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.     

Scaffold mesh size affects the osteoblastic differentiation of seeded marrow stromal cells cultured in a flow perfusion bioreactor

In this study, we cultured marrow stromal cells on titanium fiber meshes in a flow perfusion bioreactor and examined the effect of altering scaffold mesh size on cell behavior in an effort to develop a bone tissue construct composed of a scaffold, osteogenic cells, and extracellular matrix. Scaffolds of differing mesh size, that is, distance between fibers, were created by altering the diameter of the mesh fibers (20 or 40 microm) while maintaining a constant porosity. These scaffolds had a porosity of 80% and mesh sizes of 65 microm (20-microm fibers) or 119 microm (40-microm fibers). Cell/scaffold constructs were grown in static culture or under flow for up to 16 days and assayed for osteoblastic differentiation. Cellularity was higher at early time points and Ca2+ deposition was higher at later time points for flow constructs over static controls. The 20-microm mesh had reduced cellularity in static culture. Under flow conditions, mass transport limitations are mitigated allowing uniform cell growth throughout the scaffold, and there was no difference in cellularity between mesh types. There was greater alkaline phosphatase (ALP) activity, osteopontin levels, and calcium under flow at 8 days for the 40-microm mesh compared to the 20-microm mesh. However, by day 16, the trend was reversed, suggesting the time course of differentiation was dependent on scaffold mesh size under flow conditions. However, this dependence was not linear with respect to time; larger mesh size was conducive to early osteoblast differentiation while smaller mesh size was conducive to later differentiation and matrix deposition.     

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.     

Formation of three-dimensional cell/polymer constructs for bone tissue engineering in a spinner flask and a rotating wall vessel bioreactor

The aim of this study is to investigate the effect of the cell culture conditions of three-dimensional polymer scaffolds seeded with rat marrow stromal cells (MSCs) cultured in different bioreactors concerning the ability of these cells to proliferate, differentiate towards the osteoblastic lineage, and generate mineralized extracellular matrix. MSCs harvested from male Sprague-Dawley rats were culture expanded, seeded on three-dimensional porous 75:25 poly(D,L-lactic-co-glycolic acid) biodegradable scaffolds, and cultured for 21 days under static conditions or in two model bioreactors (a spinner flask and a rotating wall vessel) that enhance mixing of the media and provide better nutrient transport to the seeded cells. The spinner flask culture demonstrated a 60% enhanced proliferation at the end of the first week when compared to static culture. On day 14, all cell/polymer constructs exhibited their maximum alkaline phosphatase activity (AP). Cell/polymer constructs cultured in the spinner flask had 2.4 times higher AP activity than constructs cultured under static conditions on day 14. The total osteocalcin (OC) secretion in the spinner flask culture was 3.5 times higher than the static culture, with a peak OC secretion occurring on day 18. No considerable AP activity and OC secretion were detected in the rotating wall vessel culture throughout the 21-day culture period. The spinner flask culture had the highest calcium content at day 14. On day 21, the calcium deposition in the spinner flask culture was 6.6 times higher than the static cultured constructs and over 30 times higher than the rotating wall vessel culture. Histological sections showed concentration of cells and mineralization at the exterior of the foams at day 21. This phenomenon may arise from the potential existence of nutrient concentration gradients at the interior of the scaffolds. The better mixing provided in the spinner flask, external to the outer surface of the scaffolds, may explain the accelerated proliferation and differentiation of marrow stromal osteoblasts, and the localization of the enhanced mineralization on the external surface of the scaffolds.     

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.     

