Oscillatory Perfusion Culture of CaP-Based Tissue Engineering Bone with and without Dexamethasone

Dexamethasone, a powerful osteogenic agent for osteoblast differentiation, has been suggested to have synergistic effects when applied together with perfusion culture. As ceramic scaffolds are widely used clinically and oscillatory flow well replicates the natural physical conditions, the biological effects of dexamethasone on oscillatory perfusion culture of CaP-based tissue engineering bone were investigated in this study. Mouse osteoblast-like cells, MC 3T3-E1, were seeded onto porous ceramic scaffolds using the oscillatory perfusion method. The seeded constructs were then either cultured by a static method or an oscillatory perfusion method at different flow rates continuously for 6 days with and without dexamethasone. The cell proliferation, early osteogenic effects, and viability were subsequently evaluated. The results showed that the oscillatory flow could enhance early osteogenesis of osteoblast-like cells in three-dimensional culture on ceramic scaffolds, with a peak function at the flow rate of 0.5 mL/min. The cell viability was significantly higher and more uniform in the perfusion groups than in the static culture groups. The uniformity decreased as the perfusion rates decreased. However, dexamethasone seems to have had no significant effects in any of the groups. Our results suggest that dexamethasone is not an efficient osteogenic supplement during perfusion culture on CaP ceramic scaffolds, and predifferentiation before seeding or additional osteogenic factors should be considered for such cultures.     

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.     

Three-dimensional flow perfusion culture system for stem cell proliferation inside the critical-size beta-tricalcium phosphate scaffold

A 3-dimensional flow perfusion system has been created in our laboratory to provide continuous and homogeneous nutrient supply inside the critical-size beta-tricalcium phosphate (beta-TCP) scaffold and permit cell proliferation during long-term incubation. The critical-size porous cylindrical scaffold (14 mm in diameter, 30 mm in length) with a central tunnel was impregnated with sheep mesenchymal stem cells. In the flow perfusion group, the hybrid scaffolds were continuously perfused with complete alpha-minimum essential medium via a peristaltic pump for 7, 14, and 28 days. In the static culture group, the hybrid composites were immersed in the medium without perfusion for 14 and 28 days. The daily glucose consumption was much higher in the flow perfusion group than in the static group (p < 0.001). In the flow perfusion group, glucose consumption increased dramatically in the first 14 days, and the increase slowed in the last 14 days. In the static group, the increase occurred only in the first 14 days. Cell viability via MTT colorimetry increased with time, which coincided with the results of glucose consumption. Histological study showed that the cells proliferated through the whole scaffolds under the flow perfusion culture. While under the static culture, the cells survived and proliferated only inside the first to third rows of the macropores under the scaffold surface. The cell quantity increased with time under flow perfusion culture. The results suggest that flow perfusion culture is superior to static culture for mesenchymal stem cell proliferation in the critical-size porous scaffold. This perfusion culture system permits a constant nutrition supply into the center of a large-scale scaffold for at least 4 weeks. Determination of D-glucose in the culture medium is a noninvasive way to survey cell proliferation in this system.     

Application of a polyelectrolyte complex coacervation method to improve seeding efficiency of bone marrow stromal cells in a 3D culture system

High seeding efficiency with homogenous distribution of limited cell sources such as bone marrow stromal cells (BMSCs) are of clinical relevance in scaffold-based tissue engineering. Therefore, considerable research efforts have been invested to ameliorate the seeding efficiency in 3D scaffolds. Preliminary data demonstrated that indeed BMSCs were viable and were able to proliferate in a model 3D scaffold, i.e. Cytomatrix scaffold. However, the eventual practical application of BMSCs in such 3D scaffolds is limited by the low seeding efficiency of the cells within the scaffold. Here, we demonstrated that the cell seeding efficiency of BMSCs in the Cytomatrix scaffold can be improved significantly (t-test, p<0.05) by means of macroencapsulating the scaffold via the complex coacervation of a methylated collagen and terpolymer. The thickness and density of the polyeletrolyte complex can be modulated by the contact time between the methylated collagen and terpolymer to balance between cell entrapment efficacy and mass transfer impedance imparted by the complex. Porcine BMSCs were macroencapsulated in Cytomatrix scaffolds using various polyelectrolyte contact time and cultured under both static and dynamic conditions. Throughout the range of contact time investigated, macroencapsulation did not affect the viability of the porcine BMSCs in dynamic culture. However, the viability of the cells under static cultures was compromised with longer polyelectrolyte contact time. Therefore, this proposed method of macroencapsulation enables customization to achieve enhanced seeding efficiency without mass transfer impedance for different culture configurations.     

