Mesenchymal Stem Cell Extracellular Vesicles from Tissue-Mimetic System Enhance Epidermal Regeneration via Formation of Migratory Cell Sheets.

BACKGROUND: The secretome of adipose-derived mesenchymal stem cells (ASCs) offers a unique approach to understanding and treating wounds, including the critical process of epidermal regeneration orchestrated by keratinocytes. However, 2D culture techniques drastically alter the secretory dynamics of ASCs, which has led to ambiguity in understanding which secreted compounds (e.g., growth factors, exosomes, reactive oxygen species) may be driving epithelialization. METHODS: A novel tissue-mimetic 3D hydrogel system was utilized to enhance the retainment of a more regenerative ASC phenotype and highlight the functional secretome differences between 2D and 3D. Subsequently, the ASC-secretome was stratified by molecular weight and the presence/absence of extracellular vesicles (EVs). The ASC-secretome fractions were then evaluated to assess for the capacity to augment specific keratinocyte activities. RESULTS: Culture of ASCs within the tissue-mimetic system enhanced protein secretion ~ 50%, exclusively coming from the > 100 kDa fraction. The ASC-secretome ability to modulate epithelialization functions, including migration, proliferation, differentiation, and morphology, resided within the "> 100 kDa" fraction, with the 3D ASC-secretome providing the greatest improvement. 3D ASC EV secretion was enhanced two-fold and exhibited dose-dependent effects on epidermal regeneration. Notably, ASC-EVs induced morphological changes in keratinocytes reminiscent of native regeneration, including formation of stratified cell sheets. However, only 3D-EVs promoted collective cell sheet migration and an epithelial-to-mesenchymal-like transition in keratinocytes, whereas 2D-EVs contained an anti-migratory stimulus. CONCLUSION: This study demonstrates how critical the culture environment is on influencing ASC-secretome regenerative capabilities. Additionally, the critical role of EVs in modulating epidermal regeneration is revealed and their translatability for future clinical therapies is discussed.

Tailoring the secretome composition of mesenchymal stem cells to augment specific functions of epidermal regeneration: an in vitro diabetic model.

Introduction: Wound healing consists of a dynamic series of events that are highly dependent on paracrine factors for proper progression through the phases of wound healing. Inappropriate progression through the phases is associated with insufficient epidermal regeneration (i.e., re-epithelialization) of wounds and subsequent propagation of chronic wounds, such as diabetic ulcers, which are associated with increased patient morbidity. Recently, investigation into the dynamic secretome of Adipose-derived Mesenchymal Stem Cells (ASCs), have shown promise in augmenting the wound healing response of chronic diabetic wounds. However, currently utilized 2D culture techniques are known to drastically alter the regenerative phenotype of ASCs. In this study a novel tissue-mimetic 3D system was utilized as a means to culture ASCs. Methods: The capacity for the ASC secretome to augment epidermal regeneration activity was then evaluated after exposure of ASCs to "wound priming stimuli" in 2D and 3D. The priming stimuli consisted of coating the 2D and 3D systems with the wound matrix proteins, collagen type I, fibronectin, and fibrin. To understand the potential benefit of the ASC secretome in the context of diabetic wounds, keratinocytes (KCs) were exposed to super-physiological glucose levels to induce a diabetic-like phenotype (idKCs). Results: Relative to KCs, idKC exhibited a 52% and 23% decline in proliferation and migration, respectively. Subsequently, analyses of the ASC secretome were performed. ASC conditioned media (ASC-CM) from tissue-mimetic culture demonstrated a > 50% increase secretion of proteins and a 2-fold increase in secreted EVs, relative to 2D culture. Interestingly, the different priming stimuli did not alter the total amount of protein or EVs secreted within the tissue-mimetic system. However, evaluation of specific soluble proteins via ELISA revealed significant differences in key epidermal regeneration factors, such as EGF, IGF-1, FGF-2, MMP-1, TIMP-1, and TGFß-1. Additionally, the relative effect of ASC-EVs from the 2D and 3D system on idKCs epidermal regeneration functionality varied significantly, with EVs from 3D-Collagen culture providing the most significant benefit on idKC activity. Discussion: Together, these data support the utilization of tissue-mimetic culture system to enhance the adaptability and secretory activity of MSC-like populations in order to generate tailored biologics, via priming stimuli, for specific wound healing applications.

