Potential of Atlantic Codfish (Gadus morhua) Skin Collagen for Skincare Biomaterials.

Collagen is the major structural protein in extracellular matrix present in connective tissues, including skin, being considered a promising material for skin regeneration. Marine organisms have been attracting interest amongst the industry as an alternative collagen source. In the present work, Atlantic codfish skin collagen was analyzed, to evaluate its potential for skincare. The collagen was extracted from two different skin batches (food industry by-product) using acetic acid (ASColl), confirming the method reproducibility since no significant yield differences were observed. The extracts characterization confirmed a profile compatible with type I collagen, without significant differences between batches or with bovine skin collagen (a reference material in biomedicine). Thermal analyses suggested ASColl's native structure loss at 25 °C, and an inferior thermal stability to bovine skin collagen. No cytotoxicity was found for ASColl up to 10 mg/mL in keratinocytes (HaCaT cells). ASColl was used to develop membranes, which revealed smooth surfaces without significative morphological or biodegradability differences between batches. Their water absorption capacity and water contact angle indicated a hydrophilic feature. The metabolic activity and proliferation of HaCaT were improved by the membranes. Hence, ASColl membranes exhibited attractive characteristics to be applied in the biomedical and cosmeceutical field envisaging skincare.

Pre-selection of fibroblast subsets prompts prevascularization of tissue engineered skin analogues.

The papillary and reticular dermis harbors phenotypically distinct fibroblasts, whose functions such as maintenance of skin's microvasculature are also distinct. Thus, we hypothesized that pre-selection of the subpopulations of fibroblasts would benefit the generation of skin tissue engineered (TE) constructs, promoting their prevascularization in vitro. We first isolated papillary and reticular fibroblasts using fluorescence-activated cell sorting and studied the effect of their secretome and extracellular matrix (ECM) on human dermal microvascular endothelial cell (hDMEC) organization. Subsequently, we developed a bilayered 3D polymeric structure with distinct layer-associated features to house the subpopulations of fibroblasts, to generate a skin analogue. Both papillary and reticular fibroblasts were able to stimulate capillary-like network formation in a Matrigel assay. However, the secretome of the two subpopulations was substantially different, being enriched in VEGF, IGF-1, and Angio-1 in the case of papillary fibroblasts and in HGF and FGF-2 for the reticular subset. In addition, the fibroblast subpopulations deposited varied levels of ECM proteins, more collagen I and laminin was produced by the reticular subset, but these differences did not impact hDMEC organization. Vessel-like structures with lumens were observed earlier in the 3D skin analogue prepared with the sorted fibroblasts, although ECM deposition was not affected by the cell's pre-selection. Moreover, a more differentiated epidermal layer was obtained in the skin analogue formed by the sorted fibroblasts, confirming that its whole structure was not affected. Overall, we provide evidence that pre-selection of papillary and reticular fibroblasts is relevant for promoting the in vitro prevascularization of skin TE constructs.

Keratinocyte Growth Factor-Based Strategies for Wound Re-Epithelialization.

Wound re-epithelialization is a dynamic process that comprises the formation of new epithelium through an active signaling network between several growth factors (GFs) and various cell types. The main players are keratinocytes (KCs) that migrate from the wound edges over the wound bed to restore the epidermal barrier. One of the most important molecules involved in the re-epithelialization process is keratinocyte growth factor (KGF), a central player on promoting both migration and proliferation of KCs. Stromal cells, such as dermal fibroblasts, are the main producers of this factor, acting on KCs through paracrine signaling. Multiple therapeutic strategies to deliver KGF have been proposed to boost wound healing by targeting re-epithelialization. Different approaches have been explored to attain that purpose, such as topical application of this factor, controlled release of KGF from different biomaterials (hydrogels, nanoparticles, and membranes), and also gene delivery techniques. Among these strategies, KGF release via biomaterials- and genetic-based strategies shows great effectiveness in maintaining sustained KGF levels at the wound site, which is reflected in an efficient wound closure. Under this scope, this review aims not only to elucidate the potential of KGF in wound re-epithelialization but also to describe the underlying mechanism of action and further explore the therapeutic approaches using this GF. Impact statement Upon skin injury, wound re-epithelialization is one of the major milestones of the healing process. This is especially difficult to achieve on hard-to-heal wounds that are often open for long periods, as the dysregulation of the growth factors involved in this response contributes to an impaired proliferation and migration of keratinocytes. Keratinocyte growth factor (KGF) plays a central role in this problematic, as it is a potent factor that in the normal healing scenario promotes direct proliferation and migration of epidermal cells, consequently impacting re-epithelialization. Under this context, in the first part of this review, the process of wound healing and the mechanism of action of KGF are described. In the second part, various KGF delivery approaches aiming at skin re-epithelialization are reported and actively discussed. In this sense, it is herein highlighted the role of KGF in wound re-epithelialization and provided a critical overview of potential therapeutic strategies exploited so far.

