Periodic Reporting for period 2 - SkinTERM (Skin Tissue Engineering and Regenerative Medicine: From skin repair to regeneration)
Período documentado: 2022-10-01 hasta 2025-07-31
Treating skin burns and large trauma using transplantation of autologous split-thickness skin carries a number of serious drawbacks, including pain, mobility-limiting contractures and disfiguring scars. The EU-funded SkinTERM project aims to address wound healing in a completely different way: reproducing embryonic skin development in adults by aiming for regeneration rather than repair. Skin organogenesis will be induced by key elements taken from the extracellular matrix of fetal skin and from skin of species that exhibit no scarring, and by employing (stem) cells from relevant cellular origins.
The general training objective was to deliver excellent, supradisciplinary and intersectorially trained, highly employable scientists with knowledge necessary to drive this research area further towards clinical translation in Europe. Research objectives included 1) to identify important elements of the extracellular matrix involved in scarring and non-scarring systems, 2) to investigate the relevance of (stem) cells and cellular origin in scarring and non-scarring systems, 3) to investigate methodologies to regenerate skin appendages and sensory nerves in skin after wounding, as these are not restored during repair and 4) to take initial steps to translate the research results into clinical applications (medical devices and advanced therapy medicinal products).
The general training objective was to deliver excellent, supradisciplinary and intersectorially trained, highly employable scientists with knowledge necessary to drive this research area further towards clinical translation in Europe. Research objectives included 1) to identify important elements of the extracellular matrix involved in scarring and non-scarring systems, 2) to investigate the relevance of (stem) cells and cellular origin in scarring and non-scarring systems, 3) to investigate methodologies to regenerate skin appendages and sensory nerves in skin after wounding, as these are not restored during repair and 4) to take initial steps to translate the research results into clinical applications (medical devices and advanced therapy medicinal products).
The WP’s progress towards the objectives in the reporting period are summarized below.
WP2 “The regenerative matrix”, different components were prepared to be included in bioscaffolds, e.g. type III collagen and fractions of elastin hydrolysates. First prototypes of 3D collagen-based bioscaffolds were constructed and characterized. For extracellular matrix production by fibroblasts of different origins, protocols were optimized and initial characterizations performed. Three Early-stage researchers (ESRs) participated in a laboratory secondment at ADBC in Beverwijk to study the behaviour of various fibroblasts on biomaterials. Another ESR established a protocol to decellularize dermis of mouse and spiny mouse. During two distinct ESR secondments at HMGU, the basis was laid for collaborative proteomics studies. For this, the host ESR is setting up a histology-directed workflow for the molecular analysis of ECM proteins in an animal model for skin regeneration and in vitro cultured human foetal/adult fibroblasts while performing pilot experiments.
WP3 “Regenerative non-scarring cells”, one ESR has been trained spiny mouse handling and was able to adapt existing histological techniques protocols of tissue to the particularities of Acomys skin. For human foetal, adult, or eschar fibroblasts, protocols regarding e.g. RNA isolation, RT-qPCR and transcriptome sequencing were optimized for mesenchymal cell populations of different origins. To study the human fibroblast lineages accountable for scarless skin regeneration, another ESR successfully established a human induced pluripotent stem cells (iPSC)-derived skin organoid model. In addition, differentiation protocols for blood-derived monocytes to differentiate into pro-inflammatory or anti-inflammatory macrophage subsets were optimized.
WP4 “Introduction of skin appendages and sensory nerves”, focus lies on 1) human sweat gland stem cells, 2) human hair proto-follicles, and 3) human nerve cells. Human iPSC-derived skin organoids were used to isolate and propagate sweat gland stem cells, where sweat gland-specific markers proved that after long-time culture (day 120) the fate choice towards sweat gland formation was achieved. Human hair proto-follicle studies applied hair follicle dermal cell cultures in 2D and bilayered 3D spheroids. To investigate the neurovascular link, human endothelial cells and nerve cells are being included into collagen hydrogels to create a vascular link with nerve cells. For this purpose, human dermal microvascular endothelial cells were isolated and sorted from human skin biopsies and commercial human iPSCs cultures were established.
WP5 “Towards translation into medical devices and ATMPs”, prototypes of an intact three-dimensional collagen biomatrix were produced by different approaches and compounds and the most promising prototype was defined according to key opinion leader meetings, internal evaluation, and market needs. In addition, the first protocol for a melanocyte-containing tissue-engineered skin construct was designed and executed.
In WP6 “Education and training”, ESRs are trained in complementary and specific research skills. Nine training courses on personal skills and four workshops on interrelated fields such as biomaterials, skin tissue engineering, hair biology and regeneration were provided.
WP2 “The regenerative matrix”, different components were prepared to be included in bioscaffolds, e.g. type III collagen and fractions of elastin hydrolysates. First prototypes of 3D collagen-based bioscaffolds were constructed and characterized. For extracellular matrix production by fibroblasts of different origins, protocols were optimized and initial characterizations performed. Three Early-stage researchers (ESRs) participated in a laboratory secondment at ADBC in Beverwijk to study the behaviour of various fibroblasts on biomaterials. Another ESR established a protocol to decellularize dermis of mouse and spiny mouse. During two distinct ESR secondments at HMGU, the basis was laid for collaborative proteomics studies. For this, the host ESR is setting up a histology-directed workflow for the molecular analysis of ECM proteins in an animal model for skin regeneration and in vitro cultured human foetal/adult fibroblasts while performing pilot experiments.
