Regulation of autocrine fibroblast growth factor‐2 signaling by perfusion flow in 3D human mesenchymal stem cell constructs

Perfusion bioreactor systems play a crucial role in mitigating nutrient limitation as well as providing biomechanical stimuli and redistributing regulatory macromolecules that influence human mesenchymal stem cells (hMSC) fate in three‐dimensional (3D) scaffolds. As fibroblast growth factor‐2 (FGF‐2) is known to regulate hMSC phenotype, understanding the role of autocrine FGF‐2 signaling in the 3D construct under the different perfusion flow provides important insight into an optimal bioreactor design. To investigate FGF‐2 signaling inhibition in hMSC cultured in the porous poly(ethylene terephthalate) (PET) scaffolds perfused under two flow configurations, PD173074, an FGFR1 inhibitor, was added in growth media after 7 day of pre‐culture and its impact on hMSC proliferation and clonogenicity during the subsequent 7 days of cultivation was analyzed. Compared with control constructs in growth media, the addition of PD173074 resulted in significant reduction in hMSC proliferation and colony formation in both constructs with a more dramatic reduction in the parallel flow constructs. The results demonstrate that autocrine FGF‐2 plays a significant role in 3D scaffold and suggest modulation of the perfusion flow in the bioreactor as a strategy to influence autocrine actions and cell fate in the 3D scaffold. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Effects of shear stress on 3-D human mesenchymal stem cell construct development in a perfusion bioreactor system: Experiments and hydrodynamic modeling

Shear stress is an important biomechanical parameter in regulating human mesenchymal stem cell (hMSC) construct development. In this study, the biomechanical characteristics of hMSCs within highly porous 3-D poly (ethylene terephthalate) (PET) matrices in a perfusion bioreactor system were analyzed for two flow rates of 0.1 and 1.5 mL/min, respectively over a 20-day culture period. A 1.4 times higher proliferation rate, higher CFU-F formation, and more fibronectin and HSP-47 secretion at day 20 were observed at the flow rate of 0.1 mL/min compared to those at the flow rate of 1.5 mL/min. The higher flow rate of 1.5 mL/min upregulated osteogenic differentiation potential at day 20 as measured by the expression of alkaline phosphatase activity and calcium deposition in the matrix after 14 days osteogenic induction, consistent with those reported in literatures. Mathematical modeling indicated that shear stress existed in the range of 1 x 10(-5) to 1 x 10(-4) Pa in the constructs up to a depth of 70 microm due to flow penetration in the porous constructs. Analysis of oxygen transport in the constructs for the two flow rates yielded oxygen levels significantly higher than those at which cell growth and metabolism are affected (Jiang et al., 1996). This indicates that differences in convective transport have no significant influence on cell growth and metabolism for the range of flow rates studied. These results demonstrate that shear stress is an important microenvironment parameter that regulates hMSC construct development at a range significantly lower than those reported previously in the perfusion system.     

3D culture of osteoblast‐like cells by unidirectional or oscillatory flow for bone tissue engineering

A medium perfusion system is expected to be beneficial for three‐dimensional (3D) culture of engineered bone, not only by chemotransport enhancement but also by mechanical stimulation. In this study, perfusion systems with either unidirectional or oscillatory medium flow were developed, and the effects of the different flow profiles on 3D culturing of engineered bone were studied. Mouse osteoblast‐like MC 3T3‐E1 cells were 3D‐cultured with porous ceramic scaffolds in vitro for 6 days under static and hydrodynamic conditions with either a unidirectional or oscillatory flow. We found that, in the static culture, the cells proliferated only on the scaffold surfaces. In perfusion culture with the unidirectional flow, the proliferation was significantly higher than in the other groups but was very inhomogeneous, which made the construct unsuitable for transplantation. Only the oscillatory flow allowed osteogenic cells to proliferate uniformly throughout the scaffolds, and also increased the activity of alkaline phosphatase (ALP). These results suggested that oscillatory flow might be better than unidirectional flow for 3D construction of cell‐seeded artificial bone. The oscillatory perfusion system could be a compact, safe, and efficient bioreactor for bone tissue engineering. Biotechnol. Bioeng. 2009;102: 1670–1678. © 2008 Wiley Periodicals, Inc.     

