Developing Implantable Scaffolds to Enhance Neural Stem Cell Therapy for Post-Operative Glioblastoma.

Pre-clinical and clinical studies have shown that engineered tumoricidal neural stem cells (tNSCs) are a promising treatment strategy for the aggressive brain cancer glioblastoma (GBM). Yet, stabilizing human tNSCs within the surgical cavity following GBM resection is a significant challenge. As a critical step toward advancing engineered human NSC therapy for GBM, we used a preclinical variant of the clinically utilized NSC line HB1.F3.CD and mouse models of human GBM resection/recurrence to identify a polymeric scaffold capable of maximizing the transplant, persistence, and tumor kill of NSC therapy for post-surgical GBM. Using kinetic bioluminescence imaging, we found that tNSCs delivered into the mouse surgical cavity wall by direct injection persisted only 3 days. We found that delivery of tNSCs into the cavity on nanofibrous electrospun poly-l-lactic acid scaffolds extended tNSC persistence to 8 days. Modifications to fiber surface coating, diameter, and morphology of the scaffold failed to significantly extend tNSC persistence in the cavity. In contrast, tNSCs delivered into the post-operative cavity on gelatin matrices (GEMs) persisted 8-fold longer as compared to direct injection. GEMs remained permissive to tumor-tropic homing, as tNSCs migrated off the scaffolds and into invasive tumor foci both in vitro and in vivo. To mirror envisioned human brain tumor trials, we engineered tNSCs to express the prodrug/enzyme thymidine kinase (tNSCstk) and transplanted the therapeutic cells in the post-operative cavity of mice bearing resected orthotopic patient-derived GBM xenografts. Following administration of the prodrug ganciclovir, residual tumor volumes in mice receiving GEM/tNSCs were reduced by 10-fold at day 35, and median survival was extended from 31 to 46 days. Taken together, these data begin to define design parameters for effective scaffold/tNSC composites and suggest a new approach to maximizing the efficacy of tNSC therapy in human patient trials.

Primary cilia are sensors of electrical field stimulation to induce osteogenesis of human adipose-derived stem cells.

In this study, we report for the first time that the primary cilium acts as a crucial sensor for electrical field stimulation (EFS)-enhanced osteogenic response in osteoprogenitor cells. In addition, primary cilia seem to functionally modulate effects of EFS-induced cellular calcium oscillations. Primary cilia are organelles that have recently been implicated to play a crucial sensor role for many mechanical and chemical stimuli on stem cells. Here, we investigate the role of primary cilia in EFS-enhanced osteogenic response of human adipose-derived stem cells (hASCs) by knocking down 2 primary cilia structural proteins, polycystin-1 and intraflagellar protein-88. Our results indicate that structurally integrated primary cilia are required for detection of electrical field signals in hASCs. Furthermore, by measuring changes of cytoplasmic calcium concentration in hASCs during EFS, our findings also suggest that primary cilia may potentially function as a crucial calcium-signaling nexus in hASCs during EFS.-Cai, S., Bodle, J. C., Mathieu, P. S., Amos, A., Hamouda, M., Bernacki, S., McCarty, G., Loboa, E. G. Primary cilia are sensors of electrical field stimulation to induce osteogenesis of human adipose-derived stem cells.

Concise Review: Primary Cilia: Control Centers for Stem Cell Lineage Specification and Potential Targets for Cell-Based Therapies.

Directing stem cell lineage commitment prevails as the holy grail of translational stem cell research, particularly to those interested in the application of mesenchymal stem cells and adipose-derived stem cells in tissue engineering. However, elucidating the mechanisms underlying their phenotypic specification persists as an active area of research. In recent studies, the primary cilium structure has been intimately associated with defining cell phenotype, maintaining stemness, as well as functioning in a chemo, electro, and mechanosensory capacity in progenitor and committed cell types. Many hypothesize that the primary cilium may indeed be another important player in defining and controlling cell phenotype, concomitant with lineage-dictated cytoskeletal dynamics. Many of the studies on the primary cilium have emerged from disparate areas of biological research, and crosstalk amongst these areas of research is just beginning. To date, there has not been a thorough review of how primary cilia fit into the current paradigm of stem cell differentiation and this review aims to summarize the current cilia work in this context. The goal of this review is to highlight the cilium's function and integrate this knowledge into the working knowledge of stem cell biologists and tissue engineers developing regenerative medicine technologies. Stem Cells 2016;34:1445-1454.

