Impact of Suramin on Key Pathological Features of Sporadic Alzheimer's Disease-Derived Forebrain Neurons.

Background: Alzheimer's disease (AD) is characterized by disrupted proteostasis and macroautophagy (hereafter "autophagy"). The pharmacological agent suramin has known autophagy modulation properties with potential efficacy in mitigating AD neuronal pathology. Objective: In the present work, we investigate the impact of forebrain neuron exposure to suramin on the Akt/mTOR signaling pathway, a major regulator of autophagy, in comparison with rapamycin and chloroquine. We further investigate the effect of suramin on several AD-related biomarkers in sporadic AD (sAD)-derived forebrain neurons. Methods: Neurons differentiated from ReNcell neural progenitors were used to assess the impact of suramin on the Akt/mTOR signaling pathway relative to the autophagy inducer rapamycin and autophagy inhibitor chloroquine. Mature forebrain neurons were differentiated from induced pluripotent stem cells (iPSCs) sourced from a late-onset sAD patient and treated with 100µM suramin for 72 h, followed by assessments for amyloid-ß, phosphorylated tau, oxidative/nitrosative stress, and synaptic puncta density. Results: Suramin treatment of sAD-derived neurons partially ameliorated the increased p-Tau(S199)/Tau ratio, and fully remediated the increased glutathione to oxidized nitric oxide ratio, observed in untreated sAD-derived neurons relative to healthy controls. These positive results may be due in part to the distinct increases in Akt/mTOR pathway mediator p-p70S6K noted with suramin treatment of both ReNcell-derived and iPSC-derived neurons. Longer term neuronal markers, such as synaptic puncta density, were unaffected by suramin treatment. Conclusions: These findings provide initial evidence supporting the potential of suramin to reduce the degree of dysregulation in sAD-derived forebrain neurons in part via the modulation of autophagy.

Trends in bioactivity: inducing and detecting mineralization of regenerative polymeric scaffolds.

Due to limitations of biological and alloplastic grafts, regenerative engineering has emerged as a promising alternative to treat bone defects. Bioactive polymeric scaffolds are an integral part of such an approach. Bioactivity importantly induces hydroxyapatite mineralization that promotes osteoinductivity and osseointegration with surrounding bone tissue. Strategies to confer bioactivity to polymeric scaffolds utilize bioceramic fillers, coatings and surface treatments, and additives. These approaches can also favorably impact mechanical and degradation properties. A variety of fabrication methods are utilized to prepare scaffolds with requisite morphological features. The bioactivity of scaffolds may be evaluated with a broad set of techniques, including in vitro (acellular and cellular) and in vivo methods. Herein, we highlight contemporary and emerging approaches to prepare and assess scaffold bioactivity, as well as existing challenges.

Effects of Stromal Cell Conditioned Medium and Antipurinergic Treatment on Macrophage Phenotype.

The immunomodulatory capacity of the human mesenchymal stromal cell (MSC) secretome has been a critical driver for the development of cell-free MSC products, such as conditioned medium (CM), for regenerative medicine applications. This is particularly true as cell-free MSC products present several advantages over direct autologous or allogeneic MSC delivery with respect to safety, manufacturability, and defined potency. Recently, significant effort has been placed into creating novel MSC CM formulations with an immunomodulatory capacity tailored for specific regenerative contexts. For instance, the immunoregulatory nature of MSC CM has previously been tuned through a number of cytokine-priming strategies. Herein, we propose an alternate method to tailor the immunomodulatory "phenotype" of cytokine-primed MSC CM through coupling with the pharmacological agent, suramin. Suramin interferes with the signaling of purines including extracellular adenosine triphosphate (ATP), which plays a critical role in the activation of the innate immune system after injury. Toward this end, human THP-1-derived macrophages were activated to a proinflammatory phenotype and treated with (1) unprimed/native MSC CM, (2) interferon-γ/tumor necrosis factor α-primed MSC CM (primed CM), (3) suramin alone, or (4) primed MSC CM and suramin (primed CM/suramin). Markers of key macrophage functions-cytokine secretion, autophagy, oxidative stress modulation, and activation/migration-were assessed. Consistent with previous literature, primed CM elevated macrophage secretion of several proinflammatory and pleiotropic cytokines relative to native CM; whereas addition of suramin imparted consistent shifts in terms of TNFα (↓), interleukin-10 (↓), and hepatocyte growth factor (↑) irrespective of CM. In addition, both primed CM and suramin, individually and combined, increased reactive oxygen species production relative to native CM, and addition of suramin to primed CM shifted levels of CX3CL1, a factor involved in ATP-associated macrophage regulation. Varimax rotation assessment of the secreted cytokine profiles confirmed that primed CM/suramin resulted in a THP-1 phenotypic shift away from the lipopolysaccharide-activated proinflammatory state that was distinct from that of primed CM or native CM alone. This altered primed CM/suramin-associated phenotype may prove beneficial for healing in certain regenerative contexts. These results may inform future work coupling antipurinergic treatments with MSC-derived therapies in regenerative medicine applications.