Self-assembled composite matrix in a hierarchical 3-D scaffold for bone tissue engineering

It is of high clinical relevance in bone tissue engineering that scaffolds promote a high seeding efficiency of cells capable of osteogenic differentiation, such as human bone marrow-derived mesenchymal stem cells (hMSCs). We evaluated the effects of a novel polycaprolactone (PCL) scaffold on hMSC seeding efficiency, proliferation, distribution and differentiation. Porous PCL meshes prepared by fused deposition modeling (FDM) were embedded in matrix of hyaluronic acid, methylated collagen and terpolymer via polyelectrolyte complex coacervation. Scaffolds were cultured statically and dynamically in osteogenic stimulation medium for up to 28 days. Compared to naked PCL scaffolds, embedded scaffolds provided a higher cell seeding efficiency (t-test, P<0.05), a more homogeneous cell distribution and more osteogenically differentiated cells, verified by a more pronounced gene expression of the bone markers alkaline phosphatase, osteocalcin, bone sialoprotein I and bone sialoprotein II. Dynamic culture resulted in higher amounts of DNA (day 14 and day 21) and calcium (day 21 and day 28), compared to static culture. Dynamic culture and the embedding synergistically enhanced the calcium deposition of hMSC on day 21 and day 28. This in vitro study provides evidence that hybrid scaffolds made from natural and synthetic polymers improve cellular seeding efficiency, proliferation, distribution and osteogenic differentiation.     

Online measurement of oxygen consumption by goat bone marrow stromal cells in a combined cell-seeding and proliferation perfusion bioreactor

In an effort to produce clinically useful volumes of tissue engineered bone products, a direct perfusion bioreactor system was developed. Perfusion flow rate, flow direction, and the position of the bioreactor are factors that influenced the amounts and homogeneity of the cells seeded on the scaffold surface. Goat bone marrow stromal cells (GBMSCs) were dynamically seeded and proliferated in this system in relevant volumes (10 cm(3)) of small-sized macroporous biphasic calcium phosphate (BCP) scaffolds (2-6 mm). Cell load and cell distribution were shown using Methylene Blue block staining, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining was used to demonstrate the viability of the cells. Although cells were not distributed homogenously after cell seeding, the scaffolds were covered with a viable, homogeneous cell layer after 25 days of cultivation. The hybrid structures became interconnected, and a dense layer of extracellular matrix formed on and in the scaffolds. Online oxygen measurements during cultivation were correlated with proliferating GBMSCs. It was shown that the oxygen consumption could possibly be used to estimate GBMSC population doubling times during growth in this bioreactor system. On the basis of our results, we conclude that a direct perfusion bioreactor system is capable of seeding and proliferating GBMSCs on BCP ceramic scaffolds that can be monitored online during cultivation.     

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.     

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.     

Control of in vitro tissue-engineered bone-like structures using human mesenchymal stem cells and porous silk scaffolds

Natural bone consists of cortical and trabecular morphologies, the latter having variable pore sizes. This study aims at engineering different bone-like structures using scaffolds with small pores (112-224 microm) in diameter on one side and large pores (400-500 microm) on the other, while keeping scaffold porosities constant among groups. We hypothesized that tissue engineered bone-like structure resulting from silk fibroin (SF) implants is pre-determined by the scaffolds' geometry. To test this hypothesis, SF scaffolds with different pore diameters were prepared and seeded with human mesenchymal stem cells (hMSC). As compared to static seeding, dynamic cell seeding in spinner flasks resulted in equal cell viability and proliferation, and better cell distribution throughout the scaffold as visualized by histology and confocal microscopy, and was, therefore, selected for subsequent differentiation studies. Differentiation of hMSC in osteogenic cell culture medium in spinner flasks for 3 and 5 weeks resulted in increased alkaline phosphatase activity and calcium deposition when compared to control medium. Micro-computed tomography (microCT) detailed the pore structures of the newly formed tissue and suggested that the structure of tissue-engineered bone was controlled by the underlying scaffold geometry.     