The effect of the co-immobilization of human osteoprogenitors and endothelial cells within alginate microspheres on mineralization in a bone defect

Bone regeneration seems to be dependant on cell communication between osteogenic and endothelial cells arising from surrounding blood vessels. This study aims to determine whether endothelial cells can regulate the osteogenic potential of osteoprogenitor cells in vitro and in vivo, in a long bone defect, when co-immobilized in alginate microspheres. Alginate is a natural polymer widely used as a biomaterial for cell encapsulation. Human osteoprogenitors (HOP) from bone marrow mesenchymal stem cells were immobilized alone or together with human umbilical vein endothelial cells (HUVEC) inside irradiated, oxidized and RGD-grafted alginate microspheres. Immobilized cells were cultured in dynamic conditions and cell metabolic activity increased during three weeks. The gene expression of alkaline phosphatase and osteocalcin, both specific markers of the osteoblastic phenotype, and mineralization deposits were upregulated in co-immobilized HOPs and HUVECs, comparing to the immobilization of monocultures. VEGF secretion was also increased when HOPs were co-immobilized with HUVECs. Microspheres containing co-cultures were further implanted in a bone defect and bone formation was analysed by μCT and histology at 3 and 6 weeks post-implantation. Mineralization was observed inside and around the implanted microspheres containing the immobilized cells. However, when HOPs were co-immobilized with HUVECs, mineralization significantly increased. These findings demonstrate that co-immobilization of osteogenic and endothelial cells within RGD-grafted alginate microspheres provides a promising strategy 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.     

TGF-β3-induced chondrogenesis in co-cultures of chondrocytes and mesenchymal stem cells on biodegradable scaffolds

In this work, it was hypothesized that co-cultures of articular chondrocytes (ACs) and mesenchymal stem cells (MSCs) would exhibit enhanced sensitivity to chondrogenic stimuli, such as TGF-β3, and would require a reduced concentration of TGF-β3 to achieve an equivalent level of chondrogenesis compared to monocultures of each cell type. Furthermore, it was hypothesized that compared to monocultures, the chondrogenic phenotype of AC/MSC co-cultures would be more stable upon the removal of TGF-β3 from the culture medium. These hypotheses were investigated by culturing ACs and MSCs alone and in a 1:3 ratio on electrospun poly(?-caprolactone) scaffolds. All cell populations were cultured for two weeks with 0, 1, 3, or 10 ng/ml of TGF-β3. After two weeks growth factor supplementation was removed, and the constructs were cultured for two additional weeks. Cell proliferation, extracellular matrix production, and chondrogenic gene expression were evaluated after two and four weeks. The results demonstrated that co-cultures of ACs and MSCs require a reduced concentration and duration of TGF-β3 exposure to achieve an equivalent level of chondrogenesis compared to AC or MSC monocultures. Thus, the present work implicates that the promise of co-cultures for cartilage engineering is enhanced by their robust phenotype and heightened sensitivity to TGF-β3.     

一种骨种子细胞体外应力培养系统的建立

目的建立体外应力培养系统，观察应力刺激对骨种子细胞成骨分化的影响。方法选择具有明确成骨分化潜能的间充质干细胞(MSCs)作为种子细胞，以脱细胞骨基质为支架材料，以流体切应力作为对种子细胞的体外应力刺激。建立一种骨种子细胞体外三维应力培养系统——流动腔灌流体系，并利用该系统对MSCs的碱性磷酸酶(ALP)活性和骨钙素产物的影响进行评价。结果该系统可以明显促进ALP活性和骨钙素产物的表达，而细胞计数无明显改变。结论本系统为骨组织工程研究提供了一种有效的体外培养模型。     

Cultured Human Periosteal-Derived Cells Have Inducible Adipogenic Activity and Can Also Differentiate Into Osteoblasts in a Perioxisome Proliferator-Activated Receptor-Mediated Fashion