Continuous-Wave Stimulated Emission Depletion Microscope for Imaging Actin Cytoskeleton in Fixed and Live Cells.

Stimulated emission depletion (STED) microscopy provides a new opportunity to study fine sub-cellular structures and highly dynamic cellular processes, which are challenging to observe using conventional optical microscopy. Using actin as an example, we explored the feasibility of using a continuous wave (CW)-STED microscope to study the fine structure and dynamics in fixed and live cells. Actin plays an important role in cellular processes, whose functioning involves dynamic formation and reorganization of fine structures of actin filaments. Frequently used confocal fluorescence and STED microscopy dyes were employed to image fixed PC-12 cells (dyed with phalloidin- fluorescein isothiocyante) and live rat chondrosarcoma cells (RCS) transfected with actin-green fluorescent protein (GFP). Compared to conventional confocal fluorescence microscopy, CW-STED microscopy shows improved spatial resolution in both fixed and live cells. We were able to monitor cell morphology changes continuously; however, the number of repetitive analyses were limited primarily by the dyes used in these experiments and could be improved with the use of dyes less susceptible to photobleaching. In conclusion, CW-STED may disclose new information for biological systems with a proper characteristic length scale. The challenges of using CW-STED microscopy to study cell structures are discussed.

Mechanical Stimulation of Adipose-Derived Stromal/Stem Cells for Functional Tissue Engineering of the Musculoskeletal System via Cyclic Hydrostatic Pressure, Simulated Microgravity, and Cyclic Tensile Strain.

It is critical that human adipose-derived stromal/stem cell (hASC) tissue engineering therapies possess appropriate mechanical properties in order to restore the function of the load-bearing tissues of the musculoskeletal system. In an effort to elucidate hASC response to mechanical stimulation and develop mechanically robust tissue-engineered constructs, recent research has utilized a variety of mechanical loading paradigms, including cyclic tensile strain, cyclic hydrostatic pressure, and mechanical unloading in simulated microgravity. This chapter will describe the methods for applying these mechanical stimuli to hASC to direct differentiation for functional tissue engineering of the musculoskeletal system.

A biomechanical assessment of tissue-engineered polymer neo-uteri after orthotopic implantation.

OBJECTIVE: To assess the in vivo biomechanical maturation of tissue-engineered neo-uteri that have previously supported live births in a rabbit model. DESIGN: Nonclinical animal study. SETTING: University-based research laboratory. ANIMALS: Eighteen adult female rabbits. INTERVENTION: Biodegradable poly-DL-lactide-co-glycolide-coated polyglycolic acid scaffolds seeded with autologous uterine-derived endometrial and myometrial cells. Nonseeded scaffolds and seeded, tissue-engineered neo-uteri were implanted into one uterine horn of rabbits for 1, 3, or 6 months, excised, and biomechanically assessed in comparison to native uterine tissue. MAIN OUTCOME MEASURES: Tensile stress-relaxation testing, strain-to-failure testing, and viscoelastic modeling. RESULTS: By evaluating the biomechanical data with several viscoelastic models, it was revealed that tissue-engineered uteri were more mechanically robust than nonseeded scaffolds. For example, the 10% instantaneous stress of the tissue-engineered neo-uteri was 2.1 times higher than the nonseeded scaffolds at the 1-month time point, 1.6 times higher at the 3-month time point, and 1.5 times higher at the 6-month time point. Additionally, as the duration of implantation increased, the engineered constructs became more mechanically robust (e.g., 10% instantaneous stress of the tissue-engineered neo-uteri increased from 22 kPa at 1 month to 42 kPa at 6 months). Compared with native tissue values, tissue-engineered neo-uteri achieved or surpassed native tissue values by the 6-month time point. CONCLUSION: The present study evaluated the mechanical characteristics of novel tissue-engineered neo-uteri that have previously been reported to support live births in the rabbit model. We demonstrate that the biomechanics of these implants closely resemble those of native tissue, giving further credence to their development as a clinical solution to uterine factor infertility.

