Split-thickness skin grafting (STSG) is the standard of care for skin replacement therapy. While STSG is a well-established technique, it has several limitations at both the donor and recipient sites. Full-thickness skin column (FTSC) grafting is an alternative approach that involves the orthogonal harvesting of small skin columns containing the epidermis, dermis, and associated skin appendages. Peptides have been shown to promote wound repair through various reparative and regenerative mechanisms. In this study, we aimed to evaluate the extent to which FTSCs and the matrix-derived peptide TSN6, individually or in combination, influenced the rate and quality of healing, as assessed by metrics such as epithelialization, epithelial thickness, and the presence of adnexal structures. TSN6 peptide and its scrambled form was synthetized in a laboratory and mixed with a carboxymethylcellulose (CMC) hydrogel. Up to 16 standardized full-thickness excisional wounds (∅ 5 cm) were created on the dorsum of two anesthetized pigs. FTSC biopsies (∅ 1.5 mm) were harvested from donor sites located on the rump of the pig at a ratio of up to eight 1.5 mm-diameter skin columns/1 cm2. Subsequently, the wounds were randomized to receive either (1) FTSC + TSN6, (2) FTSC + scrambled peptide, (3) FTSC alone, and (4) blank CMC hydrogel. Healing was monitored for 14 or 28 days. After euthanasia, the wounds were excised and processed for histology. Additionally, non-invasive imaging systems were utilized to assess wound healing. By day 14, wounds treated with FTSC or FTSC + TSN6 were significantly more re-epithelialized compared to those treated with blank CMC hydrogel. By day 28, all FTSC-transplanted wounds were fully re-epithelialized. Notably, wounds treated with FTSC + TSN6 exhibited improved healing quality, characterized by a thicker neo-epidermis and increased rete ridges at day 28 post-transplantation. All FTSC-transplanted wounds healed better than the untransplanted controls. The TSN6 peptide further improved healing quality when applied in combination with FTSCs, particularly by enhancing epidermal maturation.

Significance: Nonhealing wounds are an ever-growing global pandemic, with mortality rates and management costs exceeding many common cancers. Although our understanding of the molecular and cellular factors driving wound healing continues to grow, standards for diagnosing and evaluating wounds remain largely subjective and experiential, whereas therapeutic strategies fail to consistently achieve closure and clinicians are challenged to deliver individualized care protocols. There is a need to apply precision medicine practices to wound care by developing evidence-based approaches, which are predictive, prescriptive, and personalized. Recent Advances: Recent developments in "advanced" wound diagnostics, namely biomarkers (proteases, acute phase reactants, volatile emissions, and more) and imaging systems (ultrasound, autofluorescence, spectral imaging, and optical coherence tomography), have begun to revolutionize our understanding of the molecular wound landscape and usher in a modern age of therapeutic strategies. Herein, biomarkers and imaging systems with the greatest evidence to support their potential clinical utility are reviewed. Critical Issues: Although many potential biomarkers have been identified and several imaging systems have been or are being developed, more high-quality randomized controlled trials are necessary to elucidate the currently questionable role that these tools are playing in altering healing dynamics or predicting wound closure within the clinical setting. Future Directions: The literature supports the need for the development of effective point-of-care wound assessment tools, such as a platform diagnostic array that is capable of measuring multiple biomarkers at once. These, along with advances in telemedicine, synthetic biology, and "smart" wearables, will pave the way for the transformation of wound care into a precision medicine. Clinical Trial Registration number: NCT03148977.

