Dental Stem Cell and Dental Repair PDF

3/3/21 Dental Stem Cell and Dental Repair Ana Angelova Volponi, DDS, PhD Lecturer in Regenerative Dentistry, Faculty of Dentistry, Oral & Craniofacial Sciences, King’s College, University of London REGENERATIVE DENTISTRY Faculty of Dentistry, Oral and Craniofacial Sciences King’s College University...

3/3/21 Dental Stem Cell and Dental Repair Ana Angelova Volponi, DDS, PhD Lecturer in Regenerative Dentistry, Faculty of Dentistry, Oral & Craniofacial Sciences, King’s College, University of London REGENERATIVE DENTISTRY Faculty of Dentistry, Oral and Craniofacial Sciences King’s College University of London 1 CLINICAL RELEVANCE Tooth loss Bone loss Periodontal disease Regenerative Dentistry Underlying principles of Biological repair / regeneration 2 1 3/3/21 3 Stem cells unspecialized cells renewing themselves Under certain physiologic or experimental conditions, they can be induced to become tissue- or organ-specific cells with special functions. sometimes after long periods of inactivity. 4 2 3/3/21 Different types of stem cells Embryonic stem cells Non-embryonic “somatic” or “adult” stem cells. Induced pluripotent stem cells. 5 Embryonic stem cells 1981 embryonic stem cells from early mouse embryos In the 3- to 5-day-old embryo, (blastocyst stage), the inner cells give rise to the entire body of the organism 1998 Human embryonic stem cells culturing Sub-culturing (passage) ES cell line (pluripotent) 6 3 3/3/21 The ID card of a ESCs Non specialised -undifferentiated (many interest expressed) Characteristics in appearance: (special marks): Particular cell surface markers (Oct4, Nanog,..) How do we identify the ESCs? In other words, they can develop into each of the more than 220 cell types of the adult body. Interests: In special conditions they form cells from three germ layers. They are PLURIPOTENT. Under defined conditions, embryonic stem cells are capable of propagating themselves indefinitely. 7 How do we stimulate ESCs to differentiate into specialized cells in vitro? Different recipes on the menu,.. -change in composition of media -alter surface of dishes -insert genes into cells 8 4 3/3/21 Where are adult stem cells found, and what do they normally do? -present in many organs and tissues -reside in stem cell niches -remain non-dividing for a long time -activated when needed to maintain tissue or disease/injury -multipotent 9 Induced Pluripotent Cells (IPSCs) Adult cells that have been genetically re-programmed to an embryonic cell-like state by introduction of certain embryonic genes into a somatic cell. cMyc, Oct4, Sox2, Klf4, Nanog, LIN28 Mouse iPSCs (2006) hIPSCs (2007) 10 5 3/3/21 Late 90’s… In 2003 Miura and collaborators identified SHED cells as highly proliferative, clonogenic cells capable of differentiating into a variety of cell types including neural cells, adipocytes, and odontoblasts, expressed Stem cells’ markers. These cells were called SHED (Stem cells from Human Exfoliated Dentition) 11 The Tooth BCMP (a treasure chest of different Stem cells) 12 6 3/3/21 Root apical papilla stem cells SCAP Apical Papilla region 13 Dental Follicular stem cells Dental follicular tissue Extracted tooth with roots in development with surrounding follicular sac 14 7 3/3/21 Gingival tissue- a source of mesenchymal and epithelial stem cell Gingival Fibroblast Stem Cells Separate! Gingival (Oral) Epithelial Stem Cells 15 The tooth – a perfect source ! Accessibility …otherwise discarded material Possibility of authologus or/and allogenic source of stem cells Different mesenchymal and/or epithelial stem cells derived from tooth/oral tissues 16 8 3/3/21 Questions to think about: Are the different dental stem cells different between themselves?! …if yes, what is the difference?! Are the stem cells grown/expanded in vitro the same as the ones in vivo? How much they keep their original properties in vitro? 