Laminin-2 immobilized on a 3D fibrous structure impacts cortical thymic epithelial cells behaviour and their interaction with thymocytes.

The tri-dimensionality of the thymic extracellular matrix (ECM) supports the crosstalk between thymocytes and thymic epithelial cells (TECs). The thymic ECM component laminin-2 is involved in the regulation of thymocytes and their interaction with cortical TECs (cTECs). Most in vitro studies use planar surfaces to study the interaction between ECM components and thymic cells. Herein, we developed a novel biofunctionalized culture system by immobilizing laminin-2 at the surface of porous and fibrous electrospun meshes. We aimed to study the interaction of cTECs with thymocytes in the presence of laminin-2 presented through this system. The results indicated that the presence of laminin-2, not its density, has a positive effect on the cell viability and proliferation of cTECs. qPCR results demonstrated that laminin-2 density influenced the expression of cTECs genes. An increased percentage of adherent CD4-CD8- thymocytes and a decreased percentage of CD4+CD8+ thymocytes were evident in higher laminin-2 concentrations. Higher concentrations decreased the expression of Il7 and Ccl25 in cTECs after thymocyte adhesion. Altogether, these results indicate that the interaction of thymocytes with the thymic cortical compartment is affected by laminin-2 density and supports the need for immobilized ECM proteins in porous and fibrous substrates for the study of thymus biology.

Highly tailorable gellan gum nanoparticles as a platform for the development of T cell activator systems.

BACKGROUND: T cell priming has been shown to be a powerful immunotherapeutic approach for cancer treatment in terms of efficacy and relatively weak side effects. Systems that optimize the stimulation of T cells to improve therapeutic efficacy are therefore in constant demand. A way to achieve this is through artificial antigen presenting cells that are complexes between vehicles and key molecules that target relevant T cell subpopulations, eliciting antigen-specific T cell priming. In such T cell activator systems, the vehicles chosen to deliver and present the key molecules to the targeted cell populations are of extreme importance. In this work, a new platform for the creation of T cell activator systems based on highly tailorable nanoparticles made from the natural polymer gellan gum (GG) was developed and validated. METHODS: GG nanoparticles were produced by a water in oil emulsion procedure, and characterized by dynamic light scattering, high resolution scanning electronic microscopy and water uptake. Their biocompatibility with cultured cells was assessed by a metabolic activity assay. Surface functionalization was performed with anti-CD3/CD28 antibodies via EDC/NHS or NeutrAvidin/Biotin linkage. Functionalized particles were tested for their capacity to stimulate CD4+ T cells and trigger T cell cytotoxic responses. RESULTS: Nanoparticles were approximately 150 nm in size, with a stable structure and no detectable cytotoxicity. Water uptake originated a weight gain of up to 3200%. The functional antibodies did efficiently bind to the nanoparticles, as confirmed by SDS-PAGE, which then targeted the desired CD4+ populations, as confirmed by confocal microscopy. The developed system presented a more sustained T cell activation over time when compared to commercial alternatives. Concurrently, the expression of higher levels of key cytotoxic pathway molecules granzyme B/perforin was induced, suggesting a greater cytotoxic potential for future application in adoptive cancer therapy. CONCLUSIONS: Our results show that GG nanoparticles were successfully used as a highly tailorable T cell activator system platform capable of T cell expansion and re-education.

