Three-dimensional biodegradable microscaffolding: scaffold characterization and cell population at single cell resolution.

Engineering artificial tissue scaffolds with a similar organization to that of the natural tissue is a key element to the successful recapitulation of function. However, three-dimensional (3-D) fabrication of tissue scaffolds containing complex microarchitectures still remains a challenge. In addition, little attention has been paid to the issue of how to incorporate cells within 3-D tissue scaffolds that contain precisely engineered architectures. Here we report a 3-D biodegradable microscaffolding (3D-BMS) technology and its process characterization as well as a microscale cellular loading technology as an efficient way to massively populate biodegradable polymers with cells at single cell resolution. In this study a particular emphasis was given to characterization of the material properties of the biodegradable polymers undergoing the 3D-BMS processes. Optimal process conditions were identified in order to avoid any unwanted change in material properties, such as crystallinity and scaffold strength, that have a direct impact on the degradation speed and physical integrity of the constructed scaffolds. For precise control of the cell distribution within the microstructured scaffolds a high precision microsieve structure was designed to localize rat hepatocytes and human articular chondrocytes in the biodegradable polymers. Cell suspensions were passed at a predetermined flow rate through biodegradable polymer layers that contained tapered microholes in a massively parallel process. This high resolution cell seeding method allows accurate manipulation of cell placement in thin layers of biodegradable polymers.

Investigating the role of hematopoietic stem and progenitor cells in regulating the osteogenic differentiation of mesenchymal stem cells in vitro.

Significant progress has been made in understanding the hematopoietic supportive capacity of both mesenchymal stem cells (MSCs) and osteogenic cells in maintaining hematopoietic stem and progenitor cells (HSPCs) in vitro. However the role of HSPCs in regulating their bone marrow niche environment through influencing the function of neighboring cell populations to complete this reciprocal relationship is not well understood. In this study, we investigated the influence of HSPCs on the osteogenic differentiation of MSCs in vitro, using a highly enriched population of hematopoietic cells with the phenotype c-Kit(+)Sca-1(+)Lineage(-)(KSL) and bone marrow derived mesenchymal stromal cells in direct contact co-culture in medium with or without the addition of the osteogenic supplement dexamethasone. The data suggest that a low dose of HSPCs in co-culture with MSCs in combination with dexamethasone treatment accelerates the osteogenic progression of MSCs, as evidenced in the earlier peak in alkaline phosphatase activity and enhanced calcium deposition compared to cultures of MSCs alone. We observed a longer persistence of functional primitive hematopoietic stem and progenitor cells in the population treated with dexamethasone, and this observation was positively correlated with enhanced osteogenic differentiation of MSCs. Therefore, our findings further support the concept that HSPCs are actively involved in regulating the development and maintenance of the stem cell niche environment in which they reside.

Elastic properties of induced pluripotent stem cells.

The recent technique of transducing key transcription factors into unipotent cells (fibroblasts) to generate pluripotent stem cells (induced pluripotent stem cells [iPSCs]) has significantly changed the stem cell field. These cells have great promise for many clinical applications, including that of regenerative medicine. Our findings show that iPSCs can be derived from human adipose-derived stromal cells (hASCs), a notable advancement in the clinical applicability of these cells. To investigate differences between two iPS cell lines (fibroblast-iPSC and hASC-iPSC), and also the gold standard human embryonic stem cell, we looked at cell stiffness as a possible indicator of cell differentiation-potential differences. We used atomic force microscopy as a tool to determine stem cell stiffness, and hence differences in material properties between cells. Human fibroblast and hASC stiffness was also ascertained for comparison. Interestingly, cells exhibited a noticeable difference in stiffness. From least to most stiff, the order of cell stiffness was as follows: hASC-iPSC, human embryonic stem cell, fibroblast-iPSC, fibroblasts, and, lastly, as the stiffest cell, hASC. In comparing hASC-iPSCs to their origin cell, the hASC, the reprogrammed cell is significantly less stiff, indicating that greater differentiation potentials may correlate with a lower cellular modulus. The stiffness differences are not dependent on cell culture density; hence, material differences between cells cannot be attributed solely to cell-cell constraints. The change in mechanical properties of the cells in response to reprogramming offers insight into how the cell interacts with its environment and might lend clues to how to efficiently reprogram cell populations as well as how to maintain their pluripotent state.

In vitro effects of direct current electric fields on adipose-derived stromal cells.