循环灌注接种方法在人脐带间充质干细胞骨组织工程中的应用

背景：课题前期设计了一套模块式三维灌注生物反应器系统,并初步将其应用于大鼠骨髓间充质干细胞接种于三维非纺型聚对苯二甲酸乙二醇酯(polyethylene terephthalate,PET)纤维片状载体的骨组织工程研究中。 目的：应用自制的循环灌注接种系统将传代人脐带间充质干细胞接种至三维无纺布PET片状载体,并与传统的静态接种方法接种的载体进行比较。 方法：培养传代人脐带间充质干细胞,流式细胞仪检测细胞表面标记,将细胞用循环灌注方法(高速率组和低速率组)和静态接种方法接种至三维聚PET无纺布片状载体。 结果与结论：传代细胞形态稳定、活力好,高标达 CD90,CD105,不表达CD14,CD45。循环灌注方法组的接种效率,细胞密度,增殖能力均优于静态接种组。循环灌注高速率组乳酸脱氢酶偏高,并且细胞的延迟期长于静态接种。循环灌注组的碱性磷酸酶活性高于静态接种组。结果表明,循环灌注接种方法更适合间充质干细胞骨组织工程的应用,进一步应用仍需对工程参数进行优化。     

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.     

Three-dimensional cell culture and tissue engineering in a T-CUP (tissue culture under perfusion)

The aim of this study was to develop and validate a simple and compact bioreactor system for perfusion cell seeding and culture through 3-dimensional porous scaffolds. The developed Tissue Culture Under Perfusion (T-CUP) bioreactor is based on the concept of controlled and confined alternating motion of scaffolds through a cell suspension or culture medium, as opposed to pumping of the fluid through the scaffolds. Via the T-CUP, articular chondrocytes and bone marrow stromal cells could be seeded into porous scaffolds of different compositions and architectures (chronOS, Hyaff-11, and Polyactive) at high efficiency (greater than 75%), uniformity (cells were well distributed throughout the scaffold pores), and viability (greater than 97%). Culture of articular chondrocytes seeded into 4-mm thick Polyactive scaffolds for 2 weeks in the T-CUP resulted in uniform deposition of cartilaginous matrix. Cultivation of freshly isolated human bone marrow nucleated cells seeded into ENGipore ceramic scaffolds for 19 days in the T-CUP resulted in stromal cell-populated constructs capable of inducing ectopic bone formation in nude mice. The T-CUP bioreactor represents an innovative approach to simple, efficient, and reliable 3D cell culture, and could be used either as a model to investigate mechanisms of tissue development or as a graft manufacturing system in the context of regenerative medicine.     

Application of porous glycosaminoglycan-based scaffolds for expansion of human cord blood stem cells in perfusion culture

In vitro expansion of hematopoietic stem cells (HSCs) has been employed to obtain sufficient numbers of stem cells for successful engraftment after HSC transplantation. A three-dimensional perfusion bioreactor system with a heparin-chitosan scaffold was designed and evaluated for its capability to support maintenance and expansion of HSCs. Porous chitosan scaffolds were fabricated by a freeze-drying technique and N-desulfated heparin was covalently immobilized within the scaffolds using carbodiimide chemistry. CD34+ HSCs isolated from umbilical cord blood by immunomagnetic separation were cultured within the porous scaffold in a perfusion bioreactor system. Control cultures were maintained on dishes coated with similar heparin-chitosan films. Oxygen uptake was measured during the culture period. After 7 days of culture, scaffolds were harvested for analysis. Cellular phenotype and HSC characteristics were evaluated via flow cytometry and colony forming unit assays. The results indicate good cell retention and proliferation within the perfused scaffolds. Oxygen consumption in the perfusion bioreactor system increased continuously during the culture, indicating steady cell growth. Cells from the perfused scaffold cultures showed higher percentages of primitive progenitors and exhibited superior colony forming unit performance as compared to cells from static cultures. In addition, perfusion culture at low oxygen (5%) enhanced the expansion of CD34+ cells and colony-forming activity compared to high oxygen (19%) cultures. The results suggest that perfusion culture of cord blood CD34+ cells under bone marrow-like conditions enhances HSC expansion compared to static cultures.     