Blood-brain barrier (BBB) endothelial cell (EC) function depends on flow conditions and on supportive cells, like pericytes and astrocytes, which have been shown to be both beneficial and detrimental for brain EC function. Most studies investigating BBB EC function lack physiological relevance, using sub-physiological shear stress magnitudes and/or omitting pericytes and astrocytes. In this study, we developed a millifluidic device compatible with standard transwell inserts to investigate BBB function. In contrast to standard polydimethylsiloxane (PDMS) microfluidic devices, this model allows for easy, reproducible shear stress exposure without common limitations of PDMS devices such as inadequate nutrient diffusion and air bubble formation. In no-flow conditions, we first used the device to examine the impact of primary human pericytes and astrocytes on human brain microvascular EC (HBMEC) barrier integrity. Astrocytes, pericytes, and a 1-to-1 ratio of both cell types increased HBMEC barrier integrity via reduced 3 and 40 kDa fluorescent dextran permeability and increased claudin-5 expression. There were differing levels of low 3 kDa permeability in HBMEC-pericyte, HBMEC-astrocyte, and HBMEC-astrocyte-pericyte co-cultures, while levels of low 40 kDa permeability were consistent across co-cultures. The 3 kDa findings suggest that pericytes provide more barrier support to the BBB model compared to astrocytes, although both supportive cell types are permeability reducers. Incorporation of 24-hour 12 dynes per cm2 flow significantly reduced dextran permeability in HBMEC monolayers, but not in the tri-culture model. These results indicate that tri-culture may exert more pronounced impact on overall BBB permeability than flow exposure. In both cases, monolayer and tri-culture, flow exposure interestingly reduced HBMEC expression of both claudin-5 and occludin. ZO-1 expression, and localization at cell-cell junctions increased in the tri-culture but exhibited no apparent change in the HBMEC monolayer. Under flow conditions, we also observed HBMEC alignment in the tri-culture but not in HBMEC monolayers, indicating supportive cells and flow are both essential to observe brain EC alignment in vitro. Collectively, these results support the necessity of physiologically relevant, multicellular BBB models when investigating BBB EC function. Consideration of the roles of shear stress and supportive cells within the BBB is critical for elucidating the physiology of the neurovascular unit.

Brain endothelial cells of the blood-brain barrier (BBB) have been shown to be regulated by supportive cells, such as pericytes and astrocytes, and shear stress exposure. However, studies investigating the impact of pericytes and astrocytes on brain endothelial cell function have identified both beneficial and detrimental results. Additionally, most studies investigating the relationship between shear stress and brain endothelial cell function lack physiological relevance via the use of sub-physiological shear stress magnitudes and/or via the absence of pericytes and astrocytes. In this study, we developed a millifluidic device compatible with standard transwell inserts to investigate BBB function. In contrast to standard polydimethylsiloxane (PDMS) microfluidic devices, this model allows for easy, reproducible shear stress exposure without common limitations of PDMS devices such as inadequate nutrient diffusion and air bubble formation. In no-flow conditions, we first used the device to examine the impact of primary human pericytes and astrocytes on human brain microvascular endothelial cell (HBMEC) barrier integrity. We found that astrocytes, pericytes, and a 1:1 ratio of both cell types increased HBMEC barrier integrity via reduced permeability to 40 kDa fluorescent dextran, which was associated with increased expression of tight junction protein, claudin-5. Interestingly, we also observed a significantly lower permeability to 3 kDa dextran in HBMEC-pericyte co-cultures compared to HBMEC-astrocyte and HBMEC-astrocyte-pericyte co-cultures. Based on these findings, we hypothesize pericytes may be providing increased barrier support to the BBB model compared to astrocytes although they both function as permeability reducers. After using the device to generate 24-hour flow at 12 dynes/cm2, we observed that shear stress exposure significantly reduced dextran permeability in HBMEC monolayers, but not in tri-culture models consisting of HBMECs, pericytes, and astrocytes. These results indicate that co-cultures may demonstrate a more pronounced impact on overall BBB permeability than flow exposure. In both cases, flow exposure was interestingly associated with reduced expression of both claudin-5 and occludin. However, ZO-1 expression, and localization at cell-cell junctions increased in the tri-culture but exhibited no apparent change in the HBMEC monolayer. Under flow conditions, we also observed alignment of HBMECs in the tri-culture while no such phenomenon was observed in HBMEC monolayers, indicating supportive cells and flow are both essential to observe brain endothelial cell alignment in vitro. Collectively, these results support the necessity of physiologically relevant, multicellular BBB models when investigating brain endothelial cell function in relation to the BBB. Additionally, our findings provide clues on the role of shear stress and supportive cells within the BBB, a critical step to elucidating the physiology of the neurovascular unit.

Ischemic stroke, a major cause of mortality in the United States, often contributes to disruption of the blood-brain barrier (BBB). The BBB along with its supportive cells, collectively referred to as the "neurovascular unit," is the brain's multicellular microvasculature that bi-directionally regulates the transport of blood, ions, oxygen, and cells from the circulation into the brain. It is thus vital for the maintenance of central nervous system homeostasis. BBB disruption, which is associated with the altered expression of tight junction proteins and BBB transporters, is believed to exacerbate brain injury caused by ischemic stroke and limits the therapeutic potential of current clinical therapies, such as recombinant tissue plasminogen activator. Accumulating evidence suggests that endothelial mechanobiology, the conversion of mechanical forces into biochemical signals, helps regulate function of the peripheral vasculature and may similarly maintain BBB integrity. For example, the endothelial glycocalyx (GCX), a glycoprotein-proteoglycan layer extending into the lumen of bloods vessel, is abundantly expressed on endothelial cells of the BBB and has been shown to regulate BBB permeability. In this review, we will focus on our understanding of the mechanisms underlying BBB damage after ischemic stroke, highlighting current and potential future novel pharmacological strategies for BBB protection and recovery. Finally, we will address the current knowledge of endothelial mechanotransduction in BBB maintenance, specifically focusing on a potential role of the endothelial GCX.