17 Isolating dental pulp stem cells in vitro and their characterisation In 2000 Grontos and collaborators first identified a population of cells isolated from the dental pulp of human third molars and termed them Dental Pulp Stem cells (DPSCs). These cells were characterized by their high proliferation and colony forming properties and when compared to Bone Marrow Stem cells (BMSCs) in vitro, shared similar immunophenotype. DPSCs are capable of differentiating into adipoctyes, osteoblasts and endoteliocytes. These cells were able to produce sporadic, but densely calcified nodules. When transplanted in vivo (using immunocompromised mice as a host), the cells derived from dental pulp (DPSCs) generated functional dental tissue in the form of dentin/pulp-like complexes. DPSCs may have a broader capacity for differentiation and this might be due to their developmental origin as neural crest derived cells. 18 9 3/3/21 Origin of the dental pulp Stem cells Dual origin of mesenchymal stem cells contributing to organ growth and repair Stem cell niches in the dental pulp By using genetic lineage tracing, it was shown that some pericytes differentiate into specialized tooth mesenchyme-derived cells-odontoblasts--during tooth growth and in response to damage in vivo. As the pericyte-derived mesenchymal cell contribution to odontoblast differentiation does not account for all cell differentiation, we identify an additional source of cells with MSClike properties that are stimulated to migrate toward areas of tissue damage and differentiate into odontoblasts. This study identified a dual origin of MSCs in a single tissue and suggests that the pericyte contribution to MSCderived mesenchymal cells in any given tissue is variable and possibly dependent on the extent of the vascularity. Directed cell migration toward tissue damage visualized by DiI labeling of incisor mesenchyme pulp cells Feng J, Mantesso A, De Bari C, Nishiyama A, Sharpe PT..Dual origin of mesenchymal stem cells contributing to organ growth and repair.Proc Natl Acad Sci U S A. 2011 Apr 19;108(16):65038. doi: 10.1073/pnas.1015449108. Epub 2011 Apr 4 19 Glial origin of mesenchymal stem cells in a tooth model system -used mouse strains allowing for permanent genetic labelling of multipotent Schwann cell precursors (SCPs) and Schwann cells. A significant population of mesenchymal stem cells during development, self-renewal and repair of a tooth are derived from peripheral nerve-associated glia. Glial cells generate multipotent mesenchymal stem cells that produce pulp cells and odontoblasts Glial origin of mesenchymal stem cells in a tooth model system. Nature. 2014 Sep 25;513(7519):551-4. Kaukua N, Shahidi MK, Konstantinidou C, Dyachuk V, Kaucka M, Furlan A, An Z, Wang L, Hultman I, Ahrlund-Richter L, Blom H, Brismar H, Lopes NA6, Pachnis V, Suter U, Clevers H, Thesleff I, Sharpe P, Ernfors P, Fried K, Adameyko I. 20 10 3/3/21 Single-cell analysis of human adult and grown teeth Characterization of cell composition across five adult and two growing human molars Colors correspond to individual datasets and indicate clustering by cell types. c Characterization of dental cell types in human teeth. Colors demonstrate 17 clusters as defined by leiden clustering. Major clusters are defined by expression of known markers. d Human dental pulp have at least six transcriptionally distinct states. Top color bar reflects colors of clusters shown in c). Top 198 genes enriched in each cluster are shown e, f Identification of apical-likemouse-incisor regions in the growing apical papilla of human molar shown by the expression of SFRP2 and SMOC2 in the growing apical papilla 21 Are the different Dental Stem Cells really different? 