Engineering of rat articular cartilage on porous sponges: effects of tgf-beta 1 and microgravity bioreactor culture

The objective of this study was to develop an engineered rat hyaline cartilage by culturing articular chondrocytes on three-dimensional (3D) macroporous poly(DL-lactic-co-glycolic acid) (PLGA) sponges under chondrogenic induction and microgravity bioreactor conditions. Experimental groups consisted of 3D static and dynamic cultures, while a single cell monolayer (2D) served as the control. The effect of seeding conditions (static vs. dynamic) on cellularization of the scaffolds was investigated. MTT assay was used to evaluate the number of viable cells in each group at different time points. Formation of a hyaline-like cartilage was evaluated for up to 4 weeks in vitro. While 2D culture resulted in cell sheets with very poor matrix production, 3D culture was in the favor of tissue formation. A higher yield of cell attachment and spatially uniform cell distribution was achieved when dynamic seeding technique was used. Dynamic culture promoted cell growth and infiltration throughout the sponge structure and showed the formation of cartilage tissue, while chondrogenesis appeared attenuated more towards the outer region of the constructs in the static culture group. Medium supplemented with TGF-beta 1 (5 ng/ml) had a positive impact on proteoglycan production as confirmed by histochemical analyses with Alcian blue and Safranin-O stainings. Formation of hyaline-like tissue was demonstrated by immunohistochemistry performed with antibodies against type II collagen and aggrecan. SEM confirmed higher level of cellularization and cartilage tissue formation in bioreactor cultures induced by TGF-beta 1. The data suggest that PLGA sponge inside rotating bioreactor with chondrogenic medium provides an environment that mediates isolated rat chondrocytes to redifferentiate and form hyaline-like rat cartilage, in vitro.     

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.     

Biological characterisation of vascular grafts cultured in a bioreactor

In this study, the development is described of a tissue-engineered construct mimicking the structure of a natural blood vessel. Smooth muscle cells (SMC) were cultured under pulsatile flow conditions in porous tubular scaffolds composed of crosslinked type I insoluble collagen and insoluble elastin. Under these dynamic culture conditions, average wall shear rate, systolic and diastolic pressures and pressure wave-forms comparable to conditions in the human carotid artery were obtained. Culturing of SMC in tubular scaffolds under dynamic conditions resulted in enhanced tissue formation compared to static conditions. Higher SMC numbers, a more homogeneous distribution of SMC throughout the scaffolds and higher collagen mRNA expression levels were found when cells were cultured under dynamic compared to static conditions. mRNA expression levels of markers of proliferation and apoptosis showed that the higher cell numbers in the scaffolds cultured under dynamic conditions can be explained by increased cell proliferation but not by decreased apoptosis. Glucose consumption and lactate formation by the cells showed that cell metabolism was more aerobic under dynamic compared to static conditions. Lining of the dynamically cultured constructs with a luminal monolayer of endothelial cells might result in vessels suitable for in vivo applications.     

Use of a rotating bioreactor toward tissue engineering the temporomandibular joint disc

This objective of this study was to determine the effects of a rotating bioreactor in temporomandibular joint (TMJ) disc tissue engineering. Porcine TMJ disc cells were seeded at a density of 20 million cells/mL onto nonwoven poly(glycolic acid) (PGA) scaffolds in spinner flasks for 1 week and then cultured either under static conditions or in a rotating bioreactor for a period of 6 weeks. A series of analyses was performed, including mechanical testing, measurement of cellularity, quantification of matrix biosynthesis with a hydroxyproline assay and enzyme-linked immunosorbent assays, and observation of matrix distribution with immunohistochemistry. Between the bioreactor and static cultures, there were marked differences in gross appearance, histological structure, and distribution of collagen types I and II. Engineered constructs from the bioreactor contracted earlier and to a greater extent, resulting in a denser matrix and cell composition. In addition, immunostaining intensity was generally uniform in static constructs, in contrast to higher intensity around the periphery of bioreactor constructs. Moreover, bioreactor constructs had higher amounts of collagen II than did static constructs. However, differences in total matrix content and compressive stiffness were generally not significant. On the basis of the results of this study there is no clear benefit from use of the rotating bioreactor, although a sequence of static culture followed by rotating bioreactor culture may prove in the future to be more beneficial than either alone.     

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.     

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.     

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.     

