Lecture 17 - Skin ATMPs

Skin advanced therapy medicinal products Laura Trujillo Cubillo; BEng, MSc, Diana C. Gonçalves; BSc, MSc, Dimitrios I. Zeugolis; BSc (Hons), MSc, PhD Regenerative, Modular & Developmental Engineering Laboratory (REMODEL), Charles Institute of Dermatology, Conway Institute of Biomole...

Skin advanced therapy medicinal products Laura Trujillo Cubillo; BEng, MSc, Diana C. Gonçalves; BSc, MSc, Dimitrios I. Zeugolis; BSc (Hons), MSc, PhD Regenerative, Modular & Developmental Engineering Laboratory (REMODEL), Charles Institute of Dermatology, Conway Institute of Biomolecular & Biomedical Research, School of Mechanical & Materials Engineering, University College Dublin (UCD), Dublin, Ireland www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Skin physiology and characteristics Skin appendages Characteristics: Skin is the largest organ of the body First line of defense against external agents Regulates body temperature Cell types: Epidermis: Keratinocytes, melanocytes, Langerhans and Merkel cells Dermis: Fibroblasts, macrophages, mast cells Hypodermis: Adipocytes Extracellular matrix: Dermis: collagens (e.g. collagen I, collagen III), fibronectin, elastin, laminin, glycosaminoglycans www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Wound healing progression: from acute wounds to tissue regeneration Stages of wound healing: 1. Haemostasis 2. Inflammation 3. Proliferation 4. Remodelling www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Wound healing progression: Coagulation / Hemostasis www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Wound healing progression: Inflammation Neutrophils: First immune response in the wound bed. Extravasate from the blood vessels attracted by damage- released signals. Their primary function is to kill microbes this is achieved by a combination of targeted ROS release and the formation of NETs Clearance of neutrophils by macrophages is essential to resolve inflammation Macrophages: Differentiate from monocytes Release chemoattractants that amplify the inflammatory response Phagocytose ECM and cell debris and signal to fibroblasts to regulate ECM deposition and remodelling In the early stages of wound healing, they display a pro- inflammatory phenotype,in later stages of wound healing, macrophages transition to “alternative activation”, promoting resolution of inflammation www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Wound healing progression: Proliferation Proliferation: Fibroblasts differentiate into myofibroblasts, which drive wound contraction. Degradation of the fibrin clot by matrix metalloproteinases Deposition of a temporal ECM (granulation tissue) abundant in fibronectin, collagen III and proteoglycans abundant Keratinocytes at the wound undergo partial epithelial to mesenchymal transition and start re-epithelialisation and basement membrane reconstruction Cell infiltration into the granulation tissue leads to angiogenesis and ECM remodelling www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Wound healing progression: Remodelling Remodelling: Replacement of the unorganised temporary ECM by aligned collagen fibres Collagen type III is replaced by collagen type I Apoptosis of myofibroblasts fibroblasts and macrophages Increase in the tensile strength, up to 80% of the original strength Failure to replace the temporal ECM leads to permanent scarring. www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Chronic wounds result from abnormal wound healing progression Characteristics: Impaired wound healing progression despite treatment Commonly associated to an underlaying condition (e.g. poor blood circulation, diabetes, weak immune system) www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Skin tissue engineering targets impaired wound healing www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Skin tissue engineering targets impaired wound healing www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Skin tissue engineering targets impaired wound healing ATMP is a regulatory category Not all tissue engineered products are considered an ATMP www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  What is an ATMP? “Advanced therapy medicinal products (ATMPs) are medicines for human use that are based on genes, tissues or cells. They offer groundbreaking new opportunities for the treatment of disease and injury.” www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  What is an ATMP? Regulatory classification “Advanced therapy medicinal products (ATMPs) are medicines for human use that are based on genes, tissues or cells. They offer groundbreaking new opportunities for the treatment of disease and injury.” Gene therapy medicines: “Contains an active substance which contains or consists of a recombinant nucleic acid used in or administered to human beings with a view to regulating, repairing, replacing, adding or deleting a genetic sequence; its therapeutic, prophylactic or diagnostic effect relates directly to the recombinant nucleic acid sequence it contains, or to the product of genetic expression of this sequence.” Directive 2001/83/EC www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  What is an ATMP? “Advanced therapy medicinal products (ATMPs) are medicines for human use that are based on genes, tissues or cells. They offer groundbreaking new opportunities for the treatment of