Perfusion bioreactor system for human mesenchymal stem cell tissue engineering: dynamic cell seeding and construct development

Human mesenchymal stem cells (hMSCs) have great potential for therapeutic applications. A bioreactor system that supports long-term hMSCs growth and three-dimensional (3-D) tissue formation is an important technology for hMSC tissue engineering. A 3-D perfusion bioreactor system was designed using non-woven poly (ethylene terepthalate) (PET) fibrous matrices as scaffolds. The main features of the perfusion bioreactor system are its modular design and integrated seeding operation. Modular design of the bioreactor system allows the growth of multiple engineered tissue constructs and provides flexibility in harvesting the constructs at different time points. In this study, four chambers with three matrices in each were utilized for hMSC construct development. The dynamic depth filtration seeding operation is incorporated in the system by perfusing cell suspensions perpendicularly through the PET matrices, achieving a maximum seeding efficiency of 68%, and the operation effectively reduced the complexity of operation and the risk of contamination. Statistical analyses suggest that the cells are uniformly distributed in the matrices. After seeding, long-term construct cultivation was conducted by perfusing the media around the constructs from both sides of the matrices. Compared to the static cultures, a significantly higher cell density of 4.22 x 10(7) cell/mL was reached over a 40-day culture period. Cellular constructs at different positions in the flow chamber have statistically identical cell densities over the culture period. After expansion, the cells in the construct maintained the potential to differentiate into osteoblastic and adipogenic lineages at high cell density. The perfusion bioreactor system is amenable to multiple tissue engineered construct production, uniform tissue development, and yet is simple to operate and can be scaled up for potential clinical use. The results also demonstrate that the multi-lineage differentiation potential of hMSCs are preserved even after extensive expansion, thus indicating the potential of hMSCs for functional tissue construct development. The system has important applications in stem cell tissue engineering.     

Three hours of perfusion culture prior to 28 days of static culture, enhances osteogenesis by human cells in a collagen GAG scaffold

In tissue engineering, bioreactors can be used to aid in the in vitro development of new tissue by providing biochemical and physical regulatory signals to cells and encouraging them to undergo differentiation and/or to produce extracellular matrix prior to in vivo implantation. This study examined the effect of short term flow perfusion bioreactor culture, prior to long‐term static culture, on human osteoblast cell distribution and osteogenesis within a collagen glycosaminoglycan (CG) scaffold for bone tissue engineering. Human fetal osteoblasts (hFOB 1.19) were seeded onto CG scaffolds and pre‐cultured for 6 days. Constructs were then placed into the bioreactor and exposed to 3 × 1 h bouts of steady flow (1 mL/min) separated by 7 h of no flow over a 24‐h period. The constructs were then cultured under static osteogenic conditions for up to 28 days. Results show that the bioreactor and static culture control groups displayed similar cell numbers and metabolic activity. Histologically, however, peripheral cell‐encapsulation was observed in the static controls, whereas, improved migration and homogenous cell distribution was seen in the bioreactor groups. Gene expression analysis showed that all osteogenic markers investigated displayed greater levels of expression in the bioreactor groups compared to static controls. While static groups showed increased mineral deposition; mechanical testing revealed that there was no difference in the compressive modulus between bioreactor and static groups. In conclusion, a flow perfusion bioreactor improved construct homogeneity by preventing peripheral encapsulation whilst also providing an enhanced osteogenic phenotype over static controls. Bioeng. 2011; 108:1203–1210. © 2010 Wiley Periodicals, Inc.     

Perfusion affects the tissue developmental patterns of human mesenchymal stem cells in 3D scaffolds

Human mesenchymal stem cells (hMSCs) developed in three‐dimensional (3D) scaffolds are significantly affected by culture conditions. We hypothesized that the hydrodynamic forces generated in perfusion bioreactors significantly affected hMSC functionality in 3D scaffolds by shaping the extracellular matrix (ECM) proteins. In this study, hMSCs were grown in 3D poly(ethylene terephthalate) (PET) scaffolds in static and a parallel perfusion system under similar initial conditions for up to 35 days. Results demonstrated that even at very low media velocities (O [10^?4 cm/sec]), perfusion cultures affected the ability of hMSCs to form an organized ECM network as illustrated by the immunostaining of collagen I and laminin fibrous structure. The change in the ECM microenvironment consequently influenced the nuclear shape. The hMSCs grown at the lower surface of static culture displayed a 15.2 times higher nuclear elongation than those at the upper surface, whereas cells grown in the perfusion bioreactor displayed uniform spherical nuclei on both surfaces. The difference in ECM organization and nuclear morphology associated with gene expression and differentiation characteristics of hMSCs. The cells exhibited lower CFU‐F colony forming ability and decreased expressions of stem‐cell genes of Rex‐1 and Oct‐4, implying a less primitive stem‐cell phenotype was maintained in the perfusion culture relative to the static culture conditions. The significantly higher expression level of osteonectin gene in the perfusion culture at day 28 indicated an upregulation of osteogenic ability of hMSCs. The study highlights the critical role of dynamic culture conditions on 3D hMSC construct development and properties. J. Cell. Physiol. 219: 421–429, 2009. © 2009 Wiley‐Liss, Inc.     

