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.     

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.     

Flow perfusion culture induces the osteoblastic differentiation of marrow stroma cell-scaffold constructs in the absence of dexamethasone

Flow perfusion culture of scaffold/cell constructs has been shown to enhance the osteoblastic differentiation of rat bone marrow stroma cells (MSCs) over static culture in the presence of osteogenic supplements including dexamethasone. Although dexamethasone is known to be a powerful induction agent of osteoblast differentiation in MSC, we hypothesied that the mechanical shear force caused by fluid flow in a flow perfusion bioreactor would be sufficient to induce osteoblast differentiation in the absence of dexamethasone. In this study, we examined the ability of MSCs seeded on titanium fiber mesh scaffolds to differentiate into osteoblasts in a flow perfusion bioreactor in both the presence and absence of dexamethasone. Scaffold/cell constructs were cultured for 8 or 16 days and osteoblastic differentiation was determined by analyzing the constructs for cellularity, alkaline phosphatase activity, and calcium content as well as media samples for osteopontin. For scaffold/cell constructs cultured under flow perfusion, there was greater scaffold cellularity, alkaline phosphatase activity, osteopontin secretion, and calcium deposition compared with static controls, even in the absence of dexamethasone. When dexamethasone was present in the cell culture medium under flow perfusion conditions, there was further enhancement of osteogenic differentiation as evidenced by lower scaffold cellularity, greater osteopontin secretion, and greater calcium deposition. These results suggest that flow perfusion culture alone induces osteogenic differentiation of rat MSCs and that there is a synergistic effect of enhanced osteogenic differentiation when both dexamethasone and flow perfusion culture are used.     

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.     

Modulation of osteogenic properties of biodegradable polymer/extracellular matrix scaffolds generated with a flow perfusion bioreactor

In this study, composite scaffolds consisting of both synthetic and natural components with controllable properties were generated by incorporating mineralized extracellular matrix (ECM) and electrospun poly(ε-caprolactone) (PCL) microfiber scaffolds. Mesenchymal stem cells (MSCs) were cultured on PCL scaffolds under flow perfusion conditions with culture medium supplemented with dexamethasone to investigate the effect of culture duration on mineralized extracellular matrix deposition. MSCs differentiated down the osteogenic lineage and produced extracellular matrix with different compositions of mineral, collagen, and glycosaminoglycan with distinct morphologies at various stages of osteogenesis. To determine whether the presence and maturity of mineralized extracellular matrix influences osteogenic differentiation in vitro, PCL/ECM constructs were decellularized to yield PCL/ECM composite scaffolds that were subsequently seeded with MSCs and cultured in the absence of dexamethasone. The presence of mineralized matrix reduced cellular proliferation while stimulating alkaline phosphatase activity with increasing amounts of calcium deposition over time. PCL/ECM composite scaffolds containing the most mature mineralized matrix resulted in the most rapid increase and highest levels of alkaline phosphatase activity and calcium deposition compared to all other scaffold groups. Therefore, we demonstrate that mineralized extracellular matrix generated under controlled flow perfusion conditions can impart osteogenic properties to an osteoconductive polymer scaffold, and that the maturity of this matrix influences osteogenic differentiation in vitro, even in the absence of dexamethasone.     

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.     

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.     

Flow perfusion culture of marrow stromal osteoblasts in titanium fiber mesh

The objective of this study was to evaluate the effect of two cell culture techniques, static and flow perfusion, on the osteogenic expression of rat bone marrow cells seeded into titanium fiber mesh for a period up to 16 days. A cell suspension of rat bone marrow stromal osteoblasts (5 x 10(5) cells/300 microL) was seeded into the mesh material. Thereafter, the constructs were cultured under static conditions or in a flow perfusion system for 4, 8, and 16 days. To evaluate cellular proliferation and differentiation, constructs were examined for DNA, calcium content, and alkaline phosphatase activity. Samples were also examined with scanning electron microscopy (SEM) and plastic-embedded histological sections. Results showed an increase in DNA from day 4 to day 8 for the flow perfusion system. At day 8, a significant enhancement in DNA content was observed for flow perfusion culture compared with static culture conditions, but similar cell numbers were found for each culture system at 16 days. Calcium measurements showed a large increase in calcium content of the meshes subjected to flow perfusion at day 16. The SEM examination revealed that the 16-day samples subjected to flow perfusion culture were completely covered with layers of cells and mineralized matrix. In addition, this matrix extended deep into the scaffolds. In contrast, meshes cultured under static conditions had only a thin sheet of matrix present on the upper surface of the meshes. Evaluation of the light microscopy sections confirmed the SEM observations. On the basis of our results, we conclude that a flow perfusion system can enhance the early proliferation, differentiation, and mineralized matrix production of bone marrow stromal osteoblasts seeded in titanium fiber mesh.     