disease and injury.” Somatic-cell therapy medicines: “Contains or consists of cells or tissues that have been subject to substantial manipulation so that biological characteristics, physiological functions or structural properties relevant for the intended clinical use have been altered, or of cells or tissues that are not intended to be used for the same essential function(s) in the recipient and the donor; is presented as having properties for, or is used in or administered to human beings with a view to treating, preventing or diagnosing a disease through the pharmacological, immunological or metabolic action of its cells or tissues.” Directive 2001/83/EC www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  What is an ATMP? “Advanced therapy medicinal products (ATMPs) are medicines for human use that are based on genes, tissues or cells. They offer groundbreaking new opportunities for the treatment of disease and injury.” Tissue-engineered medicines: “Contains or consists of engineered cells or tissues, and is presented as having properties for, or is used in or administered to human beings with a view to regenerating, repairing or replacing a human tissue A tissue engineered product may contain cells or tissues of human or animal origin, or both. The cells or tissues may be viable or non-viable. It may also contain additional substances, such as cellular products, bio-molecules, bio-materials, chemical substances, scaffolds or matrices.” Directive 1394/2007/EC www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Are this skin tissue engineered products ATMPs? Apligraf®: full-thickness skin Integra : dermal substitute, Meshed allogeneic skin graft Bioprinted skin substitutes equivalent used to treat synthetic skin replacement chronic diabetic and venous used to reconstruct wounds ulcers. after elective planned surgery, or after trauma. www.ucd.ie MEEN40630 Biomaterials www.remodel.ie Epidermal Full thickness Dermal The history of tissue engineering begins with skin tissue engineering 1975 1979 1981 www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Apligraf® is the first ever tissue engineered product to obtain FDA approval 1985 2001 Human Fibroblast-derived Dermal Substitute 1988 2017 1996 Human Fibroblast-derived Dermal Substitute 2021 1998 www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Characteristics of skin tissue engineering ATMPs Full thickness Epidermal Dermal Full thickness Full thickness Culture time: 20 days Culture time: 17 days Culture time: 17 days Culture time: - days Culture time: 24 days Matrix: Collagen lattice Matrix: Cell sheet Matrix: Polymeric scaffold Matrix: Collagen hydrogel Matrix: Collagen hydrogel Cell types: Neonatal fibroblasts Cell types: Autologous Cell types: Neonatal fibroblasts Cell types: Adult fibroblasts Cell types: Autologous and keratinocytes keratinocytes and NIKS fibroblasts and keratinocytes Price: $25,370 patient/year Price: $13,000 / %body surface Price: $24,552 patient/year Price: $4,222/film Price: - Indications: Venous leg ulcers Indications: Dermal or full Indications: Diabetic ulcers (>6 Indications: Orphan drug for Indications: Orphan drug for (VLUs) and diabetic foot ulcers thickness burn weeks); orphan drug for deep partial thickness burns treatment of burns (DFUs) epidermolysis bullosa www.ucd.ie MEEN40630 Biomaterials www.remodel.ie www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  3D Bioprinting Technique where bioinks (solution in the hydrogel form) are mixed with living cells and 3D printed www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Multicellular bioprinted skin facilitates human like skin architecture in vivo www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Bioprinted human skin constructs with six primary human cell types maintain a trilayer structure and mature in vitro Cellular viability was maintained in the constructs (live, green; dead, red) Epidermal keratinocytes undergo terminal differentiation with the superficial region showing evidence of cornification. and programmed cell death known as cornification which leads to the formation of epidermal layers. www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Bioprinted human skin constructs with six primary human cell types maintain a trilayer structure and mature in vitro www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Bioprinted skin accelerates wound closure via formation of an epidermal barrier and supports healthy ECM remodeling Masson’s trichome (MT) staining: selectively stains collagen, muscles and erythrocytes www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Bioprinted skin accelerates wound closure via formation of an epidermal barrier and supports healthy ECM remodeling Picrosirius red (PS) staining (under polarised light): Red to orange staining indicative of bundled collagen fibres www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Bioprinted skin supports skin neovascularization via formation of chimeric human/mouse microcapillaries Hematoxylin and eosin (H&E) staining: deep blue-purple: nucleic acids; pink: non-specific proteins www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Bioprinted human skin constructs integrate with regenerated skin wounds Pan-cytokeratin: epidermal formation Lamin AC: integration of human cells Vimentin: epithelial-mesenchymal transition Adiponectin: adipocytes KRT7: