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Additional Supporting Information may be found in the online version of this article.

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CNE_23162_sm_SuppFig1.tif6900KSupporting Information Figure 1. (Magenta-green version of Figure 1 for the assistance of color-blind readers.) Angiogenic reponse of dental pulp. (A) Caries-induced chronic dentinal injury and associated pulpal zone (Z1-3). (B) A healthy tooth and associated pulpal zones (H1-3). (C) Prominent hyperemic response in the injured pulp, (D) not evident in the healthy sample. (E) A terminal microvessel (arrow) past the cell-free zone in an injured pulp. Od: odontoblasts, Cf: cell-free zone, Cr: cell-rich zone. (F) Terminal microvasculature in the cell-rich zone of a healthy tooth. (G, H) Terminal microvascular loops in an injured pulp adjacent to the odontoblastic layer, (I) and associated a-SMA+ pericyte (a-SMA: green, arrowhead). (J) Microvascular loops were absent in healthy pulps. (K) Minimum distance between terminal microvasculature and odontoblasts in the injured versus healthy pulp (n=10 samples). (L) Pulpal zones associated with the dentinal injury. Note the established microvascular loops in zones 1 and 2. (M) Gene expression for HIF-1a in the injured and healthy pulpal zones (n=4 pulps). (N) Notch-1 expression in the odontoblasts of zone-3 (notch-1: green). Note the a-SMA+ (red) pericyte.
CNE_23162_sm_SuppFig2.tif20631KSupporting Information Figure 2. (Magenta-green version of Figure 2 for the assistance of color-blind readers.) Terminal intra-vascular loop formation. (A) Intra-vascular tunnel formation (arrowheads) in a terminal microvessel lined with basement membrane (collagen-IV: red). (B) Higher magnification of an early intra-vascular tunnel from Figure A. (C) Higher magnification of a more advanced stage of intra-vascular tunnel from Figure A. (D) Final configuration of a terminal intra-vascular loop. (E) A trans-capillary pillar (arrow). (F) A fused trans-capillary pillar (arrow). Note the contractile profile of the adjacent pericyte (arrowhead). (G) Higher magnification of trans-capillary pillar fusion site with multiple tight junctions (arrowhead). (H) A 3D model demonstrating endothelial ingrowth and trans-capillary pillar formation.
CNE_23162_sm_SuppFig3.tif7808KSupporting Information Figure 3. (Magenta-green version of Figure 3 for the assistance of color-blind readers.) Involvement of pericytes in terminal intra-vascular loop formation. (A-D) Finite element modelling of blood flow following vascular contraction. (E-H) An expanding terminal intra-vascular loop (laminin: green, collagen-IV: red, UEA-l: purple). Note the UEA-l- pericytes and associated cytonemes. (I-L) 3D modelling of terminal intra-vascular loop formation commencing with intra-vascular tunnel formation and followed by the expansion of the loop through activity of the pericytes (green).
