Hypoxic pre-conditioning increases the infiltration of endothelial cells into scaffolds for dermal regeneration pre-seeded with mesenchymal stem cells.

Many therapies using mesenchymal stem cells (MSC) rely on their ability to produce and release paracrine signals with chemotactic and pro-angiogenic activity. These characteristics, however, are mostly studied under standard in vitro culture conditions. In contrast, various novel cell-based therapies imply pre-seeding MSC into bio-artificial scaffolds. Here we describe human bone marrow-derived MSC seeded in Integra matrices, a common type of scaffold for dermal regeneration (SDR). We show and measured the distribution of MSC within the SDR, where cells clearly establish physical interactions with the scaffold, exhibiting constant metabolic activity for at least 15 days. In the SDR, MSC secrete VEGF and SDF-1α and induce transwell migration of CD34(+) hematopoietic/endothelial progenitor cells, which is inhibited in the presence of a CXCR4/SDF-1α antagonist. MSC in SDR respond to hypoxia by altering levels of angiogenic signals such as Angiogenin, Serpin-1, uPA, and IL-8. Finally, we show that MSC-containing SDR that have been pre-incubated in hypoxia show higher infiltration of endothelial cells after implantation into immune deficient mice. Our data show that MSC are fully functional ex vivo when implanted into SDR. In addition, our results strongly support the notion of hypoxic pre-conditioning MSC-containing SDR, in order to promote angiogenesis in the wounds.

Diabetic foot ulcer: an evidence-based treatment update.

BACKGROUND: Diabetic foot ulcers (DFUs) are extremely debilitating and difficult to treat. Multidisciplinary management, patient education, glucose control, debridement, offloading, infection control, and adequate perfusion are the mainstays of standard care endorsed by most practice guidelines. Adjunctive therapies represent new treatment modalities endorsed in recent years, though many lack significant high-powered studies to support their use as standard of care. OBJECTIVE: This update intends to identify recent, exclusively high level, evidence-based evaluations of DFU therapies. Furthermore, it suggests a direction for future research. METHODS: PubMed, Embase, Ovid Technologies, CINAHL, Cochrane, and Web of Science databases were systematically searched for recent systematic reviews published after 2004, and randomized controlled trials published in 2012-2013 that evaluated treatment modalities for DFUs. These papers are reviewed and the quality of available evidence is discussed. RESULTS: A total of 34 studies met inclusion criteria. Studied therapies include debridement, off-loading, negative pressure therapy, dressings, topical therapies, hyperbaric oxygen therapy, growth factors, bioengineered skin substitutes, electrophysical therapy, and alternative therapy. Good-quality evidence is lacking to justify the use of many of these therapies, with the exception of standard care (offloading, debridement) and possibly negative pressure wound therapy. LIMITATIONS: There is an overall lack of high-level evidence in new adjunctive management of DFU. Comparison of different treatment modalities is difficult, since existing studies are not standardized. CONCLUSIONS: Many therapeutic modalities are available to treat DFU. Quality high-level evidence exists for standard care such as off-loading. Evidence for adjunctive therapies such as negative pressure wound therapy, skin substitutes, and platelet-derived growth factor can help guide adjunctive care but limitations exist in terms of evidence quality.

Crosstalk between adrenergic and toll-like receptors in human mesenchymal stem cells and keratinocytes: a recipe for impaired wound healing.

Previous studies demonstrate that skin wounds generate epinephrine (EPI) that can activate local adrenergic receptors (ARs), impairing healing. Bacterially derived activators of Toll-like receptors (TLRs) within the wound initiate inflammatory responses and can also impair healing. In this study, we examined the hypothesis that these two pathways crosstalk to one another, using EPI and macrophage-activating lipopeptide-2 (MALP2) to activate ARs and TLR2, respectively, in human bone marrow-derived mesenchymal stem cells (BM-MSCs) and neonatal keratinocytes (NHKs). BM-MSCs exposed to EPI significantly (p < .05) increased TLR2 message (sevenfold BM-MSCs), TLR2 protein (twofold), and myeloid differentiation factor 88 (MyD88) (fourfold). Conversely, activation of TLR2 by MALP2 in these cells increased ß2-AR message (twofold in BM-MSCs, 2.7-fold in NHKs), ß2-AR protein (2.5-fold), phosphorylation of ß-AR-activated kinase (p-BARK, twofold), and induced release of EPI from both cell types (twofold). Treating cells with EPI and MALP2 together, as would be encountered in a wound, increased ß2-AR and p-BARK protein expression (sixfold), impaired cell migration (BM-MSCs- 21%↓ and NHKs- 60%↓, p < .002), and resulted in a 10-fold (BM-MSCs) and 51-fold (NHKs) increase in release of IL-6 (p < .001) responses that were remarkably reduced by pretreatment with ß2-AR antagonists. In vivo, EPI-stressed animals exhibited impaired healing, with elevated levels of TLR2, MyD88, and IL-6 in the wounds (p < .05) relative to nonstressed controls. Thus, our data describe a recipe for decreasing cell migration and exacerbating inflammation via novel crosstalk between the adrenergic and Toll-like receptor pathways in BM-MSCs and NHKs.

Galectin-3 regulates intracellular trafficking of EGFR through Alix and promotes keratinocyte migration.

