A rotating bed system bioreactor enables cultivation of primary osteoblasts on well‐characterized sponceram® regarding structural and flow properties

The development of bone tissue engineering depends on the availability of suitable biomaterials, a well‐defined and controlled bioreactor system, and on the use of adequate cells. The biomaterial must fulfill chemical, biological, and mechanical requirements. Besides biocompatibility, the structural and flow characteristics of the biomaterial are of utmost importance for a successful dynamic cultivation of osteoblasts, since fluid percolation within the microstructure must be assured to supply to cells nutrients and waste removal. Therefore, the biomaterial must consist of a three‐dimensional structure, exhibit high porosity and present an interconnected porous network. Sponceram®, a ZrO₂ based porous ceramic, is characterized in the presented work with regard to its microstructural design. Intrinsic permeability is obtained through a standard Darcy's experiment, while Young's modulus is derived from a two plates stress–strain test in the linear range. Furthermore, the material is applied for the dynamic cultivation of primary osteoblasts in a newly developed rotating bed bioreactor. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

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.     

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.     

Rotating versus perfusion bioreactor for the culture of engineered vascular constructs based on hyaluronic acid

It is generally accepted that dynamic culture conditions are required for vascular tissue engineering. We compared the effects of two dynamic culture systems, a perfusion and a rotating bioreactor, using tubular constructs based on hyaluronic acid seeded with porcine aortic smooth muscle cells (SMC), that we recently showed to be adequate for the generation of vascular tissue. In perfused constructs mechanical stimulation importantly affected cell morphology, increased the incidence of cell proliferation and reduced apoptosis. However, extracellular matrix deposition, cytoskeletal organization and mechanical properties were poor. In rotated constructs cell proliferation was also higher and apoptosis lower than in static controls. Rotated constructs showed the highest ultimate stress and the lowest elastic modulus. Our data indicate that the rotating bioreactor is more efficient than the perfusion bioreactor and we then suggest that this method can be considered a valid alternative to complex bioreactor systems described in the literature.     

NASA-approved rotary bioreactor enhances proliferation of human epidermal stem cells and supports formation of 3D epidermis-like structure

The skin is susceptible to different injuries and diseases. One major obstacle in skin tissue engineering is how to develop functional three-dimensional (3D) substitute for damaged skin. Previous studies have proved a 3D dynamic simulated microgravity (SMG) culture system as a "stimulatory" environment for the proliferation and differentiation of stem cells. Here, we employed the NASA-approved rotary bioreactor to investigate the proliferation and differentiation of human epidermal stem cells (hEpSCs). hEpSCs were isolated from children foreskins and enriched by collecting epidermal stem cell colonies. Cytodex-3 micro-carriers and hEpSCs were co-cultured in the rotary bioreactor and 6-well dish for 15 days. The result showed that hEpSCs cultured in rotary bioreactor exhibited enhanced proliferation and viability surpassing those cultured in static conditions. Additionally, immunostaining analysis confirmed higher percentage of ki67 positive cells in rotary bioreactor compared with the static culture. In contrast, comparing with static culture, cells in the rotary bioreactor displayed a low expression of involucrin at day 10. Histological analysis revealed that cells cultured in rotary bioreactor aggregated on the micro-carriers and formed multilayer 3D epidermis structures. In conclusion, our research suggests that NASA-approved rotary bioreactor can support the proliferation of hEpSCs and provide a strategy to form multilayer epidermis structure.     

Transfer of an adherent Vero cell culture method between two different rocking motion type bioreactors with respect to cell growth and metabolic rates

Rapid and simple cell and virus cultivation can currently be carried out using disposable bioreactors. The CELL-tainer^® (CELLution Biotech BV) disposable bioreactor is a rocking-type bioreactor which not only has vertical movement but horizontal displacement as well. Due to this two-directional movement relatively high mass-transfer capacities can be reached when compared with conventional rocking motion-type bioreactors.Using the design of experiments (DoE) approach we have developed models for the mixing times in both the CELL-tainer^® and the BIOSTAT^® CultiBag RM (Sartorius Stedim Biotech) bioreactor (standard rocking motion-type). The conditions for cultivation of Vero cells in the CELL-tainer^® bioreactor were chosen based on comparable mixing times.Vero cells growing adherent to Cytodex 1 microcarriers were cultivated in the CELL-tainer^® and in the BIOSTAT^® CultiBag RM. Both bioreactors were controlled with regard to temperature, pH and % dissolved oxygen. Vero cell growth in both bioreactors was comparable with respect to the growth characteristics and main metabolite production and consumption rates. Additionally, polio virus production in both bioreactors was shown to be similar.     