Ultra-High Modulus Hydrogels Mimicking Cartilage of the Human Body.

The human body is comprised of numerous types of cartilage with a range of high moduli, despite their high hydration. Owing to the limitations of cartilage tissue healing and biological grafting procedures, synthetic replacements have emerged but are limited by poorly matched moduli. While conventional hydrogels can achieve similar hydration to cartilage tissues, their moduli are substantially inferior. Herein, triple network (TN) hydrogels are prepared to synergistically leverage intra-network electrostatic repulsive and hydrophobic interactions, as well as inter-network electrostatic attractive interactions. They are comprised of an anionic 1st network, a neutral 2nd network (capable of hydrophobic associations), and a cationic 3rd network. Collectively, these interactions act synergistically as effective, yet dynamic crosslinks. By tuning the concentration of the cationic 3rd network, these TN hydrogels achieve high moduli of ≈1.5 to ≈3.5 MPa without diminishing cartilage-like water contents (≈80%), strengths, or toughness values. This unprecedented combination of properties poises these TN hydrogels as cartilage substitutes in applications spanning articulating joints, intervertebral discs (IVDs), trachea, and temporomandibular joint disc (TMJ).

Endothelial colony forming cell rolling and adhesion supported by peptide-grafted hydrogels.

The aim of this study was to investigate the ability of peptides and peptide combinations to support circulating endothelial colony forming cell (ECFC) rolling and adhesion under shear flow, informing biomaterial design in moving toward rapid cardiovascular device endothelialization. ECFCs have high proliferative capability and can differentiate into endothelial cells, making them a promising cell source for endothelialization. Both single peptides and peptide combinations designed to target integrins α4ß1 and α5ß1 were coupled to poly(ethylene glycol) hydrogels, and their performance was evaluated by monitoring velocity patterns during the ECFC rolling process, in addition to firm adhesion (capture). Tether percentage and velocity fluctuation, a parameter newly defined here, were found to be valuable in assessing cell rolling velocity patterns and when used in combination were able to predict cell capture. REDV-containing peptides binding integrin α4ß1 have been previously shown to reduce ECFC rolling velocity but not to support firm adhesion. This study finds that the performance of REDV-containing peptides in facilitating ECFC dynamic adhesion and capture can be improved by combination with α5ß1 integrin-binding peptides, which support ECFC static adhesion. Moreover, when similar in length, the peptide combinations may have synergistic effects in capturing ECFCs. With matching lengths, the peptide combinations including CRRETAWAC(cyclic)+REDV, P_RGDS+KSSP_REDV, and P_RGDS+P_REDV showed high values in both tether percentage and velocity fluctuation and improvement in ECFC capture compared to the single peptides at the shear rate of 20 s-1. These newly identified peptide combinations have the potential to be used as vascular device coatings to recruit ECFCs. STATEMENT OF SIGNIFICANCE: Restoration of functional endothelium following placement of stents and vascular grafts is critical for maintaining long-term patency. Endothelial colony forming cells (ECFCs) circulating in blood flow are a valuable cell source for rapid endothelialization. Here we identify and test novel peptides and peptide combinations that can potentially be used as coatings for vascular devices to support rolling and capture of ECFCs from flow. In addition to the widely used assessment of final ECFC adhesion, we also recorded the rolling process to quantitatively evaluate the interaction between ECFCs and the peptides, obtaining detailed performance of the peptides and gaining insight into effective capture molecule design. Peptide combinations targeting both integrin α4ß1 and integrin α5ß1 showed the highest percentages of ECFC capture.

Prokaryotic Collagen-Like Proteins as Novel Biomaterials.

