Attachment and growth of mPDLPs on root surfaces of extracted teeth in vitro. (b, c, d) Light microscopic images of mPDLPs attached to denuded first maxillary molars and cultured in vitro for 3 days before replantation in the tooth socket. Note the extension of fiber bundles and progenitor cells at the apical tip of the cultured implant (arrow, d). (a) A denuded rat first maxillary molar before treatment with mPDLPs. (e, f) The distribution and morphology of mPDLPs seeded on rat first maxillary molars after 10 days of culture using scanning electron microscopy. Note the PDL-like fibrous outgrowth of parallel-aligned and elongated PDL-like cells at the apical end of the tooth root. Histological analysis revealed fibrous attachment (arrow, h) of mPDLPs on root surfaces after 10 days (h) compared to untreated controls (g). Western blot (i) document significant changes in protein expression after PDL progenitors were exposed to micropatterned 3D surfaces. (i) Micropatterned 3D environments were created either by 3D cell culture in conjunction with micropatterned tooth root surfaces (center column: 3D) or after in vivo replantation for 8 weeks (right column: in vivo), and protein expression levels were compared to mPDLP expression levels in 2D culture without any microstructural challenge (left column: 2D). For all six proteins investigated (β1 integrin, α5 integrin, fibronectin, Rho A, F-actin, and periostin), protein levels were higher after exposure to 3D micropatterned root surfaces (i). Integrin α5β1 blockage (k) resulted in a loss of actin fibers and polarization when compared to control mPDLPs cultured on fibronectin coated plates (j). (l–o) Images compares various progenitor lineages and preosteoblast MC3T3 cells in response to microstructured surface topography. mPDLPs demonstrated highly elongated, fibroblast-like shaped cells aligned perpendicular to the microstructured apatite chips (l), whereas dental follicle progenitors (m), dental pulp cells (n), and MC3T3 cells (o) formed small and polygonal cells surrounding the microstructured apatite chips. rt, root; fib, fibers; GAPDH, glyceraldehyde 3-phophate dehydrogenase; PSTN, periostin.