Sensitization of transforming growth factor-ß signaling by multiple peptides patterned on DNA nanostructures.

We report sensitization of a cellular signaling pathway by addition of functionalized DNA nanostructures. Signaling by transforming growth factor ß (TGFß) has been shown to be dependent on receptor clustering. By patterning a DNA nanostructure with closely spaced peptides that bind to TGFß receptor, we observe increased sensitivity of NMuMG cells to TGFß ligand. This is evidenced by translocation of secondary messenger proteins to the nucleus and stimulation of an inducible luciferase reporter at lower concentrations of TGFß ligand. We believe this represents an important initial step toward realization of DNA as a self-assembling and biologically compatible material for use in tissue engineering and drug delivery.

Paracrine signals from mesenchymal cell populations govern the expansion and differentiation of human hepatic stem cells to adult liver fates.

UNLABELLED: The differentiation of embryonic or determined stem cell populations into adult liver fates under known conditions yields cells with some adult-specific genes but not others, aberrant regulation of one or more genes, and variations in the results from experiment to experiment. We tested the hypothesis that sets of signals produced by freshly isolated, lineage-dependent mesenchymal cell populations would yield greater efficiency and reproducibility in driving the differentiation of human hepatic stem cells (hHpSCs) into adult liver fates. The subpopulations of liver-derived mesenchymal cells, purified by immunoselection technologies, included (1) angioblasts, (2) mature endothelia, (3) hepatic stellate cell precursors, (4) mature stellate cells (pericytes), and (5) myofibroblasts. Freshly immunoselected cells of each of these subpopulations were established in primary cultures under wholly defined (serum-free) conditions that we developed for short-term cultures and were used as feeders with hHpSCs. Feeders of angioblasts yielded self-replication, stellate cell precursors caused lineage restriction to hepatoblasts, mature endothelia produced differentiation into hepatocytes, and mature stellate cells and/or myofibroblasts resulted in differentiation into cholangiocytes. Paracrine signals produced by the different feeders were identified by biochemical, immunohistochemical, and quantitative reverse-transcription polymerase chain reaction analyses, and then those signals were used to replace the feeders in monolayer and three-dimensional cultures to elicit the desired biological responses from hHpSCs. The defined paracrine signals were proved to be able to yield reproducible responses from hHpSCs and to permit differentiation into fully mature and functional parenchymal cells. CONCLUSION: Paracrine signals from defined mesenchymal cell populations are important for the regulation of stem cell populations into specific adult fates; this finding is important for basic and clinical research as well as industrial investigations.

Dietary calcium restriction affects mesenchymal stem cell activity and bone development in neonatal pigs.

The effects of dietary calcium (Ca) deficiency on skeletal integrity are well characterized in growing and mature mammals; however, less is known about Ca nutrition during the neonatal period. In this study, we examined the effects of neonatal Ca nutrition on bone integrity, endocrine hormones, and mesenchymal stem cell (MSC) activity. Neonatal pigs (24 ± 6 h of age) received either a Ca-adequate (1.2 g/100 g) or an ~40% Ca-deficient diet for 18 d. Ca deficiency reduced (P < 0.05) bone flexural strength and bone mineral density without major differences in plasma indicators of Ca status. There were no meaningful differences in plasma Ca, phosphate (PO(4)), parathyroid hormone, or 1,25-dihydroxycholecalciferol due to Ca nutrition throughout the study. Calcium deficiency also reduced (P < 0.05) the in vivo proliferation of MSC by ~50%. In vitro studies utilizing homologous sera demonstrated that MSC activity was affected (P < 0.05) by both the Ca status of the pig and the sera as well as by their interaction. The results indicate that neonatal Ca nutrition is crucial for bone integrity and suggest that early-life Ca restriction may have long-term effects on bone integrity via programming of MSC.

