Breathing new life into tissue engineering: exploring cutting-edge vascularization strategies for skin substitutes

Tissue-engineered skin substitutes (TESS) emerged as a new therapeutic option to improve skin transplantation. However, establishing an adequate and rapid vascularization in TESS is a critical factor for their clinical application and successful engraftment in patients. Therefore, several methods have been applied to improve the vascularization of skin substitutes including (i) modifying the structural and physicochemical properties of dermal scaffolds; (ii) activating biological scaffolds with growth factor-releasing systems or gene vectors; and (iii) developing prevascularized skin substitutes by loading scaffolds with capillary-forming cells. This review provides a detailed overview of the most recent and important developments in the vascularization strategies for skin substitutes. On the one hand, we present cell-based approaches using stem cells, microvascular fragments, adipose tissue derived stromal vascular fraction, endothelial cells derived from blood and skin as well as other pro-angiogenic stimulation methods. On the other hand, we discuss how distinct 3D bioprinting techniques and microfluidics, miRNA manipulation, cell sheet engineering and photosynthetic scaffolds like GelMA, can enhance skin vascularization for clinical applications. Finally, we summarize and discuss the challenges and prospects of the currently available vascularization techniques that may serve as a steppingstone to a mainstream application of skin tissue engineering

Immunomodulation of Skin Repair: Cell-Based Therapeutic Strategies for Skin Replacement (A Comprehensive Review)

The immune system has a crucial role in skin wound healing and the application of specific cell-laden immunomodulating biomaterials emerged as a possible treatment option to drive skin tissue regeneration. Cell-laden tissue-engineered skin substitutes have the ability to activate immune pathways, even in the absence of other immune-stimulating signals. In particular, mesenchymal stem cells with their immunomodulatory properties can create a specific immune microenvironment to reduce inflammation, scarring, and support skin regeneration. This review presents an overview of current wound care techniques including skin tissue engineering and biomaterials as a novel and promising approach. We highlight the plasticity and different roles of immune cells, in particular macrophages during various stages of skin wound healing. These aspects are pivotal to promote the regeneration of nonhealing wounds such as ulcers in diabetic patients. We believe that a better understanding of the intrinsic immunomodulatory features of stem cells in implantable skin substitutes will lead to new translational opportunities. This, in turn, will improve skin tissue engineering and regenerative medicine applications. Keywords: biomaterials; chronic wounds; immunomodulation; intrinsic immune cell signals; regenerative medicine; skin substitutes; skin tissue engineering; wound healing

Effects of an Adipose Mesenchymal Stem Cell-Derived Conditioned medium and TGF-β1 on Human Keratinocytes In Vitro

Human keratinocytes play a crucial role during skin wound healing and in skin replacement therapies. The secretome of adipose-derived stem cells (ASCs) has been shown to secrete pro-healing factors, among which include TGF-β1, which is essential for keratinocyte migration and the re-epithelialization of cutaneous wounds during skin wound healing. The benefits of an ASC conditioned medium (ASC-CM) are primarily orchestrated by trophic factors that mediate autocrine and paracrine effects in keratinocytes. Here, we evaluated the composition and the innate characteristics of the ASC secretome and its biological effects on keratinocyte maturation and wound healing in vitro. In particular, we detected high levels of different growth factors, such as HGF, FGFb, and VEGF, and other factors, such as TIMP1 and 4, IL8, PAI-1, uPA, and IGFBP-3, in the ASC-CM. Further, we investigated, using immunofluorescence and flow cytometry, the distinct effects of a human ASC-CM and/or synthetic TGF-β1 on human keratinocyte proliferation, migration, and cell apoptosis suppression. We demonstrated that the ASC-CM increased keratinocyte proliferation as compared to TGF-β1 treatment. Further, we found that the ASC-CM exerted cell cycle progression in keratinocytes via regulating the phases G1, S, and G2/M. In particular, cells subjected to the ASC-CM demonstrated increased DNA synthesis (S phase) compared to the TGF-β1-treated KCs, which showed a pronounced G0/G1 phase. Furthermore, both the ASC-CM and TGF-β1 conditions resulted in a decreased expression of the late differentiation marker CK10 in human keratinocytes in vitro, whereas both treatments enhanced transglutaminase 3 and loricrin expression. Interestingly, the ASC-CM promoted significantly increased numbers of keratinocytes expressing epidermal basal keratinocyte markers, such DLL1 and Jagged2 Notch ligands, whereas those ligands were significantly decreased in TGF-β1-treated keratinocytes. In conclusion, our findings suggest that the ASC-CM is a potent stimulator of human keratinocyte proliferation in vitro, particularly supporting basal keratinocytes, which are crucial for a successful skin coverage after transplantation. In contrast, TGF-β1 treatment decreased keratinocyte proliferation and specifically increased the expression of differentiation markers in vitro

