Role of growth factors and endothelial cells in therapeutic angiogenesis and tissue engineering

To achieve the goals of engineering large complex tissues, and possibly internal organs, vascularization of the regenerating tissue is essential. To maintain the initial volume after implantation of regenerated tissue, improved vascularization is considered to be important. Recent advances in understanding the process of blood vessel growth has offered significant tools for the neovascularization of bioengineered tissues and therapeutic angiogenesis. Several angiogenic growth factors including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF) were used for vascularization of ischemic tissues. Other approaches such as prevascularization of the scaffold, prior to cell seeding, and incorporation of endothelial cells in the bioengineered tissue showed encouraging results. In this article, we will review recent advances in angiogenic growth factors, and discuss the role of these growth factors and endothelial cells in therapeutic angiogenesis and tissue engineering.     

Principals of neovascularization for tissue engineering

The goals in tissue engineering include the replacement of damaged, injured or missing body tissues with biological compatible substitutes such as bioengineered tissues. However, due to an initial mass loss after implantation, improved vascularization of the regenerated tissue is essential. Recent advances in understanding the process of blood vessel growth has offered significant tools for therapeutic neovascularization. Several angiogenic growth factors including vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor (bFGF) were used for vascularization of ischemic tissues. Three approaches have been used for vascularization of bioengineered tissue: incorporation of angiogenic factors in the bioengineered tissue, seeding endothelial cells with other cell types and prevascularization of matrices prior to cell seeding. This paper reviews the process of blood vessel growth and tissue vascularization, and discuss strategies for efficient vascularization of engineered tissues.     

Effect of different cell sheet ECM microenvironment on the formation of vascular network

The repair and reconstruction of large bone defects remains as a significant clinical challenge mainly due to the insufficient vascularization. The prefabrication of vascular network based on cell sheet technique brings a promising potential for sufficient vascularization due to rich extracellular matrix (ECM) of cell sheets. However, the effect of different cell sheet ECM micro-environment on the formation of a vascular network has not been well understood. Here our goal is to study the effect of different cell sheets on the formation of a vascular network. First we cultured human bone marrow mesenchymal stem cells (hBMSCs) under two culture conditions to obtain osteogenic differentiated cell sheet (ODCS) and undifferentiated cell sheet (UDCS), respectively. Then the human umbilical vein endothelial cells (HUVECs) were seeded onto the surface of the two sheets at different seeding densities to fabricate pre-vascularized cell sheets. Our results indicated that the two sheets facilitated the alignment of HUVECs and promoted the formation of vascular networks. Quantitative analysis showed that the number of networks in ODCS was higher than that in the UDCS. The ECM of the two sheets was remodeled and rearranged during the tubulogenesis process. Furthermore, results showed that the optimal seeding density of HUVECs was 5 × 10⁴ cell/cm². In summary, these results suggest that the vascularized ODCS has a promising potential to construct pre-vascularized tissue for bone repair.     

Engineering vascularized skeletal muscle tissue

One of the major obstacles in engineering thick, complex tissues such as muscle is the need to vascularize the tissue in vitro. Vascularization in vitro could maintain cell viability during tissue growth, induce structural organization and promote vascularization upon implantation. Here we describe the induction of endothelial vessel networks in engineered skeletal muscle tissue constructs using a three-dimensional multiculture system consisting of myoblasts, embryonic fibroblasts and endothelial cells coseeded on highly porous, biodegradable polymer scaffolds. Analysis of the conditions for induction and stabilization of the vessels in vitro showed that addition of embryonic fibroblasts increased the levels of vascular endothelial growth factor expression in the construct and promoted formation and stabilization of the endothelial vessels. We studied the survival and vascularization of the engineered muscle implants in vivo in three different models. Prevascularization improved the vascularization, blood perfusion and survival of the muscle tissue constructs after transplantation.     

Cell Proliferation and Oxygen Diffusion in a Vascularising Scaffold

The supply of oxygen to proliferating cells within a scaffold is a key factor for the successful building of new tissue in soft tissue engineering applications. A recent in vivo model, where an arteriovenous loop is placed in a scaffold, allows a vascularising network to form within a scaffold, establishing an oxygen source within, rather than external, to the scaffold. A one-dimensional model of oxygen concentration, cell proliferation and cell migration inside such a vascularising scaffold is developed and investigated. In addition, a vascularisation model is presented, which supports a vascularisation front which moves at a constant speed. The effects of vascular growth, homogenous and heterogenous seeding, diffusion of cells and critical hypoxic oxygen concentration are considered. For homogenous seeding, a relationship between the speed of the vascular front and a parameter defining the rate of oxygen diffusion relative to the rate of oxygen consumption determines whether a hypoxic region exists at some time. In particular, an estimate of the length of time that a fixed point in the scaffold will remain under hypoxic conditions is determined. For heterogenous seeding, a Fisher-like travelling wave of cells is established behind the vascular front. These findings provide a fundamental understanding of the important interplay between the parameters and allows for a theoretical assessment of a seeding strategy in a vascularising scaffold.     

