In vivo bone regeneration using tubular perfusion system bioreactor cultured nanofibrous scaffolds.

The use of bioreactors for the in vitro culture of constructs for bone tissue engineering has become prevalent as these systems may improve the growth and differentiation of a cultured cell population. Here we utilize a tubular perfusion system (TPS) bioreactor for the in vitro culture of human mesenchymal stem cells (hMSCs) and implant the cultured constructs into rat femoral condyle defects. Using nanofibrous electrospun poly(lactic-co-glycolic acid)/poly(ε-caprolactone) scaffolds, hMSCs were cultured for 10 days in vitro in the TPS bioreactor with cellular and acellular scaffolds cultured statically for 10 days as a control. After 3 and 6 weeks of in vivo culture, explants were removed and subjected to histomorphometric analysis. Results indicated more rapid bone regeneration in defects implanted with bioreactor cultured scaffolds with a new bone area of 1.23 ± 0.35 mm(2) at 21 days compared to 0.99 ± 0.43 mm(2) and 0.50 ± 0.29 mm(2) in defects implanted with statically cultured scaffolds and acellular scaffolds, respectively. At the 21 day timepoint, statistical differences (p<0.05) were only observed between defects implanted with cell containing scaffolds and the acellular control. After 42 days, however, defects implanted with TPS cultured scaffolds had the greatest new bone area with 1.72 ± 0.40 mm(2). Defects implanted with statically cultured and acellular scaffolds had a new bone area of 1.26 ± 0.43 mm(2) and 1.19 ± 0.33 mm(2), respectively. The increase in bone growth observed in defects implanted with TPS cultured scaffolds was statistically significant (p<0.05) when compared to both the static and acellular groups at this timepoint. This study demonstrates the efficacy of the TPS bioreactor to improve bone tissue regeneration and highlights the benefits of utilizing perfusion bioreactor systems to culture MSCs for bone tissue engineering.

Bioreactors to influence stem cell fate: augmentation of mesenchymal stem cell signaling pathways via dynamic culture systems.

BACKGROUND: Mesenchymal stem cells (MSCs) are a promising cell source for bone and cartilage tissue engineering as they can be easily isolated from the body and differentiated into osteoblasts and chondrocytes. A cell based tissue engineering strategy using MSCs often involves the culture of these cells on three-dimensional scaffolds; however the size of these scaffolds and the cell population they can support can be restricted in traditional static culture. Thus dynamic culture in bioreactor systems provides a promising means to culture and differentiate MSCs in vitro. SCOPE OF REVIEW: This review seeks to characterize key MSC differentiation signaling pathways and provides evidence as to how dynamic culture is augmenting these pathways. Following an overview of dynamic culture systems, discussion will be provided on how these systems can effectively modify and maintain important culture parameters including oxygen content and shear stress. Literature is reviewed for both a highlight of key signaling pathways and evidence for regulation of these signaling pathways via dynamic culture systems. MAJOR CONCLUSIONS: The ability to understand how these culture systems are affecting MSC signaling pathways could lead to a shear or oxygen regime to direct stem cell differentiation. In this way the efficacy of in vitro culture and differentiation of MSCs on three-dimensional scaffolds could be greatly increased. GENERAL SIGNIFICANCE: Bioreactor systems have the ability to control many key differentiation stimuli including mechanical stress and oxygen content. The further integration of cell signaling investigations within dynamic culture systems will lead to a quicker realization of the promise of tissue engineering and regenerative medicine. This article is part of a Special Issue entitled Biochemistry of Stem Cells.

Tubular perfusion system culture of human mesenchymal stem cells on poly-L-lactic acid scaffolds produced using a supercritical carbon dioxide-assisted process.

In vitro human mesenchymal stem cell (hMSC) proliferation and differentiation is dependent on scaffold design parameters and specific culture conditions. In this study, we investigate how scaffold microstructure influences hMSC behavior in a perfusion bioreactor system. Poly-L-lactic acid (PLLA) scaffolds are fabricated using supercritical carbon dioxide (SC-CO(2)) gel drying. This production method results in scaffolds fabricated with nanostructure. To introduce a microporous structure, porogen leaching was used in addition to this technique to produce scaffolds of average pore size of 100, 250, and 500 µm. These scaffolds were then cultured in static culture in well plates or dynamic culture in the tubular perfusion system (TPS) bioreactor. Results indicated that hMSCs were able to attach and maintain viability on all scaffolds with higher proliferation in the 250 µm and 500 µm pore sizes of bioreactor cultured scaffolds and 100 µm pore size of statically cultured scaffolds. Osteoblastic differentiation was enhanced in TPS culture as compared to static culture with the highest alkaline phosphatase expression observed in the 250 µm pore size group. Bone morphogenetic protein-2 was also analyzed and expression levels were highest in the 250 µm and 500 µm pore size bioreactor cultured samples. These results demonstrate cellular response to pore size as well as the ability of dynamic culture to enhance these effects.