CD146 expression profile in human skin and pre-vascularized dermo-epidermal skin substitutes in vivo

Background CD146 is a cell adhesion molecule whose expression profile in human skin has not yet been elucidated. Here, we characterize CD146 expression pattern in human skin, in particular in blood endothelial cells (BECs) and lymphatic endothelial cells (LECs), which constitute human dermal microvascular endothelial cells (HDMECs), as well as in perivascular cells. Results We demonstrated that CD146 is a specific marker of BECs, but not of LECs. Moreover, we found CD146 expression also in human pericytes surrounding blood capillaries in human skin. In addition, we demonstrated that CD146 expression is up-regulated by the TNFα-IL-1β/NF-kB axis in both BECs and pericytes. Finally, we engineered 3D collagen hydrogels composed of HDMECs, CD146+ pericytes, and fibroblasts which developed, in vitro and in vivo, a complete microvasculature network composed of blood and lymphatic capillaries with pericytes investing blood capillaries. Conclusions Overall, our results proved that CD146 is a specific marker of BECs and pericytes, but not LECs in human skin. Further, the combination of CD146+ pericytes with HDMECs in skin substitutes allowed to bioengineer a comprehensive 3D in vitro and in vivo model of the human dermal microvasculature

Bio-engineering a prevascularized human tri-layered skin substitute containing a hypodermis

Severe injuries to skin including hypodermis require full-thickness skin replacement. Here, we bioengineered a tri-layered human skin substitute (TLSS) containing the epidermis, dermis, and hypodermis. The hypodermal layer was generated by differentiation of human adipose stem cells (ASC) in a collagen type I hydrogel and combined with a prevascularized dermis consisting of human dermal microvascular endothelial cells and fibroblasts, which arranged into a dense vascular network. Subsequently, keratinocytes were seeded on top to generate the epidermal layer of the TLSS. The differentiation of ASC into adipocytes was confirmed in vitro on the mRNA level by the presence of adiponectin, as well as by the expression of perilipin and FABP-4 proteins. Moreover, functional characteristics of the hypodermis in vitro and in vivo were evaluated by Oil Red O, BODIPY, and AdipoRed stainings visualizing intracellular lipid droplets. Further, we demonstrated that both undifferentiated ASC and mature adipocytes present in the hypodermis influenced the keratinocyte maturation and homeostasis in the skin substitutes after transplantation. In particular, an enhanced secretion of TGF-β1 by these cells affected the epidermal morphogenesis as assessed by the expression of key proteins involved in the epidermal differentiation including cytokeratin 1, 10, 19 and cornified envelope formation such as involucrin. Here, we propose a novel functional hypodermal-dermo-epidermal tri-layered skin substitute containing blood capillaries that efficiently promote regeneration of skin defects. Statement of significance The main objective of this study was to develop and assess the usefulness of a tri-layered human prevascularized skin substitute (TLSS) containing an epidermis, dermis, and hypodermis. The bioengineered hypodermis was generated from human adipose mesenchymal stem cells (ASC) and combined with a prevascularized dermis and epidermis. The TLSS represents an exceptional model for studying the role of cell-cell and cell-matrix interactions in vitro and in vivo. In particular, we observed that enhanced secretion of TGF-β1 in the hypodermis exerted a profound impact on fibroblast and keratinocyte differentiation, as well as epidermal barrier formation and homeostasis. Therefore, improved understanding of the cell-cell interactions in such a physiological skin model is essential to gain insights into different aspects of wound healing

The Role of CD200–CD200 Receptor in Human Blood and Lymphatic Endothelial Cells in the Regulation of Skin Tissue Inflammation

