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Biomedical Engineering - Technology for Regenerative Medicine

Skin

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Skin burns tissue regeneration Among the conventional therapy for skin burns of grade II-III in clinical use, there is the regeneration of epidermis and dermis (for lesions < 30 cm 2 in dimensions). In a new research, the lesion is surgically prepared and covered with a new biodegradable synthetic membrane, that progressively degrades and is repopulated by fibroblasts and endothelial cells that migrate from the underlying tissue. Keratinocytes then migrate from the peri-lesional tissue to re- epithelialize the lesion. Finally, the silicone membrane, that was placed as a protection cover, progressively detaches from the lesion. Epithelial cells (composed by keratinocytes and their progenitors) were isolated and then expanded in culture flasks. In order to promote their proliferation, they were seeded on the polymeric membrane according to two methodologies: 1. The membrane with a diameter of 1 cm is placed into each well of a six-well plate. Then, culture medium containing cells is dropped on the surface of the polymeric membrane in each well in order to permit adequate seeding. The device is maintained at 37°C. 2. Cells in a normal culture medium are sandwiched between two polymeric membranes; this structure is placed on a temperature-responsive dish at 37°C. After the in vitro culture and expansion, rats were subjected to a surgical procedure during which a circular full-thickness layer of skin measuring 1 cm in diameter was removed. The polymeric membranes populated with epithelial cells were implanted in rats and placed so that the defect was secured using a polydioxanone suture. Answer briefly to the questions in the table: What is the therapeutic product? Cells (keratinocytes and their progenitors) grown on the polymeric membrane What are the elements that identify the therapeutic product as a PTC? The main therapeutic agent are the cells , obtained after "non-minimal manipulation" consisting in cell isolation, expansion and seeding In what phase of the regulatory process for new PTCs can this PTC be localized? Animal trial What are the standards that apply in this stage of the PTC process of development? Good Manufacturing Practi ce (GMP) for its production; Good Clinical Practice (GCP) for the animal trial What are the risks associated with this PTC(immunogenic, tumour, teratoma, infection, toxicity)? Why can they occur? Immunological rejection : YES . The cell source is not specified. It may be non-autologous. Tumor formation: YES. It could be due to the polymeric membrane and to the progenitor cells. Teratoma formation: YES, ONLY if pluripotent cells differentiated into epithelial cells are used. Transmission of infections: YES, from the donor material or from any manipulations Administration of toxic contaminants: YES, from the donor material or from any manipulation s. Classify all the cell sources potentially usable in human patients for this therapy Cell source (based on immunogenicity) Cell type/s Advantages Criticalities is it USABLE ? (Y/N) Autologous keratinocytes and their progenitors isolated from the patient and expanded Immunological compatibility. Limited availability. Limited expandability. Invasive test. Risk of tumor (due to the progenitors) YES Acceptable even if risk of tumor formation Autologous iPS from somatic cells of the patient, differentiated into epithelial cells Immunological Compatibility. Limitations in redifferentiation protocols Risk of teratoma (due to the pluripotency of iPS ) NO because of the risk of teratoma Syngeneic Embryonic stem cells isolated from clones of the patient and differentiated into epithelial cells Immunological compatibility with the exception of the mitochondrial DNA. Ethical, technical and regulatory limitations for cloned cells. Limitations in redifferentiation protocols. Risk of teratoma (due to the pluripotency of ESC ). NO because of the risk of teratoma Allogeneic keratinocytes and their progenitors isolated from a human donor and expanded Available from HLA-matched donors. Can be Industrialized. Immunogenicity. Risk of infection Risk of tumor (due to the progenitors) NO Because they are highly immunogenic. Xenogeneic keratinocytes and their progenitors isolated from a nonhuman donor and expanded Largely available from animal livestock. Can be industrialized. Risk of acute immune rejection. Risk of xeno- zoonoses. Limited biological Functionality. Risk of tumor (due to the progenitors) NO Because of the immune rejection and tumor formation EXERCISES: A) Establish the time necessary for cells to proliferate in the first culture phase in flasks assuming the final number of cells (������������ ������������) for the seeding on the grafts equal to ������������.������������ ⋅ ������������������������ ������������������������������������������������������������������������ and the cell division time (t d) equal to 12 h. Cells are extracted thanks to a 1 ml biopsy (������������ ������������) with a density of 1 ⋅10 6 ������������������������������������������������������������ ������������������������ (������������ ������������). Data X f=2.8*10 7 cells t d=12h Vb=1ml Nb=10 6 ������������������������������������������������������������ ������������������������ Solution: The number of initial cells (������������ 0) harvested from the biopsy is: ������������ 0= ������������ ������������������������������������=1 ������������������������∗10 6������������������������������������������������������������ ������������������������ =10 6������������������������������������������������������������ The number of divisions ������������ is given by: ������������= ln ������������ ������������ ������������0 ln 2 =ln (( 2.8∗10 7 ) 10 6 ) ln2 =ln (28) ln2 =3 .33 0.69 =4.8 The time necessary for the first proliferation of cells in culture flasks: ������������=������������∗������������ ������������=12 ℎ 4.8=57.6 ℎ=2.4 ������������������������������������ Expansion time t = d*t d Number of passages d= ln(X f/X i)/ln 2 Number of cells X=N*V B Determine the oxygen concentration profiles and plot it in the two cases discussed above, with the assumption of linear diffusion. ������������ Steady state: ������������������������ ������������������������ =0 No convection (v∙∇)������������=0 No consumption: ν=0 No production P=0 0=������������������������ 2������������ Cartesian Coordinates 0=D( ������������2������������ ������������������������ 2+ ������������2������������ ������������������������ 2+ ������������2������������ ������������������������) Reaction - Diffusion equation: ������������������������ ������������������������ = &� 6? F 8 + 2 – (v ∙∇)������������ The Laplacian operator in Cartesian coordinates: ∇ 2= ������������2 ������������������������ 2+ ������������2 ������������������������ 2+ ������������2 ������������������������ Linear diffusion ∇ 2= ������������2 ������������������������ 2 I Fick’s law ������������= −������������∇c X=s Polymeric membrane X=0 Fick’s second law (= pure molecular diffusion) governs oxygen diffusion. With the hypothesis of linear diffusion, the Reaction- Diffusion equation is reduced to : 0=������������������������ 2������������ ������������������������ 2 Control volume: 0