细胞联合培养在血管构建中的作用

组织工程三大要素为种子细胞、支架材料和信号分子,干细胞因其多分化潜能成为热门的种子细胞。血管化问题是制约工程化组织应用于临床的问题之一。利用干细胞构建组织工程血管的手段之一是在分离培养得到足够的种子细胞后,通过生长因子、细胞外基质、外力作用、其他细胞等的调控实现内皮向分化。只有实现了成功的血管构建,工程化组织才能正常的发挥作用。近年来不少国内外专家学者通过细胞联合培养的方法,观察细胞间的相互作用对血管构建的影响,结果表明,细胞联合培养在血管的形成、存活、稳定方面起到了重要的作用,为组织工程血管化提供了有效的途径,本文就部分细胞联合培养在血管构建中的作用作一综述。     

Effects of Cell-Attachment and Extracellular Matrix on Bone Formation In Vivo in Collagen-Hydroxyapatite Scaffolds

Cell-based tissue engineering can be used to replace missing or damaged bone, but the optimal methods for delivering therapeutic cells to a bony defect have not yet been established. Using transgenic reporter cells as a donor source, two different collagen-hydroxyapatite (HA) scaffolds, and a critical-size calvarial defect model, we investigated the effect of a cell-attachment period prior to implantation, with or without an extracellular matrix-based seeding suspension, on cell engraftment and osteogenesis. When quantitatively compared, the in-house scaffold implanted immediately had a higher mean radiopacity than in-house scaffolds incubated overnight. Both scaffold types implanted immediately had significantly higher area fractions of donor cells, while the in-house collagen-HA scaffolds implanted immediately had higher area fractions of the mineralization label compared with groups incubated overnight. When the cell loading was compared in vitro for each delivery method using the in-house scaffold, immediate loading led to higher numbers of delivered cells. Immediate loading may be preferable in order to ensure robust bone formation in vivo. The use of a secondary ECM carrier improved the distribution of donor cells only when a pre-attachment period was applied. These results have improved our understanding of cell delivery to bony defects in the context of in vivo outcomes.     

In silico study of bone tissue regeneration in an idealised porous hydrogel scaffold using a mechano-regulation algorithm

Mechanical stimulation, in the form of fluid perfusion or mechanical strain, enhances osteogenic differentiation and overall bone tissue formation by mesenchymal stems cells cultured in biomaterial scaffolds for tissue engineering applications. In silico techniques can be used to predict the mechanical environment within biomaterial scaffolds, and also the relationship between bone tissue regeneration and mechanical stimulation, and thereby inform conditions for bone tissue engineering experiments. In this study, we investigated bone tissue regeneration in an idealised hydrogel scaffold using a mechano-regulation model capable of predicting tissue differentiation, and specifically compared five loading cases, based on known experimental bioreactor regimes. These models predicted that low levels of mechanical loading, i.e. compression (0.5% strain), pore pressure of 10 kPa and a combination of compression (0.5%) and pore pressure (10 kPa), could induce more osteogenic differentiation and lead to the formation of a higher bone tissue fraction. In contrast greater volumes of cartilage and fibrous tissue fractions were predicted under higher levels of mechanical loading (i.e. compression strain of 5.0% and pore pressure of 100 kPa). The findings in this study may provide important information regarding the appropriate mechanical stimulation for in vitro bone tissue engineering experiments.     

Adipose tissue‐derived progenitors for engineering osteogenic and vasculogenic grafts