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.     

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。     

Cell-nanofiber-based cartilage tissue engineering using improved cell seeding, growth factor, and bioreactor technologies

Biodegradable nanofibrous scaffolds serving as an extracellular matrix substitute have been shown to be applicable for cartilage tissue engineering. However, a key challenge in using nanofibrous scaffolds for tissue engineering is that the small pore size limits the infiltration of cells, which may result in uneven cell distribution throughout the scaffold. This study describes an effective method of chondrocyte loading into nanofibrous scaffolds, which combines cell seeding, mixing, and centrifugation to form homogeneous, packed cell-nanofiber composites (CNCs). When the effects of different growth factors are compared, CNCs cultured in medium containing a combination of insulin-like growth factor-1 and transforming growth factor-beta1 express the highest mRNA levels of collagen type II and aggrecan. Radiolabeling analyses confirm the effect on collagen and sulfated-glycosaminoglycans (sGAG) production. Histology reveals chondrocytes with typical morphology embedded in lacuna-like space throughout the entire structure of the CNC. Upon culturing using a rotary wall vessel bioreactor, CNCs develop into a smooth, glossy cartilage-like tissue, compared to a rough-surface tissue when maintained in a static environment. Bioreactor-grown cartilage constructs produce more total collagen and sGAG, resulting in greater gain in net tissue weight, as well as express cartilage-associated genes, including collagen types II and IX, cartilage oligomeric matrix protein, and aggrecan. In addition, dynamic culture enhances the mechanical property of the engineered cartilage. Taken together, these results indicate the applicability of nanofibrous scaffolds, combined with efficient cell loading and bioreactor technology, for cell-based cartilage 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.     

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.     

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.     

A Dynamically Cultured Collagen/Cells-Incorporated Elastic Scaffold for Small-Diameter Vascular Grafts

Abstract

There is an essential demand for tissue-engineered autologous small-diameter vascular grafts, which offer temporary supports and guides for vascular tissue organization, repair and remodeling. This study reports on the effect of collagen/smooth muscle cells (SMCs) mixtures under dynamic cultures and SMC-endothelial cell (ECs) co-culture on cell proliferation, uniform cell distribution, extracellular matrix deposition, and endothelial cells monolayer formation in tissue-engineered tubular arterial constructs of 4 mm inner diameter. Rabbit aortic SMCs were infiltrated with collagen solution in poly(L-lactide-co-?-caprolactone) (PLCL) scaffolds under vacuum to form collagenous gel and subjected to dynamic strain by culturing them in a dynamic perfusion bioreactor. The construct lumen was subsequently seeded with ECs and experiments were completed to create ECs–SMCs co-culture constructs. The collagen/SMCs incorporated elastic scaffold cultured under dynamic culture conditions promoted matrix deposition, leading to the development of tissue-engineered vascular constructs, and induced SMC to have more uniform cell distribution. Scanning electron microscopic examination and von Willebrand Factor staining demonstrated the presence of ECs spread over the lumen. Quantitative analysis of elastin contents demonstrated that the engineered vessels acquired similar elastin contents as native arteries. The collagen/SMCs/ECs incorporated PLCL scaffolds under dynamic culture conditions can be used as a scaffold for tissue engineering to facilitate small-diameter vascular-tissue formation.     

旋转灌注式生物反应器系统构建及在骨组织工程中的应用

构建一种旋转灌注式生物反应器系统，并设计制造具有相互连通微管道结构的大段支架。生物反应器在旋转的同时实现对大段支架的灌注，系统内外气体的实时交换通过安装在反应器两端的半透膜和可透气的蠕动泵软管实现。容器内的氧气和营养物质在得到充分混合之后，连续不断地输送到支架微管道内。使黏附在微管道内的细胞获得充足营养的同时受到一定流体剪应力的刺激，调节细胞功能的发挥。该系统克服了静态培养中存在的各种缺点，改善了培养环境，增加了培养过程的可控性，有助于促进黏附在微管道内部的细胞增殖、分化和产生大量基质。将成骨细胞／支架结构体在该生物反应器系统中培养14d后，用扫描电镜观察细胞在大段支架微管道内的生长情况，结果表明，支架微管道内有大量细胞长入。     

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 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.     