Tissue-mimetic culture enhances mesenchymal stem cell secretome capacity to improve regenerative activity of keratinocytes and fibroblasts in vitro.

Mesenchymal stem/stromal cells (MSCs) are a heterogenous population of multipotent and highly secretory cells currently being investigated in the field of wound healing for their ability to augment tissue responses. The adaptive response of MSC populations to the rigid substrate of current 2D culture systems has been considered to result in a deterioration of regenerative 'stem-like' properties. In this study, we characterise how the improved culture of adipose-derived mesenchymal stem cells (ASCs) within a tissue-mimetic 3D hydrogel system, that is mechanically similar to native adipose tissue, enhances their regenerative capabilities. Notably, the hydrogel system contains a porous microarchitecture that permits mass transport, enabling efficient collection of secreted cellular compounds. By utilising this 3D system, ASCs retained a significantly higher expression of ASC 'stem-like' markers while demonstrating a significant reduction in senescent populations, relative to 2D. Additionally, culture of ASCs within the 3D system resulted in enhanced secretory activity with significant increases in the secretion of proteinaceous factors, antioxidants and extracellular vesicles (EVs) within the conditioned media (CM) fraction. Lastly, treatment of wound healing cells, keratinocytes (KCs) and fibroblasts (FBs), with ASC-CM from the 2D and 3D systems resulted in augmented functional regenerative activity, with ASC-CM from the 3D system significantly increasing KC and FB metabolic, proliferative and migratory activity. This study demonstrates the potential beneficial role of MSC culture within a tissue-mimetic 3D hydrogel system that more closely mimics native tissue mechanics, and subsequently how the improved phenotype augments secretory activity and potential wound healing capabilities of the MSC secretome.

Novel hydrogel system eliminates subculturing and improves retention of nonsenescent mesenchymal stem cell populations.

Aim: To compare the physiological behavior of mesenchymal stem/stromal cells (MSCs) within an expandable tissue-mimetic 3D system relative to in vitro expansion in a traditional 2D system. Methods: Adipose-derived MSCs (ASCs) were continuously cultured for 6 weeks on either 2D culture plastic or in a 3D hydrogel system that eliminated subculturing. ASCs were assessed for senescence, 'stem-like' MSC markers, and ability for their secretome to augment a secondary cell population. Results: The 3D hydrogel system resulted in an enhanced retention of more regenerative, nonsenescent ASC populations that exhibited increased expression of 'stem-like' MSC surface markers. Conclusion: This study introduces a proof-of-concept design for a novel modular 3D system that can improve in vitro expansion of stem-like cell populations for future regenerative therapies.

Evaluating polymeric biomaterials to improve next generation wound dressing design.

Wound healing is a dynamic series of interconnected events with the ultimate goal of promoting neotissue formation and restoration of anatomical function. Yet, the complexity of wound healing can often result in development of complex, chronic wounds, which currently results in a significant strain and burden to our healthcare system. The advancement of new and effective wound care therapies remains a critical issue, with the current therapeutic modalities often remaining inadequate. Notably, the field of tissue engineering has grown significantly in the last several years, in part, due to the diverse properties and applications of polymeric biomaterials. The interdisciplinary cohesion of the chemical, biological, physical, and material sciences is pertinent to advancing our current understanding of biomaterials and generating new wound care modalities. However, there is still room for closing the gap between the clinical and material science realms in order to more effectively develop novel wound care therapies that aid in the treatment of complex wounds. Thus, in this review, we discuss key material science principles in the context of polymeric biomaterials, provide a clinical breadth to discuss how these properties affect wound dressing design, and the role of polymeric biomaterials in the innovation and design of the next generation of wound dressings.

Improved porosity of electrospun poly (Lactic-Co-Glycolic) scaffolds by sacrificial microparticles enhances cellular infiltration compared to sacrificial microfiber.