keratinocytes Mel5: melanocytes (melanin synthesis) www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Autologous bioprinted skin supports healthy epidermal and dermal skin repair and regulates expression of ECM remodelling genes in full thickness porcine skin wounds EGF: endothelial growth factor COL14A1: collagen type XIV, alpha 1 MMP9: matrix metalloproteinase-9 TGFB1: transforming growth factor beta 1 TGFB3: transforming growth factor beta 1 www.ucd.ie MEEN40630 Biomaterials www.remodel.ie www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Improving cutaneous wound healing in diabetic mice using naturally derived tissue-engineered biological dressing produced under serum-free conditions ASCs: adipose-derived mesenchymal stem cells; SFM: serum-free medium www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  ASC-based biological dressing accelerated mice wound closure www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  ASC sheets supported complete reepithelisation within 20 days Masson’s trichrome staining Untreated mice Treated mice www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  ASC sheets promoted formation of dense and homogeneous granulation tissue and improved wound healing Untreated mice Treated mice www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  ASC sheets displayed high total collagen content with similar proportions of collagen fibers Untreated mice Picrosirius red staining Treated mice www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  ASC sheets promoted neovascularisation identified by the formation of long organised tubular structures Untreated mice Treated mice Hoechst: nuclear DNA CD31: capillary structures (neodermis) www.ucd.ie MEEN40630 Biomaterials www.remodel.ie ASC sheets secreted a panel of bioactive molecules involved in fibroblast recruitment and proliferation and angiogenesis HGF: hepatocyte growth factor PAI-1: plasminogen activator inhibitor-1 VEGF: vascular endothelial growth factor Ang-1: angiopoietin-1 bFGF: basic fibroblast growth factor www.ucd.ie MEEN40630 Biomaterials www.remodel.ie www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Electrospinning ▪ Voltage-driven fabrication process capable to generate homogeneous ultrathin fibres ▪ High voltage is used to eject a polymeric solution through a syringe pump www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Cell genetic manipulation A. Expression vector containing VEGF fragment and hygromycin (antibiotic) B. Electrophoresis: PCR amplified target gene fragment (620 bp of VEGF) Transfection C. Cloning confirmation www.ucd.ie MEEN40630 Biomaterials www.remodel.ie Overexpression of VEGF in dermal fibroblast cells accelerates the angiogenesis and wound healing function: in vitro and in vivo studies PU-CA: polyurethane- cellulose acetate BCA assay: total MTT assay: cellular protein concentration metabolic activity FTIR: Fourier Transform Infrared Spectroscopy SDS-PAGE: sodium SEM: scanning electron dodecyl-sulfate microscopy polyacrylamide gel electrophoresis www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Successfully transfected fibroblasts expressed EGFP (green fluorescence) and exhibited high expression of VEGF 1. Control 2. Transfected cells 3. Untransfected cells Hu02: fibroblast cell line www.ucd.ie MEEN40630 Biomaterials www.remodel.ie Fibroblasts seeded on scaffolds maintained their viability and manipulated cells showed high expression of VEGF, SGPL- 1 and DDR2 genes TCPS: tissue culture polystyrene SGPL1: sphingosine-1- phosphate lyase 1 DDR2: discoidin domain receptor tyrosine kinase 2 www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  Manipulated cells seeded on scaffolds promoted wound closure within 15 days www.ucd.ie MEEN40630 Biomaterials www.remodel.ie Scaffold containing manipulated cells promoted angiogenesis, reephitelisation and scar and connective tissue formation H&E staining Black arrow: neutrophil infiltration Arrow head: angiogenesis (formation of blood vessels) Yellow arrow: connective tissue formation Green stars: collagen deposition Yellow star: re-ephitelisation Black star: hemorrhage Red star: scar tissue formation www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  High accumulation of collagen organised and few angiogenesis was observed in manipulated cells group Arrow: collagen bundles generation Arrow: angiogenesis www.ucd.ie MEEN40630 Biomaterials www.remodel.ie  References / Further Reading Skin wound healing: Peña, O. A., & Martin, P. (2024). Cellular and molecular mechanisms of skin wound healing. Nature Reviews Molecular Cell Biology, 1-18. Tottoli, E. M., Dorati, R., Genta, I., Chiesa, E., Pisani, S., & Conti, B. (2020). Skin wound healing process and new emerging technologies for skin wound care and regeneration. Pharmaceutics, 12(8), 735. Biofabrication of skin substitutes: Hosseini, M., Koehler, K. R., & Shafiee, A. (2022). Biofabrication of human skin with its appendages. Advanced healthcare materials, 11(22), 2201626. Sierra-Sánchez, Á., Kim, K. H., Blasco-Morente, G., & Arias-Santiago, S. (2021). Cellular human tissue-engineered skin substitutes investigated for deep and difficult to heal injuries. NPJ Regenerative Medicine, 6(1), 35. Hosseini, M., & Shafiee, A. (2021). Engineering bioactive scaffolds for skin regeneration. Small, 17(41), 2101384. www.ucd.ie MEEN40630 Biomaterials www.remodel.ie