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.     

Chondrogenesis on BMP‐6 loaded chitosan scaffolds in stationary and dynamic cultures

We originally investigated the suitability of chitosan scaffolds loaded with bone morphogenetic protein 6 (BMP‐6) in both stationary and dynamic conditions for cartilage tissue engineering. In the first part of the present study, ATDC5 murine chondrogenic cells were seeded in chitosan and BMP‐6 loaded chitosan scaffolds and cultured for 28 days under static conditions. In the following part, we examined the influence of dynamic cultivation conditions over BMP‐6 loaded chitosan scaffolds by using rotating bioreactor with perfusion (RCMW?). Tissue engineered constructs were characterized by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazoliumbromide (MTT) assay, scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) and biochemical assays for glycosaminoglycans (GAG) deoxyribonucleic acid (DNA) and collagen Type II quantification. At the end of 4 weeks static incubation period high levels of GAG (21.22 mg/g dry weight), DNA amounts (1.37 mg/g dry weight) and collagen Type II amounts (1.94 µg/g dry weight) were achieved for BMP‐6 loaded chitosan scaffolds compared to chitosan scaffolds. However, the results obtained from morphological observations suggested hypertrophic differentiation of ATDC5 cells in the presence of BMP‐6 under stationary conditions. The influence of mechanical stimulation appeared significantly with differentiated cells, cultured under dynamic conditions, showing the effect of retaining their phenotypes without hypertrophy. Biotechnol. Bioeng. 2009; 104: 601–610 © 2009 Wiley Periodicals, Inc.     

Recellularization of Decellularized Lung Scaffolds Is Enhanced by Dynamic Suspension Culture

Strategies are needed to improve repopulation of decellularized lung scaffolds with stromal and functional epithelial cells. We demonstrate that decellularized mouse lungs recellularized in a dynamic low fluid shear suspension bioreactor, termed the rotating wall vessel (RWV), contained more cells with decreased apoptosis, increased proliferation and enhanced levels of total RNA compared to static recellularization conditions. These results were observed with two relevant mouse cell types: bone marrow-derived mesenchymal stromal (stem) cells (MSCs) and alveolar type II cells (C10). In addition, MSCs cultured in decellularized lungs under static but not bioreactor conditions formed multilayered aggregates. Gene expression and immunohistochemical analyses suggested differentiation of MSCs into collagen I-producing fibroblast-like cells in the bioreactor, indicating enhanced potential for remodeling of the decellularized scaffold matrix. In conclusion, dynamic suspension culture is promising for enhancing repopulation of decellularized lungs, and could contribute to remodeling the extracellular matrix of the scaffolds with subsequent effects on differentiation and functionality of inoculated cells.     

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.     

A bioreactor test system to mimic the biological and mechanical environment of oral soft tissues and to evaluate substitutes for connective tissue grafts

Gingival cells of the oral connective tissue are exposed to complex mechanical forces during mastication, speech, tooth movement and orthodontic treatments. Especially during wound healing following surgical procedures, internal and external forces may occur, creating pressure upon the newly formed tissue. This clinical situation has to be considered when developing biomaterials to augment soft tissue in the oral cavity. In order to pre‐evaluate a collagen sponge intended to serve as a substitute for autogenous connective tissue grafts (CTGs), a dynamic bioreactor system was developed. Pressure and shear forces can be applied in this bioreactor in addition to a constant medium perfusion to cell‐material constructs. Three‐dimensional volume changes and stiffness of the matrices were analyzed. In addition, cell responses such as cell vitality and extracellular matrix (ECM) production were investigated. The number of metabolic active cells constantly increased under fully dynamic culture conditions. The sponges remained elastic even after mechanical forces were applied for 14 days. Analysis of collagen type I and fibronectin revealed a statistically significant accumulation of these ECM molecules (P < 0.05–0.001) when compared to static cultures. An increased expression of tenascin‐c, indicating tissue remodeling processes, was observed under dynamic conditions only. The results indicate that the tested in vitro cell culture system was able to mimic both the biological and mechanical environments of the clinical situation in a healing wound. Biotechnol. Bioeng. 2010;107: 1029–1039. © 2010 Wiley Periodicals, Inc.     

