Light from Afield: Fast, High-Resolution, and Layer-Free Deep Vat 3D Printing.

Harnessing light for cross-linking of photoresponsive materials has revolutionized the field of 3D printing. A wide variety of techniques leveraging broad-spectrum light shaping have been introduced as a way to achieve fast and high-resolution printing, with applications ranging from simple prototypes to biomimetic engineered tissues for regenerative medicine. Conventional light-based printing techniques use cross-linking of material in a layer-by-layer fashion to produce complex parts. Only recently, new techniques have emerged which deploy multidirection, tomographic, light-sheet or filamented light-based image projections deep into the volume of resin-filled vat for photoinitiation and cross-linking. These Deep Vat printing (DVP) approaches alleviate the need for layer-wise printing and enable unprecedented fabrication speeds (within a few seconds) with high resolution (>10 µm). Here, we elucidate the physics and chemistry of these processes, their commonalities and differences, as well as their emerging applications in biomedical and non-biomedical fields. Importantly, we highlight their limitations, and future scope of research that will improve the scalability and applicability of these DVP techniques in a wide variety of engineering and regenerative medicine applications.

Synergizing Algorithmic Design, Photoclick Chemistry and Multi-Material Volumetric Printing for Accelerating Complex Shape Engineering.

The field of biomedical design and manufacturing has been rapidly evolving, with implants and grafts featuring complex 3D design constraints and materials distributions. By combining a new coding-based design and modeling approach with high-throughput volumetric printing, a new approach is demonstrated to transform the way complex shapes are designed and fabricated for biomedical applications. Here, an algorithmic voxel-based approach is used that can rapidly generate a large design library of porous structures, auxetic meshes and cylinders, or perfusable constructs. By deploying finite cell modeling within the algorithmic design framework, large arrays of selected auxetic designs can be computationally modeled. Finally, the design schemes are used in conjunction with new approaches for multi-material volumetric printing based on thiol-ene photoclick chemistry to rapidly fabricate complex heterogeneous shapes. Collectively, the new design, modeling and fabrication techniques can be used toward a wide spectrum of products such as actuators, biomedical implants and grafts, or tissue and disease models.

Enzymatically Crosslinked Collagen as a Versatile Matrix for In Vitro and In Vivo Co-Engineering of Blood and Lymphatic Vasculature.

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.

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-1ß/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.

Human fetal skin derived merkel cells display distinctive characteristics in vitro and in bio-engineered skin substitutes in vivo.

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.

Filamented Light (FLight) Biofabrication of Highly Aligned Tissue-Engineered Constructs.

Cell-laden hydrogels used in tissue engineering generally lack sufficient 3D topographical guidance for cells to mature into aligned tissues. A new strategy called filamented light (FLight) biofabrication rapidly creates hydrogels composed of unidirectional microfilament networks, with diameters on the length scale of single cells. Due to optical modulation instability, a light beam is divided optically into FLight beams. Local polymerization of a photoactive resin is triggered, leading to local increase in refractive index, which itself creates self-focusing waveguides and further polymerization of photoresin into long hydrogel microfilaments. Diameter and spacing of the microfilaments can be tuned from 2 to 30 µm by changing the coherence length of the light beam. Microfilaments show outstanding cell instructive properties with fibroblasts, tenocytes, endothelial cells, and myoblasts, influencing cell alignment, nuclear deformation, and extracellular matrix deposition. FLight is compatible with multiple types of photoresins and allows for biofabrication of centimeter-scale hydrogel constructs with excellent cell viability within seconds (<10 s per construct). Multidirectional microfilaments are achievable within a single hydrogel construct by changing the direction of FLight projection, and complex multimaterial/multicellular tissue-engineered constructs are possible by sequentially exchanging the cell-laden photoresin. FLight offers a transformational approach to developing anisotropic tissues using photo-crosslinkable biomaterials.

Human Basal and Suprabasal Keratinocytes Are Both Able to Generate and Maintain Dermo-Epidermal Skin Substitutes in Long-Term In Vivo Experiments.

The basal layer of human interfollicular epidermis has been described to harbour both quiescent keratinocyte stem cells and a transit amplifying cell population that maintains the suprabasal epidermal layers. We performed immunofluorescence analyses and revealed that the main proliferative keratinocyte pool in vivo resides suprabasally. We isolated from the human epidermis two distinct cell populations, the basal and the suprabasal keratinocytes, according to the expression of integrin ß4 (iß4). We compared basal iß4+ or suprabasal iß4- keratinocytes with respect to their proliferation and colony-forming ability and their Raman spectral properties. In addition, we generated dermo-epidermal substitutes using freshly isolated and sorted basal iß4+ or suprabasal iß4- keratinocytes and transplanted them on immuno-compromised rats. We show that suprabasal iß4- keratinocytes acquire a similar proliferative capacity as basal iß4+ keratinocytes after two weeks of culture in vitro, with expression of high levels of iß4 and downregulation of K10 expression. In addition, both basal iß4+ and suprabasal iß4- keratinocytes acquire authentic self-renewing properties during the in vitro 3D-culture phase and are able to generate and maintain a fully stratified epidermis for 16 weeks in vivo. Therefore, against the leading dogma, we propose that human suprabasal keratinocytes can retro-differentiate into true basal stem cells in a wound situation and/or when in contact with the basement membrane.

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.

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.

Expression Profile of CD157 Reveals Functional Heterogeneity of Capillaries in Human Dermal Skin.

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.

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-ß1 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-ß1 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.

Mechanical stimulation induces rapid fibroblast proliferation and accelerates the early maturation of human skin substitutes.

The clinical treatment of large, full-thickness skin injuries with tissue-engineered autologous dermo-epidermal skin substitutes is an emerging alternative to split-thickness skin grafting. However, their production requires about one month of in vitro cell and tissue culture, which is a significant drawback for the treatment of patients with severe skin defects. With the aim to reduce the production time, we developed a new dynamic bioreactor setup that applies cyclic biaxial tension to collagen hydrogels for skin tissue engineering. By reliably controlling the time history of mechanical loading, the dynamic culturing results in a three-fold increase in collagen hydrogel stiffness and stimulates the embedded fibroblasts to enter the cell cycle. As a result, the number of fibroblasts is increased by 75% compared to under corresponding static culturing. Enhanced fibroblast proliferation promotes expression of dermal extracellular matrix proteins, keratinocyte proliferation, and the early establishment of the epidermis. The time required for early tissue maturation can therefore be reduced by one week. Analysis of the separate effects of cyclic loading, matrix stiffening, and interstitial fluid flow indicates that cyclic deformation is the dominant biophysical factor determining fibroblast proliferation, while tissue stiffening plays a lesser role. Local differences in the direction of deformation (in-plane equibiaxial vs. uniaxial strain) influence fibroblast orientation but not proliferation, nor the resulting tissue properties. Importantly, dynamic culturing does not activate fibroblast differentiation into myofibroblasts. The present work demonstrates that control of mechanobiological cues can be very effective in driving cell response toward a shorter production time for human skin substitutes.