Mechanomodulatory biomaterials prospects in scar prevention and treatment.

Scarring is a major clinical issue that affects a considerable number of patients. The associated problems go beyond the loss of skin functionality, as scars bring aesthetic, psychological, and social difficulties. Therefore, new strategies are required to improve the process of healing and minimize scar formation. Research has highlighted the important role of mechanical forces in the process of skin tissue repair and scar formation, in addition to the chemical signalling. A more complete understanding of how engineered biomaterials can modulate these mechanical stimuli and modify the mechanotransduction signals in the wound microenvironment is expected to enable scar tissue reduction. The present review aims to provide an overview of our current understanding of skin biomechanics and mechanobiology underlying wound healing and scar formation, with an emphasis on the development of novel mechanomodulatory wound dressings with the capacity to offload mechanical tension in the wound environment. Furthermore, a broad overview of current challenges and future perspectives of promising mechanomodulatory biomaterials for this application are provided. STATEMENT OF SIGNIFICANCE: Scarring still is one of the biggest challenges in cutaneous wound healing. Beyond the loss of skin functionality, pathological scars, like keloids and hypertrophic, are associated to aesthetic, psychological, and social distress. Nonetheless, the understanding of the pathophysiology behind the formation of those scars remains elusive, which has in fact hindered the development of effective therapeutics. Therefore, in this review we provide an overview of our current understanding of skin biomechanics and mechanobiology underlying wound healing and scar formation, with an emphasis on the development of novel mechanomodulatory wound dressings with the capacity to offload mechanical tension in the wound environment.

Interfollicular epidermal stem-like cells for the recreation of the hair follicle epithelial compartment.

BACKGROUND: Hair follicle (HF) development and growth are dependent on epithelial-mesenchymal interactions (EMIs). Dermal papilla (DP) cells are recognized as the key inductive mesenchymal player, but the ideal source of receptive keratinocytes for human HF regeneration is yet to be defined. We herein investigated whether human interfollicular epidermal keratinocytes with stem-like features (EpSlKCs), characterized by a α6bri/CD71dim expression, can replace human hair follicular keratinocytes (HHFKCs) for the recreation of the HF epithelium and respective EMIs. METHODS: The α6bri/CD71dim cellular fraction was selected from the whole interfollicular keratinocyte population through fluorescence-activated cell sorting and directly compared with follicular keratinocytes in terms of their proliferative capacity and phenotype. The crosstalk with DP cells was studied in an indirect co-culture system, and EpSlKC hair forming capacity tested in a hair reconstitution assay when combined with DP cells. RESULTS: EpSlKCs exhibited a phenotypic profile similar to follicular keratinocytes and were capable of increasing DP cell proliferation and, for short co-culture times, the number of alkaline phosphatase-active cells, suggesting an improvement of their inductivity. Moreover, the recreation of immature HFs and sebaceous glands was observed after EpSlKC and DP cell co-grafting in nude mice. CONCLUSIONS: Our results suggest that EpSlKCs are akin to follicular keratinocytes and can crosstalk with DP cells, contributing to HF morphogenesis in vivo, thus representing an attractive epithelial cell source for hair regeneration strategies.

Rescuing key native traits in cultured dermal papilla cells for human hair regeneration.