Periodic harvesting of embryonic stem cells from a hollow‐fiber membrane based four‐compartment bioreactor

Different types of stem cells have been investigated for applications in drug screening and toxicity testing. In order to provide sufficient numbers of cells for such in vitro applications a scale‐up of stem cell culture is necessary. Bioreactors for dynamic three‐dimensional (3D) culture of growing cells offer the option for culturing large amounts of stem cells at high densities in a closed system. We describe a method for periodic harvesting of pluripotent stem cells (PSC) during expansion in a perfused 3D hollow‐fiber membrane bioreactor, using mouse embryonic stem cells (mESC) as a model cell line. A number of 100 × 10⁶ mESC were seeded in bioreactors in the presence of mouse embryonic fibroblasts (MEF) as feeder cells. Over a cultivation interval of nine days cells were harvested by trypsin perfusion and mechanical agitation every second to third culture day. A mean of 380 × 10⁶ mESC could be removed with every harvest. Subsequent to harvesting, cells continued growing in the bioreactor, as determined by increasing glucose consumption and lactate production. Immunocytochemical staining and mRNA expression analysis of markers for pluripotency and the three germ layers showed a similar expression of most markers in the harvested cells and in mESC control cultures. In conclusion, successful expansion and harvesting of viable mESC from bioreactor cultures with preservation of sterility was shown. The present study is the first one showing the feasibility of periodic harvesting of adherent cells from a continuously perfused four‐compartment bioreactor including further cultivation of remaining cells. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:141–151, 2016     

Growth and differentiation of alveolar bone cells in tissue-engineered constructs and monolayer cultures

The ability to enhance bone regeneration by implanting autologous osteoblasts in combination with an appropriate scaffold would be of great clinical interest. The aim of our study was to compare the growth and differentiation of alveolar bone cells in tissue-engineered constructs and in monolayer cultures, as the basis for developing procedures for routine preparation of bone-like tissue constructs. Alveolar bone tissue was obtained from four human donors and explant cultures of the cells were established. Expanded cells were seeded on macroporous hydroxyapatite granules, and cultured in medium supplemented with osteogenic differentiation factors for up to 3 weeks. Control monolayer cultures were established in parallel, and cultured in media with or without osteogenic supplements. Cell proliferation, alkaline phosphatase (AP) activity and gene expression of AP, osteopontin and osteocalcin were determined under different culture conditions at weekly intervals. Cells in tissue constructs exhibited growth patterns similar to those in control monolayer cultures: enhanced proliferation was noted during the first 2 weeks of cultivation, followed by a decrease in cell numbers. AP activity at 3 weeks was higher in all cultures in osteogenic medium than in control medium. Gene expression levels were stable in monolayer cultures in both types of media whereas, in tissue constructs, they exhibited patterns of osteogenic differentiation. Light and scanning electron microscopy examination of the cell-seeded constructs showed uniform cell distribution, as well as cell attachment and growth into the interior region of the hydroxyapatite granules. Our results show that bone-like constructs with viable cells exhibiting differentiated phenotype can be prepared by cultivation of alveolar-bone cells on the tested hydroxyapatite granules.     

Pellet culture elicits superior chondrogenic redifferentiation than alginate‐based systems

Although pellet culture and encapsulation of chondrocytes into gel‐like biomaterials have lead to major advances in cartilage tissue engineering, a quantitative comparative characterization of cellular differentiation behavior during those cultivation procedures has not yet been performed. Our study therefore aimed at answering the following question: is the redifferentiation pathway of chondrocytes altered by slight changes in the type of alginate biomaterial (pure alginate, alginate‐fibrin, alginate‐chitosan) and how do the cells behave in comparison to biomaterial‐free (pellet) three‐dimensional culturing? Monolayer‐expanded chondrocytes from healthy adult porcine knee joints were cultivated in alginate, alginate‐chitosan, alginate‐fibrin beads and as pellets up to 4 weeks. Quantitative PCR and Immunohistology were used to assess chondrogenic markers. Alginate‐fibrin—encapsulated chondrocytes behaved almost like monolayer chondrocytes. Alginate‐ and alginate‐chitosan encapsulation lead to a low chondrogenic marker gene expression. Although all 3D‐cultured chondrocytes showed a considerable amount of Sox9 expression, only pellet cultivation lead to a sufficient Collagen II expression. This puts the usage of alginate‐cultivated cartilage tissue engineering constructs under question. Fibrin addition is not beneficial for chondrogenic differentiation. Sox9 and Collagen II behave differently, depending upon the surrounding 3D‐environment. © 2009 American Institute of Chemical Engineers AIChE J, 2009     

Cuttlefish bone scaffold for tissue engineering: a novel hydrothermal transformation, chemical-physical, and biological characterization