Collagens are the major structural component in animal extracellular matrices and are critical signaling molecules in various cell-matrix interactions. Its unique triple helical structure is enabled by tripeptide Gly-X-Y repeats. Understanding of sequence requirements for animal-derived collagen led to the discovery of prokaryotic collagen-like protein in the early 2000s. These prokaryotic collagen-like proteins are structurally similar to mammalian collagens in many ways. However, unlike the challenges associated with recombinant expression of mammalian collagens, these prokaryotic collagen-like proteins can be readily expressed in E. coli and are amenable to genetic modification. In this review article, we will first discuss the properties of mammalian collagen and provide a comparative analysis of mammalian collagen and prokaryotic collagen-like proteins. We will then review the use of prokaryotic collagen-like proteins to both study the biology of conventional collagen and develop a new biomaterial platform. Finally, we will describe the application of Scl2 protein, a streptococcal collagen-like protein, in thromboresistant coating for cardiovascular devices, scaffolds for bone regeneration, chronic wound dressing and matrices for cartilage regeneration.

Modeling Sympathetic Hyperactivity in Alzheimer's Related Bone Loss.

BACKGROUND: A significant subset of patients with Alzheimer's disease (AD) exhibit low bone mineral density and are therefore more fracture-prone, relative to their similarly aged neurotypical counterparts. In addition to chronic immune hyperactivity, behavioral dysregulation of effector peripheral sympathetic neurons-which densely innervate bone and potently modulate bone remodeling-is implicated in this pathological bone reformation. OBJECTIVE: Thus, there exists a pressing need for a robust in vitro model which allows interrogation of the paracrine interactions between the putative mediators of AD-related osteopenia: sympathetic neurons (SNs) and mesenchymal stem cells (MSCs). METHODS: Toward this end, activated SN-like PC12 cells and bone marrow derived MSCs were cultured in poly(ethylene glycol) diacrylate (PEGDA) hydrogels in the presence or absence of the AD-relevant inflammatory cytokine tumor necrosis factor alpha (TNF-α) under mono- and co-culture conditions. RESULTS: PC12s and MSCs exposed separately to TNF-α displayed increased expression of pro-inflammatory mediators and decreased osteopontin (OPN), respectively. These data indicate that TNF-α was capable of inducing a dysregulated state in both cell types consistent with AD. Co-culture of TNF-α-activated PC12s and MSCs further exacerbated pathological behaviors in both cell types. Specifically, PC12s displayed increased secretion of interleukin 6 relative to TNF-α stimulated monoculture controls. Similarly, MSCs demonstrated a further reduction in osteogenic capacity relative to TNF-α stimulated monoculture controls, as illustrated by a significant decrease in OPN and collagen type I alpha I chain. CONCLUSION: Taken together, these data may indicate that dysregulated sympathetic activity may contribute to AD-related bone loss.

Cannabidiol-Driven Alterations to Inflammatory Protein Landscape of Lipopolysaccharide-Activated Macrophages In Vitro May Be Mediated by Autophagy and Oxidative Stress.

Background: The nonpsychotropic phytocannabinoid cannabidiol (CBD) presents itself as a potentially safe and effective anti-inflammatory treatment relative to clinical standards. In this present study, we compare the capacity of CBD to the corticosteroid dexamethasone (Dex) in altering the secreted protein landscape of activated macrophages and speculate upon the mechanism underpinning these alterations. Materials and Methods: Human THP-1 monocytes were differentiated into macrophages (THP-1 derived macrophages [tMACs]), activated with lipopolysaccharide (LPS), and then treated with 5, 10, 25, 50, or 100 µM CBD or 10 µM Dex for 24 h. Following treatment, cytotoxicity of CBD and protein expression levels from culture supernatants and from whole cell lysates were assessed for secreted and intracellular proteins, respectively. Results: High concentration (50 and 100 µM) CBD treatments exhibit a cytotoxic effect on LPS-activated tMACs following the 24-h treatment. Relative to the LPS-activated and untreated control (M[LPS]), both 25 µM CBD and 10 µM Dex reduced expression of pro-inflammatory markers-tumor necrosis factor alpha, interleukin 1 beta, and regulated on activation, normal T cell expressed and secreted (RANTES)-as well as the pleiotropic marker interleukin-6 (IL-6). A similar trend was observed for anti-inflammatory markers interleukin-10 and vascular endothelial growth factor (VEGF). Dex further reduced secreted levels of monocyte chemoattractant protein-1 in addition to suppressing IL-6 and VEGF beyond treatments with CBD. The anti-inflammatory capacity of 25 µM CBD was concurrent with reduction in levels of phosphorylated mammalian target of rapamycin Ser 2448, endothelial nitric oxide synthase, and induction of cyclooxygenase 2 relative to M(LPS). This could suggest that the observed effects on macrophage immune profile may be conferred through inhibition of mammalian target of rapamycin complex 1 and ensuing induction of autophagy. Conclusion: Cumulatively, these data demonstrate cytotoxicity of high concentration CBD treatment. The data reported herein largely agree with other literature demonstrating the anti-inflammatory effects of CBD. However, there is discrepancy within literature surrounding efficacious concentrations and effects of CBD on specific secreted proteins. These data expand upon previous work investigating the effects of CBD on inflammatory protein expression in macrophages, as well as provide insight into the mechanism by which these effects are conferred.