Electrospinning is a technique used to fabricate nano-/microfiber scaffolds for tissue engineering applications. However, a major limitation of electrospun scaffolds is the high packing density of fibers that leads to poor cellular infiltration. Thus, incorporation of a water soluble sacrificial porogen, polyethylene oxide (PEO), was utilized to fine-tune the porous fraction of the scaffolds and decrease fiber packing density. Poly(lactic-co-glycolic) acid (PLGA) scaffolds were either co-electrospun with sacrificial PEO microfibers or co-electrosprayed with sacrificial PEO microparticles at three different extrusion rates to control the relative morphology and dose of PEO. A dose-dependent response in PLGA scaffold bulk porosity and pore area was noted as PEO content was increased. Notably, PLGA scaffolds after removal of sacrificial PEO microparticles significantly increased the porous fraction and pore area approximately 8, 10, and 14% and 46, 20, and 33 µm2, respectively, relative to the analogous PEO microfiber scaffold. The tensile properties of the more porous PLGA scaffolds after PEO microparticle removal, remained stable for all extrusion rates of PEO tested, relative to the PLGA scaffolds after PEO microfiber removal. Histological analysis revealed that removal of PEO microparticles significantly increased the depth of cellular migration through the PLGA scaffolds, relative to PEO microfiber scaffolds, with maximum migratory depths of 1120 µm versus 150 µm over 28 days, respectively. Additionally, depth of cellular infiltration responded dose-dependently in the PEO microparticle scaffolds, whereas in the PEO microfiber scaffolds there was no correlation. Further analysis with Masson's Trichrome staining and electron microscopy revealed that collagen density and depth of deposition substantially increased in PLGA scaffolds after removal of PEO microparticles relative to PEO microfibers. Thus, this study demonstrates an effective strategy to control the porous fraction of electrospun scaffolds via the incorporation of sacrificial PEO microparticles, without significant decreases in mechanical properties, thereby enhancing cellular infiltration and subsequent extracellular matrix deposition.

Novel insights into negative pressure wound healing from an in situ porcine perspective.

Negative pressure wound therapy (NPWT) is used clinically to promote tissue formation and wound closure. In this study, a porcine wound model was used to further investigate the mechanisms as to how NPWT modulates wound healing via utilization of a form of NPWT called the vacuum-assisted closure. To observe the effect of NPWT more accurately, non-NPWT control wounds containing GranuFoam™ dressings, without vacuum exposure, were utilized. In situ histological analysis revealed that NPWT enhanced plasma protein adsorption throughout the GranuFoam™, resulting in increased cellular colonization and tissue ingrowth. Gram staining revealed that NPWT decreased bacterial dissemination to adjacent tissue with greater bacterial localization within the GranuFoam™. Genomic analysis demonstrated the significant changes in gene expression across a number of genes between wounds treated with non-NPWT and NPWT when compared against baseline tissue. However, minimal differences were noted between non-NPWT and NPWT wounds, including no significant differences in expression of collagen, angiogenic, or key inflammatory genes. Similarly, significant increases in immune cell populations were observed from day 0 to day 9 for both non-NPWT and NPWT wounds, though no differences were noted between non-NPWT and NPWT wounds. Furthermore, histological analysis demonstrated the presence of a foreign body response (FBR), with giant cell formation and encapsulation of GranuFoam™ particles. The unique in situ histological evaluation and genomic comparison of non-NPWT and NPWT wounds in this pilot study provided a never-before-shown perspective, offering novel insights into the physiological processes of NPWT and the potential role of a FBR in NPWT clinical outcomes.

Surfactant location and internal phase volume fraction dictate emulsion electrospun fiber morphology and modulate drug release and cell response.

Emulsion electrospinning is a versatile technique used to create fibrous meshes for applications in drug delivery and tissue engineering. In this study, the effects of surfactant and increasing internal phase volume fraction on emulsion electrospun fiber morphology were investigated. The fiber diameter, surface topography, internal architecture, mesh hydrophobicity, and fiber volume fraction were all characterized and the resulting effects on model drug release and cell response were determined. Surfactant relocation to the fiber surface resulted in alterations to fiber surface topography and internal morphology, increased rate of water adsorption into the mesh, and reduced burst effects of drug release. Increasing the internal phase volume fraction within the emulsion resulted in minimal change to fiber diameter, surface morphology, fiber volume fraction, and rate of water adsorption illustrating the ability to increase drug loading without affecting fiber properties. Lastly, all meshes promoted cell adhesion and good viability with a trend of increased MTT absorbance from cells on the surfactant and emulsion fibers possibly suggesting that an increase in surface area via smaller fiber diameter and fiber volume fraction increases metabolic activity. Overall, these studies indicate that fiber morphology and mesh hydrophobicity can be tuned by controlling surfactant location within fibers and internal phase volume fraction. Modulating fiber properties within the emulsion electrospun mesh is important to achieve controlled drug release and cell response for tissue engineering applications.