Introduction: The dermal papilla (DP) represents the major regulatory entity within the hair follicle (HF), inducing hair formation and growth through reciprocal interactions with epithelial cells. However, human DP cells rapidly lose their hair inductive ability when cultured in an epithelium-deficient environment. Objectives: To determine if the conditioned medium collected from interfollicular keratinocytes (KCs-CM) is capable of improving DP cell native properties and inductive phenotype. Methods: DP cells were cultured with KCs-CM both in 2D and 3D culture conditions (spheroids). Further, the hair-inductive capacity of DP cells precultured with KCs-CM was tested in a hair reconstitution assay, after co-grafting with human keratinocytes in nude mice. Results: We demonstrate that KCs-CM contributes to restore the inductivity of cultured human DP cells in a more effective mode than the conventional 3D-cultures. This is supported by the higher active alkaline phosphatase (ALP) levels in DP cells, the improved self-aggregative capacity and the reduced expression of α-SMA and the V1-isoform of versican. Moreover, DP cells cultured with KCs-CM displayed a secretome profile (VEGF, BMP2, TGF- ß1, IL-6) that matches the one observed during anagen. KCs-CM also enhanced DP cell proliferation, while preventing cells to undergo morphological changes characteristic of high passage cells. In opposition, the amount of collagenous and non-collagenous proteins deposited by DP cells was lower in the presence of KCs-CM. The improvement in ALP activity was maintained in 3D spheroidal cultures, even after KCs-CM retrieval, being superior to the effect of the gold-standard culture conditions. Moreover, DP cells cultured with KCs-CM and grafted with human keratinocytes supported the formation of HF- and sebaceous gland-like structures in mice. Conclusion: The proposed strategy encourages future cell-based strategies for HF regeneration not only in the context of hair-associated disorders, but also in the management of wounds to aid in restoring critical skin regulatory appendages.

Correction: In vitro vascularization of tissue engineered constructs by non-viral delivery of pro-angiogenic genes.

Correction for 'In vitro vascularization of tissue engineered constructs by non-viral delivery of pro-angiogenic genes' by Helena R. Moreira et al., Biomater. Sci., 2021, DOI: 10.1039/d0bm01560a.

In vitro vascularization of tissue engineered constructs by non-viral delivery of pro-angiogenic genes.

Vascularization is still one of the major challenges in tissue engineering. In the context of tissue regeneration, the formation of capillary-like structures is often triggered by the addition of growth factors which are associated with high cost, bolus release and short half-life. As an alternative to growth factors, we hypothesized that delivering genes-encoding angiogenic growth factors to cells in a scaffold microenvironment would lead to a controlled release of angiogenic proteins promoting vascularization, simultaneously offering structural support for new matrix deposition. Two non-viral vectors, chitosan (Ch) and polyethyleneimine (PEI), were tested to deliver plasmids encoding for vascular endothelial growth factor (pVEGF) and fibroblast growth factor-2 (pFGF2) to human dermal fibroblasts (hDFbs). hDFbs were successfully transfected with both Ch and PEI, without compromising the metabolic activity. Despite low transfection efficiency, superior VEGF and FGF-2 transgene expression was attained when pVEGF was delivered with PEI and when pFGF2 was delivered with Ch, impacting the formation of capillary-like structures by primary human dermal microvascular endothelial cells (hDMECs). Moreover, in a 3D microenvironment, when PEI-pVEGF and Ch-FGF2 were delivered to hDFbs, cells produced functional pro-angiogenic proteins which induced faster formation of capillary-like structures that were retained in vitro for longer time in a Matrigel assay. The dual combination of the plasmids resulted in a downregulation of the production of VEGF and an upregulation of FGF-2. The number of capillary-like segments obtained with this system was inferior to the delivery of plasmids individually but superior to what was observed with the non-transfected cells. This work confirmed that cell-laden scaffolds containing transfected cells offer a novel, selective and alternative approach to impact the vascularization during tissue regeneration. Moreover, this work provides a new platform for pathophysiology studies, models of disease, culture systems and drug screening.

Interfollicular Epidermal Stem Cells: Boosting and Rescuing from Adult Skin.

Epidermal stem cells (EpSCs) isolation struggle remains, mainly due to the yet essential requirement of well-defined approaches and markers. The herein proposed methodology integrates an assemblage of strategies to accomplish the enrichment of the interfollicular EpSCs multipotent fraction and their subsequent separation from the remaining primary human keratinocytes (hKC) culture. Those include rapid adherence of freshly isolated hKC to collagen type IV through the ß1-integrin ligand and Rho-associated protein kinase inhibitor (Rocki) Y-27632 administration to the cultures, followed by an immunomagnetic separation to obtain populations based in the combined CD49fbri/CD71dim expression. Flow cytometry is the supporting method to analyze the effect of the treatments over the expression rate of early epidermal markers keratins19/5/14 and in correlation to CD49fbri/CD71dim sub-populations. The step-by-step methodology herein described indulges the boosting and consecutive purification and separation of interfollicular epidermal stem cells, from human keratinocytes cultures.