CD200 is a cell membrane glycoprotein that interacts with its structurally related receptor (CD200R) expressed on immune cells. We characterized CD200–CD200R interactions in human adult/juvenile (j/a) and fetal (f) skin and in in vivo prevascularized skin substitutes (vascDESS) prepared by co-culturing human dermal microvascular endothelial cells (HDMEC), containing both blood (BEC) and lymphatic (LEC) EC. We detected the highest expression of CD200 on lymphatic capillaries in j/a and f skin as well as in vascDESS in vivo, whereas it was only weakly expressed on blood capillaries. Notably, the highest CD200 levels were detected on LEC with enhanced Podoplanin expression, while reduced expression was observed on Podoplanin-low LEC. Further, qRT-PCR analysis revealed upregulated expression of some chemokines, including CC-chemokine ligand 21 (CCL21) in j/aCD200+ LEC, as compared to j/aCD200− LEC. The expression of CD200R was mainly detected on myeloid cells such as granulocytes, monocytes/macrophages, T cells in human peripheral blood, and human and rat skin. Functional immunoassays demonstrated specific binding of skin-derived CD200+ HDMEC to myeloid CD200R+ cells in vitro. Importantly, we confirmed enhanced CD200–CD200R interaction in vascDESS in vivo. We concluded that the CD200–CD200R axis plays a crucial role in regulating tissue inflammation during skin wound healing

Combining bioengineered human skin with bioprinted cartilage for ear reconstruction

Microtia is a congenital disorder that manifests as a malformation of the external ear leading to psychosocial problems in affected children. Here, we present a tissue-engineered treatment approach based on a bioprinted autologous auricular cartilage construct (EarCartilage) combined with a bioengineered human pigmented and prevascularized dermo-epidermal skin substitute (EarSkin) tested in immunocompromised rats. We confirmed that human-engineered blood capillaries of EarSkin connected to the recipient’s vasculature within 1 week, enabling rapid blood perfusion and epidermal maturation. Bioengineered EarSkin displayed a stratified epidermis containing mature keratinocytes and melanocytes. The latter resided within the basal layer of the epidermis and efficiently restored the skin color. Further, in vivo tests demonstrated favorable mechanical stability of EarCartilage along with enhanced extracellular matrix deposition. In conclusion, EarCartilage combined with EarSkin represents a novel approach for the treatment of microtia with the potential to circumvent existing limitations and improve the aesthetic outcome of microtia reconstruction

Bioprinting and plastic compression of large pigmented and vascularized human dermo-epidermal skin substitutes by means of a new robotic platform

Extensive availability of engineered autologous dermo-epidermal skin substitutes (DESS) with functional and structural properties of normal human skin represents a goal for the treatment of large skin defects such as severe burns. Recently, a clinical phase I trial with this type of DESS was successfully completed, which included patients own keratinocytes and fibroblasts. Yet, two important features of natural skin were missing: pigmentation and vascularization. The first has important physiological and psychological implications for the patient, the second impacts survival and quality of the graft. Additionally, accurate reproduction of large amounts of patient’s skin in an automated way is essential for upscaling DESS production. Therefore, in the present study, we implemented a new robotic unit (called SkinFactory) for 3D bioprinting of pigmented and pre-vascularized DESS using normal human skin derived fibroblasts, blood- and lymphatic endothelial cells, keratinocytes, and melanocytes. We show the feasibility of our approach by demonstrating the viability of all the cells after printing in vitro, the integrity of the reconstituted capillary network in vivo after transplantation to immunodeficient rats and the anastomosis to the vascular plexus of the host. Our work has to be considered as a proof of concept in view of the implementation of an extended platform, which fully automatize the process of skin substitution: this would be a considerable improvement of the treatment of burn victims and patients with severe skin lesions based on patients own skin derived cells

Characterization of Distinct Chondrogenic Cell Populations of Patients Suffering from Microtia Using Single-Cell Micro-Raman Spectroscopy

Microtia is a congenital condition of abnormal development of the outer ear. Tissue engineering of the ear is an alternative treatment option for microtia patients. However, for this approach, the identification of high regenerative cartilage progenitor cells is of vital importance. Raman analysis provides a novel, non-invasive, label-free diagnostic tool to detect distinctive biochemical features of single cells or tissues. Using micro-Raman spectroscopy, we were able to distinguish and characterize the particular molecular fingerprints of differentiated chondrocytes and perichondrocytes and their respective progenitors isolated from healthy individuals and microtia patients. We found that microtia chondrocytes exhibited lower lipid concentrations in comparison to healthy cells, thus indicating the importance of fat storage. Moreover, we suggest that collagen is a useful biomarker for distinguishing between populations obtained from the cartilage and perichondrium because of the higher spectral contributions of collagen in the chondrocytes compared to perichondrocytes from healthy individuals and microtia patients. Our results represent a contribution to the identification of cell markers that may allow the selection of specific cell populations for cartilage tissue engineering. Moreover, the observed differences between microtia and healthy cells are essential for gaining better knowledge of the cause of microtia. It can be useful for designing novel treatment options based on further investigations of the discovered biochemical substrate alterations