Seeding cells into needled felt scaffolds for tissue engineering applications

Tissue engineering methods are under development that will enable the repair or replacement of a variety of tissues, including articular cartilage and bone. To engineer functional tissue it is necessary that scaffolds initially be seeded with a large number of cells distributed evenly throughout the scaffold structure. It previously has been shown that, compared to static seeding conditions, seeding scaffolds under dynamic conditions facilitates high seeding densities and even distributions of cells (Li et al., Biotechnology Progress 2001;17:935-944). The efficiency of seeding HOSTE85 cells and bovine chondrocytes into needled felt scaffolds following agitation at different speeds was determined. Seeding efficiency was determined using the Hoechst 33258 assay, and cell viability was assessed using the Alamar Blue trade mark assay. The distribution of cells within the scaffolds was imaged using scanning electron microscopy. It was found that the optimum seeding conditions varied for HOSTE85 cells and bovine chondrocytes, with different agitation speeds leading to different seeding efficiencies, cell viabilities, and distributions of cells within scaffolds. The optimum agitation speeds for seeding a high number of viable cells into scaffolds so that they were arranged evenly were 300 rpm for HOSTE85 cells and 200 rpm for bovine chondrocytes.     

Three-dimensional plotted scaffolds with controlled pore size gradients: Effect of scaffold geometry on mechanical performance and cell seeding efficiency

Scaffolds produced by rapid prototyping (RP) techniques have proved their value for tissue engineering applications, due to their ability to produce predetermined forms and structures featuring fully interconnected pore architectures. Nevertheless, low cell seeding efficiency and non-uniform distribution of cells remain major limitations when using such types of scaffold. This can be mainly attributed to the inadequate pore architecture of scaffolds produced by RP and the limited efficiency of cell seeding techniques normally adopted. In this study we aimed at producing scaffolds with pore size gradients to enhance cell seeding efficiency and control the spatial organization of cells within the scaffold. Scaffolds based on blends of starch with poly(ε-caprolactone) featuring both homogeneously spaced pores (based on pore sizes of 0.75 and 0.1 mm) and pore size gradients (based on pore sizes of 0.1-0.75-0.1 and 0.75-0.1-0.75 mm) were designed and produced by three-dimensional plotting. The mechanical performance of the scaffolds was characterized using dynamic mechanical analysis (DMA) and conventional compression testing under wet conditions and subsequently characterized using scanning electron microscopy and micro-computed tomography. Osteoblast-like cells were seeded onto such scaffolds to investigate cell seeding efficiency and the ability to control the zonal distribution of cells upon seeding. Scaffolds featuring continuous pore size gradients were originally produced. These scaffolds were shown to have intermediate mechanical and morphological properties compared with homogenous pore size scaffolds. The pore size gradient scaffolds improved seeding efficiency from ～35% in homogeneous scaffolds to ～70% under static culture conditions. Fluorescence images of cross-sections of the scaffolds revealed that scaffolds with pore size gradients induce a more homogeneous distribution of cells within the scaffold.     

Comparative study of seeding methods for three-dimensional polymeric scaffolds

Development of tissue-engineered devices may be enhanced by combining cells with porous absorbable polymeric scaffolds before implantation. The cells are seeded throughout the scaffolds and allowed to proliferate in vitro for a predetermined amount of time. The distribution of cells throughout the porous material is one critical component determining success or failure of the tissue-engineered device. This can influence both the successful integration of the device with the host tissue as well as the development of a vascularized network throughout the entire scaffold volume. This research sought to compare different seeding and proliferation methods to select an ideal method for a polyglycolide/aortic endothelial cell system. Two seeding environments, static and dynamic, and three proliferation environments, static, dynamic, and bioreactor, were analyzed, for a total of six possible methods. The six seeding and proliferation combinations were analyzed following a 1-week total culture time. It was determined that for this specific system, dynamic seeding followed by a dynamic proliferation phase is the least promising method and dynamic seeding followed by a bioreactor proliferation phase is the most promising.     

