Tissue engineering and regenerative medicine an other way of anti-aging? Inducing autologous production of stemcells in human by using PRP

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1 Tissue engineering and regenerative medicine an other way of anti-aging? Inducing autologous production of stemcells in human by using PRP a change of paradigm and therapeutical perspectives 1

2 Tissue Engineering Attempts to answer the question : Once disease or injury has occured, how regenerate the damaged tissues? Implicit in this aim is the understanding that the regeneration, or the reconstitution of the form and function of the injured tissues, is very different from repair, which often naturally occurs in the body following injury. Repair is the restoration of tissue continuity whithout its original architecture OR function however The Basic principles of Tissue Engineering : the final product of regeneration is to produce a tissue, and to restore the tissue to its original state and possesses physical and mechanical properties with characteristics indistinguishable from the original 2

3 Mesenchymal stem cells ( MSC) : definition by the International Society Cell Therapies Osteogenic differentiation Adipogenic differentiation Chondrogenic differentiation 3

4 MSC$sources$ Dental pulp Skin Umbilical blood Wharton s jelly Peripheral blood Transversale section of umbilical cord Bone marrow Adipose tissue Placenta Spleen, pancreas, lung, kidneys Amniotic fluid 4$ 4

5 MSC$proper)es! Hematopoie)c$support$ Immunomodula)on$ MSC! Differen)a)on,$ Tissue$repair$ 5

6 PARACRINE(EFFECTS(OF(MSC!!!!!!!!!!!!!!!!!!!!!!!!!!!Da!Silva!Meirelles!L,!Cytokine!and!growth!factor!reviews!2009! 6! 6

7 The Main cell source used in autologous regenerative medicine : Bone marrow ( BM-MSC) & Adipose tissue (ADSC) CELL$SPECIFIC)DIFFERENCES)BETWEEN)ADSC)AND)BM$MSC) FREQUENCY+ BM!1:10 4!to!1:10 6! nucleated!cells! AT!!!0,5!10 4! to!2!10 5 /g! Number)of)CFU$F/10 6 ) plated)cells) 800) 600) 400) 200) 0) BM) AT) PROLIFERATION+ CELL+MORPHOLOGY+!!!!!!!BM$MSC!!!!!!!!!!!!!!!!!!!!!!ADSC! IMMUNOPHENOTYPE+ 7!!!!!!!!!!!!!!!!!!!!!!!!!!Zhang!HT!et!al,!Cytotherapy!2012! Noel!D!et!al,!Exp!Cell!Res!2008!! 7!

8 Tissue Engineering : a triad association CELLS (MSC) Scaffold bone marrow PRF, bone mineral synthetics Signalling molecules growth factors adhesins,.. Tissue engineering, combines three key elements + time + appropriate environment 8

9 Tissue Engineering aim to restore the biological and physiological functions of tissues damaged by injury or degeneration Rejuvenetion medicine aim to restore the biological and physiological functions of tissues damaged by age Tissue Engineering is an other way to decrease damages bind to age by using cells with specific characteristics 9

10 Stem Cell-based approach therapy : a challenging task Nub of the problem Lower level of Stem cells in human body need critical mass to achieve success requiring in vitro expansion for clinical application in vitro expansion culture media passage number cell density replicative senescence alteration of phenotype telomere length shortening DNA methylation immunomosuppressive activity production of cytokines global gene expression pattern differentiation potential divergent profile & heterogenicity : less reliable in clinical use 10

11 Stem Cell-based approach therapy: a common strategy in the field of some lethal diseases a promising strategy in the field of regenerative medicine to safe the life of the patient to improve the life of the patient autologous cell transfer or heterologous cell transfer ( allograft) need culture cell expansion immunocompatibility required as most as possible GvHD as major complication autologous cell transfer avoid cell culture expansion keep all characteristics immunomodulation differentiation tissue repair 11

12 Limitating factors that impact T.E clinical applications Autologous material : cells and matrix is the «golden standard» procedure to avoid cell graft rejection /and /or GVHD :graft versus host disease Association : scaffold/growth factors that control and direct endogenous tissue formation : which is the best sequence of application? Development new procedure to produce autologous stem or projenitor cells to reach the minimum threshold of cell amount that induce T.E Understanding, how, when, and where these different progenitor cell population are operating Pre commitment stem or progenitor cells to direct in vivo final cell line differentiation 12

13 The supreme goal of TE in regenerative medicine should be : fully secure easy access and process reliable.one possible approach to achieve self production of autologous stem cells Scaffold = bone marrow PRF(platelet rich fibrin) autologous growths factors Tissue engineering, combines three key elements + time + appropriate environment 13

14 Autologous methodology seems to be the best way for T.E. in no compromise patient : a challenging task today Gold Standard Potential future autologous bone transplantation limited by: -Donor site morbidity -Material availability -Tissue in growth uncontrolled healing process Autologous Cells: pluripotent cells precursor to stimulate bone regeneration in ex vivo and in vivo with positive correlation between applied cells number and healing results); Bio Matrix: cells seeded in tri dimensional scaffold (osteoconductive and osteoinductive); Autologous Growth Factors (PRP) - target reactive cell population, inducing - proliferation and commitment of cells; - Early revascularization induced by neoangiogenesis. 14

15 Inducing human bone marrow to safety autologous bioreactor suitable for bone reconstruction surgery or cell banking Philippart,P. Meuleman,N. Stamatopoulos,B. Mehdi,N.Pieters, K.De Bruyn, C. Bron,D. Lagneaux,L. Tissue Engineering, parta,vol 20, Using human bone marrow as a physiological bioreactor to produce autologous SC by injection of autologous platelet-rich plasma (PRP) added with recombinant human soluble tissue factor (rhstf) in iliac crest. A trial on 13 healthy volunteers showed the feasibility and harmlessness of the procedure. This self-stimulation system of bone marrow seems thus to be a promising feasible process for clinical cell therapy applications,by production of MSC as well as EPC 15