The evaluation of a multiphasic 3D-bioplotted scaffold seeded with adipose derived stem cells to repair osteochondral defects in a porcine model.

There is a need for the development of effective treatments for focal articular cartilage injuries. We previously developed a multiphasic 3D-bioplotted osteochondral scaffold design that can drive site-specific tissue formation when seeded with adipose-derived stem cells (ASC). The objective of this study was to evaluate this scaffold in a large animal model. Osteochondral defects were generated in the trochlear groove of Yucatan minipigs and repaired with scaffolds that either contained or lacked an electrospun tidemark and were either unseeded or seeded with ASC. Implants were monitored via computed tomography (CT) over the course of 4 months of in vivo implantation and compared to both open lesions and autologous explants. ICRS II evaluation indicated that defects with ASC-seeded scaffolds had healing that most closely resembled the aulogous explant. Scaffold-facilitated subchondral bone repair mimicked the structure of native bone tissue, but cartilage matrix staining was not apparent within the scaffold. The open lesions had the highest volumetric infill detected using CT analysis (p < 0.05), but the repair tissue was largely disorganized. The acellular scaffold without a tidemark had significantly more volumetric filling than either the acellular or ASC seeded groups containing a tidemark (p < 0.05), suggesting that the tidemark limited cell infiltration into the cartilage portion of the scaffold. Overall, scaffold groups repaired the defect more successfully than an open lesion but achieved limited repair in the cartilage region. With further optimization, this approach holds potential to treat focal cartilage lesions in a highly personalized manner using a human patient's own ASC cells.

Investigation of multiphasic 3D-bioplotted scaffolds for site-specific chondrogenic and osteogenic differentiation of human adipose-derived stem cells for osteochondral tissue engineering applications.

Osteoarthritis is a degenerative joint disease that limits mobility of the affected joint due to the degradation of articular cartilage and subchondral bone. The limited regenerative capacity of cartilage presents significant challenges when attempting to repair or reverse the effects of cartilage degradation. Tissue engineered medical products are a promising alternative to treat osteochondral degeneration due to their potential to integrate into the patient's existing tissue. The goal of this study was to create a scaffold that would induce site-specific osteogenic and chondrogenic differentiation of human adipose-derived stem cells (hASC) to generate a full osteochondral implant. Scaffolds were fabricated using 3D-bioplotting of biodegradable polycraprolactone (PCL) with either ß-tricalcium phosphate (TCP) or decellularized bovine cartilage extracellular matrix (dECM) to drive site-specific hASC osteogenesis and chondrogenesis, respectively. PCL-dECM scaffolds demonstrated elevated matrix deposition and organization in scaffolds seeded with hASC as well as a reduction in collagen I gene expression. 3D-bioplotted PCL scaffolds with 20% TCP demonstrated elevated calcium deposition, endogenous alkaline phosphatase activity, and osteopontin gene expression. Osteochondral scaffolds comprised of hASC-seeded 3D-bioplotted PCL-TCP, electrospun PCL, and 3D-bioplotted PCL-dECM phases were evaluated and demonstrated site-specific osteochondral tissue characteristics. This technique holds great promise as cartilage morbidity is minimized since autologous cartilage harvest is not required, tissue rejection is minimized via use of an abundant and accessible source of autologous stem cells, and biofabrication techniques allow for a precise, customizable methodology to rapidly produce the scaffold.

Expression of proinflammatory cytokines by human mesenchymal stem cells in response to cyclic tensile strain.