Heterotypic spheroids are used to study mesenchymal-epithelial interactions important for skin development and regeneration. In order to investigate the behavior and interaction of human hair follicle dermal papilla cells (DPC) and keratinocytes during in vitro three-dimensional (3D) co-culture, we labeled DPC and keratinocytes with two different fluorescent cell tracking dyes and prepared spheroids in DPC medium by hanging-drop technique. After incubation for 1 or 2 days, spheroids were live embedded in an agarose cylinder and image stacks were generated in multi-view mode from several angles using dual-sided LSFM. In presence of 100 μg/ml rat tail collagen I, cells aggregated into a single 300-500 μm spheroid, whereas multiple spheroids of various sizes formed in absence of collagen. Cells appeared randomly distributed after 1 day, but after 2 days the vast majority of DPC were located in the core with most keratinocytes distributed in the periphery, with or without collagen. LSFM can be used to study self-assembly of DPC and keratinocytes in spheroids in order to gain insights into factors regulating organogenesis during skin regeneration and hair follicle neogenesis. The sub-micrometer resolution of the LSFM microscope in x-y dimension in conjunction with multi-view imaging allows for detailed quantification of distribution of cell populations in 3D space.

Background Endothelial-dependent atherosclerosis develops in a non-random pattern in regions of vessel bending and bifurcations, where blood flow exhibits disturbed flow (DF) patterns. In contrast, uniform flow (UF), normal endothelium, and healthy vessel walls co-exist within straight vessels. In clarifying how flow protectively or atherogenically regulates endothelial cell behavior, involvement of the endothelial surface glycocalyx has been suggested due to reduced expression in regions of atherosclerosis development. Here, we hypothesized that pro-atherosclerotic endothelial dysfunction occurs as a result of DF-induced reduction in glycocalyx expression and subsequently impairs endothelial sensitivity to flow. Specifically, we propose that glycocalyx degradation can induce pro-atherosclerotic endothelial dysfunction through decreased caveolin-1 and endothelial nitric oxide synthase expression and localization. Methods We studied endothelial cells in atherosclerotic-prone DF and atherosclerotic-resistant UF conditions in parallel plate flow culture and in C57Bl/6 mice. The effects of flow conditioning on endothelial cell behavior were quantified using immunocytochemistry. The glycocalyx was fluorescently labeled for wheat germ agglutinin, which serves as a general glycocalyx label, and heparan sulfate, a major glycocalyx component. Additionally, mechanosensitivity was assessed by immunocytochemical fluorescence expression and function of caveolin-1, the protein that forms the mechanosignaling caveolar invaginations on the endothelial surface, total endothelial-type nitric oxide synthase (eNOS), which synthesizes nitric oxide, and serine 1177 phosphorylated eNOS (eNOS-pS1177), which is the active form of eNOS. Caveolin function and eNOS expression and activation were correlated to glycocalyx expression. Heparinase III enzyme was used to degrade a major glycocalyx component, HS, to identify the role of the glycocalyx in caveoin-1 and eNOS-pS1177 regulation. Results Results confirmed that DF reduces caveolin-1 expression and abolishes most of its subcellular localization preferences, when compared to the effect of UF. DF down-regulates caveolin-1 mechanosignaling, as indicated by its reduced colocalization with serine 1177 phosphorylated endothelial-type nitric oxide synthase (eNOS-pS1177), a vasoregulatory signaling molecule whose activity is regulated by its residence in caveolae. As expected, DF inhibited glycocalyx expression compared to UF. In the absence of heparan sulfate, a major glycocalyx component, UF-conditioned endothelial cells exhibited near DF-like caveolin-1 expression, localization, and colocalization with eNOS-pS1177. Conclusions This is the first demonstration of a flow-defined role of the glycocalyx in caveolae expression and function related to vasculoprotective endothelial mechanosensitivity that defends against atherosclerosis. The results suggest that a glycocalyx-based therapeutic targeted to areas of atherosclerosis development could prevent disease initiation and progression.