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.

Formation of an aggregated alginate construct in a tubular perfusion system.

Tissue engineering strategies are often limited by in vitro culture techniques of three dimensional scaffolds. Here we develop a method to form an aggregated cell-containing construct in vitro in a bioreactor system. Human mesenchymal stem cells (hMSCs) are cultured in individual alginate beads in a tubular perfusion system (TPS) bioreactor and then aggregated to form a single large construct. Mechanical evaluation of this construct demonstrated that aggregated alginate constructs (AACs) made from beads with 2.15 mm diameters had a Young's modulus of 85.6±15.8 kPa, a tensile strength of 3.24±0.55 kPa and a yield strength of 1.44±0.27 kPa. These mechanical properties were shown to be dependent on the bead size used to fabricate the AACs with smaller bead sizes resulting in stronger constructs. Analysis of metabolic activity revealed that hMSCs encapsulated in alginate exposed to AAC treatment sustained metabolic activity while live dead staining indicated cells remain viable. These results demonstrate the formation of AACs in the TPS bioreactor as an elegant method to create tissue engineering constructs in vitro.

Tubular perfusion system for the long-term dynamic culture of human mesenchymal stem cells.

In vitro culture techniques must be improved to increase the feasibility of cell-based tissue engineering strategies. To enhance nutrient transport we have developed a novel bioreactor, the tubular perfusion system (TPS), to culture human mesenchymal stem cells (hMSCs) in three-dimensional scaffolds. This system utilizes an elegant design to create a more effective environment for cell culture. In our design, hMSCs in the TPS bioreactor are encapsulated in alginate beads that are tightly packed in a tubular growth chamber. The medium is perfused by a peristaltic pump through the growth chamber and around the tightly packed scaffolds enhancing nutrient transfer while exposing the cells to shear stress. Results demonstrate that bioreactor culture supports early osteoblastic differentiation of hMSCs as shown by alkaline phosphatase gene expression. After 14 and 28 days of culture significant increases in the gene expression levels of osteocalcin, osteopontin, and bone morphogenetic protein-2 were observed with bioreactor culture, and expression of these markers was shown to increase with media flow rate. These results demonstrate the TPS bioreactor as an effective means to culture hMSCs and provide insight to the effect of long-term shear stresses on differentiating hMSCs.

Bone tissue engineering bioreactors: dynamic culture and the influence of shear stress.

A bone tissue engineering strategy involving the in vitro expansion of cells on a scaffold before implantation into the body represents a promising alternative to current clinical treatments. To improve in vitro culture, bioreactor systems have been widely researched for bone tissue engineering purposes. Spinner flask, rotating wall bioreactors, and perfusion systems have all been the focus of experiments, and each system has advantages and disadvantages. This review seeks to summarize these efforts and provide the current status of research in this area. Research using spinner flasks and rotating wall bioreactors is discussed, but focus is placed on perfusion bioreactor systems. While spinner flasks and rotating wall bioreactors have been shown to improve in vitro culture conditions by increasing homogeneity of nutrients in the media, perfusion systems expose cells to shear stress and more efficiently enhance nutrient transfer. Enhanced mineralized matrix deposition and enhancement of osteoblastic signal expression in response to culture in these systems have been widely reported. This review provides analysis of the causes of these changes in signal expression as well as reports on bioreactor systems that have been commercialized.

Stereolithographic bone scaffold design parameters: osteogenic differentiation and signal expression.

