Year: 2019
Type: dissertation
Abstract: Evaluation of cellularized skin substitutes before implantationCultured skin substitutes belong to the Advanced Therapy Medicinal Products and should therefore comply with the regulations set by the involved authorities (e.g.European Medicines Agency, Federal Drug Administration) 79,104 .The products are tested mainly on safety and only to limited extent on functionality, before being implanted onto the patient 80,81 .Besides a general histological analysis with respect to epidermal coverage 1 19 and structure of the ECM, the proliferative capacity of the cells seeded in the skin can be assessed as well 81,95,105 .Unfortunately, these assays are mainly qualitative or semi-quantitative and do not provide information on the functionality of the seeded cells with respect to ECM deposition. Thesis aims and outlinesMany skin substitutes have been developed to restore the barrier function of wounded skin, and many techniques are being used to qualitatively evaluate their performance.However, no ideal skin substitute exists that stimulates perfect skin regeneration.Current evaluation of skin substitutes often lacks objectivity.Methods focusing on gene expression and protein analysis of ECM molecules could give insight in the regenerative capacity of cells seeded into the skin substitute and on the wound healing process in general.Increased understanding of skin wound healing and regeneration will ultimately lead to the design of the ideal skin substitute that meets the standard quality of life.This thesis addresses different approaches to tissue engineering of the skin and analyses hereof, by evaluation of the functionality of cells in a cultured skin substitute, or in regenerated skin after implantation.In Chapter 2, the use of protein based assays and gene expression analysis is investigated as a tool to quantitatively evaluate the quality of a cultured skin substitute prior to implantation.Given the importance of the extracellular matrix for regeneration of the skin, the focus is on components of the ECM.A novel technique to detect and localize newly synthesized collagen by cells seeded in a collagen biomaterial is presented in Chapter 3.With this technique, the functionality of the cells in the biomaterial prior to implantation can be assessed.Chapter 4 describes the application of a gene expression microarray analysis for unbiased evaluation of skin substitutes.A detailed approach to isolate high quality RNA from small human skin biopsies is presented for use in gene expression microarrays.Furthermore, the novel skin substitute Novomaix is compared to the standard treatment of burn wounds in patients, based on gene expression levels and gene ontology terms.The use of microarray gene expression analysis in addition to the more qualitative histological analysis of skin regeneration is discussed.The potential of collagen scaffolds functionalized with heparin and the growth factors VEGF and FGF2 to induce skin growth after implantation in full-thickness fetal skin defects CHAPTER 1 20 in lambs is investigated in Chapter 5. Extensive qualitative and quantitative analyses are performed to investigate the long-term effect on the regenerated skin at 1 and 6 months after delivery.In Chapter 6 the use of growth factor mimetics, the so-called small molecules, is studied.The influence of three small molecules on essential cell processes during wound healing (proliferation, differentiation and migration) in fibroblasts and keratinocytes is evaluated.A general discussion and future outlook are provided in Chapter 7. Finally, the overall findings are summarized in Chapter 8.
Author: Corien Oostendorp
Language: English
Topic: Wound Healing and Treatments
Subfield: Surgery
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2999814562