Donor organ shortage remains a clear problem for many end-stage organ patients around the world. The number of available donor organs pales in comparison with the number of patients in need of these organs. The field of tissue engineering proposes a plausible solution. Using stem cells, a patient’s autologous cells, or allografted cells to seed-engineered scaffolds, tissue-engineered constructs can effectively supplement the donor pool and bypass other problems that arise when using donor organs, such as who receives the organ first and whether donor organ rejection may occur. However, current research methods and technologies have been unable to successfully engineer and vascularize large volume tissue constructs. This review examines the current perfusion methods for ex vivo organ systems, defines the different types of vascularization in organs, explores various strategies to vascularize ex vivo organ systems, and discusses challenges and opportunities for the field of tissue engineering.

In the body, tissues are formed to maintain order, organization, and optimization of function. This is particularly demonstrated in the retina, where the arrangement of cells into layers and arrays is a key element for efficient visual processing. Vision loss due to neuronal cell death represents one of the most difficult challenges in regenerative medicine, as these cells display little to no regeneration capacity. Although pharmacological therapies aimed at halting the progression of cell loss are being investigated, cell-based therapy is a promising strategy to not only rescue but also restore lost cells. With the promising development of a new source of retinal cells for transplantation, successful vision restoration is closer to becoming a reality. However, cell-based therapies have been limited by the poor survival of grafted cells upon transplantation to the subretinal space. Deterioration of the retinal microenvironment in …

Nonhealing cutaneous ulcers that occur in diabetes, vascular insufficiency, and other chronic conditions, are a global health concern and continue to increase in annual incidence. Despite the volume of preclinical and clinically significant findings regarding either the molecular and cellular mechanisms driving wound chronicity or treatment strategies that aim to couple wound diagnostics with treatment strategies, existing therapeutics have not matched the enormous, unmet global need for advancements in chronic wound care. In this chapter, we briefly review the factors precipitating chronic wounds, the therapeutic options currently in use, the need for high-quality biomarkers to standardize wound evaluation and care, and how concepts of synthetic biology can serve as the basis of advanced, point-of-care devices that couple molecular diagnostics with programmed therapeutic responses in real time.

Non-healing wounds are a major global health concern and account for the majority of non-traumatic limb amputations worldwide. However, compared to standard care practices, few advanced therapeutics effectively resolve these injuries stemming from cardiovascular disease, aging, and diabetes-related vasculopathies. While matrix turnover is disrupted in these injuries, debriding enzymes may promote healing by releasing matrix fragments that induce cell migration, proliferation, and morphogenesis, and plasma products may also stimulate these processes. Thus, we created matrix- and plasma-derived peptides, Comb1 and UN3, which induce cellular injury responses in vitro, and accelerate healing in rodent models of non-healing wounds. However, the effects of these peptides in non-healing wounds in diabetes are not known. Here, we interrogated whether these peptides stimulate healing in a diabetic porcine model highly reminiscent of human healing impairments in type 1 and type 2-diabetes.

Vascular endothelial cells are known to respond to a range of biochemical and time-varying mechanical cues that can promote blood vessel sprouting termed angiogenesis. It is less understood how these cells respond to sustained (i.e., static) mechanical cues such as the deformation generated by other contractile vascular cells, cues which can change with age and disease state. Here we demonstrate that static tensile strain of 10%, consistent with that exerted by contractile microvascular pericytes, can directly and rapidly induce cell cycle re-entry in growth-arrested microvascular endothelial cell monolayers. S-phase entry in response to this strain correlates with absence of nuclear p27, a cyclin-dependent kinase inhibitor. Furthermore, this modest strain promotes sprouting of endothelial cells, suggesting a novel mechanical 'angiogenic switch'. These findings suggest that static tensile strain can directly stimulate pathological angiogenesis, implying that pericyte absence or death is not necessarily required of endothelial cell re-activation.