CNE_23162_sm_SuppFig4.tif15287KSupporting Information Figure 4. (Magenta-green version of Figure 4 for the assistance of color-blind readers.) The molecular mechanisms underlying intra-vascular loop formation. (A) Expression of Ki-67 (green) by a proliferating endothelial cell surrounded by a-SMA+ (red) pericyte. (B) VEGF-A (green) expression at discrete loci along the remodelling microvaculature (a-SMA: red). (C, D) VEGF-A (green) expression by an a-SMA+ (red) pericyte. (E) Expression of sonic hedgehog (green) by endothelial cells adjacent to a-SMA+ (red) pericytes. (F) Multiplication of basement membrane (arrow) adjacent to a pericyte (green) in a remodelling microvasculature. (G) Thickness of lamina lucida and lamina densa of the endothelial and the pericytic basement membrane in quiescent vessels of healthy pulps and active vessels of injured pulps (n=50 vessels, 8 samples). (H) An endothelial cell (arrow) and a pericyte (green) involved in remodelling the corresponding basement membranes. (I) Gene expression for laminin isoforms by the microvasculature of the injured compared to healthy pulps. (J) Semi-thin resin section demonstrating the expression of notch-1 (green) by the a-SMA+ (red) pericytes. (K-N) Co-localized expression of notch-1 (green) and MMP-2 (turquoise) by the UEA-l+ endothelial cells at discrete loci along the vessel. (O) Semi-thin resin section demonstrating the expression of MMP-2 localized to inner surface of trans-capillary pillar. (P) Semi-thin resin section demonstrating peripheral redistribution of MMP-2 after intra-vascular tunnel formation. (Q) TEM micrograph demonstrating a perivascular cell and an adjacent remodelling microvessel. (R) Semi-thin resin section demonstrating the expression of MT1-MMP (green) by a pericyte (arrow) and an adjacent perivascular cell and MMP-2 (red) by endothelium in a micro-vessel with a trans-capillary pillar formation. (S) Semi-thin resin section demonstrating the expression of MT1-MMP (green) by a pericytes and MMP-2 (red) by endothelium. (T) Laminin+ (green) pericytes and a MHC-II+(red) perivascular microglia in an injured pulp. Note the intimacy of pericytic end-feet and microglia (inset). (U) a-SMA+ (green) pericytes and MHC-II+(red) perivascular microglia in an injured pulp. (V) An MHC-II+(red)/CD 163+ (green) microglia adjacent to a UEA-l+ microvessel (purple).
CNE_23162_sm_SuppFig5.tif7361KSupporting Information Figure 5. (Magenta-green version of Figure 5 for the assistance of color-blind readers.) Schematic representation of the interplay between pericytes, perivascular microglia and endothelial cells in remodelling the terminal blood-barrier microvasculature.
CNE_23162_sm_SuppFig6.tif18756KSupporting Information Figure 6. (Magenta-green version of Figure 6 for the assistance of color-blind readers.) Structural analysis of the kinetics of terminal intra-vascular loop expansion. (A-C) Involvement of MHC-II+ perivascular microglia (red) and UEA-l-/laminin+ pericyte (UEA-l: purple, laminin: green) in the expansion of a terminal microvascular loop. (D) 3D reconstruction of Figure A. (E) TEM image demonstrating the early stages of formation of endothelial lamellopodia via intra-cellular cavitation (arrowhead). (F) Higher magnification of Figure E. Note the multiple layers of basement membrane and intracellular intermediate filament arrangement indicating active remodelling by endothelial cells. (G) TEM micrograph of a more advanced stage of remodelling endothelium. Lamellation of endothothelial cells is followed by intussusception of lamellopodia separated by layers of basement membrane. (H) Higher magnification view of Figure H. (I) Post-intussusception stage of endothelial remodelling. Note the detachment of endothelial cell (green) covered in basement membrane alongside a perivascular cell (brown). (J) Higher magnification of Figure I. Note the thick outer basement membrane and multiple layers of inner basement membrane. (K) Higher magnification of Figure I showing the characteristic tight junctions between two remodelling endothelial cells. (L). A 3D model demonstrating the kinetics of endothelial remodelling while maintaining the blood-barrier phenotype.