Mesenchymal stem cells produce proinflammatory cytokines during their normal growth. Direct or indirect regulation of bone resorption by these cytokines has been reported. However, the effects of osteogenic conditions-chemical and/or mechanical-utilized during in vitro bone tissue engineering on expression of cytokines by hMSCs have not been studied. In this study, we investigated the effects of cyclic tensile strain, culture medium (with and without dexamethasone), and culture duration on the expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and interleukin-8 (IL-8) by bone marrow derived human mesenchymal stem cells (hMSCs). Human MSCs seeded in three-dimensional Type I collagen matrices were subjected to 0%, 10%, and 12% uniaxial cyclic tensile strains at 1 Hz for 4 h/day for 7 and 14 days in complete growth or dexamethasone-containing osteogenic medium. Viability of hMSCs was maintained irrespective of strain level and media conditions. Expression of either TNF-alpha or IL-1 beta was not observed in hMSCs under any of the conditions investigated in this study. Expression of IL-6 was dependent on culture medium. An increase in IL-6 expression was caused by both 10% and 12% strain levels. Both 10% and 12% strain levels caused an increase in IL-8 production by hMSCs that was dependent on the presence of dexamethasone. IL-6 and IL-8 expressions by hMSCs were induced by cyclic tensile strain and osteogenic differentiating media, indicating that IL-6 and IL-8 may be functioning as autocrine signals during osteogenic differentiation of hMSCs.

Composite tissue engineering on polycaprolactone nanofiber scaffolds.

Tissue engineering has largely focused on single tissue-type reconstruction (such as bone); however, the basic unit of healing in any clinically relevant scenario is a compound tissue type (such as bone, periosteum, and skin). Nanofibers are submicron fibrils that mimic the extracellular matrix, promoting cellular adhesion, proliferation, and migration. Stem cell manipulation on nanofiber scaffolds holds significant promise for future tissue engineering. This work represents our initial efforts to create the building blocks for composite tissue reflecting the basic unit of healing. Polycaprolactone (PCL) nanofibers were electrospun using standard techniques. Human foreskin fibroblasts, murine keratinocytes, and periosteal cells (4-mm punch biopsy) harvested from children undergoing palate repair were grown in appropriate media on PCL nanofibers. Human fat-derived mesenchymal stem cells were osteoinduced on PCL nanofibers. Cell growth was assessed with fluorescent viability staining; cocultured cells were differentiated using antibodies to fibroblast- and keratinocyte-specific surface markers. Osteoinduction was assessed with Alizarin red S. PCL nanofiber scaffolds supported robust growth of fibroblasts, keratinocytes, and periosteal cells. Cocultured periosteal cells (with fibroblasts) and keratinocytes showed improved longevity of the keratinocytes, though growth of these cell types was randomly distributed throughout the scaffold. Robust osteoinduction was noted on PCL nanofibers. Composite tissue engineering using PCL nanofiber scaffolds is possible, though the major obstacles to the trilaminar construct are maintaining an appropriate interface between the tissue types and neovascularization of the composite structure.

Industrial-scale fabrication of an osteogenic and antibacterial PLA/silver-loaded calcium phosphate composite with significantly reduced cytotoxicity.

In this study, we report an industrial-scale fabrication method of a multifunctional polymer composite as a scaffold material for bone tissue engineering. This study successfully demonstrated the potential of applying industrial polymer processing technologies to produce specially functionalized tissue engineering scaffolds. With the inclusion of a newly synthesized multifunctional additive, silver-doped-calcium phosphate (silver-CaP), the composite material exhibited excellent osteogenic inducibility of human adipose-derived stem cells (hASC) and satisfactory antibacterial efficacy against Escherichia coli and Staphylococcus aureus. Also, relative to previously reported methods of direct loading silver particles into polymeric materials, our composite exhibited significantly reduced silver associated cytotoxicity. The enhanced biocompatibility could be a significant advantage for materials to be used for regenerative medicine applications where clinical safety is a major consideration. The impact of different silver loading methodologies on hASC' osteogenic differentiation was also studied. Overall, the results of this study indicate a promising alternative approach to produce multifunctional scaffolds at industrial-scale with higher throughput, lower cost, and enhanced reproducibility. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 900-910, 2019.

Evidence for Aryl hydrocarbon Receptor-Mediated Inhibition of Osteoblast Differentiation in Human Mesenchymal Stem Cells.