Debridement, the removal of diseased, nonviable tissue, is critical for clinicians to readily assess wound status and prepare the wound bed for advanced therapeutics or downstream active healing. Removing necrotic slough and eschar through surgical or mechanical methods is less specific and may be painful for patients. Enzymatic debridement agents, such as Clostridial collagenase, selectively and painlessly degrade devitalized tissue. In addition to its debriding activities, highly-purified Clostridial collagenase actively promotes healing, and our past studies reveal that extracellular matrices digested with this enzyme yield peptides that activate cellular migratory, proliferative and angiogenic responses to injury in vitro, and promote wound closure in vivo. Intriguingly, while collagenase Santyl® ointment, a sterile preparation containing Clostridial collagenases and other non-specific proteases, is a well-accepted enzymatic debridement agent, its role as an active healing entity has never been established. Based on our previous studies of pure Clostridial collagenase, we now ask whether the mixture of enzymes contained within Santyl® produces matrix-derived peptides that promote cellular injury responses in vitro and stimulate wound closure in vivo. Here, we identify novel collagen fragments, along with collagen-associated peptides derived from thrombospondin-1, multimerin-1, fibronectin, TGFβ-induced protein ig-h3 and tenascin-C, generated from Santyl® collagenase-digested human dermal capillary endothelial and fibroblastic matrices, which increase cell proliferation and angiogenic remodeling in vitro by 50-100% over controls. Using an established model of impaired healing, we further demonstrate a specific dose of collagenase from Santyl® ointment, as well as the newly-identified and chemically-synthesized ECM-derived peptides significantly increase wound re-epithelialization by 60-100% over saline-treated controls. These results not only confirm and extend our earlier studies using purified collagenase- and matrix-derived peptides to stimulate healing in vitro and in vivo, but these Santyl®-generated, matrix-derived peptides may also represent exciting new opportunities for creating advanced wound healing therapies that are enabled by enzymatic debridement and potentially go beyond debridement.

Microvascular endothelial cell-mural cell interactions are instrumental in modulating both physiological and pathologic angiogenesis. Pericyte-endothelial cell communication through direct physical associations and secreted effectors comprises a bidirectional signal array that regulates vascular maturation and integrity. As endothelial cell proliferation, migration, and morphogenesis are key elements of vascular growth and remodeling during angiogenesis, we have developed novel preclinical systems for studying the roles of endothelial-mural cell dynamics on cell cycle entry and angiogenic activity in vitro. These coculture models not only enable evaluation of endothelial cell-pericyte "cross talk" but also allow for the quantitative analysis of both heterotypic contact-dependent and contact-independent cell cycle progression in either cell population, as well as angiogenic sprouting in three-dimensional vascular networks. Cells actively proliferating in two-dimensional assays can be labeled via incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into their DNA. Additionally, each cell population can be vitally labeled with a variety of cell-specific and/or membrane-permeant lipophilic dyes prior to coculture, such as DiO, or through immunofluorescence of mural or endothelial cell-specific markers after cellular fixation and/or permeabilization. Ultimately, this experimental approach can be used to investigate cellular contact-dependent and soluble mechanisms mediating mural-endothelial cell interactions, which may be instrumental in microvascular development and remodeling in vivo.

PURPOSE:To establish the regulatory roles that pericytes have in coordinating retinal endothelial cell (EC) growth and angiogenic potential.METHODS:Pericytes were derived from donor diabetic (DHuRP) or normal (NHuRP) human retinae, and characterized using vascular markers, coculture, contraction, morphogenesis, and proliferation assays. To investigate capillary "cross-talk," pericyte-endothelial coculture growth, and connexin-43 (Cx43) expression assays were performed. Paracrine effects were examined via treating EC with pericyte-derived conditioned media (CM) in proliferation, angiogenesis, and angiocrine assays. The effects of sphingosine 1-phosphate (S1P) were assessed using receptor antagonists.RESULTS:The DHuRP exhibit unique proliferative and morphologic properties, reflecting distinctive cytoskeletal and isoactin expression patterns. Unlike NHuRP, DHuRP are unable to sustain EC growth arrest in coculture and display reduced Cx43 expression. Further, CM from DHuRP (DPCM) markedly stimulates EC proliferation and tube formation. Treatment with S1P receptor antagonists mitigates DPCM growth-promotion in EC and S1P-mediated pericyte contraction. Angiocrine assays on normal and diabetic pericyte secretomes reveal factors involved in angiogenic control, inflammation, and metabolism.CONCLUSIONS:Effects from the diabetic microenvironment appear sustainable in cell culture: pericytes derived from diabetic donor eyes seemingly possess a "metabolic memory" in vitro, which may be linked to original donor health status. Diabetes- and pericyte-dependent effects on EC growth and angiogenesis may reflect alterations in bioactive lipid, angiocrine, and chemomechanical signaling. Altogether, our results suggest that diabetes alters pericyte contractile phenotype and cytoskeletal signaling, which ultimately may serve as a key, initiating event required for retinal endothelial reproliferation, angiogenic activation, and the pathological neovascularization accompanying proliferative diabetic retinopathy.