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.     

Flow Perfusion Co-culture of Human Mesenchymal Stem Cells and Endothelial Cells on Biodegradable Polymer Scaffolds

In this study, we investigated the effect of flow perfusion culture on the mineralization of co-cultures of human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs). Osteogenically precultured hMSCs were seeded onto electrospun scaffolds in monoculture or a 1:1 ratio with HUVECs, cultured for 7 or 14 days in osteogenic medium under static or flow perfusion conditions, and the resulting constructs were analyzed for cellularity, alkaline phosphatase (ALP) activity and calcium content. In flow perfusion, constructs with monocultures of hMSCs demonstrated higher cellularity and calcium content, but lower ALP activity compared to corresponding static controls. ALP activity was enhanced in co-cultures under flow perfusion conditions, compared to hMSCs alone; however unlike the static controls, the calcium content of the co-cultures in flow perfusion was not different from the corresponding hMSC monocultures. The data suggest that co-cultures of hMSCs and HUVECs did not contribute to enhanced mineralization compared to hMSCs alone under the flow perfusion conditions investigated in this study. However, flow perfusion culture resulted in an enhanced spatial distribution of cells and matrix compared to static cultures, which were limited to a thin surface layer.     

Flow perfusion culture of human mesenchymal stem cells on silicate-substituted tricalcium phosphate scaffolds

Autologous bone grafts are currently the gold standard for treatment of large bone defects, but their availability is limited due to donor site morbidity. Different substitutes have been suggested to replace these grafts, and this study presents a bone tissue engineered alternative using silicate-substituted tricalcium phosphate (Si-TCP) scaffolds seeded with human bone marrow-derived mesenchymal stem cells (hMSC). The cells were seeded onto the scaffolds and cultured either statically or in a perfusion bioreactor for up to 21 days and assessed for osteogenic differentiation by alkaline phosphatase activity assays and by quantitative real-time RT-PCR on bone markers. During culture, cells from the flow cultured constructs demonstrated improved proliferation and osteogenic differentiation verified by a more pronounced expression of several bone markers, e.g. alkaline phosphatase, osteopontin, Runx2, bone sialoprotein II, and bone morphogenetic protein 2. Cells and matrix were distributed homogeneously throughout the entire scaffold in flow culture, whereas only a peripheral layer was obtained after static culture. A viable and homogenous ex vivo bone construct with superior osteogenic properties was produced in dynamic culture and may provide a replacement for autologous grafts.     

Effect of convection on osteoblastic cell growth and function in biodegradable polymer foam scaffolds

Culture of seeded osteoblastic cells in three-dimensional osteoconductive scaffolds in vitro is a promising approach to produce an osteoinductive material for repair of bone defects. However, culture of cells in scaffolds sufficiently large to bridge critical-sized defects is a challenge for tissue engineers. Diffusion may not be sufficient to supply nutrients into large scaffolds and consequently cells may grow preferentially at the periphery under static culture conditions. Three alternative culturing schemes that convect media were considered: a spinner flask, a rotary vessel, and a perfusion flow system. Poly(DL-lactic-co-glycolic acid) (PLGA) foam discs (12.7 mm diameter, 6.0 mm thick, 78.8% porous) were seeded with osteoblastic marrow stromal cells and cultured in the presence of dexamethasone and L-ascorbic acid for 7 and 14 days. Cell numbers per foam were found to be similar with all culturing schemes indicating that cell growth could not be enhanced by convection, but histological analysis indicated that the rotary vessel and flow system produced a more uniform distribution of cells throughout the foams. Alkaline phosphatase (ALP) activity per cell was higher with culture in the flow system and spinner flask after 7 days, while no differences in osteocalcin (OC) activity per cell were observed among culturing methods after 14 days in culture. Based on the higher ALP activity and better cell uniformity throughout the cultured foams, the flow system appears to be the superior culturing method, although equally important is the fact that in none of the tests did any of the alternative culturing techniques underperform the static controls. Thus, this study demonstrates that culturing techniques that utilize fluid flow, and in particular the flow perfusion system, improve the properties of the seeded cells over those maintained in static culture.     

