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

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

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

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

Sulfated hydrogels as primary intervertebral disc cell culture systems

INTRODUCTION: Intervertebral disc (IVD) degeneration is a key contributor for low back pain, a leading cause of disability worldwide1. During degeneration, IVD aging is accelerated, leading to progressive structural changes, including blood vessel and nerve ingrowth that promote discogenic pain2. In vitro studies require novel biomaterials that mimic the IVD extracellular matrix (ECM) to provide mechanical support and a reservoir of cytokines and growth factors. As proteoglycans with their attached sulfated glycosaminoglycans (GAGs) are one of the major components of the ECM, the ECM’s sulfation state could be a key factor for IVD cell-fate3. Thus, we aim to explore human NP cell fate using a novel sulfated alginate model with varying degrees of sulfation (DS). METHODS: Primary human NP cells were expanded, mixed with solutions of i) 2.5% of standard alginate, ii) 0.1 DS, and iii) 0.2 DS alginate (4 x 106 cells/ml) and casted in 27 l cylindrical-shaped carriers (4 mm diameter, 2 mm height). Carriers were cultured for two weeks for phenotype recovery and were collected with the culture media on day 0, 7 and 14. RESULTS: A significant decrease of cell density (p<0.05) was observed in 0.2 DS alginate after 7 and 14 days of culture. Similarly, cell viability was significantly reduced (p<0.05) in 0.2 DS alginate after 7 days of culture (N=4). In addition, cell metabolic activity tended to be decreased in 0.2 DS alginate compared to standard alginate after 14 days of culture. Surprisingly, ECM remodeling factors such as MMP2 and TIMP1 were slightly upregulated in the 0.1 DS group (N=1), whereas catabolic cytokines were downregulated in the 0.1% DS group. DISCUSSION & CONCLUSIONS: We demonstrate significant cellular differences between 0.2 DS alginate vs standard alginate and 0.1 DS alginate. Particularly, a significant decrease in cell density, metabolic activity and viability were observed in the 0.2 DS alginate after 7 days of culture. According to the secretome, the sulfated alginate group seems to possess increased catabolic ECM remodeling with lower secretion of catabolic factors, suggesting less responsive NP cells to ECM structural changes. Overall, standard alginate seems to be the best option for NP cell 3D culture models. ACKNOWLEDGEMENTS: This project was supported by the Marie Skłodowska Curie International Training Network “disc4all” under the grant agreement #955735. REFERENCES: 1FY. Wang et al (2020) JOR Spine 5:1186. 2P. Bermudez-Lekerika et al (2022) Front Cell Dev Biol 29(10):924692. 3E. Lazarus et al (2021) Cells 10(12):3568. Keywords: Hydrogels and injectable systems, In vitro microenvironment

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

Biological and Biomechanical Testing of Bioengineered, Vascularized 3D Skin Substitutes Based on Crosslinked Collagen Hydrogels

Extensive soft tissue defects, such as burn and chronic wounds, frequently require the transplantation of bioengineered skin substitutes. Tissue engineering using autologous patient-derived cells represents the most promising approach towards restoring the full functionality of the affected areas. However, the successful translation of current bioengineered dermo-epidermal skin models to restore all skin functions to the full extent is hampered by several challenges. Firstly, although many engineered scaffolds enable the ingrowth of peripheral sensory neurons from the host, little to no attention has been given to the inclusion of mechano-sensitive epidermal cells required for fine touch sensation. This is particularly true in the case of spina bifida during fetal development, where the use of acellular scaffolds to close the tissue defect might negatively impact the restoration of sensory functions following a surgical intervention. Merkel cells are of special interest as they transduce tactile stimuli and the relative ease with which they can be isolated and cultured makes them an attractive alternative to other skin-resident sensory corpuscles. The present work described for the first time the isolation and in vitro characterization of fetal Merkel cells. These cells were incorporated into collagen type I-based avascular bioengineered dermo-epidermal skin substitutes, and subsequently transplanted using an in vivo wound healing model. Our results showed that the phenotype and functionality of Merkel cells can be retained both in vitro and in vivo, thus providing the basis for a full restoration of fine touch sensation following certain surgical interventions in utero. The production time required for such dermo-epidermal skin substitutes can last up to 4 weeks. This constitutes a significant drawback and might hinder their translation into clinics. We addressed the problem of slow in vitro maturation of bioengineered skin in a second study, using a novel bioreactor setup that applied cyclic stretch to bioengineered, physically crosslinked collagen scaffolds. Application of cyclic stretch significantly increased both fibroblast proliferation and production of extracellular matrix proteins, thereby reducing the pre-conditioning time required for optimal keratinocyte adhesion and subsequent formation of a stable epidermal layer. Successful translation of bioengineered tissue is crucially dependent on its successful perfusion with blood in order to provide nutrients and oxygen to the transplant and mediate inflammatory processes to aid in the wound healing response. Recently, the important role of lymphatics in modulating the immune response and restoring tissue homeostasis during wound healing has gained more attention as well. Most bioengineered skin models, however, still lack a well-developed and mature blood and lymphatic plexus. The second part of this thesis thus aimed at the co-engineering of blood and lymphatic microvascular networks, and characterization of their role following the host immune response. To this end, in a third study we focused on developing a novel sorting strategy which allowed us to isolate blood (BECs) from lymphatic endothelial cells (LECs) using fluorescence activated cell sorting. Furthermore, we demonstrated consistent expression of the immunomodulatory membrane glycoprotein CD200 on BECs and LECs, both in vitro and in vivo. In a related study, we successfully characterized the immunomodulatory activity of the cell adhesion molecule CD157 on a mixed population consisting of BECs and LECs called human dermal microvascular endothelial cells (HDMECs). Compared to CD200+ endothelial cells, prolonged in vitro culturing of CD157+ endothelial cells resulted in rapid downregulation of the marker expression. Our analysis showed that engineered microvascular networks enriched for either cell type led to improved immune cell trafficking as well as innate immune responses in vivo. Lastly, in a fifth study, we aimed at improving the versatility of native collagen type I hydrogels for the purpose of vascular tissue engineering. To this end, a covalent modification of the collagen backbone was introduced that enabled bio-orthogonal enzymatic crosslinking using the two enzymes sortase A (SrtA) and Factor XIII (FXIII). Bulk hydrogels that were produced by exploiting the enzymatic activity of SrtA supported the tunable neovascularization with blood and lymphatic vascular networks by modulating the hydrogel stiffness or by using variable input ratios of BECs and LECs, respectively. Furthermore, the use of FXIII as an orthogonal crosslinking enzyme was successfully used to demonstrate the concomitant tethering of the proangiogenic QK peptide to the collagen backbone during hydrogel formation, thus providing a means for vascular outgrowth in the absence of exogenous growth factors. Furthermore, collagen hydrogels with spatially defined polymer compositions or microporosity based on secondary annealing of microgels demonstrated the broad versatility of orthogonally crosslinked collagen for the bioengineering of blood and lymphatic microcapillary networks

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

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.ISSN:0935-9648ISSN:1521-409