Multipotent mesenchymal stem cells (MSCs) maintain the ability to differentiate into adipogenic, chondrogenic, or osteogenic cell lineages. There is increasing concern that exposure to environmental agents such as aryl hydrocarbon receptor (AhR) ligands, may perturb the osteogenic pathways responsible for normal bone formation. The objective of the current study was to evaluate the potential of the prototypic AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to disrupt osteogenic differentiation of human bone-derived MSCs (hBMSCs) in vitro. Primary hBMSCs from three donors were exposed to 10 nM TCDD and differentiation was interrogated using select histological, biochemical, and transcriptional markers of osteogenesis. Exposure to 10 nM TCDD resulted in an overall consistent attenuation of alkaline phosphatase (ALP) activity and matrix mineralization at terminal stages of differentiation in primary hBMSCs. At the transcriptional level, the transcriptional regulator DLX5 and additional osteogenic markers (ALP, OPN, and IBSP) displayed attenuated expression; conversely, FGF9 and FGF18 were consistently upregulated in each donor. Expression of stem cell potency markers SOX2, NANOG, and SALL4 decreased in the osteogenic controls, whereas expression in TCDD-treated cells resembled that of undifferentiated cells. Coexposure with the AhR antagonist GNF351 blocked TCDD-mediated attenuation of matrix mineralization, and either fully or partially rescued expression of genes associated with osteogenic regulation, extracellular matrix, and/or maintenance of multipotency. Thus, experimental evidence from this study suggests that AhR transactivation likely attenuates osteoblast differentiation in multipotent hBMSCs. This study also underscores the use of primary human MSCs to evaluate osteoinductive or osteotoxic potential of chemical and pharmacologic agents in vitro.

LRP receptors in chondrocytes are modulated by simulated microgravity and cyclic hydrostatic pressure.

Mechanical loading is essential for the maintenance of musculoskeletal homeostasis. Cartilage has been demonstrated to be highly mechanoresponsive, but the mechanisms by which chondrocytes respond to mechanical stimuli are not clearly understood. The goal of the study was to determine how LRP4, LRP5, and LRP6 within canonical Wnt-signaling are regulated in simulated microgravity and cyclic hydrostatic pressure, and to investigate the potential role of LRP 4/5/6 in cartilage degeneration. Rat chondrosacroma cell (RCS) pellets were stimulated using either cyclic hydrostatic pressure (1Hz, 7.5 MPa, 4hr/day) or simulated microgravity in a rotating wall vessel (RWV) bioreactor (11RPM, 24hr/day). LRP4/5/6 mRNA expression was assessed by RT-qPCR and LRP5 protein expression was determined by fluorescent immunostaining. To further evaluate our in vitro findings in vivo, mice were subjected to hindlimb suspension for 14 days and the femoral heads stained for LRP5 expression. We found that, in vitro, LRP4/5/6 mRNA expression is modulated in a time-dependent manner by mechanical stimulation. Additionally, LRP5 protein expression is upregulated in response to both simulated microgravity and cyclic hydrostatic pressure. LRP5 is also upregulated in vivo in the articular cartilage of hindlimb suspended mice. This is the first study to examine how LRP4/5/6, critical receptors within musculoskeletal biology, respond to mechanical stimulation. Further elucidation of this mechanism could provide significant clinical benefit for the identification of pharmaceutical targets for the maintenance of cartilage health.

Primary Cilia Exhibit Mechanosensitivity to Cyclic Tensile Strain and Lineage-Dependent Expression in Adipose-Derived Stem Cells.

Non-motile primary cilia are dynamic cellular sensory structures and are expressed in adipose-derived stem cells (ASCs). We have previously shown that primary cilia are involved in chemically-induced osteogenic differentiation of human ASC (hASCs) in vitro. Further, we have reported that 10% cyclic tensile strain (1 Hz, 4 hours/day) enhances hASC osteogenesis. We hypothesize that primary cilia respond to cyclic tensile strain in a lineage dependent manner and that their mechanosensitivity may regulate the dynamics of signaling pathways localized to the cilium. We found that hASC morphology, cilia length and cilia conformation varied in response to culture in complete growth, osteogenic differentiation, or adipogenic differentiation medium, with the longest cilia expressed in adipogenically differentiating cells. Further, we show that cyclic tensile strain both enhances osteogenic differentiation of hASCs while it suppresses adipogenic differentiation as evidenced by upregulation of RUNX2 gene expression and downregulation of PPARG and IGF-1, respectively. This study demonstrates that hASC primary cilia exhibit mechanosensitivity to cyclic tensile strain and lineage-dependent expression, which may in part regulate signaling pathways localized to the primary cilium during the differentiation process. We highlight the importance of the primary cilium structure in mechanosensing and lineage specification and surmise that this structure may be a novel target in manipulating hASC for in tissue engineering applications.

