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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.

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.

Adequate vascularization is required for the successful translation of many in vitro engineered tissues. This study presents a novel collagen derivative that harbors multiple recognition peptides for orthogonal enzymatic crosslinking based on sortase A (SrtA) and Factor XIII (FXIII). SrtA-mediated crosslinking enables the rapid co-engineering of human blood and lymphatic microcapillaries and mesoscale capillaries in bulk hydrogels. Whereas tuning of gel stiffness determines the extent of neovascularization, the relative number of blood and lymphatic capillaries recapitulates the ratio of blood and lymphatic endothelial cells originally seeded into the hydrogel. Bioengineered capillaries readily form luminal structures and exhibit typical maturation markers both in vitro and in vivo. The secondary crosslinking enzyme Factor XIII is used for in situ tethering of the VEGF mimetic QK peptide to collagen. This approach supports the formation of blood and lymphatic capillaries in the absence of exogenous VEGF. Orthogonal enzymatic crosslinking is further used to bioengineer hydrogels with spatially defined polymer compositions with pro- and anti-angiogenic properties. Finally, macroporous scaffolds based on secondary crosslinking of microgels enable vascularization independent from supporting fibroblasts. Overall, this work demonstrates for the first time the co-engineering of mature micro- and meso-sized blood and lymphatic capillaries using a highly versatile collagen derivative.

Amniotic fluid has been proposed as an easily available source of cells for numerous applications in regenerative medicine and tissue engineering. The use of amniotic fluid cells in biomedical applications necessitates their unequivocal characterization; however, the exact cellular composition of amniotic fluid and the precise tissue origins of these cells remain largely unclear. Using cells cultured from the human amniotic fluid of fetuses with spina bifida aperta and of a healthy fetus, we performed single-cell RNA sequencing to characterize the tissue origin and marker expression of cultured amniotic fluid cells at the single-cell level. Our analysis revealed nine different cell types of stromal, epithelial and immune cell phenotypes, and from various fetal tissue origins, demonstrating the heterogeneity of the cultured amniotic fluid cell population at a single-cell resolution. It also identified cell types of neural origin in amniotic fluid from fetuses with spina bifida aperta. Our data provide a comprehensive list of markers for the characterization of the various progenitor and terminally differentiated cell types in cultured amniotic fluid. This study highlights the relevance of single-cell analysis approaches for the characterization of amniotic fluid cells in order to harness their full potential in biomedical research and clinical applications.

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.

Human skin contains specialized neuroendocrine Merkel cells responsible for fine touch sensation. In the present study, we performed in-depth analysis of Merkel cells in human fetal back skin. We revealed that these Merkel cells expressed cytokeratin 20 (CK20), were positive for the neuroendocrine markers synaptophysin and chromogranin A, and the mechanosensitive ion channel Piezo2. Further, we demonstrated that Merkel cells were present in freshly isolated human fetal epidermal cells in vitro, and in tissue-engineered human dermo-epidermal skin substitutes 4 weeks after transplantation on immune-compromised rats. Merkel cells retained the expression of CK20, synaptophysin, chromogranin A, and Piezo2 after isolation and in culture, and in the skin substitutes after transplantation. Interestingly, we observed that in fetal skin and in skin substitutes, only Merkel cells were positive for CK8, while in culture, also non-Merkel cells showed positivity for CK8. In summary, human fetal Merkel cells showed phenotypical features confirming their cell identity. This findings are of pivotal importance for the future application of fetal tissue-engineered skin in clinics.

The evidence for fibroblast heterogeneity is continuously increasing, and recent work has shed some light on the existence of different sub-populations of fibroblasts in the human skin. Although we now have a more precise understanding of their distribution in the human body, we do not know whether their properties are predictive of where these cells derive from or whether these sub-types have functional consequences. In this study, we employed single-cell transcriptomics (10X Genomics) to study gene expression and segregate fibroblast sub-populations based on their genetic signature. We report the differential expression of a defined set of genes in fibroblasts from human skin, which may contribute to their dynamicity in vivo and in vitro. We show that the sub-population of fibroblasts expressing cytoskeletal markers, such as ANXA2, VIM, ACTB, are enriched in an adult skin sample. Interestingly, this sub-population of fibroblasts is not enriched in a neonatal skin sample but becomes predominant when neonatal fibroblasts are cultivated. On the other hand, the fibroblast sub-populations expressing COL1A1 and ELN are enriched in neonatal skin but are reduced in the adult skin and in fibroblasts from neonatal skin that are cultured in vitro. Our results indicate that fibroblasts are a dynamic cell type, and while their genetic make-up changes markedly, only a handful of genes belonging to the same functional pathway govern this alteration. The gene expression pattern of cytoskeletal markers may help in identifying whether the fibroblasts were isolated from an adult or an infant or whether they were cultivated, and this information could be useful for quality control in clinics and in cell banking. Furthermore, this study opens additional avenues to investigate the role of these markers in defining the complexity of human dermal fibroblasts.

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.

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.

It is still unknown whether the human interfollicular epidermis harbors a reservoir of melanocyte precursor cells. Here, we clearly distinguish between three distinct types of melanocytes in human interfollicular epidermis: (1) cKit+CD90-, (2) cKit+CD90+, and (3) cKit-CD90+. Importantly, we identify the Kit tyrosine kinase receptor (cKit) as a marker expressed specifically in mature, melanin-producing melanocytes. Thus, both cKit+CD90- and cKit+CD90+ cells represent polydendritic, pigmented mature melanocytes, whereas cKit-CD90+ cells display bipolar, non-dendritic morphology with reduced melanin content. Additionally, using tissue-engineered pigmented dermo-epidermal skin substitutes (melDESSs), we reveal that the cKit expression also plays an important role during melanogenesis in melDESS in vivo. Interestingly, cKit-CD90+ cells lack the expression of markers such as HMB45, TYR, and TRP1 in vitro and in vivo. However, they co-express neural-crest progenitor markers and demonstrate multilineage differentiation potential in vitro. Hence, we propose that cKit-CD90+ cells constitute the precursor melanocyte reservoir in human interfollicular epidermis. Keywords: dermo-epidermal skin substitutes; human epidermis; mature melanocytes; melanin synthesis; melanocyte stem cells; neural-crest progenitor cells; skin pigmentation; tissue engineering.

CD157 acts as a receptor, regulating leukocyte trafficking and the binding of extracellular matrix components. However, the expression pattern and the role of CD157 in human blood (BEC) and the lymphatic endothelial cells (LEC) of human dermal microvascular cells (HDMEC), remain elusive. We demonstrated constitutive expression of CD157 on BEC and LEC, in fetal and juvenile/adult skin, in situ, as well as in isolated HDMEC. Interestingly, CD157 epitopes were mostly localized on BEC, co-expressing high levels of CD31 (CD31High), as compared to CD31Low BEC, whereas the podoplanin expression level on LEC did not affect CD157. Cultured HDMEC exhibited significantly higher numbers of CD157-positive LEC, as compared to BEC. Interestingly, separated CD157− and CD157+ HDMEC demonstrated no significant differences in clonal expansion in vitro, but they showed distinct expression levels of cell adhesion molecules, before and after cytokine stimulation in vitro. In particular, we proved the enhanced and specific adherence of CD11b-expressing human blood myeloid cells to CD157+ HDMEC fraction, using an in vitro immune-binding assay. Indeed, CD157 was also involved in chemotaxis and adhesion of CD11b/c monocytes/neutrophils in prevascularized dermo–epidermal skin substitutes (vascDESS) in vivo. Thus, our data attribute specific roles to endothelial CD157, in the regulation of innate immunity during inflammation.