We investigated the adipogenic activity of cultured human periosteal-derived cells and studied perioxisome proliferator-activated receptor (PPAR) ligand-mediated differentiation of cultured human periosteal-derived cells into osteoblasts. Periosteal-derived cells expressed adipogenic markers, including CCAAT/enhancer binding protein α (C/EBP- α), C/EBP-δ, aP2, leptin, LPL, and PPARγ. Lipid vesicles were formed in the cytoplasm of periosteal-derived cells. Thus, periosteal-derived cells have potential adipogenic activity. The PPARα and PPARγ agonists, WY14643 and pioglitazone, respectively, did not modulate alkaline phosphatase (ALP) activity in periosteal-derived cells during induced osteoblastic differentiation, however, the PPARα and PPARγ antagonists, GW6471 and T0070907, respectively, both decreased ALP activity in these cells. WY14643 did not affect, whereas pioglitazone enhanced, alizarin red-positive mineralization and calcium content in the periosteal-derived cells. GW6471 and T0070907 both decreased mineralization and calcium content. By RT-PCR, pioglitazone significantly increased ALP expression in periosteal-derived cells between culture day 3 and 2 weeks. Pioglitazone increased Runx2 expression after 3 days, which declined thereafter, but did not alter osteocalcin expression. Both of GW6471 and T0070907 decreased ALP mRNA expression. These results suggest that pioglitazone enhances osteoblastic differentiation of periosteal-derived cells by increasing Runx2 and ALP mRNA expression, and increasing mineralization. GW6471 and T0070907 inhibit osteoblastic differentiation of the periosteal-derived cells by decreasing ALP expression and mineralization in the periosteal-derived cells.In conclusion, although further study will be needed to clarify the mechanisms of PPAR-regulated osteogenesis, our results suggest that PPARγ agonist stimulates osteoblastic differentiation of cultured human periosteal-derived cells and PPARα and PPARγ antagonists inhibit osteoblastic differentiation in these cells.     

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.     

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.     

Bioactive starch-based scaffolds and human adipose stem cells are a good combination for bone tissue engineering

Silicon is known to have an influence on calcium phosphate deposition and on the differentiation of bone precursor cells. This study explores the effect of the incorporation of silanol (Si-OH) groups into polymeric scaffolds on the osteogenic differentiation of human adipose stem cells (hASC) cultured under dynamic and static conditions. A blend of corn starch with polycaprolactone (30/70wt.%, SPCL) was used to produce three-dimensional fibre meshes scaffolds by the wet-spinning technique, and a calcium silicate solution was used as a non-solvent to develop an in situ functionalization with Si-OH groups. In vitro assessment, using hASC, of functionalized and non-functionalized scaffolds was evaluated in either α-MEM or osteogenic medium under static and dynamic conditions (provided by a flow perfusion bioreactor). The functionalized materials, SPCL-Si, exhibit the capacity to sustain cell proliferation and induce their differentiation into the osteogenic lineage. The formation of mineralization nodules was observed in cells cultured on the SPCL-Si materials. Culturing under dynamic conditions using a flow perfusion bioreactor was shown to enhance the hASC proliferation and differentiation and a better distribution of cells within the material. The present work demonstrates the potential of these functionalized materials for future applications in bone tissue engineering. Additionally, these results highlight the simplicity, economic and reliable production process of those materials.     

体外冲击波通过三磷酸腺苷诱导人骨髓间充质干细胞向成骨细胞分化

目的 研究体外冲击波是否通过三磷酸腺苷（ATP）激活P2X7受体,诱导人骨髓间充质干细胞(human mesenchymal stem cells,hMSCs)向成骨细胞分化。方法 培养hMSCs细胞,检测冲击波是否引起其向外释放ATP;通过检测碱性磷酸酶(ALP)活性、骨钙素表达和钙结节形成,判断骨化形成和钙质沉积;用实时定量PCR检测P2X7受体的mRNA表达;用ATP水解酶、P2X7受体的siRNA以及 P2受体的抑制剂评估ATP释放和P2X7受体在冲击波诱导hMSCs成骨分化中的作用。结果 冲击波可引起细胞内ATP向外释放,冲击波和细胞外ATP能够诱导hMSCs向成骨分化,采用ATP水解酶、P2X7受体的siRNA和抑制剂能够抑制冲击波引起的hMSCs成骨化作用。结论 冲击波通过引起细胞内ATP向外释放,激活P2X7受体传导信号通路,促进hMSCs向成骨细胞分化。本研究结果为冲击波促进骨折愈合和治疗骨不连疗法提供了理论依据。     