22 11 3/3/21 A.A. Volponi et al. J DENT RES 2015;94:1568-1574 Copyright © by International & Am erican Associations for Dental Research 23 Alizarin red staining of different Dental Stem Cells DPSCs SCAP GFSCs BCMP SHED PDL A.A. Volponi et al. J DENT RES 2015;94:1568-1574 24 12 3/3/21 Representative Raman spectra and mineral to matrix ratios for native dental tissues and material formed by dental stem cells. A.A. Volponi et al. J DENT RES 2015;94:1568-1574 Copyright © by International & Am erican Associations for Dental Research 25 Ways to regenerate and replace Regenerating/repairing dental tissues Whole tooth replacement Dentin Regeneration Pulp Regeneration PDL regeneration Bone regeneration Tissue repair and engineering Organ engineering 26 13 3/3/21 Tissue REPAIR /Engineering 27 Always consider,… Specificity of the tissue -cellular events: presence/absence of cells Response upon injury -capacity to limited repair -re-vascularization Stimulating a repair responce What are the limits of the repair of certain tissues/organs 28 14 3/3/21 Dental pulp has the potential of repairing the Injury- extend of injury dentin Subsequent cellular events 29 Extend of the injury Subsequent cellular events Early stage of injury Relatively low grade in nature Pulp tissue up-regulates the secretory activity of the odontoblasts at the site of injury Focal deposition of “reactionary dentin” at the pulp dentin border Formed by the existing primary odontoblasts, this dentin has a regular tubular structure and tubular “continuity” with the existing dentin Progression of the disease or intense injury Local death of the odontoblasts at the site of injury and inflammatory process intensify!! Focal deposition of “reparative” at the pulp dentin border 30 15 3/3/21 Strategies in recruiting cells to become odontoblasts Recruiting from: in or outside the dental pulp?! Wound Healing ? Engineering!? 31 Switching on natural healing pathways Wnt signalling pathway Wnt signaling is activated by wounding and participates in every subsequent stage of the healing process from the control of inflammation and programmed cell death, to the mobilization of stem cell reservoirs within the wound site. 32 16 3/3/21 Triggering the WnT signalling in tissue repair The activation of Wnt/βCat signalling as a universal immediate-early response to tissue damage provides a potential route for enhancing natural repair by overstimulating this pathway Promotion of natural tooth repair by small molecule GSK3 antagonists. Neves VC, Babb R, Chandrasekaran D1, Sharpe PT. Sci Rep. 2017 Jan 9;7:39654. 33 MicroCT analysis of mineral deposition 34 17 3/3/21 Periodontal repair and regeneration 35 Computer-Based designed and fabricated Scaffolds Hybrid scaffolds for periodontal cell and gene delivery. 36 18 3/3/21 Personalised scaffolding technologies 37 Organ Engineering Making a bio-tooth 38 19 3/3/21 …while some animals can do it easily,… Humans are still struggling,… 39 Whole tooth generation as a model 40 20 3/3/21 Nature combines….. Epithelium Mesenchyme 41 42 21 3/3/21 Ana Angelova Volponi , Yvonne Pang , Paul T. Sharpe “Stem cell-based biological tooth repair and regeneration” Trends in Cell Biology, Volume 20, Issue 12, 2010, 715 - 722 43 After being implanted in SCID mouse KC for 2 weeks, half of the kidney was processed for CT and histology After 2 weeks a CT Scan was done and tooth like mineralized structures revelead. 44 22 3/3/21 After 4 weeks implantation A CT scan revealed developed tooth 45 …how different we are?.... 46 23 3/3/21 Different Biological Clock Carnegie stage 17 Mouse tooth development: 5 days Carnegie stage 19 Human tooth development: 42 days Carnegie stage 23F1 8 times slower F1-F2 47 Of Mice AND Men HGEP cells grown as clusters recombined with MM (E14.5) Human adult gingival epithelial cells Mouse embryonal tooth mesenchyme Tooth Buds (E14.5) HGEP P1 Low Binding Surface HGEP P1 Grown on PCT medium on low binding surface HGEP P1 HGEP P1 After 3 days in culture formed clusters Cell clusters were harvested and used for recombinat ion ME MM Cultured in collagen After 6 weeks implantation microCT scan Histology analysis 48 24 3/3/21 MicroCT analysis A. Angelova Volponi et al. JDR, March 2013 49 DSPP immuno-staining marking odontoblasts (Ob) A. Angelova Volponi et al. JDR, March 2013 50 25 3/3/21 D - dentin DP - dentin pulp BV - blood vessel ES - enamel space Malassez cells (black/white arrows) C - cementum A. Angelova Volponi et al. JDR, March 2013 51 A. Angelova Volponi et al. JDR, March 2013 52 26 3/3/21 53 Directions for Future ?! THANK YOU! [email protected] 54 27