Gellan gum-hydroxyapatite composite spongy-like hydrogels for bone tissue engineering.

Osteoinductive biomaterials represent a promising approach to advance bone grafting. Despite promising, the combination of sustained biodegradability, mechanical strength, and biocompatibility in a unique biomaterial that can also support cell performance and bone formation in vivo is demanding. Herein, we developed gellan gum (GG)-hydroxyapatite (HAp) spongy-like hydrogels to mimic the organic (GG) and inorganic (HAp) phases of the bone. HAp was successfully introduced within the GG polymeric networks, as determined by FTIR and XRD, without compromising the thermostability of the biomaterials, as showed by TGA. The developed biomaterials showed sustained degradation, high swelling, pore sizes between 200 and 300 µm, high porosity (>90%) and interconnectivity (<60%) that was inversely proportional to the total polymeric amount and to CaCl2 crosslinker. CaCl2 and HAp reinforced the mechanical properties of the biomaterials from a storage modulus of 40 KPa to 70-80 KPa. This study also showed that HAp and CaCl2 favored the bioactivity and that cells were able to adhere and spread within the biomaterials up to 21 days of culture. Overall, the possibility to tailor spongy-like hydrogels properties by including calcium as a crosslinker and by varying the amount of HAp will further contribute to understand how these features influence bone cells performance in vitro and bone formation in vivo. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 479-490, 2018.

Cell sheet engineering using the stromal vascular fraction of adipose tissue as a vascularization strategy.

Current vascularization strategies for Tissue Engineering constructs, in particular cell sheet-based, are limited by time-consuming and expensive endothelial cell isolation and/or by the complexity of using extrinsic growth factors. Herein, we propose an alternative strategy using angiogenic cell sheets (CS) obtained from the stromal vascular fraction (SVF) of adipose tissue that can be incorporated into more complex constructs. Cells from the SVF were cultured in normoxic and hypoxic conditions for up to 8days in the absence of extrinsic growth factors. Immunocytochemistry against CD31 and CD146 revealed spontaneous organization in capillary-like structures, more complex after hypoxic conditioning. Inhibition of HIF-1α pathway hindered capillary-like structure formation in SVF cells cultured in hypoxia, suggesting a role of HIF-1α. Moreover, hypoxic SVF cells showed a trend for increased secretion of angiogenic factors, which was reflected in increased network formation by endothelial cells cultured on matrigel using that conditioned medium. In vivo implantation of SVF CS in a mouse hind limb ischemia model revealed that hypoxia-conditioned CS led to improved restoration of blood flow. Both in vitro and in vivo data suggest that SVF CS can be used as simple and cost-efficient tools to promote functional vascularization of TE constructs. STATEMENT OF SIGNIFICANCE: Neovascularization after implantation is a major obstacle for producing clinically viable cell sheet-based tissue engineered constructs. Strategies using endothelial cells and extrinsic angiogenic growth factors are expensive and time consuming and may raise concerns of tumorigenicity. In this manuscript, we describe a simplified approach using angiogenic cell sheets fabricated from the stromal vascular fraction of adipose tissue. The strong angiogenic behavior of these cell sheets, achieved without the use of external growth factors, was further stimulated by low oxygen culture. When implanted in an in vivo model of hind limb ischemia, the angiogenic cell sheets contributed to blood flux recovery. These cell sheets can therefore be used as a straightforward tool to increase the neovascularization of cell sheet-based thick constructs.

In vivo osteogenic differentiation of stem cells inside compartmentalized capsules loaded with co-cultured endothelial cells.