A biaxial rotating bioreactor for the culture of fetal mesenchymal stem cells for bone tissue engineering

The generation of effective tissue engineered bone grafts requires efficient exchange of nutrients and mechanical stimulus. Bioreactors provide a manner in which this can be achieved. We have recently developed a biaxial rotating bioreactor with efficient fluidics through in-silico modeling. Here we investigated its performance for generation of highly osteogenic bone graft using polycaprolactone–tricalcium phosphate (PCL–TCP) scaffolds seeded with human fetal mesenchymal stem cell (hfMSC). hfMSC scaffolds were cultured in either bioreactor or static cultures, with assessment of cellular viability, proliferation and osteogenic differentiation in vitro and also after transplantation into immunodeficient mice. Compared to static culture, bioreactor-cultured hfMSC scaffolds reached cellular confluence earlier (day 7 vs. day 28), with greater cellularity (2×, p < 0.01), and maintained high cellular viability in the core, which was 2000 μm from the surface. In addition, bioreactor culture was associated with greater osteogenic induction, ALP expression (1.5× p < 0.01), calcium deposition (5.5×, p < 0.001) and bony nodule formation on SEM, and in-vivo ectopic bone formation in immunodeficient mice (3.2×, p < 0.001) compared with static-cultured scaffolds. The use of biaxial bioreactor here allowed the maintenance of cellular viability beyond the limits of conventional diffusion, with increased proliferation and osteogenic differentiation both in vitro and in vivo, suggesting its utility for bone tissue engineering applications.     

Flow perfusion culture of human mesenchymal stem cells on coralline hydroxyapatite scaffolds with various pore sizes

Bone grafts are widely used in orthopaedic reconstructive surgery, but harvesting of autologous grafts is limited due to donor site complications. Bone tissue engineering is a possible alternative source for substitutes, and to date, mainly small scaffold sizes have been evaluated. The aim of this study was to obtain a clinically relevant substitute size using a direct perfusion culture system. Human bone marrowderived mesenchymal stem cells were seeded on coralline hydroxyapatite scaffolds with 200 μm or 500 μm pores, and resulting constructs were cultured in a perfusion bioreactor or in static culture for up to 21 days and analysed for cell distribution and osteogenic differentiation using histological stainings, alkaline phosphatase activity assay, and real-time RT-PCR on bone markers. We found that the number of cells was higher during static culture at most time points and that the final number of cells was higher in 500 μm constructs as compared with 200 μm constructs. Alkaline phosphatase enzyme activity assays and real time RT-PCR on seven osteogenic markers showed that differentiation occurred primarily and earlier in statically cultured constructs with 200 μm pores compared with 500 μm ones. Adhesion and proliferation of the cells was seen on both scaffold sizes, but the vitality and morphology of cells changed unfavorably during perfusion culture. In contrast to previous studies using spinner flask that show increased cellularity and osteogenic properties of cells when cultured dynamically, the perfusion culture in our study did not enhance the osteogenic properties of cell/scaffold constructs. The statically cultured constructs showed increasing cell numbers and abundant osteogenic differentiation probably because of weak initial cell adhesion due to the surface morphology of scaffolds. Our conclusion is that the specific scaffold surface microstructure and culturing system flow dynamics has a great impact on cell distribution and proliferation and on osteogenic differentiation, and the data presented warrant careful selection of in vitro culture settings to meet the specific requirements of the scaffolds and cells, especially when natural biomaterials with varying morphology are used.     