16 Targeted cell Type First in vitro piece of evidence: PRP solution induces MSC proliferation Lucarelli et al. Biomaterials

17 Type/concentration of PRP activation trigger Wanted caracteristics: 1. Strict control of coagulation kinetics 2. Maximum signalling effects Soluble Tissue factor (GMP production, safety, stable, ) First in vitro piece of evidence (Daubie et al. 2006) 17

18 Clinical study design requirements Health volunteers PRP Preparation Magellan Machine Arteriocyte USA Counting done with automated hematology analyzer (Advia120, Siemens, Brussels, BE) Outcome 13 cases: 8 females+5 males : 20ti 62 Y Whole Blood PRP Platelets concentration: 10 3 /micro L solution preparation 6ml PRP+4ml physio sol+ 334microG rstf/pl/cacl2 common bone marrow needle extraction, usual, safe, painless inj. with local anesthesia Compulsory requirement: clinical protocol for Ethical committee: Approval/ protocol number Eudract: 1443 Faisability Study: 1. Intra bone marrow injection under local anesthesia? 2. Pain for the patient? 3. Need of high pressure to inject? 18

19 Design of the clinical study: 19

20 Unchanged parameters : Blood cells Hematological cells analysis : results day 3 & 7 after the procedure bone marrow blood stream WBC Blood Bone Marrow 7 20 (White Blood 7 15 Cells)!! WBC count (106 cellules/ml) 7 7 WBC count (106 cellules/ml) D0 D+3 D+7 0 Left D0 Left D+7 Right D+7 Coagulation!! 100, ,0000 HC (Hematological Prog. Cells) % of positive cells 75, , ,0000 0,0000 CD45 CD31 CD34 CD133 CD146 KDR CD DAY 0 DAY 3 DAY 7 LEFT! LEFT DAY DAY 0 7! RIGHT DAY 7 % of positive cells 75, , ,0000 0,0000 CD45 CD31 CD34 CD133 CD146 KDR CD166 fundamental parameter for clinical use : no status of hyper or hypo coagulation

21 Changed parameters : Endothelial Progenitor Cells (EPC) mobilization of EPC in the Blood Flow at Day 3without depletion in the Bone Marrow DAY 0 DAY 3 21

22 Changed parameters : Endothelial Progenitor Cells (EPC) Angiogenic properties of EPC in vitro, medium culture with VEGF added T0 T:0,5h T:2h T:4h T: 6h T : 24 h EPC (Endothelial Progenitors Cells) Blood Bone Marrow CONCLUSION " " Dense mobilization in the blood flow over 7 days No depleation in the bonne marrow Proliferation angiogenic properties under VEGF stimulation 22

23 Changed parameters : CFU-F= MSCs committed to osteogenic differentiation increased level of CFU-F (MSCs) in the bonne marrow after activation Increased level of CFU-F (MSCs): committed to osteogenic differentiaition potential after activation LEVEL OF CFU-F IN BM MONONUCLEAR CELLS Number of CFU-F (for 106 MNC) 22,00 16,50 11,00 5,50 0,00 LEFT DAY 0 LEFT DAY 3 RIGHT DAY 3 * ALP ACTIVITY * 22,0000 LEFT DAY 0 LEFT DAY 3 RIGHT DAY 3 16,5000 CFU-F (x103) 11,0000 5,5000 0,0000 PRIMOCULTURE CALCIUM LEVEL * 250,000 LEFT DAY 0 LEFT DAY 3 RIGHT DAY 3 187,500 CFU-F (x103) 125,000 62,500 0,000 PASSAGE 1 23

24 Changed parameters : CFU-F= MSCs committed to osteogenic differentiation ALIZARIN RED STAINING RT - PCR LEFT DAY 0 RIGHT DAY LEFT DAY 3 3 DAY 7 DAY 14 DAY 21 Osteocalcin LEFT DAY 0 Osteopontin LEFT DAY 3 βactin RIGHT DAY 3 MSCs Blood Bone Marrow! " Comments Proliferation and commitment to osteogenic pathway 24

25 Changed Parameters: Level of Cytokines in blood Cytokine level in blood * * * * Cytokine level in bone marrow Blood Bone Marrow Comments Cytokine "! implementation recovery capacities support angiogenic commitment of EPC to blood supply formation 25

26 Summary : Parameters assessment Blood Bone Marrow Comments WBC (White Blood Cells)!! Coagulation!! Invariability of blood properties. Critical requirement for downstream clinical application HC Hematological stem cells EPC Endothelial progenitor cells MSCs Mesenchymal Stem Cells!! " "! " Additional evidence of the unchanged blood features. dense mobilization in blood flow over 7days no depletionin the bone marrow proliferation angiogenic properties Proliferation and commitment to osteogenic pathway Cytokines "! Implementation recovery capacities Commitment of EPC to form new vessels 26

27 Acknowledgements... Hopitaux IRIS SUD IRIS ZUID Ziekenhuizen Philippart P MD,PhD,DDS Laboratoire de Thérapie Cellulaire Clinique [Laboratory of Clinical Cell Therapy] (LTCC) Faculty of Medicine - Campus Erasme ( ULB 721 UNIT) Free University of Brussels ( ULB ) Lagneaux L., PhD, Meuleman N., MD, PhD Stamatopoulos B., PhD Bron D., MD, PhD, De Bruyn C., PhD, Delforge A., PhD, Id Boufker H., PhD st., Najar M., PhD st. Racevic G., PhD st. Thank you for your attention 27