Flow Perfusion Improves Seeding of Tissue Engineering Scaffolds with Different Architectures

Engineered bone grafts have been generated in static and dynamic systems by seeding and culturing osteoblastic cells on 3-D scaffolds. Seeding determines initial cellularity and cell spatial distribution throughout the scaffold, and affects cell–matrix interactions. Static seeding often yields low seeding efficiencies and poor cell distributions; thus creating a need for techniques that can improve these parameters. We have evaluated the effect of oscillating flow perfusion on seeding efficiency and spatial distribution of MC3T3-E1 pre-osteoblastic cells in fibrous polystyrene matrices (20, 35 and 50-μm fibers) and foams prepared by salt leaching, using as controls statically seeded scaffolds. An additional control was investigated where static seeding was followed by unidirectional perfusion. Oscillating perfusion resulted in the most efficient technique by yielding higher seeding efficiencies, more homogeneous distribution and stronger cell–matrix interactions. Cell surface density increased with inoculation cell number and then reached a maximum, but significant detachment occurred at greater flow rates. Oxygen plasma treatment of the fibers greatly improved seeding efficiency. Having similar porosity and dimensions, fibrous matrices yielded higher cell surface densities than foams. Fluorescence microscopy and histological analyses in polystyrene and PLLA scaffolds demonstrated that perfusion seeding produced more homogeneous cell distribution, with fibrous matrices presenting greater uniformity than the foams.     

一体化灌注法体外构建活化人工骨的优化研究

目的探讨人胚成骨细胞在多孔β-磷酸三钙（β-TCP）支架内灌注性接种及培养的影响因素及其作用机制,对一体化灌注法体外构建活化人工骨进行优化研究。方法应用自行设计的灌注式生物反应器进行多孔β-TCP支架内人胚成骨细胞的一体化灌注性接种和培养,以静态接种为对照。通过细胞活力（Mrrr法）测定、活细胞接种率、组织形态学观察和计量学分析等分别检测细胞接种时间、接种密度、灌注速率等因素对支架内细胞黏附和生长的作用。结果细胞灌注接种效果优于静态接种;灌注接种-灌注培养法细胞生长及分布优于静态接种-灌注培养法。接种时间、接种密度、灌注速率等因素均显著影响细胞接种及培养效果。灌注法构建活化人工骨的最优条件：接种时间12h～24h,接种密度2×10^5／ml一5×10^5／ml,灌注接种速率1ml／min,灌注培养速率0．5ml／min～2ml／min（灌注初期24h）及2ml／min（灌注24h后）。结论一体化灌注法利于多孔β-TCP支架内人胚成骨细胞的黏附和生长。接种时间、接种密度、灌注速率等因素均影响活化人工骨体外构建效果,对其进行优化有助于人工骨移植物的临床转化和推广应用。     

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.     

Engineered channels enhance cellular density in perfused scaffolds

Scaffold-based tissue engineering provides cells with an engineered matrix to enhance and direct cell attachment, proliferation and differentiation. One critical limitation to current tissue engineering approaches is the inability to create densely populated constructs thicker than a few 100 μm. We hypothesized that development of porous, channeled scaffolds would increase cell density and uniformity of their spatial distribution through scaffold channel perfusion. Patterned polyurethane sheets were fabricated using a sprayed phase separation technique and laminated together to form 1.5 mm thick channeled scaffolds. Hydraulic permeability testing confirmed the presence of functional channels throughout the multilaminate construct. A continuous flow bioreactor was used to perfuse the construct with medium during the culture period. Cross-sectional cell densities and spatial uniformities were measured in channeled and nonchanneled scaffolds under different seeding and culture conditions. Channeled scaffolds were found to have higher densities of human mesenchymal stem cells than nonchanneled samples. Perfused scaffolds had more uniform spatial distribution of cells within the scaffold compared to statically cultured scaffolds. In conclusion, we have shown the channeled scaffolds to be a promising approach toward creating thick tissue-engineered constructs.