Intrinsic osteoinductivity of PCL-DA/PLLA semi-IPN shape memory polymer scaffolds.

Engineering osteoinductive, self-fitting scaffolds offers a potential treatment modality to repair irregularly shaped craniomaxillofacial bone defects. Recently, we innovated on osteoinductive poly(ε-caprolactone)-diacrylate (PCL-DA) shape memory polymers (SMPs) to incorporate poly-L-lactic acid (PLLA) into the PCL-DA network, forming a semi-interpenetrating network (semi-IPN). Scaffolds formed from these PCL-DA/PLLA semi-IPNs display stiffnesses within the range of trabecular bone and accelerated degradation relative to scaffolds formed from slowly degrading PCL-DA SMPs. Herein, we demonstrate for the first time that PCL-DA/PLLA semi-IPN SMP scaffolds show increased intrinsic osteoinductivity relative to PCL-DA. We also confirm that application of a bioinspired polydopamine (PD) coating further improves the osteoinductive capacity of these PCL-DA/PLLA semi-IPN SMPs. In the absence of osteogenic supplements, protein level assessment of human mesenchymal stem cells (h-MSCs) cultured in PCL-DA/PLLA scaffolds revealed an increase in expression of osteogenic markers osterix, bone morphogenetic protein-4 (BMP-4), and collagen 1 alpha 1 (COL1A1), relative to PCL-DA scaffolds and osteogenic medium controls. Likewise, the expression of runt-related transcription factor 2 (RUNX2) and BMP-4 was elevated in the presence of PD-coating. In contrast, the chondrogenic and adipogenic responses associated with the scaffolds matched or were reduced relative to osteogenic medium controls, indicating that the scaffolds display intrinsic osteoinductivity.

The three NADH dehydrogenases of Pseudomonas aeruginosa: Their roles in energy metabolism and links to virulence.

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen which relies on a highly adaptable metabolism to achieve broad pathogenesis. In one example of this flexibility, to catalyze the NADH:quinone oxidoreductase step of the respiratory chain, P. aeruginosa has three different enzymes: NUO, NQR and NDH2, all of which carry out the same redox function but have different energy conservation and ion transport properties. In order to better understand the roles of these enzymes, we constructed two series of mutants: (i) three single deletion mutants, each of which lacks one NADH dehydrogenase and (ii) three double deletion mutants, each of which retains only one of the three enzymes. All of the mutants grew approximately as well as wild type, when tested in rich and minimal medium and in a range of pH and [Na+] conditions, except that the strain with only NUO (ΔnqrFΔndh) has an extended lag phase. During exponential phase, the NADH dehydrogenases contribute to total wild-type activity in the following order: NQR > NDH2 > NUO. Some mutants, including the strain without NQR (ΔnqrF) had increased biofilm formation, pyocyanin production, and killed more efficiently in both macrophage and mouse infection models. Consistent with this, ΔnqrF showed increased transcription of genes involved in pyocyanin production.

Hyperosmolar Ionic Solutions Modulate Inflammatory Phenotype and sGAG Loss in a Cartilage Explant Model.

