osteoblast-like cells on porous beta-tricalcium phosphate scaffolds

Perfusion culture systems have proven to be effective bioreactors for constructing tissue engineered bone in vitro, but existing circuit-based perfusion systems are complicated and costly for conditioned culture due to the large medium volume required. A compact perfusion system for artificial bone fabrication using oscillatory flow is described here. Mouse osteoblast-like MC 3T3-E1 cells were seeded at 1.5 x 10(6) cells/100 microL and cultured for 6 days in porous ceramic beta-tricalcium phosphate scaffolds (10 mm in diameter, 8 mm in height) by only 1.5 mL culture media per scaffold. The seeding efficiency, cell proliferation, distribution and viability, and promotion of early osteogenesis by both a static and an oscillatory perfusion method were evaluated. The oscillatory perfusion method generated higher seeding efficiency, alkaline phosphatase activity, and scaffold cellularity (by DNA content) after 6 days of culture. Stereomicroscopic observation of 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide staining and Calcein-AM/propidium iodide double staining also demonstrated homogeneous seeding, proliferation, and viability of cells throughout the scaffolds in the oscillatory perfusion system. By contrast, the static culture yielded polarized seeding and proliferation favoring the outer and upper scaffold surfaces, with only dead cells in the center of the scaffolds. Thus, these results suggest that the oscillatory flow condition not only allow a better seeding efficiency and homogeneity, but also facilitates uniform culture and early osteogenic differentiation. The oscillatory perfusion system could be a simple and effective bioreactor for bone tissue engineering.     

磷酸钙及硫酸钙支架在骨组织工程中的研究进展

BACKGROUND: It is a hotspot that calcium phosphate and calcium sulphate as the main ingredients are combined with one or more other materials to improve or increase the performance of bone tissue engineering scaffolds. OBJECTIVE: To introduce the research advance of these two kinds of scaffolds in bone tissue engineering. METHODS: The articles related to the bone tissue engineering published during January 2000 to June 2015 were retrieved from CNKI and PubMed databases by computer. The key words were “bone tissue engineering, scaffold, calcium phosphate, calcium sulphate, vascularization” in Chinese and English, respectively. ESULTS AND CONCLUSION: Calcium phosphate and calcium sulfate are characterized as having good biocompatibility, biodegradability, osteoconductivity and complete bone substitutability. However, single use of calcium phosphate or calcium sulfate scaffold has certain disadvantages, both of which are difficult to fully meet the requirements of the bone defect repair. Improvement can be acquired in the mechanical strength, injectability and biodegradability, as well as drug-loading and pro-angiogenesis of the scaffold in combination with other materials. In the basal and clinical research, we should explore and develop ideal scaffolds in on the basis of therapeutic aim. However, most of the scaffold studies are still at the extracorporeal and animal experiment stage, and the comparative studies on composite scaffolds and optimal proportion of those composite scaffolds still need to be further investigated.