Capsules coated with polyelectrolytes and co-encapsulating adipose stem (ASCs) and endothelial (ECs) cells with surface modified microparticles are developed. Microparticles and cells are freely dispersed in a liquified core, responsible to maximize the diffusion of essential molecules and allowing the geometrical freedom for the autonomous three-dimensional (3D) organization of cells. While the membrane wraps all the instructive cargo elements within a single structure, the microparticles provide a solid 3D substrate for the encapsulated cells. Our hypothesis is that inside this isolated biomimetic 3D environment, ECs would lead ASCs to differentiate into the osteogenic lineage to ultimately generate a mineralized tissue in vivo. For that, capsules encapsulating only ASCs (MONO capsules) or co-cultured with ECs (CO capsules) are subcutaneously implanted in nude mice up to 6weeks. Capsules implanted immediately after production or after 21days of in vitro osteogenic stimulation are tested. The most valuable outcome of the present study is the mineralized tissue in CO capsules without in vitro pre-differentiation, with similar levels compared to the pre-stimulated capsules in vitro. We believe that the proposed bioencapsulation strategy is a potent self-regulated system, which might find great applicability in bone tissue engineering. STATEMENT OF SIGNIFICANCE: The diffusion efficiency of essential molecules for cell survival is a main issue in cell encapsulation. Former studies reported the superior biological outcome of encapsulated cells within liquified systems. However, most cells used in TE are anchorage-dependent, requiring a solid substrate to perform main cellular processes. We hypothesized that liquified capsules encapsulating microparticles are a promising attempt. Inspired by the multiphenotypic cellular environment of bone, we combine the concept of liquified capsules with co-cultures of stem and endothelial cells. After implantation, results show that co-cultured capsules without in vitro stimulation were able to form a mineralized tissue in vivo. We believe that the present ready-to-use TE strategy requiring minimum in vitro manipulation will find great applicability in bone tissue engineering.

Growth Factor-Free Pre-vascularization of Cell Sheets for Tissue Engineering.

The therapeutic efficacy of tissue-engineered constructs is often compromised by inadequate inosculation and neo-vascularization. This problem is considered one of the biggest hurdles in the field and finding a solution is currently the focus of a great fraction of the research community. Many of the methodologies designed to address this issue propose the use of endothelial cells and angiogenic growth factors, or combinations of both, to accelerate neo-vascularization after transplantation. However, an adequate solution is still elusive. In this context, we describe a methodology that combines the use of the stromal vascular fraction (SVF) isolated from adipose tissue with low oxygen culture to produce pre-vascularized cell sheets as angiogenic tools for Tissue Engineering. The herein proposed approach takes advantage of the SVF angiogenic nature conferred by adipose stem cells, endothelial progenitors, endothelial and hematopoietic cells, and pericytes and further potentiates it using low oxygen, or hypoxic, culture. Freshly isolated nucleated SVF cells are cultured in hyperconfluent conditions under hypoxia (pO2 = 5 %) for up to 5 days in medium without extrinsic growth factors enabling the generation of contiguous sheets as described by the cell sheet engineering technique. Flow cytometry and immunocytochemistry allow confirming the phenotype of the different cell types composing the cell-sheets as well the organization of the CD31(+) cells in branched and highly complex tube-like structures. Overall, a simple and flexible approach to promote growth factor-free pre-vascularization of cell sheets for tissue engineering (TE) applications is described.

Neovascularization Induced by the Hyaluronic Acid-Based Spongy-Like Hydrogels Degradation Products.

Neovascularization has been a major challenge in many tissue regeneration strategies. Hyaluronic acid (HA) of 3-25 disaccharides is known to be angiogenic due to its interaction with endothelial cell receptors. This effect has been explored with HA-based structures but a transitory response is observed due to HA burst biodegradation. Herein we developed gellan gum (GG)-HA spongy-like hydrogels from semi-interpenetrating network hydrogels with different HA amounts. Enzymatic degradation was more evident in the GG-HA with high HA amount due to their lower mechanical stability, also resulting from the degradation itself, which facilitated the access of the enzyme to the HA in the bulk. GG-HA spongy-like hydrogels hyaluronidase-mediated degradation lead to the release of HA oligosaccharides of different amounts and sizes in a HA content-dependent manner which promoted in vitro proliferation of human umbilical cord vein endothelial cells (HUVECs) but not their migration. Although no effect was observed in human dermal microvascular endothelial cells (hDMECs) in vitro, the implantation of GG-HA spongy-like hydrogels in an ischemic hind limb mice model promoted neovascularization in a material-dependent manner, consistent with the in vitro degradation profile. Overall, GG-HA spongy-like hydrogels with a sustained release of HA oligomers are valuable options to improve tissue vascularization, a critical issue in several applications in the tissue engineering and regenerative medicine field.