Effect of seeding technique and scaffold material on bone formation in tissue-engineered constructs

The aim of the present study was to test the hypothesis that both scaffold material and the type of cell culturing contribute to the results of in vivo osteogenesis in tissue-engineered constructs in an interactive manner. CaCO3 scaffolds and mineralized collagen scaffolds were seeded with human trabecular bone cells at a density of 5 x 10(6) cells/cm(3) and were left to attach under standard conditions for 24 h. Subsequently, they were submitted to static and dynamic culturing for 14 days (groups III and IV, respectively). Dynamic culturing was carried out in a continuous flow perfusion bioreactor. Empty scaffolds and scaffolds that were seeded with cells and kept under standard conditions for 24 h served as controls (groups I and II, respectively). Five scaffolds of each biomaterial and from each group were implanted into the gluteal muscles of rnu rats for 6 weeks. Osteogenesis was assessed quantitatively by histomorphometry and expression of osteocalcin (OC) and vascular endothelial growth factor (VEGF) was determined by immunohistochemistry. CaCO3 scaffolds exhibited 15.8% (SD 3.1) of newly formed bone after static culture and 22.4% (SD 8.2) after dynamic culture. Empty control scaffolds did not show bone formation, and scaffolds after 24 h of standard conditions produced 8.2% of newly formed bone (SD 4.0). Differences between the controls and the scaffolds cultured for 14 days were significant, but there was no significant difference between static and dynamic culturing. Mineralized collagen scaffolds did not show bone formation in any group. There was a significant difference in the expression of OC within the scaffolds submitted to static versus dynamic culturing in the CaCO3 scaffolds. VEGF expression did not show significant differences between static and dynamic culturing in the two biomaterials tested. It is concluded that within the limitations of the study the type of biomaterial had the dominant effect on in vivo bone formation in small tissue-engineered scaffolds. The culture period additionally affected the amount of bone formed, whereas the type of culturing may have had a positive effect on the expression of osteogenic markers but not on the quantity of bone formation.     

A rapid and efficient method for expansion of human mesenchymal stem cells

During the past decade, there has been much interest in the use of human mesenchymal stem cells (hMSCs) in bone tissue engineering. HMSCs can be obtained relatively easily and expanded rapidly in culture, but for clinical purposes large numbers are often needed and the cost should be kept to a minimum. A rapid and efficient culturing protocol would therefore be beneficial. In this study, we examined the effect of different medium compositions on the expansion and osteogenic differentiation of bone marrow-derived hMSCs from 19 donors. We also investigated the effect of low seeding density and dexamethasone on both hMSCs expansion and their in vitro and in vivo osteogenic differentiation capacity. HMSCs seeded at a density of 100 cells/cm2 had a significantly higher growth rate than at 5000 cell/cm2, which was further improved by the addition of dexamethasone. Expanded hMSCs were characterized in vitro on the basis of positive staining for CD29, CD44, CD105, and CD166. The in vitro osteogenic potential of expanded hMSCs was assessed by flow cytometric staining for alkaline phosphatase. In vivo bone-forming potential of the hMSCs was assessed by seeding the cells in ceramic scaffolds, followed by subcutaneous implantation in nude mice and histopathologic assessment of de novo bone formation after 6-week implantation. Expanded hMSCs from all donors displayed similar osteogenic potential independent of the culture conditions. On the basis of these results we have developed an efficient method to culture hMSCs by seeding the cells at 100 cells/cm2 in an alpha-minimal essential medium-based medium containing dexamethasone.     