Purpose: Natural resources are receiving growing interest because of their possible conversion from a cheap and easily available material into a biomedical product. Cuttlefish bone from Sepia Officinalis was investigated in order to obtain an hydroxyapatite porous scaffold using hydrothermal transformation. Methods: Complete conversion of the previous calcium carbonate (aragonite) phase into a calcium phosphate (hydroxyapatite) phase was performed with an hydrothermal transformation at 200 °C (~ 15 atm), for four hours, with an aqueous solution of KH2PO4 in order to set the molar ratio Ca/P = 10/6 in a reactor (Parr 4382). The complete conversion was then analyzed by TGA, ATR-FTIR, x-ray diffraction, and SEM. Moreover, the material was biologically investigated with MC3T3-E1 in static cultures, using both osteogenic and maintenance media. The expression of osteogenic markers as ALP and osteocalcin and the cell proliferation were investigated. Results: Cuttlefish bone has been successfully transformed from calcium carbonate into calcium phosphate. Biological characterization revealed that osteogenic markers are expressed using both osteogenic and maintenance conditions. Cell proliferation is influenced by the static culture condition used for this three-dimensional scaffold. Conclusions: The new scaffold composed by hydroxyapatite and derived for a natural source presents good biocompatibility and can be used for further investigations using dynamic cultures in order to improve cell proliferation and differentiation for bone tissue engineering.     

Growth behavior of Chinese hamster ovary cells in a compact loop bioreactor: 1. Effects of physical and chemical environments.

Chinese hamster ovary (CHO) cells were cultivated in a compact loop bioreactor using MEM-alpha medium supplemented with 10% fetal calf serum. Effects of physical and chemical environments, i.e., pH in the medium, stirring speed of impellers, temperature and partial pressure of oxygen (pO2) upon growth of suspended cells in the bioreactor were determined in batch cultures. Growth behavior was characterized by specific rates of growth (mu), glucose consumption (qG) and lactate production (qL), and the yield coefficients (cell yield from glucose, YX/G, and lactate yield from glucose, YL/G). An effect of medium osmolality was also evaluated with T-flask monolayer cultivation. The best growth was observed at pH 7.6, 37 degrees C, 400 rpm, 50-100% saturation with oxygen and 320 mOsmol kg-1. Corresponding to the previous work with a human melanoma cell line, the sophisticated cultivation and process control systems have been improved for CHO cells.     

Production of PEX protein from QM7 cells cultured in polymer scaffolds in a Taylor–Couette bioreactor

In recent decades, many practical applications were developed with regard to the Taylor–Couette device, for example, reaction, filtration, extraction and bioreactor. In this study, the Taylor–Couette bioreactor was used to culture cells seeded in a biodegradable porous scaffold and produce PEX protein. Two different cell lines (NIH/3T3 and QM7) were seeded into PLGA sponges, which were fabricated using a solvent-free supercritical gas foaming method, and then cultured in the Taylor–Couette bioreactor. Cell proliferation was characterized using Quant-iT™ PicoGreen^® dsDNA assay and the results indicated that high mass transfer rate in the Taylor–Couette bioreactor enhanced cell proliferation. Qualitative distribution of live/dead cells was characterized using LIVE/DEAD^® Viability/Cytotoxicity assay and SEM and the results showed that cells cultured in static control mainly proliferated on the outer surface while the cells of Taylor-vortex bioreactor group could penetrate into the scaffold. The production yield of PEX protein, from QM7 cells transfected with pM9PEX, was quantified using PEX ELISA and the results showed a much higher PEX mass per scaffold for bioreactor than the control. As such, there is potential for the use of Taylor–Couette bioreactor in the mass production of PEX protein.     

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.     

Design and validation of a dynamic flow perfusion bioreactor for use with compliant tissue engineering scaffolds

In tissue engineering, flow perfusion bioreactors can be used to enhance nutrient diffusion while mechanically stimulating cells to increase matrix production. The goal of this study was to design and validate a dynamic flow perfusion bioreactor for use with compliant scaffolds. Using a non-permanent staining technique, scaffold perfusion was verified for flow rates of 0.1-2.0 mL/min. Flow analysis revealed that steady, pulsatile and oscillatory flow profiles were effectively transferred from the pump to the scaffold. Compared to static culture, bioreactor culture of osteoblast-seeded collagen-GAG scaffolds led to a 27-34% decrease in cell number but stimulated an 800-1200% increase in the production of prostaglandin E(2), an early-stage bone formation marker. This validated flow perfusion bioreactor provides the basis for optimisation of bioreactor culture in tissue engineering applications.