Cytoskeletal and Focal Adhesion Influences on Mesenchymal Stem Cell Shape, Mechanical Properties, and Differentiation Down Osteogenic, Adipogenic, and Chondrogenic Pathways

Mesenchymal stem cells (MSCs) hold great potential for regenerative medicine and tissue-engineering applications. They have multipotent differentiation capabilities and have been shown to differentiate down various lineages, including osteoblasts, adipocytes, chondrocytes, myocytes, and possibly neurons. The majority of approaches to control the MSC fate have been via the use of chemical factors in the form of growth factors within the culture medium. More recently, it has been understood that mechanical forces play a significant role in regulating MSC fate. We and others have shown that mechanical stimuli can control MSC lineage specification. The cytoskeleton is known to play a large role in mechanotransduction, and a growing number of studies are showing that it can also contribute to MSC differentiation. This review analyzes the significant contribution of actin and integrin distribution, and the smaller role of microtubules, in regulating MSC fate. Osteogenic differentiation is more prevalent in MSCs with a stiff, spread actin cytoskeleton and greater numbers of focal adhesions. Both adipogenic differentiation and chondrogenic differentiation are encouraged when MSCs have a spherical morphology associated with a dispersed actin cytoskeleton with few focal adhesions. Different mechanical stimuli can be implemented to alter these cytoskeletal patterns and encourage MSC differentiation to the desired lineage

Our Fat Future: Translating Adipose Stem Cell Therapy

Human adipose stem cells (hASCs) have the potential to treat patients with a variety of clinical conditions. Recent advancements in translational research, regulatory policy, and industry have positioned hASCs on the threshold of clinical translation. We discuss the progress and challenges of bringing adipose stem cell therapy into mainstream clinical use

Mechanical and Vascular Cues Synergistically Enhance Osteogenesis in Human Mesenchymal Stem Cells

Development and maintenance of a vascular network are critical for bone growth and homeostasis; strategies that promote vascular function are critical for clinical success of tissue-engineered bone constructs. Co-culture of endothelial cells (ECs) with mesenchymal stem cells (MSCs) and exposure to 10% cyclic tensile strain have both been shown to regulate osteogenesis in isolation, but potential synergistic effects have yet to be explored. The objective of this study was to expose an MSC-EC co-culture to 10% cyclic tensile strain to examine the role of this mechanical stimulus on MSC-EC behavior. We hypothesized that paracrine signaling from ECs would stimulate osteogenesis of MSCs, and exposure to 10% cyclic tensile strain would enhance this anabolic signal. Human umbilical vein ECs and human bone marrow-derived MSCs were either monocultured or co-cultured at a 1:1 ratio in a mixed osteo/angiogenic medium, exposed to 10% cyclic tensile strain at 1 Hz for 4 h/day for 2 weeks, and biochemically and histologically analyzed for endothelial and osteogenic markers. While neither 10% cyclic tensile strain nor co-culture alone had a significant effect on osteogenesis, the concurrent application of strain to an MSC-EC co-culture resulted in a significant increase in calcium accretion and mineral deposition, suggesting that co-culture and strain synergistically enhance osteogenesis. Neither co-culture, 10% cyclic tensile strain, nor a combination of these stimuli affected endothelial markers, indicating that the endothelial phenotype remained stable, but unresponsive to the stimuli evaluated in this study. This study is the first to investigate the role of cyclic tensile strain on the complex interplay between ECs and MSCs in co-culture. The results of this study provide key insights into the synergistic effects of 10% cyclic tensile strain and co-culture on osteogenesis. Understanding mechanobiological factors affecting MSC-EC crosstalk will help enhance strategies for creating vascularized tissues in tissue engineering and regenerative medicine

Evaluation of Silver Ion-Releasing Scaffolds in a 3D Coculture System of MRSA and Human Adipose-Derived Stem Cells for Their Potential Use in Treatment or Prevention of Osteomyelitis

Bone infection, also called osteomyelitis, can result when bacteria invade a bone. Treatment of osteomyelitis usually requires surgical debridement and prolonged antimicrobial therapy. The rising incidence of infection with multidrug-resistant bacteria, in particular methicillin-resistant staphylococcus aureus (MRSA), however, limits the antimicrobial treatment options available. Silver is well known for its antimicrobial properties and is highly toxic to a wide range of microorganisms. We previously reported our development of biocompatible, biodegradable, nanofibrous scaffolds that released silver ions in a controlled manner. The objective of this study was to determine the efficacy of these scaffolds in treating or preventing osteomyelitis. To achieve this objective, antimicrobial efficacy was determined using a 3D coculture system of human adipose-derived stem cells (hASC) and MRSA. Human ASC were seeded on the scaffolds and induced to undergo osteogenic differentiation in both the absence and presence of MRSA. Our results indicated that the silver ion-releasing scaffolds not only inhibited biofilm formation, but also supported osteogenesis of hASC. Our findings suggest that these biocompatible, degradable, silver ion-releasing scaffolds can be used at an infection site to treat osteomyelitis and/or to coat bone implants as a preventative measure against infection postsurgery