Semipermeable Capsules Wrapping a Multifunctional and Self-regulated Co-culture Microenvironment for Osteogenic Differentiation.

A new concept of semipermeable reservoirs containing co-cultures of cells and supporting microparticles is presented, inspired by the multi-phenotypic cellular environment of bone. Based on the deconstruction of the "stem cell niche", the developed capsules are designed to drive a self-regulated osteogenesis. PLLA microparticles functionalized with collagen I, and a co-culture of adipose stem (ASCs) and endothelial (ECs) cells are immobilized in spherical liquified capsules. The capsules are coated with multilayers of poly(L-lysine), alginate, and chitosan nano-assembled through layer-by-layer. Capsules encapsulating ASCs alone or in a co-culture with ECs are cultured in endothelial medium with or without osteogenic differentiation factors. Results show that osteogenesis is enhanced by the co-encapsulation, which occurs even in the absence of differentiation factors. These findings are supported by an increased ALP activity and matrix mineralization, osteopontin detection, and the up regulation of BMP-2, RUNX2 and BSP. The liquified co-capsules also act as a VEGF and BMP-2 cytokines release system. The proposed liquified capsules might be a valuable injectable self-regulated system for bone regeneration employing highly translational cell sources.

Boosting and rescuing epidermal superior population from fresh keratinocyte cultures.

Epidermal stem cells (EpSCs) hold great expectations in a regenerative medicine context, but innovative methods that permit to obtain a significant yield of EpSCs or stem-like epidermal cells are still required. We propose a two-step strategy to obtain a superior epidermal stem-like cell fraction among primary keratinocytes (KCs) isolated from adult human skin. The approach is based on the combination of rapid adherence to collagen IV with the rock-associated kinase inhibitor (ROCKi) treatment, and the subsequent immunomagnetic separation of the α6(high)/CD71(dim) cell subset. The combined collagen IV and ROCKi treatment showed not only to enhance cells clonogenic capacity, but also to induce an early epidermal phenotypic signature, along with the diminished expression of late differentiation-associated markers. More importantly, collagen IV and the ROCKi efficiently promoted a synergized effect over α6(high)/CD71(dim) expression, boosting the number of highly proliferative KCs stem-like cells as demonstrated by the expression of ki67. This cell fraction showed a superior ability to generate a 3D stratified epithelium formed by cells with successive differentiation phenotypes. Overall, this strategy indulged the possibility to uncover, among adult KCs, a superior epidermal cell population with stem-like proliferation capacity and early differentiation degree to be used in numerous skin regeneration approaches.

Human skin cell fractions fail to self-organize within a gellan gum/hyaluronic acid matrix but positively influence early wound healing.

Split-thickness autografts still are the current gold standard to treat skin, upon severe injuries. Nonetheless, autografts are dependent on donor site availability and often associated to poor quality neoskin. The generation of dermal-epidermal substitutes by tissue engineering is seen as a promising strategy to overcome this problematic. However, solutions that can be safely and conveniently transplanted in one single surgical intervention are still very challenging as their production normally requires long culture time, and graft survival is many times compromised by delayed vascularization upon transplantation. This work intended to propose a strategy that circumvents the prolonged and laborious preparation period of skin substitutes and allows skin cells self-organization toward improved healing. Human dermal/epidermal cell fractions were entrapped directly from isolation within a gellan gum/hyaluronic acid (GG-HA) spongy-like hydrogel formed from an off-the-shelf dried polymeric network. Upon transplantation into full-thickness mice wounds, the proposed constructs accelerated the wound closure rate and re-epithelialization, as well as tissue neovascularization. A synergistic effect of the GG-HA matrix and the transplanted cells over those processes was demonstrated at early time points. Despite the human-derived and chimeric blood vessels found, the proposed matrix did not succeed in prolonging cells residence time and in sustaining the self-organization of transplanted human cells possibly due to primitive degradation. Despite this, the herein proposed approach open the opportunity to tackle wound healing at early stages contributing to re-epithelialization and neovascularization.