Osteogenic differentiation of human bone marrow stromal cells on partially demineralized bone scaffolds in vitro

Tissue engineering has been used to enhance the utility of biomaterials for clinical bone repair by the incorporation of an osteogenic cell source into a scaffold followed by the in vitro promotion of osteogenic differentiation before host implantation. In this study, three-dimensional, partially demineralized bone scaffolds were investigated for their ability to support osteogenic differentiation of human bone marrow stromal cells (BMSCs) in vitro. Dynamic cell seeding resulted in homogeneous cell attachment and infiltration within the matrix and produced significantly higher seeding efficiencies when compared with a conventional static seeding method. Dynamically seeded scaffolds were cultured for 7 and 14 days in the presence of dexamethasone and evaluated on biochemical, molecular, and morphological levels for osteogenic differentiation. Significant elevation in alkaline phosphatase activity was observed versus controls over the 14-day culture, with a transient peak indicative of early mineralization on day 7. On the basis of RT-PCR, dexamethasone-treated samples showed elevations in alkaline phosphatase and osteocalcin expression levels at 7 and 14 days over nontreated controls, while bone sialoprotein was produced only in the presence of dexamethasone at 14 days. Scanning electron microscopy evaluation of dexamethasone-treated samples at 14 days revealed primarily cuboidal cells indicative of mature osteoblasts, in contrast to nontreated controls displaying a majority of cells with a fibroblastic cell morphology. These results demonstrate that partially demineralized bone can be successfully used with human BMSCs to support osteogenic differentiation in vitro. This osseous biomaterial may offer new potential benefits as a tool for clinical bone replacement.     

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。     

Flow Perfusion Enhances the Calcified Matrix Deposition of Marrow Stromal Cells in Biodegradable Nonwoven Fiber Mesh Scaffolds

In this study, we report on the ability of resorbable poly(L-lactic acid) (PLLA) nonwoven scaffolds to support the attachment, growth, and differentiation of marrow stromal cells (MSCs) under fluid flow. Rat MSCs were isolated from young male Wistar rats and expanded using established methods. The cells were then seeded on PLLA nonwoven fiber meshes. The PLLA nonwoven fiber meshes had 99% porosity, 17 m fiber diameter, 10 mm scaffold diameter, and 1.7-mm thickness. The nonwoven PLLA meshes were seeded with a cell suspension of 5 × 10⁵ cells in 300 l, and cultured in a flow perfusion bioreactor and under static conditions. Cell/polymer nonwoven scaffolds cultured under flow perfusion had significantly higher amounts of calcified matrix deposited on them after 16 days of culture. Microcomputed tomography revealed that the in vitro generated extracellular matrix in the scaffolds cultured under static conditions was denser at the periphery of the scaffold while in the scaffolds cultured in the perfusion bioreactor the extracellular matrix demonstrated a more homogeneous distribution. These results show that flow perfusion accelerates the proliferation and differentiation of MSCs, seeded on nonwoven PLLA scaffolds, toward the osteoblastic phenotype, and improves the distribution of the in vitro generated calcified extracellular matrix.     

Enhanced ectopic bone formation using a combination of plasmid DNA impregnation into 3-D scaffold and bioreactor perfusion culture

The objective of this study is to enhance in vivo ectopic bone formation by combination of plasmid DNA impregnation into three-dimensional (3-D) cell scaffolds and a developed in vitro culture method. Gelatin was cationized by introducing spermine (Sm) to the carboxyl groups for complexation with the plasmid DNA. As the MSC scaffold, collagen sponge reinforced by incorporation of poly(glycolic acid) (PGA) fibers was used. A complex of the cationized gelatin and plasmid DNA of BMP-2 was impregnated into the scaffold. MCS were seeded into each scaffold and cultured by a static and perfusion methods. When MSC were cultured in the PGA-reinforced collagen sponge, the level of BMP-2 expression was significantly enhanced by the perfusion culture compared with static method. When the osteoinduction activity of the PGA-reinforced collagen sponges seeded with PBS, MSC, naked plasmid DNA-BMP-2, cationized gelatin-plasmid DNA-BMP-2 complex, and transfected MSC by static and perfusion method, were studied following the implantation into the back subcutis of rats in terms of histological and biochemical examinations, homogeneous bone formation was histologically observed throughout the sponges seeded with cationized gelatin-plasmid DNA of BMP-2 complex and transfected MSC by perfusion method, although the extent of bone formation was higher for the later one. The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of sponges seeded with transfected MSC by perfusion method were significantly high compared with those seeded with other agents. We conclude that combination of plasmid DNA-impregnated PGA-reinforced collagen sponge and the perfusion method was promising to promote the in vitro gene expression for MSC and in vivo ectopic bone formation.