Optimizing the medium perfusion rate in bone tissue engineering bioreactors

There is a critical need to increase the size of bone grafts that can be cultured in vitro for use in regenerative medicine. Perfusion bioreactors have been used to improve the nutrient and gas transfer capabilities and reduce the size limitations inherent to static culture, as well as to modulate cellular responses by hydrodynamic shear. Our aim was to understand the effects of medium flow velocity on cellular phenotype and the formation of bone‐like tissues in three‐dimensional engineered constructs. We utilized custom‐designed perfusion bioreactors to culture bone constructs for 5 weeks using a wide range of superficial flow velocities (80, 400, 800, 1,200, and 1,800 µm/s), corresponding to estimated initial shear stresses ranging from 0.6 to 20 mPa. Increasing the flow velocity significantly affected cell morphology, cell–cell interactions, matrix production and composition, and the expression of osteogenic genes. Within the range studied, the flow velocities ranging from 400 to 800 µm/s yielded the best overall osteogenic responses. Using mathematical models, we determined that even at the lowest flow velocity (80 µm/s) the oxygen provided was sufficient to maintain viability of the cells within the construct. Yet it was clear that this flow velocity did not adequately support the development of bone‐like tissue. The complexity of the cellular responses found at different flow velocities underscores the need to use a range of evaluation parameters to determine the quality of engineered bone. Bioeng. 2011; 108:1159–1170. © 2010 Wiley Periodicals, Inc.     

Osteoinduction and survival of osteoblasts and bone-marrow stromal cells in 3D biphasic calcium phosphate scaffolds under static and dynamic culture conditions

In many tissue engineering approaches, the basic difference between in vitro and in vivo conditions for cells within three‐dimensional (3D) constructs is the nutrition flow dynamics. To achieve comparable results in vitro, bioreactors are advised for improved cell survival, as they are able to provide a controlled flow through the scaffold. We hypothesize that a bioreactor would enhance long‐term differentiation conditions of osteogenic cells in 3D scaffolds. To achieve this either primary rat osteoblasts or bone marrow stromal cells (BMSC) were implanted on uniform‐sized biphasic calcium phosphate (BCP) scaffolds produced by a 3D printing method. Three types of culture conditions were applied: static culture without osteoinduction (Group A); static culture with osteoinduction (Group B); dynamic culture with osteoinduction (Group C). After 3 and 6 weeks, the scaffolds were analysed by alkaline phosphatase (ALP), dsDNA amount, SEM, fluorescent labelled live‐dead assay, and real‐time RT‐PCR in addition to weekly alamarBlue assays. With osteoinduction, increased ALP values and calcium deposition are observed; however, under static conditions, a significant decrease in the cell number on the biomaterial is observed. Interestingly, the bioreactor system not only reversed the decreased cell numbers but also increased their differentiation potential. We conclude from this study that a continuous flow bioreactor not only preserves the number of osteogenic cells but also keeps their differentiation ability in balance providing a suitable cell‐seeded scaffold product for applications in regenerative medicine.     

Novel mini β-TCP 3D perfusion bioreactor for proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

Applications of bioreactors in combination with scaffolding materials are promising in tissue-engineering fields. To rapidly produce bone mesenchymal stem cells (MSCs) suitable for osteogenic differentiation (OSD), we developed a novel technique for β-TCP (β-tricalcium phosphate) scaffolds preparation and employed these scaffolds to build a new type of mini three dimensional (3D) perfusion bioreactor. Compared to the 2D static culture, MSCs acquired much higher amplification rate and alkaline phosphatase (ALP) activity over a 24-day culture period. Interestingly, the Specific ALP activity was independent of the growth rate under adequate osteogenic inducement, suggesting there may be an OSD bottleneck at a single-cell level. Furthermore, cells on scaffolds exhibited gradually reduced total migration rate (MR) and relatively constant local MR, both ideal for bone regeneration. Excellent adhesion of MSCs to the smooth scaffolds surface, especially the layer structures, was seen on scanning electron microscopy images, demonstrating fine compatibility between scaffold and cells. Our results indicate this simplified, integrated and potentially modularizable 3D bioreactor could enable the osteocytes producing from MSCs for expected applications.     