WP3 “Regenerative non-scarring cells”, one ESR has been trained spiny mouse handling and was able to adapt existing histological techniques protocols of tissue to the particularities of Acomys skin. For human foetal, adult, or eschar fibroblasts, protocols regarding e.g. RNA isolation, RT-qPCR and transcriptome sequencing were optimized for mesenchymal cell populations of different origins. To study the human fibroblast lineages accountable for scarless skin regeneration, another ESR successfully established a human induced pluripotent stem cells (iPSC)-derived skin organoid model. In addition, differentiation protocols for blood-derived monocytes to differentiate into pro-inflammatory or anti-inflammatory macrophage subsets were optimized.
WP4 “Introduction of skin appendages and sensory nerves”, focus lies on 1) human sweat gland stem cells, 2) human hair proto-follicles, and 3) human nerve cells. Human iPSC-derived skin organoids were used to isolate and propagate sweat gland stem cells, where sweat gland-specific markers proved that after long-time culture (day 120) the fate choice towards sweat gland formation was achieved. Human hair proto-follicle studies applied hair follicle dermal cell cultures in 2D and bilayered 3D spheroids. To investigate the neurovascular link, human endothelial cells and nerve cells are being included into collagen hydrogels to create a vascular link with nerve cells. For this purpose, human dermal microvascular endothelial cells were isolated and sorted from human skin biopsies and commercial human iPSCs cultures were established.
WP5 “Towards translation into medical devices and ATMPs”, prototypes of an intact three-dimensional collagen biomatrix were produced by different approaches and compounds and the most promising prototype was defined according to key opinion leader meetings, internal evaluation, and market needs. In addition, the first protocol for a melanocyte-containing tissue-engineered skin construct was designed and executed.
In WP6 “Education and training”, ESRs are trained in complementary and specific research skills. Nine training courses on personal skills and four workshops on interrelated fields such as biomaterials, skin tissue engineering, hair biology and regeneration were provided.
One of the most notable achievements has been learning from scarless skin regeneration models. By comparing wound healing in Acomys (spiny mouse) and Mus (ordinary mouse), researchers identified key extracellular matrix (ECM) components and cellular behaviours that underpin regenerative healing. These insights have directly informed the design of biofunctional scaffolds aimed at reducing scarring and improving tissue regeneration.
The project also saw the successful development and testing of collagen-based scaffolds enriched with regenerative agents such as elastin, type III collagen, hyaluronan, tenascin-X, and the heparan sulfate mimetic OTR4120. These scaffolds demonstrated enhanced vascularization, reduced contraction, and improved wound closure in both in vitro and in vivo models, marking a significant advance in biomaterial design.
In parallel, SkinTERM researchers explored the integration of skin appendages and sensory nerves into engineered constructs. Novel proto-follicle models and innervated, prevascularised skin substitutes were developed and transplanted into animal models. These constructs showed promising signs of dermal-epidermal interactivity and functional integration, laying the groundwork for future regenerative therapies that restore not only skin structure but also its sensory and appendage functions.
A major innovation during this period was the establishment of human induced pluripotent stem cell (iPSC)-derived skin organoid models. These organoids were used to study key aspects of skin development and regeneration, including sweat gland formation, fibroblast differentiation, and immune interactions. Sweat gland organoids were successfully induced and characterized, while pharmacological modulation of fibroblast-related pathways revealed critical regulators of differentiation. Furthermore, organoids were integrated with macrophages and neurons to investigate immune and neurovascular dynamics. Hair follicle spheroids were also incorporated into organoids and skin constructs, demonstrating sustained viability and functionality, and offering a powerful platform for future therapeutic applications.
Another milestone was the development of MelSkin™, a GMP-compatible pigmented skin substitute. This bioengineered construct was successfully tested in vitro and represents a major step toward clinical translation, particularly for the treatment of full-thickness wounds and burn injuries.
These findings are being disseminated through peer-reviewed publications, contributing to both scientific understanding and future therapeutic development.
The project also saw the successful development and testing of collagen-based scaffolds enriched with regenerative agents such as elastin, type III collagen, hyaluronan, tenascin-X, and the heparan sulfate mimetic OTR4120. These scaffolds demonstrated enhanced vascularization, reduced contraction, and improved wound closure in both in vitro and in vivo models, marking a significant advance in biomaterial design.
In parallel, SkinTERM researchers explored the integration of skin appendages and sensory nerves into engineered constructs. Novel proto-follicle models and innervated, prevascularised skin substitutes were developed and transplanted into animal models. These constructs showed promising signs of dermal-epidermal interactivity and functional integration, laying the groundwork for future regenerative therapies that restore not only skin structure but also its sensory and appendage functions.
A major innovation during this period was the establishment of human induced pluripotent stem cell (iPSC)-derived skin organoid models. These organoids were used to study key aspects of skin development and regeneration, including sweat gland formation, fibroblast differentiation, and immune interactions. Sweat gland organoids were successfully induced and characterized, while pharmacological modulation of fibroblast-related pathways revealed critical regulators of differentiation. Furthermore, organoids were integrated with macrophages and neurons to investigate immune and neurovascular dynamics. Hair follicle spheroids were also incorporated into organoids and skin constructs, demonstrating sustained viability and functionality, and offering a powerful platform for future therapeutic applications.
Another milestone was the development of MelSkin™, a GMP-compatible pigmented skin substitute. This bioengineered construct was successfully tested in vitro and represents a major step toward clinical translation, particularly for the treatment of full-thickness wounds and burn injuries.
These findings are being disseminated through peer-reviewed publications, contributing to both scientific understanding and future therapeutic development.