Finite element modeling of 3D human mesenchymal stem cell-seeded collagen matrices exposed to tensile strain.

The use of human mesenchymal stem cells (hMSCs) in tissue engineering is attractive due to their ability to extensively self-replicate and differentiate into a multitude of cell lineages. It has been experimentally established that hMSCs are influenced by chemical and mechanical signals. However, the combined chemical and mechanical in vitro culture conditions that lead to functional tissue require greater understanding. In this study, finite element models were created to evaluate the local loading conditions on bone marrow-derived hMSCs seeded in three-dimensional collagen matrices exposed to cyclic tensile strain. Mechanical property and geometry data used in the models were obtained experimentally from a previous study in our laboratory and from mechanical testing. Eight finite element models were created to simulate three-dimensional hMSC-seeded collagen matrices exposed to different levels of cyclic tensile strain (10% and 12%), culture media (complete growth and osteogenic differentiating), and durations of culture (7 and 14 days). Through finite element analysis, it was determined that globally applied uniaxial tensile strains of 10% and 12% resulted in local strains up to 18.3% and 21.8%, respectively. Model results were also compared to experimental studies in an attempt to explain observed differences between hMSC response to 10% and 12% cyclic tensile strain.

Cyclic tensile strain increases interactions between human epidermal keratinocytes and quantum dot nanoparticles.

The effects of quantum dots (QD) on cell viability have gained increasing interest due to many recent developments utilizing QD for pharmaceutical and biomedical applications. The potential use of QD nanoparticles as diagnostic, imaging, and drug delivery agents has raised questions about their potential for cytotoxicity. The objective of this study was to investigate the effects of applied strain on QD uptake by human epidermal keratinocytes (HEK). It was hypothesized that introduction of a 10% average strain to cell cultures would increase QD uptake. HEK were seeded at a density of 150,000 cells/mL on collagen-coated Flexcell culture plates (Flexcell Intl.). QD were introduced at a concentration of 3 nM and a 10% average strain was applied to the cells. After 4h of cyclic strain, the cells were examined for cell viability, QD uptake, and cytokine production. The results indicate that addition of strain results in an increase in cytokine production and QD uptake, resulting in irritation and a negative impact on cell viability. Application of physiological load conditions can increase cell membrane permeability, thereby increasing the concentration of QD nanoparticles in cells.

Cellular incorporation into electrospun nanofibers: retained viability, proliferation, and function in fibroblasts.

Nanofibers are an emerging scaffold for tissue engineering. To date no one has reported cell incorporation into nanofibers. Human foreskin fibroblasts and human adipose-derived adult stem cells (hADAS) were grown to confluence, resuspended in phosphate-buffered saline, and then solubilized in polyvinyl alcohol (PVA). Nanofibers were created using an electrospinning technique across an electric potential of 20 kV. Cell interaction with nanofibers was assessed with optical microscopic imaging and scanning electron microscopy. PVA nanofibers with incorporated cells were then solubilized in phosphate-buffered saline; cell viability was assessed by trypan blue exclusion. Viable cells were allowed to proliferate. Chondrogenesis in fibroblasts was induced with TGF-beta1. Both fibroblasts and hADAS survived the electrospinning process and were incorporated into PVA nanofibers. hADAS cell proliferation was negligible; however, fibroblasts proliferated and showed retained ability to undergo chondrogenesis. Cells can be incorporated into nanofibers, with maintained viability, proliferation, and function.