Three-dimensional dynamic culture of pre-osteoblasts seeded in HA-CS/Col/nHAP composite scaffolds and treated with α-ZAL

Pre-osteoblast MC3T3-E1 cells were cultured in hyaluronic acid-modified chitosan/collagen/nano-hydroxyapatite (HA-CS/Col/nHAP) composite scaffolds and treated with phytoestrogen α-zearalanol (α-ZAL) to improve bone tissue formation for bone tissue engineering. Perfusion and dynamic strain were applied to three-dimensional (3D) cultured cells, which simulates mechanical microenvironment in bone tissue and solves mass transfer issues. The morphology of cell-scaffold constructs in vitro was then examined and markers of osteogenesis were assessed by immunohistochemistry staining and western blotting. The results showed that cells expanded their pseudopodia in an irregular manner and dispersed along the walls in 3D-dynamic culture. Osteogenic phenotype was increased or maintained by enhanced collagen I (COLI) levels, decreased osteopontin expression and having little effect on osteocalcin expression during the 12 days of in vitro culture. In response to α-ZAL, the cell-scaffold constructs showed inhibited cellular proliferation, enhanced the alkaline phosphatase (ALP) activity and increased ratio of osteoprotegerin to receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL). Application of perfusion and dynamic strain to cells-scaffold constructs treated with α-ZAL represents a promising approach in the studies of osteogenesis stimulation of bone tissue engineering.     

Protein and mineral composition of osteogenic extracellular matrix constructs generated with a flow perfusion bioreactor

This study investigated the temporal composition of an osteogenic extracellular matrix construct generated by culturing mesenchymal stem cells in an electrospun biodegradable poly(ε-caprolactone) fiber mesh scaffold within a flow perfusion bioreactor. Constructs of different extracellular matrix maturities were analyzed for their protein and mineral composition at several culture durations by liquid chromatography-tandem mass spectrometry, scanning electron microscopy, energy-dispersive X-ray diffraction, X-ray diffraction, and calcium and phosphate assays. The analysis revealed that at short culture durations the cells deposited cellular adhesion proteins as a prerequisite protein network for further bone formation. At the later culture durations, the extracellular matrix was composed of collagen 1, hydroxyapatite, matrix remodeling proteins, and regulatory proteins. These results suggest that the later culture duration constructs would allow for improved bone regeneration due to the ability to mineralize and the capabilities for future remodeling.     

Effects of oxygen transport on 3-d human mesenchymal stem cell metabolic activity in perfusion and static cultures: experiments and mathematical model

Human mesenchymal stem cells (hMSCs) have unique potential to develop into functional tissue constructs to replace a wide range of tissues damaged by disease or injury. While recent studies have highlighted the necessity for 3-D culture systems to facilitate the proper biological, physiological, and developmental processes of the cells, the effects of the physiological environment on the intrinsic tissue development characteristics in the 3-D scaffolds have not been fully investigated. In this study, experimental results from a 3-D perfusion bioreactor system and the static culture are combined with a mathematical model to assess the effects of oxygen transport on hMSC metabolism and proliferation in 3-D constructs grown in static and perfusion conditions. Cells grown in the perfusion culture had order of magnitude higher metabolic rates, and the perfusion culture supports higher cell density at the end of cultivation. The specific oxygen consumption rate for the constructs in the perfusion bioreactor was found to decrease from 0.012 to 0.0017 micromol/10(6) cells/h as cell density increases, suggesting intrinsic physiological change at high cell density. BrdU staining revealed the noneven spatial distribution of the proliferating cells in the constructs grown under static culture conditions compared to the cells that were grown in the perfusion system. The hypothesis that the constructs in static culture grow under oxygen limitation is supported by higher Y(L/G) in static culture. Modeling results show that the oxygen tension in the static culture is lower than that of the perfusion unit, where the cell density was 4 times higher. The experimental and modeling results show the dependence of cell metabolism and spatial growth patterns on the culture environment and highlight the need to optimize the culture parameters in hMSC tissue engineering.     