A novel perfusion bioreactor providing a homogenous milieu for tissue regeneration

We developed a novel perfusion bioreactor that is capable of cultivating multiple 3-dimensional (3D) cellular constructs in one flow chamber with a total cross-section area of 20 cm(2). Two unique features integrated into the bioreactor provided a homogenous fluid flow along the bioreactor cross-section and maximal exposure of the cellular constructs to the perfusing medium. Mathematical modeling of the fluid flow regime in the perfusion bioreactor showed that integrating a flow-distributing mesh 1.5 cm upstream from the construct compartment imposed an equal medium flow and shear stress of 0.6 dynes /cm(2) along the entire cell construct cross-section area. The design of 95.8%open-pore-area fixing nets enabled the exposure of 99.88% of the cell construct volume to the perfusing medium. Cardiac cell constructs seeded with physiologically relevant cell density (0.7 x 10(8) cells/cm(3)) in alginate scaffolds developed into homogenous compacted cardiac tissue, as judged using cell staining with fluorescein diacetate and hematoxylin-eosin histology. The cell constructs maintained 80% viability for nearly 2 weeks, whereas in static-cultivated cell constructs, only 50% of the initial cells remained, as determined according to total DNA content and MTT viability assay. Medium perfusion resulted in better cell viability, presumably due to the convective-diffusive transport of oxygen, compared with oxygen diffusion within the static-cultivated cell constructs, as well as due to efficient removal of harmful cell secretions. It is envisioned that this bioreactor would be useful for 3D cultivation of different mammalian cells for purposes of tissue engineering or production of valuable biologicals.     

Effect of 3D scaffold and dynamic culture condition on the global gene expression profile of mouse embryonic stem cells

We have previously demonstrated that mouse embryonic stem (ES) cells differentiated on three-dimensional (3D), highly porous, tantalum-based scaffolds (Cytomatrix™) have significantly higher hematopoietic differentiation efficiency than those cultured under conventional two-dimensional (2D) tissue culture conditions. In addition, ES cell-seeded scaffolds cultured inside spinner bioreactors showed further enhancement in hematopoiesis compared to static conditions. In the present study, we evaluated how these various biomaterial-based culture conditions, e.g. 2D vs. 3D scaffolds and static vs. dynamic, influence the global gene expression profile of differentiated ES cells. We report that compared to 2D tissue culture plates, cells differentiated on porous, Cytomatrix™ scaffolds possess significantly higher expression levels of extracellular matrix (ECM)-related genes, as well as genes that regulate cell growth, proliferation and differentiation. In addition, these differences in gene expression were more pronounced in 3D dynamic culture compared to 3D static culture. We report specific genes that are either uniquely expressed under each condition or are quantitatively regulated, i.e. over expressed or inhibited by a specific culture environment. We conclude that that biomaterial-based 3D cultures, especially under dynamic conditions, might favor efficient hematopoietic differentiation of ES cells by stimulating increased expression of specific ECM proteins, growth factors and cell adhesion related genes while significantly down-regulating genes that act to inhibit expression of these molecules.     

Bone Tissue Engineering Using Human Mesenchymal Stem Cells: Effects of Scaffold Material and Medium Flow

We report studies of bone tissue engineering using human mesenchymal stem cells (MSCs), a protein substrate (film or scaffold; fast degrading unmodified collagen, or slowly degrading cross-linked collagen and silk), and a bioreactor (static culture, spinner flask, or perfused cartridge). MSCs were isolated from human bone marrow, characterized for the expression of cell surface markers and the ability to undergo chondrogenesis and osteogenesis in vitro, and cultured for 5 weeks. MSCs were positive for CD105/endoglin, and had a potential for chondrogenic and osteogenic differentiation. In static culture, calcium deposition was similar for MSC grown on collagen scaffolds and films. Under medium flow, MSC on collagen scaffolds deposited more calcium and had a higher alcaline phosphatase (AP) activity than MSC on collagen films. The amounts of DNA were markedly higher in constructs based on slowly degrading (modified collagen and silk) scaffolds than on fast degrading (unmodified collagen) scaffolds. In spinner flasks, medium flow around constructs resulted in the formation of bone rods within the peripheral region, that were interconnected and perpendicular to the construct surface, whereas in perfused constructs, individual bone rods oriented in the direction of fluid flow formed throughout the construct volume. These results suggest that osteogenesis in cultured MSC can be modulated by scaffold properties and flow environment.