The EGFR-mediated signaling pathways are important in a variety of cellular processes, including cell migration and wound re-epithelialization. Intracellular trafficking of EGFR is critical for maintaining EGFR surface expression. Galectin-3, a member of an animal lectin family, has been implicated in a number of physiological and pathological processes. Through studies of galectin-3-deficient mice and cells isolated from these mice, we demonstrated that the absence of galectin-3 impairs keratinocyte migration and skin wound re-epithelialization. We have linked this pro-migratory function to a crucial role of cytosolic galectin-3 in controlling intracellular trafficking and cell surface expression of EGFR after EGF stimulation. Without galectin-3, the surface levels of EGFR are markedly reduced, and the receptor accumulates diffusely in the cytoplasm. This is associated with reduced rates of both endocytosis and recycling of the receptor. We have provided evidence that this previously unreported function of galectin-3 may be mediated through interaction with its binding partner Alix, which is a protein component of the ESCRT (endosomal sorting complex required for transport) machinery. Our results suggest that galectin-3 is potentially a critical regulator of a number of important cellular responses through its intracellular control of trafficking of cell surface receptors.

Toll-like receptors and diabetes: a therapeutic perspective.

Diabetes is a mutifactorial metabolic disorder that leads to a number of complications. Diabetes is estimated to affect 36 million people in the U.S.A., and the prevalence of diagnosed and undiagnosed diabetes is at 9.3% and continues to rise. Evidence from experimental animal models as well as humans has indicated that systemic inflammation plays a role in the pathophysiological processes of diabetes and is facilitated by innate immune responses. TLRs (Toll-like receptors) are key innate immune receptors that recognize conserved PAMPs (pathogen-associated molecular patterns), induce inflammatory responses essential for host defences and initiate an adaptive immune response. Although TLR expression is increased in a plethora of inflammatory disorders, the effects of metabolic aberrations on TLRs and their role in diabetes and its complications is still emerging. In the present paper, we provide a systematic review on how TLRs play a detrimental role in the pathogenic processes [increased blood sugar, NEFAs (non-esterified 'free' fatty acids), cytokines and ROS (reactive oxygen species)] that manifest diabetes. Furthermore, we will highlight some of the therapeutic strategies targeted at decreasing TLRs to abrogate inflammation in diabetes that may eventually result in decreased complications.

Label-retaining cells of the bladder: candidate urothelial stem cells.

Adult tissue stem cells replicate infrequently, retaining DNA nucleotide label (BrdU) for much longer periods than mature, dividing cells in which the label is diluted during a chase period. Those "label-retaining cells" (LRCs) have been identified as the tissue stem cells in skin, cornea, intestine, and prostate. However, in the urinary tract uroepithelial stem cells have not yet been identified. In this study, BrdU administration identified urothelial LRCs in the rat bladder with 9% of the epithelial basal cells retaining BrdU label 1 yr after its administration. Markers for stem cells in other tissues, Bcl, p63, cytokeratin 14, and beta1 integrin, were immunolocalized in the basal bladder epithelium in or near urothelial LRCs, but not uniquely limited to these cells. Flow cytometry demonstrated that urothelial LRCs were small, had low granularity, and were uniquely beta4 integrin bright. Urothelium from long-term labeled bladders was cultured and LRCs were found to be significantly more clonogenic and proliferative, characteristics of stem cells, than unlabeled urothelial cells. Thus, this work demonstrates that LRCs in the bladder localize to the basal layer, are small, low granularity, uniquely beta4 integrin rich, slowly cycling and demonstrate superior clonogenic and proliferative ability compared with unlabeled epithelial cells. We propose that LRCs represent putative urothelial stem cells.

Beta1 integrin expression pattern in transitional urothelium does not allow for efficient stem cell enrichment as in other epithelia.

With a lack of distinct stem cell markers, isolation of tissue-specific stem cells for tissue engineering and gene therapy is a great challenge. Beta (beta)(1) integrin expression has been used as a way of enriching for putative epithelial stem cells through rapid adhesion to collagen IV or flow cytometry. This is a first report of enrichment of putative urothelial stem cells using rapid adhesion and flow cytometric methods. We assessed our success by determining the clonogenic and proliferative potential of the isolated cells. We demonstrated that enrichment based on beta(1) integrin expression with flow cytometry yields highly clonogenic and proliferative urothelial cells, whereas the rapid adhesion method is not as efficient, possibly because of the unique nature of urothelium, a transitional epithelium, compared to results reported in stratified and columnar epithelia.

Rat urothelium: improved techniques for serial cultivation, expansion, freezing and reconstitution onto acellular matrix.

PURPOSE: The rat has been a cost-effective model for the evaluation of bladder development, cancer and stromal-epithelial interactions. Serial cultivation of rat urothelium has been difficult. We developed a reliable protocol for the harvest, serial cultivation and cryopreservation of rat urothelium. We investigated the differentiation markers of in vivo bladder urothelium compared with cells reconstituted onto an acellular bladder matrix. MATERIALS AND METHODS: Epithelial harvest techniques using trypsin and collagenase were compared. Medium and conditions were optimized for serial culture and growth characteristics were calculated. Cultured cells were cryopreserved, and then recovered and grown on acellular bladder matrices. Morphology and markers of differentiation were compared between normal bladder and engineered grafts using scanning electron microscopy (SEM) and immunohistochemistry. RESULTS: Atraumatic enzymatic removal of urothelium with trypsin yielded more cells with greater viability than collagenase. Cells could be reliably grown beyond 10 passages using fibroblast conditioned medium and a 3T3 feeder layer during initial passages. Cryopreserved cells were successfully recovered and incorporated onto acellular matrices. Immunostaining and SEM of engineered grafts demonstrated early markers of differentiation, such as surface microvilli and cytokeratin 17, on polygonal cells with typical tight junctions. CONCLUSIONS: Rat urothelium can be reliably grown using fibroblast conditioned medium and a 3T3 feeder layer during primary culture. Serially passaged cells can survive cryopreservation and they are able to reconstitute epithelium on an acellular bladder matrix. Cells that are incorporated into the matrix express markers of early differentiation and demonstrate typical morphological characteristics by SEM. These culture techniques and this in vitro organoid model should facilitate the use of rat urothelium.