The current need for bone grafts in orthopedic and reconstructive surgery cannot be satisfied by autologous tissue transplant due to its limited availability and significant associated morbidity. Tissue engineering approaches could supply sufficient amounts of bone substitutes by exploiting the ability to harvest autologous osteogenic progenitors associated with suitable porous materials. However, the generation of clinically relevant‐sized constructs is critically hampered by limited vascularization, with consequent engraftment and survival only of a thin outer shell, upon in vivo implantation. To overcome this limitation, different non‐mutually exclusive approaches have recently been developed to promote or accelerate graft vascularization, from angiogenic growth factor gene delivery to surgical pre‐vascularization of the construct before implantation. A simple, promising strategy involves the co‐culture of vasculogenic cells to form an intrinsic vascular network inside the graft in vitro, which can rapidly anastomose with the host blood vessels in vivo. Recent data have shown that adipose tissue‐derived stromal vascular fraction (SVF) may provide an efficient, convenient, and autologous source for both osteogenic and endothelial cells. When SVF progenitors were cultured in appropriate bioreactor systems and ectopically implanted, a functional vascular network connected to the host was formed concomitantly to bone formation. Future studies should aim at demonstrating that this approach effectively supports survival of scaled up cell‐based bone grafts at an orthotopic site. The procedure should also be adapted to become compatible with an intra‐operative timeline and complemented with the definition of suitable potency markers, to facilitate its development into a simplified, reproducible, and cost‐effective clinical treatment. J. Cell. Physiol. 225: 348–353, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Adipose tissue and the vascularization of biomaterials: Stem cells,microvascular fragments and nanofat—a review

Tissue defects in the human body after trauma and injury require precise reconstruction to regain function. Hence, there is a great demand for clinically translatable approaches with materials that are both biocompatible and biodegradable. They should also be able to adequately integrate within the tissue through sufficient vascularization. Adipose tissue is abundant and easily accessible. It is a valuable tissue source in regenerative medicine and tissue engineering, especially with regard to its angiogenic potential. Derivatives of adipose tissue, such as microfat, nanofat, microvascular fragments, stromal vascular fraction and stem cells, are commonly used in research, but also clinically to enhance the vascularization of implants and grafts at defect sites. In plastic surgery, adipose tissue is harvested via liposuction and can be manipulated in three ways (macro-, micro- and nanofat) in the operating room, depending on its ultimate use. Whereas macro- and microfat are used as a filling material for soft tissue injuries, nanofat is an injectable viscous extract that primarily induces tissue remodeling because it is rich in growth factors and stem cells. In contrast to microfat that adds volume to a defect site, nanofat has the potential to be easily combined with scaffold materials due to its liquid and homogenous consistency and is particularly attractive for blood vessel formation. The same is true for microvascular fragments that are easily isolated from adipose tissue through collagenase digestion. In preclinical animal models, it has been convincingly shown that these vascular fragments inosculate with host vessels and subsequently accelerate scaffold perfusion and host tissue integration. Adipose tissue is also an ideal source of stem cells. It yields larger quantities of cells than any other source and is easier to access for both the patient and doctor compared with other sources such as bone marrow. They are often used for tissue regeneration in combination with biomaterials. Adipose-derived stem cells can be applied unmodified or as single cell suspensions. However, certain pretreatments, such as cultivation under hypoxic conditions or three-dimensional spheroids production, may provide substantial benefit with regard to subsequent vascularization in vivo due to induced growth factor production. In this narrative review, derivatives of adipose tissue and the vascularization of biomaterials are addressed in a comprehensive approach, including several sizes of derivatives, such as whole fat flaps for soft tissue engineering, nanofat or stem cells, their secretome and exosomes. Taken together, it can be concluded that adipose tissue and its fractions down to the molecular level promote, enhance and support vascularization of biomaterials. Therefore, there is a high potential of the individual fat component to be used in regenerative medicine.     

Concentric cylinder bioreactor for production of tissue engineered cartilage: effect of seeding density and hydrodynamic loading on construct development

A concentric cylinder bioreactor has been developed to culture tissue engineered cartilage constructs under hydrodynamic loading. This bioreactor operates in a low shear stress environment, has a large growth area for construct production, allows for dynamic seeding of constructs, and provides for a uniform loading environment. Porous poly-lactic acid constructs, seeded dynamically in the bioreactor using isolated bovine chondrocytes, were cultured for 4 weeks at three seeding densities (60, 80, 100 x 10(6) cells per bioreactor) and three different shear stresses (imposed at 19, 38, and 76 rpm) to characterize the effect of chondrocyte density and hydrodynamic loading on construct growth. Construct seeding efficiency with chondrocytes is greater than 95% within 24 h. Extensive chondrocyte proliferation and matrix deposition are achieved so that after 28 days in culture, constructs from bioreactors seeded at the highest cell densities contain up to 15 x 10(6) cells, 2 mg GAG, and 3.5 mg collagen per construct and exhibit morphology similar to that of native cartilage. Bioreactors seeded with 60 million chondrocytes do not exhibit robust proliferation or matrix deposition and do not achieve morphology similar to that of native cartilage. In cultures under different steady hydrodynamic loading, the data demonstrate that higher shear stress suppresses matrix GAG deposition and encourages collagen incorporation. In contrast, under dynamic hydrodynamic loading conditions, cartilage constructs exhibit robust matrix collagen and GAG deposition. The data demonstrate that the concentric cylinder bioreactor provides a favorable hydrodynamic environment for cartilage construct growth and differentiation. Notably, construct matrix accumulation can be manipulated by hydrodynamic loading. This bioreactor is useful for fundamental studies of construct growth and to assess the significance of cell density, nutrients, and hydrodynamic loading on cartilage development. In addition, studies of cartilage tissue engineering in the well-characterized, uniform environment of the concentric cylinder bioreactor will develop important knowledge of bioprocessing parameters critical for large-scale production of engineered tissues.     