Gene expression profile of human mesenchymal stem cells during osteogenesis in three-dimensional thermoreversible gelation polymer

This study attempted to characterize the ability of thermoreversible gelation polymer (TGP) to induce differentiation of human mesenchymal stem cells (hMSC) into osteoblasts. Using a long oligo microarray system consisting of 3760 genes, we compared the expression profiles of the cells in 2-dimensional (2D) culture, 3D culture in collagen gel, and 3D culture in TGP with or without osteogenic induction. Compared to 2D culture, the gene expression profile of hMSC showed almost the same pattern in TGP without osteogenic induction, but 72% of genes (2701/3760) were up-regulated in collagen gel. With osteogenic induction, hMSC showed higher ALP activity and osteocalcin production in TGP as compared to 2D culture. Moreover, up-regulation and down-regulation of osteogenic genes were augmented in 3D culture in TGP as compared to 2D culture. As TGP is chemically synthesized and completely free from pathogen such as prion in bovine spongiform encephalopathy, these results suggest that TGP could be applied clinically to induce osteogenic differentiation of hMSC.     

Human mesenchymal stem cell position within scaffolds influences cell fate during dynamic culture

Cell-based tissue engineering is limited by the size of cell-containing constructs that can be successfully cultured in vitro. This limit is largely a result of the slow diffusion of molecules such as oxygen into the interior of three-dimensional scaffolds in static culture. Bioreactor culture has been shown to overcome these limits. In this study we utilize a tubular perfusion system (TPS) bioreactor for the three-dimensional dynamic culture of human mesenchymal stem cells (hMSCs) in spherical alginate bead scaffolds. The goal of this study is to examine the effect of shear stress in the system and then quantify the proliferation and differentiation of hMSCs in different radial annuli of the scaffold. Shear stress was shown to have a temporal effect on hMSC osteoblastic differentiation with a strong correlation of shear stress, osteopontin, and bone morphogenic protein-2 occurring on day 21, and weaker correlation occurring at early timepoints. Further results revealed an approximate 2.5-fold increase in cell number in the inner annulus of TPS cultured constructs as compared to statically cultured constructs after 21 days. This result demonstrated a nutrient transfer limitation in static culture which can be mitigated by dynamic culture. A significant increase (P < 0.05) in mineralization in the inner and outer annuli of bioreactor cultured 4 mm scaffolds occurred on day 21 with 79 ± 29% and 53 ± 25% mineralization area, respectively, compared to 6 ± 4% and 19 ± 6% mineralization area, respectively, in inner and outer annuli of 4 mm statically cultured scaffolds. Surprising lower mineralization area was observed in 2 mm bioreactor cultured beads which had the highest levels of proliferation. These results may demonstrate a relationship between scaffold position and stem cell fate. In addition the decreased proliferation and matrix production in statically cultured scaffolds compared to bioreactor cultured constructs demonstrate the need for bioreactor systems and the effectiveness of the TPS bioreactor in promoting hMSC proliferation and differentiation in three-dimensional scaffolds.     

Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs

Low oxygen tension is thought to be an integral component of the human mesenchymal stem cell (hMSC) native bone marrow microenvironment. HMSC were cultured under physiologically relevant oxygen environments (2% O2) in three-dimensional (3D) constructs for up to 1 month in order to investigate the combined effects of chronic hypoxia and 3D architecture on hMSC tissue-development patterns. Hypoxic hMSC exhibited an extended lag phase in order to acclimatize to culture conditions. However, they subsequently proliferated continuously throughout the culture period, while maintaining significantly higher colony-forming unit capabilities and expressing higher levels of stem cell genes than hMSC cultured at 20% O2 (normoxic) conditions. Upon induction, hypoxic hMSC also expressed higher levels of osteoblastic and adipocytic differentiation markers than normoxic controls. Hypoxia induced increased total protein levels in hMSC throughout the culture period, as well as significantly different fibronectin expression patterns suggesting that oxygen levels can significantly affect tissue-development patterns. Importantly, hMSC maintained the ability to thrive in prolonged hypoxic conditions suggesting that hypoxia may be an essential element of the in vivo hMSC niche. Further studies are required to determine how variations in cellular characteristics and ECM expression impact on the physiological properties of the engineered tissue, yet these results strongly indicate that oxygen tension is a key parameter that influences the in vitro characteristics of hMSC and their development into tissues.     