CNE_23162_sm_SuppFig7.tif12909KSupporting Information Figure 7. (Magenta-green version of Figure 7 for the assistance of color-blind readers.) Anastomosis formation between adjacent terminal microvascular loops. (A) An a-SMA+ (green) migrating pericyte and associated cytoneme. Note adjacent MHC-II+(red) perivascular microglia parallel to the pericyte. (B) A laminin+ (green) migrating pericyte and the associated MHC-II+ perivascular microglia (red). (C) TEM micrograph of early pericyte (P) migration. Multiplication of the basement membrane between the pericyte and the endothelium (arrowhead) is noteworthy. Inset shows the higher magnification of multilayered basement membrane from the area marked by the arrowhead. (D) TEM micrograph demonstrating detachment of a pericyte (P) from the associated microvessel. The pericyte is still enclosed in basement membrane and attachment to the main vessel is maintained through basement membrane. Inset shows the higher magnification of the pericyte tail (black arrow). Note the associated basement membrane (white arrowhead). (E) A migrating pericyte close to a perivascular microglial cell (purple). Condensation of collagen fibers between the vessel and perivascular microglia is notable. (F) A migrating UEA-l-(purple)/laminin+(green) pericyte connecting together the two adjacent vessels. (G, H) A UEA-l-(purple)/laminin+(green) pericyte anticipating a UEA-l+/laminin+ endothelial cell and the attachment to the target vessel. (I-L) A UEA-l-(purple)/collagen-IV+(red)/laminin+(green) pericyte is about to fuse to the target vessel. (M) A MHC-II+ perivascular microglia (red) forming a template for guiding the anastomosis (UEA-l: purple, laminin: green). (N) A MHC-II+ perivascular microglia (red) adjacent to terminal microvasculature (UAE-l: purple) and expressing FGF-8 (arrowhead) in a polarized manner. (O) Pericyte-pericyte connection established by a cytoneme (laminin: green). (P) The pericyte-pericyte connection through a cytoneme is later occupied by migrating endothelium (UEA-l: purple). (Q) A 3D reconstruction of a terminal microvascular loop. (R) Finite element analysis demonstrated lower velocity of blood flow in the terminal loop. (S) A section through middle of the loop. (T) 3D reconstruction and finite element analysis of hemodynamic following the establishment of anastomoses. Note the enhanced blood flow in the terminal loops.
CNE_23162_sm_SuppFig8.tif10177KSupporting Information Figure 8. (Magenta-green version of Figure 8 for the assistance of color-blind readers.) Structural transformation of S100+ telacytes. (A) A stellate S100+ telacyte (green) in a healthy pulp (arrowhead). (B) Injury-induced radial transformation of S100+ telacyte (green). (C) Expression of endothelin-1 (arrowhead, red) in the vicinity of a stellate S100+ telacyte (green). (D) Gene expression for PDGF-A in different zones of injured pulps (n=4 pulps) compared to healthy pulps (n=4 pulps).
CNE_23162_sm_SuppFig9.tif13007KSupporting Information Figure 9. (Magenta-green version of Figure 9 for the assistance of color-blind readers.) Distribution of microglia and telacytes. (A-C) S100+ telacytes (green) and MHC-II+ microglia (red) are intimately associated in the injured pulp. Note the parallel orientation of these cells. (D-F) Higher magnification view of S100+ telacytes (green) and MHC-II+ microglia (red) and the associated UEA-l+ microvasculature (purple). Note the intimacy of spatial association (arrowhead). (G) 3D reconstruction of Figure F. The inset shows a different perspective. (H) A polar histogram demonstrating the orientation and distribution of microglia in healthy (red dots) and injured (blue dots) pulps (n=4 pulps). (I) Correlation of microglia orientation with the distance from the odontoblasts in healthy (red dots) and injured (blue dots) pulps (n=4 pulps). (J) A polar histogram demonstrating the orientation and distribution of telacytes in healthy (red dots) and injured (blue dots) pulps (n=4 pulps). (K) Correlation of telacyte orientation with the distance from the odontoblasts in injured (blue dots) pulps (n=4 pulps). (L) The method for calculation of the association of microglia and telacytes. The region bound by the white dotted line (An) represents the surface area between microglia and the closest neighboring telacyte. The region bound by the yellow dotted line (Ad) represents the surface area between microglia and the second closest neighboring telacyte. (M) Subsequent telacyte-dependent distribution analysis of microglia as per methods. The top graphs demonstrate distribution of microglia between two adjacent telacytes. Note the skewed distribution pattern compatible with an Inverse Gaussian distribution. The bottom graphs confirm the predictable power of an Inverse Gaussian distribution.
CNE_23162_sm_SuppFig10.tif8658KSupporting Information Figure 10. (Magenta-green version of Figure 1 for the assistance of color-blind readers.) Schematic representation of angiogenic remodelling of the blood-barrier microvasculature. Intra-vascular loop formation (left), through combined activity of endothelial cells, pericytes, perivascular micoglia and telacytes, is followed by expansion of the loop (middle) and pericyte-guided formation of vascular anastomosis (right). Endothelium: gray, pericyte: green, microglia: yellow, telacyte: orange, odontoblasts: violet.

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