The interplay between tissue growth and scaffold degradation in engineered tissue constructs

In vitro tissue engineering is emerging as a potential tool to meet the high demand for replacement tissue, caused by the increased incidence of tissue degeneration and damage. A key challenge in this field is ensuring that the mechanical properties of the engineered tissue are appropriate for the in vivo environment. Achieving this goal will require detailed understanding of the interplay between cell proliferation, extracellular matrix (ECM) deposition and scaffold degradation. In this paper, we use a mathematical model (based upon a multiphase continuum framework) to investigate the interplay between tissue growth and scaffold degradation during tissue construct evolution in vitro. Our model accommodates a cell population and culture medium, modelled as viscous fluids, together with a porous scaffold and ECM deposited by the cells, represented as rigid porous materials. We focus on tissue growth within a perfusion bioreactor system, and investigate how the predicted tissue composition is altered under the influence of (1) differential interactions between cells and the supporting scaffold and their associated ECM, (2) scaffold degradation, and (3) mechanotransduction-regulated cell proliferation and ECM deposition. Numerical simulation of the model equations reveals that scaffold heterogeneity typical of that obtained from $\mu $ CT scans of tissue engineering scaffolds can lead to significant variation in the flow-induced mechanical stimuli experienced by cells seeded in the scaffold. This leads to strong heterogeneity in the deposition of ECM. Furthermore, preferential adherence of cells to the ECM in favour of the artificial scaffold appears to have no significant influence on the eventual construct composition; adherence of cells to these supporting structures does, however, lead to cell and ECM distributions which mimic and exaggerate the heterogeneity of the underlying scaffold. Such phenomena have important ramifications for the mechanical integrity of engineered tissue constructs and their suitability for implantation in vivo.     

Hybrid cellular automaton modeling of nutrient modulated cell growth in tissue engineering constructs

Mathematic models help interpret experimental results and accelerate tissue engineering developments. We develop in this paper a hybrid cellular automata model that combines the differential nutrient transport equation to investigate the nutrient limited cell construct development for cartilage tissue engineering. Individual cell behaviors of migration, contact inhibition and cell collision, coupled with the cell proliferation regulated by oxygen concentration were carefully studied. Simplified two-dimensional simulations were performed. Using this model, we investigated the influence of cell migration speed on the overall cell growth within in vitro cell scaffolds. It was found that intense cell motility can enhance initial cell growth rates. However, since cell growth is also significantly modulated by the nutrient contents, intense cell motility with conventional uniform cell seeding method may lead to declined cell growth in the final time because concentrated cell population has been growing around the scaffold periphery to block the nutrient transport from outside culture media. Therefore, homogeneous cell seeding may not be a good way of gaining large and uniform cell densities for the final results. We then compared cell growth in scaffolds with various seeding modes, and proposed a seeding mode with cells initially residing in the middle area of the scaffold that may efficiently reduce the nutrient blockage and result in a better cell amount and uniform cell distribution for tissue engineering construct developments.     

骨组织工程血管化的研究进展

血管化是组织工程骨成活的关键,组织工程骨的血管化过程与生理情况下的血管发生相似,但又有其独特性,并受多种因素影响。基于对组织工程骨血管化过程的认识,研究者通过联合细胞培养、促血管化生长因子、显微外科等方法重建血运。本文综述了当前骨组织工程的血管化研究进展。     

Tumor Necrosis Factor Improves Vascularization in Osteogenic Grafts Engineered with Human Adipose-Derived Stem/Stromal Cells