Inhibition of histone acetylation as a tool in bone tissue engineering

Our approach to bone tissue engineering is the in vitro expansion and osteogenic differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) and their subsequent implantation on porous ceramic materials. Current osteogenic differentiation protocols use dexamethasone to initiate the osteogenic process, thus ignoring the multiple signaling pathways that control osteogenesis in vivo. Supporting osteogenesis at multiple stages might further enhance the bone-forming capacity of hMSCs. As reported previously, inhibition of so-called histone deacetylases (HDACs) stimulates osteoblast maturation, and in this report, we investigated whether trichostatin A (TSA), a widely used HDAC inhibitor, can be implemented in bone tissue engineering. We confirmed that TSA treatment of hMSCs results in increased expression of alkaline phosphatase (ALP) with concomitant increase in mineralization. Flow cytometry demonstrated that TSA increases the percentage of ALP-positive hMSCs as well as their average ALP expression level, but the robustness of the response differs between donors. Unfortunately, TSA has a profound negative effect on cell proliferation, so we investigated whether hMSCs respond to TSA after reaching confluence. Confluent hMSCs on tissue culture plastic displayed enhanced ALP expression. Therefore, we seeded TSA-treated hMSCs onto ceramic particles and analyzed ectopic bone formation upon implantation in immune-deficient mice. Unfortunately, TSA-treated hMSCs did not display better bone formation in vivo than control cells. Finally, we observed that TSA treatment strongly enhanced bone formation of ex vivo cultured mouse calvaria, which warrants further exploration of TSA in bone tissue engineering.     

Study of osteoblastic cells in a microfluidic environment

Bone tissue engineering consists of culturing osteoblastic cells onto synthetic three-dimensional (3D) porous scaffolds. The organization of bone cells into 3D scaffolds is crucial for ex vivo tissue formation. Diffusional rates of nutrients could be greatly improved by perfusing media through the 3D microporous scaffolds. However, bone cells cultured in vitro are responsive to a variety of different mechanical signals including fluid flow and shear stresses. In this work, we attempt to study osteoblastic cells behaviour cultured within microdevices allowing continuous and homogenous feeding of cells. We have fabricated polydimethylsiloxane PDMS microdevices with a 3D microstructured channel network. Mouse calvarial osteoblastic cells MC3T3-E1 were seeded at 2x10(6)cells/ml and cultured into the microdevices under flow rates of 0, 5, 35 microl/min. Cells attached and proliferated well in the designed microdevices. Cell viability was found around 85% up to 1 to 2 weeks for shear stress value under 5 mPa. The alkaline phosphatase (ALP) activity was enhanced 3- and 7.5-fold inside the microdevices under static and dynamic flow of 5 microl/min as compared to flat static cultures in PDMS coated Petri dishes. Therefore, osteoblastic cells could be successfully cultured inside the microdevices under dynamic conditions and their ALP activity was enhanced. These results are promising for bone cell growth and differentiation as well as future tissue regeneration using larger 3D microfluidic microdevices.     

Monitoring Bone Tissue Engineered (BTE) Constructs Based on the Shifting Metabolism of Differentiating Stem Cells

Ever-increasing demand for bone grafts necessitates the realization of clinical implementation of bone tissue engineered constructs. The predominant hurdle to implementation remains to be securing FDA approval, based on the lack of viable methods for the rigorous monitoring of said constructs. The study presented herein details a method for such monitoring based on the shifting metabolism of mesenchymal stem cells (MSCs) as they differentiate into osteoblasts. To that end, rat MSCs seeded on 85% porous spunbonded poly(L-lactic acid) scaffolds were cultured in flow perfusion bioreactors with baseline or osteoinductive media, and levels of key physio-metabolic markers (oxygen, glucose, osteoprotegerin, and osteocalcin) were monitored throughout culture. Comparison of these non-destructively obtained values and current standard destructive analyses demonstrated key trends useful for the concurrent real-time monitoring of construct cellularity and maturation. Principle among these is the elucidation of the ratio of the rates of oxygen uptake to glucose consumption as a powerful quality marker. This ratio, supported on a physiological basis, has been shown herein to be reliable in the determination of both construct maturation (defined as osteoblastic differentiation and accompanying mineralization) and construct cellularity. Supplementary monitoring of OPG and OCN are shown to provide further validation of such metrics.     

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.