Endogenous electric fields play an important role in embryogenesis, regeneration, and wound repair and previous studies have shown that many populations of cells, leukocytes, fibroblasts, epithelial cells, and endothelial cells, exhibit directed migration in response to electric fields. As regenerative therapies continue to explore ways to control mesenchymal progenitor cells to recreate desirable tissues, it is increasingly necessary to characterize the vast nature of biological responses imposed by physical phenomena. Murine adipose-derived stromal cells (mASCs) migrated toward the cathode in direct current (DC) fields of physiologic strength and show a dose dependence of migration rate to stronger fields. Electric fields also caused mASCs to orient perpendicularly to the field vector and elicited a transient increase in cytosolic calcium. Additionally, their galvanotactic response appears to share classic chemotactic signaling pathways that are involved in the migration of other cell types. Galvanotaxis is one predominant result of electric fields on mASCs and it may be exploited to engineer adult stem cell concentrations and locations within implanted grafts or toward sites of wound repair.

Pulsed direct current electric fields enhance osteogenesis in adipose-derived stromal cells.

Adipose-derived stromal cells (ASCs) constitute a promising source of cells for regenerative medicine applications. Previous studies of osteogenic potential in ASCs have focused on chemicals, growth factors, and mechanical stimuli. Citing the demonstrated role electric fields play in enhancing healing in bone fractures and defects, we investigated the ability of pulsed direct current electric fields to drive osteogenic differentiation in mouse ASCs. Employing 50 Hz direct current electric fields in concert with and without osteogenic factors, we demonstrated increased early osteoblast-specific markers. We were also able to establish that commonly reported artifacts of electric field stimulation are not the primary mediators of the observed effects. The electric fields caused marked changes in the cytoskeleton. We used atomic force microscopy-based force spectroscopy to record an increase in the cytoskeletal tension after treatment with electric fields. We abolished the increased cytoskeletal stresses with the rho-associated protein kinase inhibitor, Y27632, and did not see any decrease in osteogenic gene expression, suggesting that the pro-osteogenic effects of the electric fields are not transduced via cytoskeletal tension. Electric fields may show promise as candidate enhancers of osteogenesis of ASCs and may be incorporated into cell-based strategies for skeletal regeneration.

Inhibition of histone deacetylase activity in reduced oxygen environment enhances the osteogenesis of mouse adipose-derived stromal cells.

Recent studies suggest that oxygen tension has a great impact on the osteogenic differentiation capacity of mesenchymal cells derived from adipose tissue: reduced oxygen impedes osteogenesis. We have found that expansion of mouse adipose-derived stromal cells (mASCs) in reduced oxygen tension (10%) results in increased cell proliferation along with induction of histone deacetylase (HDAC) activity. In this study, we utilized two HDAC inhibitors (HDACi), sodium butyrate (NaB) and valproic acid (VPA), and studied their effects on mASCs expanded in various oxygen tensions (21%, 10%, and 1% O(2)). Significant growth inhibition was observed with NaB or VPA treatment in each oxygen tension. Osteogenesis was enhanced by treatment with NaB or VPA, particularly in reduced oxygen tensions (10% and 1% O(2)). Conversely, adipogenesis was decreased with treatments of NaB or VPA at all oxygen tensions. Finally, NaB- or VPA-treated, reduced oxygen tension-exposed (1% O(2)) ASCs were grafted into surgically created mouse tibial defects and resulted in significantly increased bone regeneration. In conclusion, HDACi significantly promote the osteogenic differentiation of mASCs exposed to reduced oxygen tension; HDACi may hold promise for future clinical applications of ASCs for skeletal regeneration.

The construction of three-dimensional micro-fluidic scaffolds of biodegradable polymers by solvent vapor based bonding of micro-molded layers.

It is increasingly important to control cell growth into and within artificial scaffolds. Tissues such as skin, blood vessels, and cartilage have multi-layer structures with different cells in each layer. With the aid of micro-fabrication technology, a novel scaffolding method for biodegradable polymers such as polylactic acid (PLA), polyglycolic acid (PGA), and the copolymers poly(lactide-co-glycolide)(PLGA), was developed to construct three-dimensional multi-layer micro-fluidic tissue scaffolds. The method emphasizes micro-fluidic interconnections between layers within the scaffolds and maintenance of high-resolution geometries during the bonding process for the creation of multi-layered scaffolds. Micro-holes (10-100 microm), micro-channels, and micro-cavities were all created by micro-molding. Solvent-vapor based bonding of micro-molded layers preserved 20 microm sized structures. Sample scaffolds were constructed for purposes such as channel-directed cell growth and size-based cell sorting. Further extension of these techniques to create a micro-vascular network within or between layers is possible. Culturing of human coronary artery endothelial cells (HCAECs) on the sample scaffolds demonstrated the biocompatibility of the developed process and the strong influence of high-resolution micro-geometries on HCAEC growth.