Skin Tissue Engineering for the Infected Wound Site: Biodegradable PLA Nanofibers and a Novel Approach for Silver Ion Release Evaluated in a 3D Coculture System of Keratinocytes and Staphylococcus aureus

Wound infection presents a challenging and growing problem. With the increased prevalence and growth of multidrug-resistant bacteria, there is a mounting need to reduce and eliminate wound infections using methodologies that limit the ability of bacteria to evolve into further drug-resistant strains. A well-known strategy for combating bacterial infection and preventing wound sepsis is through the delivery of silver ions to the wound site. High surface area silver nanoparticles (AgNPs) allowing extensive silver ion release have therefore been explored in different wound dressings and/or skin substitutes. However, it has been recently shown that AgNPs can penetrate into the stratum corneum of skin or diffuse into the cellular plasma membrane, and may interfere with a variety of cellular mechanisms. The goal of this study was to introduce and evaluate a new type of high surface area metallic silver in the form of highly porous silver microparticles (AgMPs). Polylactic acid (PLA) nanofibers were successfully loaded with either highly porous AgMPs or AgNPs and the antimicrobial efficacy and cytotoxicity of the two silver-based wound dressings were assessed and compared. To better mimic the physiological environment in vivo where both human cells and bacteria are present, a novel coculture system combining human epidermal keratinocytes and Staphylococcus aureus bacteria was designed to simultaneously evaluate human skin cell cytotoxicity with antimicrobial efficacy in a three-dimensional environment. We found that highly porous AgMPs could be successfully incorporated in nanofibrous wound dressings, and exhibited comparable antimicrobial efficacy and cytotoxicity to AgNPs. Further, PLA nanofibers containing highly porous AgMPs exhibited steady silver ion release, at a greater rate of release, than nanofibers containing AgNPs. The replacement of AgNPs with the newly introduced AgMPs overcomes concerns regarding the use of nanoparticles and holds great promise as skin substitutes or wound dressings for infected wound sites

Fabrication of novel high surface area mushroom gilled fibers and their effects on human adipose derived stem cells under pulsatile fluid flow for tissue engineering applications

Abstract The fabrication and characterization of novel high surface area hollow gilled fiber tissue engineering scaffolds via industrially relevant, scalable, repeatable, high speed, and economical nonwoven carding technology is described. Scaffolds were validated as tissue engineering scaffolds using human adipose derived stem cells (hASC) exposed to pulsatile fluid flow (PFF). The effects of fiber morphology on the proliferation and viability of hASC, as well as effects of varied magnitudes of shear stress applied via PFF on the expression of the early osteogenic gene marker runt related transcription factor 2 (RUNX2) were evaluated. Gilled fiber scaffolds led to a significant increase in proliferation of hASC after seven days in static culture, and exhibited fewer dead cells compared to pure PLA round fiber controls. Further, hASC-seeded scaffolds exposed to 3 and 6 dyn/cm resulted in significantly increased mRNA expression of RUNX2 after one hour of PFF in the absence of soluble osteogenic induction factors. This is the first study to describe a method for the fabrication of high surface area gilled fibers and scaffolds. The scalable manufacturing process and potential fabrication across multiple nonwoven and woven platforms makes them promising candidates for a variety of applications that require high surface area fibrous materials. Statement of Significance We report here for the first time the successful fabrication of novel high surface area gilled fiber scaffolds for tissue engineering applications. Gilled fibers led to a significant increase in proliferation of human adipose derived stem cells after one week in culture, and a greater number of viable cells compared to round fiber controls. Further, in the absence of osteogenic induction factors, gilled fibers led to significantly increased mRNA expression of an early marker for osteogenesis after exposure to pulsatile fluid flow. This is the first study to describe gilled fiber fabrication and their potential for tissue engineering applications. The repeatable, industrially scalable, and versatile fabrication process makes them promising candidates for a variety of scaffold-based tissue engineering applications. Graphical abstrac