In silico multi‐scale model of transport and dynamic seeding in a bone tissue engineering perfusion bioreactor

Computer simulations can potentially be used to design, predict, and inform properties for tissue engineering perfusion bioreactors. In this work, we investigate the flow properties that result from a particular poly‐L ‐lactide porous scaffold and a particular choice of perfusion bioreactor vessel design used in bone tissue engineering. We also propose a model to investigate the dynamic seeding properties such as the homogeneity (or lack of) of the cellular distribution within the scaffold of the perfusion bioreactor: a pre‐requisite for the subsequent successful uniform growth of a viable bone tissue engineered construct. Flows inside geometrically complex scaffolds have been investigated previously and results shown at these pore scales. Here, it is our aim to show accurately that through the use of modern high performance computers that the bioreactor device scale that encloses a scaffold can affect the flows and stresses within the pores throughout the scaffold which has implications for bioreactor design, control, and use. Central to this work is that the boundary conditions are derived from micro computed tomography scans of both a device chamber and scaffold in order to avoid generalizations and uncertainties. Dynamic seeding methods have also been shown to provide certain advantages over static seeding methods. We propose here a novel coupled model for dynamic seeding accounting for flow, species mass transport and cell advection‐diffusion‐attachment tuned for bone tissue engineering. The model highlights the timescale differences between different species suggesting that traditional homogeneous porous flow models of transport must be applied with caution to perfusion bioreactors. Our in silico data illustrate the extent to which these experiments have the potential to contribute to future design and development of large‐scale bioreactors. Biotechnol. Bioeng. 2013; 110: 1221–1230. © 2012 Wiley Periodicals, Inc.     

3-D computational modeling of media flow through scaffolds in a perfusion bioreactor

Media perfusion bioreactor systems have been developed to improve mass transport throughout three-dimensional (3-D) tissue-engineered constructs cultured in vitro. In addition to enhancing the exchange of nutrients and wastes, these systems simultaneously deliver flow-mediated shear stresses to cells seeded within the constructs. Local shear stresses are a function of media flow rate and dynamic viscosity, bioreactor configuration, and porous scaffold microarchitecture. We have used the Lattice-Boltzmann method to simulate the flow conditions within perfused cell-seeded cylindrical scaffolds. Microcomputed tomography imaging was used to define the scaffold microarchitecture for the simulations, which produce a 3-D fluid velocity field throughout the scaffold porosity. Shear stresses were estimated at various media flow rates by multiplying the symmetric part of the gradient of the velocity field by the dynamic viscosity of the cell culture media. The shear stress algorithm was validated by modeling flow between infinite parallel plates and comparing the calculated shear stress distribution to the analytical solution. Relating the simulation results to perfusion experiments, an average surface shear stress of 5x10(-5)Pa was found to correspond to increased cell proliferation, while higher shear stresses were associated with upregulation of bone marker genes. This modeling approach can be used to compare results obtained for different perfusion bioreactor systems or different scaffold microarchitectures and may allow specific shear stresses to be determined that optimize the amount, type, or distribution of in vitro tissue growth.     

Effects of oxygen on engineered cardiac muscle

Concentration gradients associated with the in vitro cultivation of engineered tissues that are vascularized in vivo result in the formation of only a thin peripheral tissue-like region (e.g., approximately 100 microm for engineered cardiac muscle) around a relatively cell-free interior. We previously demonstrated that diffusional gradients within engineered cardiac constructs can be minimized by direct perfusion of culture medium through the construct. In the present study, we measured the effects of medium perfusion rate and local oxygen concentration (p(O2)) on the in vitro reconstruction of engineered cardiac muscle. Neonatal rat cardiomyocytes were seeded onto biodegradable polymer scaffolds (fibrous discs, 1.1 cm diameter x 2 mm thick, made of polyglycolic acid, 24 x 10(6) cells per scaffold). The resulting cell-polymer constructs were cultured for a total of 12 days in serially connected cartridges (n = 1-8), each containing one construct directly perfused with culture medium at a flow rate of 0.2-3.0 mL/min. In all groups, oxygen concentration decreased due to cell respiration, and depended on construct position in the series and medium flow rate. Higher perfusion rates and higher p(O2) correlated with more aerobic cell metabolism, and higher DNA and protein contents. Constructs cultured at p(O2) of 160 mm Hg had 50% higher DNA and protein contents, markedly higher expression of sarcomeric alpha-actin, better organized sarcomeres and cell junctions, and 4.5-fold higher rate of cell respiration as compared to constructs cultured at p(O2) of 60 mm Hg. Contraction rates of the corresponding cardiac cell monolayers were 40% higher at p(O2) of 160 than 60 mm Hg. The control of oxygen concentration in cell microenvironment can thus improve the structure and function of engineered cardiac muscle. Experiments of this kind can form a basis for controlled studies of the effects of oxygen on the in vitro development of engineered tissues.