OBJECTIVE: The objective of this study was to compare the effects of hyperosmolar sodium (Na+), lithium (Li+) and potassium (K+) on catabolic and inflammatory osteoarthritis (OA) markers and sulfated glycosaminoglycan (sGAG) loss in TNF-α-stimulated cartilage explants. METHODS: Explants from bovine stifle joints were stimulated with TNF-α for 1 day to induce cartilage degradation followed by supplementation with 50 mM potassium chloride (KCl), 50 mM lithium chloride (LiCl), 50 mM sodium chloride (NaCl), or 100 nM dexamethasone for an additional 6 days. We assessed the effect of TNF-α stimulation and hyperosmolar ionic treatment on sGAG loss and expression of OA-associated proteins: ADAMTS-5, COX-2, MMP-1, MMP-13, and VEGF. RESULTS: TNF-α treatment increased sGAG loss (P < 0.001) and expression of COX-2 (P = 0.018), MMP-13 (P < 0.001), and VEGF (P = 0.017) relative to unstimulated controls. Relative to activated controls, LiCl and dexamethasone treatment attenuated sGAG loss (P = 0.008 and P = 0.042, respectively) and expression of MMP-13 (P = 0.005 and P = 0.036, respectively). In contrast, KCl treatment exacerbated sGAG loss (P = 0.032) and MMP-1 protein expression (P = 0.010). NaCl treatment, however, did not alter sGAG loss or expression of OA-related proteins. Comparing LiCl and KCl treatment shows a potent reduction (P < 0.05) in catabolic and inflammatory mediators following LiCl treatment. CONCLUSION: These results suggest that these ionic species elicit varying responses in TNF-α-stimulated explants. Cumulatively, these findings support additional studies of hyperosmolar ionic solutions for potential development of novel intraarticular injections targeting OA.

The Role of Chronic Inflammatory Bone and Joint Disorders in the Pathogenesis and Progression of Alzheimer's Disease.

Late-onset Alzheimer's Disease (LOAD) is a devastating neurodegenerative disorder that causes significant cognitive debilitation in tens of millions of patients worldwide. Throughout disease progression, abnormal secretase activity results in the aberrant cleavage and subsequent aggregation of neurotoxic Aß plaques in the cerebral extracellular space and hyperphosphorylation and destabilization of structural tau proteins surrounding neuronal microtubules. Both pathologies ultimately incite the propagation of a disease-associated subset of microglia-the principle immune cells of the brain-characterized by preferentially pro-inflammatory cytokine secretion and inhibited AD substrate uptake capacity, which further contribute to neuronal degeneration. For decades, chronic neuroinflammation has been identified as one of the cardinal pathophysiological driving features of AD; however, despite a number of works postulating the underlying mechanisms of inflammation-mediated neurodegeneration, its pathogenesis and relation to the inception of cognitive impairment remain obscure. Moreover, the limited clinical success of treatments targeting specific pathological features in the central nervous system (CNS) illustrates the need to investigate alternative, more holistic approaches for ameliorating AD outcomes. Accumulating evidence suggests significant interplay between peripheral immune activity and blood-brain barrier permeability, microglial activation and proliferation, and AD-related cognitive decline. In this work, we review a narrow but significant subset of chronic peripheral inflammatory conditions, describe how these pathologies are associated with the preponderance of neuroinflammation, and posit that we may exploit peripheral immune processes to design interventional, preventative therapies for LOAD. We then provide a comprehensive overview of notable treatment paradigms that have demonstrated considerable merit toward treating these disorders.

Enhanced Osteogenic Potential of Phosphonated-Siloxane Hydrogel Scaffolds.

In a material-guided approach, instructive scaffolds that leverage potent chemistries may efficiently promote bone regeneration. A siloxane macromer has been previously shown to impart osteoinductivity and bioactivity when included in poly(ethylene glycol) diacrylate (PEG-DA) hydrogel scaffolds. Herein, phosphonated-siloxane macromers were evaluated for enhancing the osteogenic potential of siloxane-containing PEG-DA scaffolds. Two macromers were prepared with different phosphonate pendant group concentrations, poly(diethyl(2-(propylthio)ethyl)phosphonate methylsiloxane) diacrylate (PPMS-DA) and 25%-phosphonated analogue (PPMS-DA 25%). Macroporous, templated scaffolds were prepared by cross-linking these macromers with PEG-DA at varying mol % (15:85, 30:70, and 45:55 PPMS-DA to PEG-DA; 30:70 PPMS-DA 25% to PEG-DA). Other scaffolds were also prepared by combining PEG-DA with PDMS-MA (i.e., no phosphonate) or with vinyl phosphonate (i.e., no siloxane). Scaffold material properties were thoroughly assessed, including pore morphology, hydrophobicity, swelling, modulus, and bioactivity. Scaffolds were cultured with human bone marrow-derived mesenchymal stem cells (normal media) and calcium deposition and protein expression were assessed at 14 and 28 days.