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

Sensitization of Transforming Growth Factor-β 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 β (TGFβ) has been shown to be dependent on receptor clustering. By patterning a DNA nanostructure with closely spaced peptides that bind to TGFβ we observe increased sensitivity of NMuMG cells to TGFβ ligand. This is evidenced by translocation of secondary messenger proteins to the nucleus and stimulation of an inducible luciferase reporter at lower concentrations of TGFβ ligand. We believe this represents an important initial step towards realization of DNA as a self assembling and biologically compatible material for use in tissue engineering and drug delivery

Electrical Cell-Substrate Impedance Spectroscopy Can Monitor Age-Grouped Human Adipose Stem Cell Variability During Osteogenic Differentiation

: Human adipose stem cells (hASCs) are an attractive cell source for bone tissue engineering applications. However, a critical issue to be addressed before widespread hASC clinical translation is the dramatic variability in proliferative capacity and osteogenic potential among hASCs isolated from different donors. The goal of this study was to test our hypothesis that electrical cell-substrate impedance spectroscopy (ECIS) could track complex bioimpedance patterns of hASCs throughout proliferation and osteogenic differentiation to better understand and predict variability among hASC populations. Superlots composed of hASCs from young (aged 24-36 years), middle-aged (aged 48-55 years), and elderly (aged 60-81 years) donors were seeded on gold electrode arrays. Complex impedance measurements were taken throughout proliferation and osteogenic differentiation. During osteogenic differentiation, four impedance phases were identified: increase, primary stabilization, drop phase, and secondary stabilization. Matrix deposition was first observed 48-96 hours after the impedance maximum, indicating, for the first time, that ECIS can identify morphological changes that correspond to late-stage osteogenic differentiation. The impedance maximum was observed at day 10.0 in young, day 6.1 in middle-aged, and day 1.3 in elderly hASCs, suggesting that hASCs from younger donors require a longer time to differentiate than do hASCs from older donors, but young hASCs proliferated more and accreted more calcium long-term. This is the first study to use ECIS to predict osteogenic potential of multiple hASC populations and to show that donor age may temporally control onset of osteogenesis. These findings could be critical for development of patient-specific bone tissue engineering and regenerative medicine therapies. SIGNIFICANCE: Human adipose stem cells (hASCs) are an appealing cell source for bone tissue engineering and regenerative medicine applications because they can be obtained in high quantities via liposuction procedures and can differentiate down musculoskeletal lineages. However, a major barrier to clinical translation of hASCs is that cells from different donors have varying capacities to proliferate and differentiate. This study used electrical impedance spectroscopy to noninvasively track osteogenic differentiation of age-grouped donors in real time, showing that age-grouped hASCs have distinct complex impedance patterns. This method could be used to improve understanding of the biology that causes variability among hASC populations and to provide quantitative quality control standards for hASC populations in stem cell manufacturing and bone tissue engineering applications

Electrospun nanofibrous scaffolds increase the efficacy of stem cell-mediated therapy of surgically resected glioblastoma

Engineered stem cell (SC)-based therapy holds enormous promise for treating the incurable brain cancer glioblastoma (GBM). Retaining the cytotoxic SCs in the surgical cavity after GBM resection is one of the greatest challenges to this approach. Here, we describe a biocompatible electrospun nanofibrous scaffold (bENS) implant capable of delivering and retaining tumor-homing cytotoxic stem cells that suppress recurrence of post-surgical GBM. As a new approach to GBM therapy, we created poly(l-lactic acid) (PLA) bENS bearing drug-releasing human mesenchymal stem cells (hMSCs). We discovered that bENS-based implant increased hMSC retention in the surgical cavity 5-fold and prolonged persistence 3-fold compared to standard direct injection using our mouse model of GBM surgical resection/recurrence. Time-lapse imaging showed cytotoxic hMSC/bENS treatment killed co-cultured human GBM cells, and allowed hMSCs to rapidly migrate off the scaffolds as they homed to GBMs. In vivo, bENS loaded with hMSCs releasing the anti-tumor protein TRAIL (bENSsTR) reduced the volume of established GBM xenografts 3-fold. Mimicking clinical GBM patient therapy, lining the post-operative GBM surgical cavity with bENSsTR implants inhibited the re-growth of residual GBM foci 2.3-fold and prolonged post-surgical median survival from 13.5 to 31 days in mice. These results suggest that nanofibrous-based SC therapies could be an innovative new approach to improve the outcomes of patients suffering from terminal brain cancer