The innate immune response following bone injury plays an important role in promoting cellular recruitment, revascularization, and other repair mechanisms. Tumor necrosis factor-α (TNF) is a prominent pro-inflammatory cytokine in this cascade, and has been previously shown to improve bone formation and angiogenesis in a dose- and timing-dependent manner. This ability to positively impact both osteogenesis and vascular growth may benefit bone tissue engineering, as vasculature is essential to maintaining cell viability in large grafts after implantation. Here, we investigated the effects of exogenous TNF on the induction of adipose-derived stem/stromal cells (ASCs) to engineer pre-vascularized osteogenic tissue in vitro with respect to dose, timing, and co-stimulation with other inflammatory mediators. We found that acute (2-day), low-dose exposure to TNF promoted vascularization, whereas higher doses and continuous exposure inhibited vascular growth. Co-stimulation with platelet-derived growth factor (PDGF), another key factor released following bone injury, increased vascular network formation synergistically with TNF. ASC-seeded grafts were then cultured within polycaprolactone-fibrin composite scaffolds and implanted in nude rats for 2 weeks, resulting in further tissue maturation and increased angiogenic ingrowth in TNF-treated grafts. VEGF-A expression levels were significantly higher in TNF-treated grafts immediately prior to implantation, indicating a long-term pro-angiogenic effect. These findings demonstrate that TNF has the potential to promote vasculogenesis in engineered osteogenic grafts both in vitro and in vivo. Thus, modulation and/or recapitulation of the immune response following bone injury may be a beneficial strategy for bone tissue engineering.     

Scaffold-based bone engineering by using genetically modified cells

The first generation of clinically applied tissue engineering concepts in the area of skin, cartilage and bone marrow regeneration was based on the isolation, expansion and implantation of cells from the patient's own tissue. Although successful in selective treatments, tissue engineering needs to overcome major challenges to allow widespread clinical application with predictable outcomes. One challenge is to present the cells in a matrix to the implantation site to allow the cells to survive the wound healing contraction forces, tissue remodeling in certain tissues such as bone and biomechanical loading. Hence, several tissue engineering strategies focus on the development of load-bearing scaffold/cell constructs. From a cell source point of view, bone engineers face challenges to isolate and expand cells with the highest potential to form osseous tissue along with harvesting tissue without extensive donor site morbidity. A major hurdle to tissue engineering is de-differentiation and limited ability to control cell phenotype following in vitro expansion. Due to early successes with genetic engineering, bone tissue engineers have used different strategies to genetically alter various types of mesenchymal cells to enhance the mineralization capacity of tissue-engineered scaffold/cell constructs. Although the development of multi-component scaffold/osteogenic cell constructs requires a combination of interdisciplinary research strategies, the following review is limited to describe the general aspects of bone engineering and to present overall directions of technology platforms, which include a genetic engineering component. This paper reviews the most recent work in the field and discusses the concepts developed and executed by a collaborative effort of the multi-disciplinary teams of the two authors.     

Analysis of cell growth and diffusion in a scaffold for cartilage tissue engineering

Developments in tissue engineering over the past decade have offered promising future for the repair and reconstruction of damaged tissues. To regenerate three dimensional and weight-bearing implants, advances in biomaterials and manufacturing technologies prompted cell cultivations with natural or artificial scaffolds, in which cells are allowed to proliferate, migrate, and differentiate in vitro. In this article, we develop a mathematical model for cell growth in a porous scaffold. By treating the cell-scaffold construct as a porous medium, a continuum model is set up based on basic principles of mass conservation. In addition to cell growth kinetics, we incorporate cell diffusion in the model to describe the effects of cell random walks. Computational results are compared to experimental data found in the literature. With this model, we are able to investigate cell motility, heterogeneous cell distributions, and non-uniform seeding for tissue engineering applications. Results show that random walks tend to enhance uniform cell spreads in space, which in turn increases the probabilities for cells to acquire nutrients; therefore random walks are likely to be a positive contribution to the overall cell growth on scaffolds.     

Current and emerging vascularization strategies in skin tissue engineering

Vascularization is a key process in skin tissue engineering, determining the biological function of artificial skin implants. Hence, efficient vascularization strategies are a major prerequisite for the safe application of these implants in clinical practice. Current approaches include (i) modification of structural and physicochemical properties of dermal scaffolds, (ii) biological scaffold activation with growth factor-releasing systems or gene vectors, and (iii) generation of prevascularized skin substitutes by seeding scaffolds with vessel-forming cells. These conventional approaches may be further supplemented by emerging strategies, such as transplantation of adipose tissue-derived microvascular fragments, 3D bioprinting and microfluidics, miRNA modulation, cell sheet engineering, and fabrication of photosynthetic scaffolds. The successful translation of these vascularization strategies from bench to bedside may pave the way for a broad clinical implementation of skin tissue engineering.     

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.