Scaffold design parameters including porosity, pore size, interconnectivity, and mechanical properties have a significant influence on osteogenic signal expression and differentiation. This review evaluates the influence of each of these parameters and then discusses the ability of stereolithography (SLA) to be used to tailor scaffold design to optimize these parameters. Scaffold porosity and pore size affect osteogenic cell signaling and ultimately in vivo bone tissue growth. Alternatively, scaffold interconnectivity has a great influence on in vivo bone growth but little work has been done to determine if interconnectivity causes changes in signaling levels. Osteogenic cell signaling could be also influenced by scaffold mechanical properties such as scaffold rigidity and dynamic relationships between the cells and their extracellular matrix. With knowledge of the effects of these parameters on cellular functions, an optimal tissue engineering scaffold can be designed, but a proper technology must exist to produce this design to specification in a repeatable manner. SLA has been shown to be capable of fabricating scaffolds with controlled architecture and micrometer-level resolution. Surgical implantation of these scaffolds is a promising clinical treatment for successful bone regeneration. By applying knowledge of how scaffold parameters influence osteogenic cell signaling to scaffold manufacturing using SLA, tissue engineers may move closer to creating the optimal tissue engineering scaffold.

Macroporous hydrogels upregulate osteogenic signal expression and promote bone regeneration.

The objective of this work was to investigate the effects of macroporous hydrogel architecture on the osteogenic signal expression and differentiation of human mesenchymal stem cells (hMSCs). In particular, we have proposed a tissue engineering approach for orbital bone repair based on a cyclic acetal biomaterial formed from 5-ethyl-5-(hydroxymethyl)-beta,beta-dimethyl-1,3-dioxane-2-ethanol diacrylate (EHD) and poly(ethylene glycol) diacrylate (PEGDA). The EHD monomer and PEGDA polymer may be fabricated into macroporous EH-PEG hydrogels by radical polymerization and subsequent porogen leaching, a novel technique for hydrophilic gels. We hypothesized that EH-PEG hydrogel macroporosity facilitates intercellular signaling among hMSCs. To investigate this phenomenon, hMSCs were loaded into EH-PEG hydrogels with varying pore size and porosity. The viability of hMSCs, the expression of bone morphogenetic protein-2 (BMP-2), BMP receptor type 1A, and BMP receptor type 2 by hMSCs, and the differentiation of hMSCs were then assessed. Results demonstrate that macroporous EH-PEG hydrogels support hMSCs and that this macroporous environment promotes a dramatic increase in BMP-2 expression by hMSCs. This upregulation of BMP-2 expression is associated by a more rapid hMSC differentiation, as measured by alkaline phosphatase expression. Altering hMSC interactions with the EH-PEG hydrogel surface, by the addition of fibronectin, did not appear to augment BMP-2 expression. We therefore speculate that EH-PEG hydrogel macroporosity facilitates autocrine and paracrine signaling by localizing endogenously expressed factors within the hydrogel's pores and thus promotes hMSC osteoblastic differentiation and bone regeneration.

Meta-analysis of microarray studies reveals a novel hematopoietic progenitor cell signature and demonstrates feasibility of inter-platform data integration.

Microarray-based studies of global gene expression (GE) have resulted in a large amount of data that can be mined for further insights into disease and physiology. Meta-analysis of these data is hampered by technical limitations due to many different platforms, gene annotations and probes used in different studies. We tested the feasibility of conducting a meta-analysis of GE studies to determine a transcriptional signature of hematopoietic progenitor and stem cells. Data from studies that used normal bone marrow-derived hematopoietic progenitors was integrated using both RefSeq and UniGene identifiers. We observed that in spite of variability introduced by experimental conditions and different microarray platforms, our meta-analytical approach can distinguish biologically distinct normal tissues by clustering them based on their cell of origin. When studied in terms of disease states, GE studies of leukemias and myelodysplasia progenitors tend to cluster with normal progenitors and remain distinct from other normal tissues, further validating the discriminatory power of this meta-analysis. Furthermore, analysis of 57 normal hematopoietic stem and progenitor cell GE samples was used to determine a gene expression signature characteristic of these cells. Genes that were most uniformly expressed in progenitors and at the same time differentially expressed when compared to other normal tissues were found to be involved in important biological processes such as cell cycle regulation and hematopoiesis. Validation studies using a different microarray platform demonstrated the enrichment of several genes such as SMARCE, Septin 6 and others not previously implicated in hematopoiesis. Most interestingly, alpha-integrin, the only common stemness gene discovered in a recent comparative murine analysis (Science 302(5644):393) was also enriched in our dataset, demonstrating the usefulness of this analytical approach.