Assessment of Enrichment of Human Mesenchymal Stem Cells Based on Plasma and Mitochondrial Membrane Potentials.

Background: Human mesenchymal stem cells (hMSCs) are utilized preclinically and clinically as a candidate cell therapy for a wide range of inflammatory and degenerative diseases. Despite promising results in early clinical trials, consistent outcomes with hMSC-based therapies have proven elusive in many of these applications. In this work, we attempt to address this limitation through the design of a stem cell therapy to enrich hMSCs for desired electrical and ionic properties with enhanced stemness and immunomodulatory/regenerative capacity. Materials and Methods: In this study, we sought to develop initial protocols to achieve electrically enriched hMSCs (EE-hMSCs) with distinct electrical states and assess the potential relationship with respect to hMSC state and function. We sorted hMSCs based on fluorescence intensity of tetramethylrhodamine ethyl ester (TMRE) and investigated phenotypic differences between the sorted populations. Results: Subpopulations of EE-hMSCs exhibit differential expression of genes associated with senescence, stemness, immunomodulation, and autophagy. EE-hMSCs with low levels of TMRE, indicative of depolarized membrane potential, have reduced mRNA expression of senescence-associated markers, and increased mRNA expression of autophagy and immunomodulatory markers relative to EE-hMSCs with high levels of TMRE (hyperpolarized). Conclusions : This work suggests that the utilization of EE-hMSCs may provide a novel strategy for cell therapies, enabling live cell enrichment for distinct phenotypes that can be exploited for different therapeutic outcomes.

Interferon-Gamma Stimulated Murine Macrophages In Vitro: Impact of Ionic Composition and Osmolarity and Therapeutic Implications.

Background: Injections of osmolytes are promising immunomodulatory treatments for medical benefit, although the rationale and underlying mechanisms are often lacking. The goals of the present study were twofold: (1) to clarify the anti-inflammatory role of the potassium ion and (2) to begin to decouple the effects that ionic strength, ionic species, and osmolarity have on macrophage biology. Materials and Methods: RAW 264.7 murine macrophages were encapsulated in three-dimensional, poly(ethylene glycol) diacrylate hydrogels and activated with interferon-gamma to yield M(IFN). Gene and protein profiles were made of M(IFN) exposed to different hyperosmolar treatments (80 mM potassium gluconate, 80 mM sodium gluconate, and 160 mM sucrose). Results: Relative to M(IFN), all hyperosmolar treatments suppressed expression of pro-inflammatory markers (nitric oxide synthase-2 [NOS-2], tumor necrosis factor-alpha, monocyte chemoattractant protein-1 [MCP-1]) and increased messenger RNA (mRNA) expression of the pleiotropic and angiogenic markers interleukin-6 (IL-6) and vascular endothelial growth factor-A (VEGF), respectively. Ionic osmolytes also demonstrated a greater level of change compared to the nonionic treatments, with mRNA levels of IL-6 the most significantly affected. M(IFN) exposed to K+ exhibited the lowest levels of NOS-2 and MCP-1, and this ion limited IL-6 release induced by osmolarity. Conclusion: Cumulatively, these data suggest that osmolyte composition, ionic strength, and osmolarity are all parameters that can influence therapeutic outcomes. Future work is necessary to further decouple and mechanistically understand the influence that these biophysical parameters have on cell biology, including their impact on other macrophage functions, intracellular osmolyte composition, and cellular and organellular membrane potentials.

Dual Bioelectrical Assessment of Human Mesenchymal Stem Cells Using Plasma and Mitochondrial Membrane Potentiometric Probes.

Background: Bioelectrical properties are known to impact stem cell fate, state, and function. However, assays that measure bioelectrical properties are generally limited to the plasma membrane potential. In this study, we propose an assay to simultaneously assess cell plasma membrane and mitochondrial membrane potentials. Materials and Methods: Mesenchymal stem cell (MSC) plasma and mitochondrial membrane potentials were measured using flow cytometry and a combination of tetramethylrhodamine, methyl ester (TMRM), and bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC) dyes. We investigated the shifts in the bioelectrical phenotype of MSCs due to extended culture in vitro, activation with interferon-gamma (IFN-γ), and aggregate conditions. Results: MSCs subjected to extended culture in vitro acquired plasma and mitochondrial membrane potentials consistent with a hyperpolarized bioelectrical phenotype. Activation with IFN-γ shifted MSCs toward a state associated with increased levels of both DiBAC and TMRM. MSCs in aggregate conditions were associated with a decrease in TMRM levels, indicating mitochondrial depolarization. Conclusions: Our proposed assay described distinct MSC bioelectrical transitions due to extended in vitro culture, exposure to an inflammatory cytokine, and culture under aggregate conditions. Overall, our assay enables a more complete characterization of MSC bioelectrical properties within a single experiment, and its relative simplicity enables researchers to apply it in variety of settings.

Incorporation of a silicon-based polymer to PEG-DA templated hydrogel scaffolds for bioactivity and osteoinductivity.

A scaffold that is inherently bioactive, osteoinductive and osteoconductive may guide mesenchymal stem cells (MSCs) to regenerate bone tissue in the absence of exogenous growth factors. Previously, we established that hydrogel scaffolds formed by crosslinking methacrylated star poly(dimethylsiloxane) (PDMSstar-MA) with diacrylated poly(ethylene glycol) (PEG-DA) promote bone bonding by induction of hydroxyapatite formation ("bioactive") and promote MSC lineage progression toward osteoblast-like fate ("osteoinductive"). Herein, we have combined solvent induced phase separation (SIPS) with a fused salt template to create PDMSstar-PEG hydrogel scaffolds with controlled PDMSstar-MA distribution as well as interconnected macropores of a tunable size to allow for subsequent cell seeding and neotissue infiltration ("osteoconductive"). Scaffolds were prepared with PDMSstar-MA of two number average molecular weights (Mns) (2k and 7k) with varying PDMSstar-MA:PEG-DA ratios and template salt sizes. The distribution of PDMSstar-MA within the hydrogels was examined as well as pore size, percent interconnectivity, dynamic and static moduli, hydration, degradation and in vitro bioactivity (i.e. mineralization when exposed to simulated body fluid, SBF). Finally, cell culture with seeded human bone marrow-derived MSCs (hBMSCs) was used to confirm non-cytotoxicity and characterize osteoinductivity. Tunable, interconnected macropores were achieved by utilization of a fused salt template of a specified salt size during fabrication. Distribution of PDMSstar-MA within the PEG-DA matrix improved for the lower Mn and contributed to differences in specific material properties (e.g. local modulus) and cellular response. However, all templated SIPS PDMSstar-PEG hydrogels were confirmed to be bioactive, non-cytotoxic and displayed PDMSstar-MA dose-dependent osteogenesis. STATEMENT OF SIGNIFICANCE: A tissue engineering scaffold that can inherently guide mesenchymal stem cells (MSCs) to regenerate bone tissue without growth factors would be a more cost-effective and safe strategy for bone repair. Typically, glass/ceramic fillers are utilized to achieve this through their ability to induce hydroxyapatite formation ("bioactive") and promote MSC differentiation to an osteoblast-like fate ("osteoinductive"). Herein, we have fabricated an interconnected, macroporous PEG-DA hydrogel scaffold that utilizes PDMSstar-MA as a bioactive and osteoinductive scaffold component. We were able to show that these PDMSstar-PEG hydrogels maintain several key material characteristics for bone repair. Further, bioactivity and osteoinductivity were simultaneously achieved in human bone marrow-derived MSC culture, representing a notable achievement for an exclusively material-based strategy.

Elucidating the role of graft compliance mismatch on intimal hyperplasia using an ex vivo organ culture model.

There is a growing clinical need to address high failure rates of small diameter (<6 mm) synthetic vascular grafts. Although there is a strong empirical correlation between low patency rates and low compliance of synthetic grafts, the mechanism by which compliance mismatch leads to intimal hyperplasia is poorly understood. To elucidate this relationship, synthetic vascular grafts were fabricated that varied compliance independent of other graft variables. A computational model was then used to estimate changes in fluid flow and wall shear stress as a function of graft compliance. The effect of compliance on arterial remodeling in an ex vivo organ culture model was then examined to identify early markers of intimal hyperplasia. The computational model prediction of low wall shear stress of low compliance grafts and clinical control correlated well with alterations in arterial smooth muscle cell marker, extracellular matrix, and inflammatory marker staining patterns at the distal anastomoses. Conversely, high compliance grafts displayed minimal changes in fluid flow and arterial remodeling, similar to the sham control. Overall, this work supports the intrinsic link between compliance mismatch and intimal hyperplasia and highlights the utility of this ex vivo organ culture model for rapid screening of small diameter vascular grafts. STATEMENT OF SIGNIFICANCE: We present an ex vivo organ culture model as a means to screen vascular grafts for early markers of intimal hyperplasia, a leading cause of small diameter vascular graft failure. Furthermore, a computational model was used to predict the effect of graft compliance on wall shear stress and then correlate these values to changes in arterial remodeling in the organ culture model. Combined, the ex vivo bioreactor system and computational model provide insight into the mechanistic relationship between graft-arterial compliance mismatch and the onset of intimal hyperplasia.

Refined assessment of the impact of cell shape on human mesenchymal stem cell differentiation in 3D contexts.

Numerous studies have demonstrated that the differentiation potential of human mesenchymal stem cells (hMSCs) can be modulated by chemical and physical cues. In 2D contexts, inducing different cell morphologies, by varying the shape, area and/or curvature of adhesive islands on patterned surfaces, has significant effects on hMSC multipotency and the onset of differentiation. In contrast, in vitro studies in 3D contexts have suggested that hMSC differentiation does not directly correlate with cell shape. However, in 3D, the effects of cell morphology on hMSC differentiation have not yet been clearly established due to the chemical and physical properties being intertwined in 3D matrices. In this work, we studied the effects of round or elongated cell morphologies on hMSC differentiation independently of scaffold composition, modulus, crosslink density and cell-mediated matrix remodeling. The effects of cell shape on hMSC lineage progression were studied using three different cell culture media compositions and two values of scaffold rigidity. Differences in cell shape were achieved using interpenetrating polymer networks (IPNs). The mechanical and diffusional properties of the scaffolds and cell-matrix interactions were characterized. In addition, cell responses were evaluated in terms of cell spreading via gene and protein expression of differentiation markers. Cumulative results support, and extend upon previous work indicating that cell shape alone in 3D contexts does not significantly modulate hMSC differentiation, at least for the scaffold chemistry, range of modulus and culture conditions explored in this study. STATEMENT OF SIGNIFICANCE: In 2D contexts, inducing different cell shapes, by varying the curvature, area size and shape of a patterned surface, has significant effects on hMSC multipotency and the onset of cell differentiation. In contrast, in vitro studies in 3D contexts have suggested that hMSC differentiation does not directly correlate with cell shape. However, in 3D, the effects of cell morphology on hMSC differentiation have not yet been clearly established due to the chemical and physical properties being intertwined in 3D matrices. In this work, we studied the effects of round or elongated cell morphologies on the differentiation of hMSCs independently of scaffold composition, modulus, crosslink density and cell mediated matrix remodeling. Cumulative results support, and extend upon previous work indicating that cell shape alone in 3D contexts does not significantly modulate hMSCs differentiation commitment.

Elucidation of Endothelial Cell Hemostatic Regulation with Integrin-Targeting Hydrogels.

Despite advances in the development of materials for cardiovascular devices, current strategies generally lack the thromboresistance of the native endothelium both in terms of efficacy and longevity. To harness this innate hemostatic regulation and improve long-term hemocompatibility, biohybrid devices are designed to promote endothelialization. Much of the research effort to date has focused on the use of extracellular matrix (ECM)-mimics and coatings to promote endothelial cell adhesion and migration with less attention given to the effect of the supported ECM binding events on hemostatic regulation. In this study, we developed integrin-targeted hydrogels to investigate the individual and combined effects of integrin binding events supported by many ECM-based coatings (α1ß1, α2ß1, α5ß1, αvß3). Targeted endothelial cell integrin interactions were first confirmed with antibody blocking studies and then correlated with gene expression of hemostatic regulators and a functional assay of platelet attachment and activation. Surfaces that targeted integrins α1ß1 and α2ß1 resulted in an endothelial cell layer that exhibited a thromboresistant phenotype with an associated reduction in platelet attachment and activation. It is anticipated that identification of specific integrins that promote endothelial cell adhesion as well as thromboresistance will enable the design of cardiovascular materials with improved long-term hemocompatibility.