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. 2014 May 28;6(238):238ra69.
doi: 10.1126/scitranslmed.3008234.

Photoactivation of Endogenous Latent Transforming Growth factor-β1 Directs Dental Stem Cell Differentiation for Regeneration

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Free PMC article

Photoactivation of Endogenous Latent Transforming Growth factor-β1 Directs Dental Stem Cell Differentiation for Regeneration

Praveen R Arany et al. Sci Transl Med. .
Free PMC article

Abstract

Rapid advancements in the field of stem cell biology have led to many current efforts to exploit stem cells as therapeutic agents in regenerative medicine. However, current ex vivo cell manipulations common to most regenerative approaches create a variety of technical and regulatory hurdles to their clinical translation, and even simpler approaches that use exogenous factors to differentiate tissue-resident stem cells carry significant off-target side effects. We show that non-ionizing, low-power laser (LPL) treatment can instead be used as a minimally invasive tool to activate an endogenous latent growth factor complex, transforming growth factor-β1 (TGF-β1), that subsequently differentiates host stem cells to promote tissue regeneration. LPL treatment induced reactive oxygen species (ROS) in a dose-dependent manner, which, in turn, activated latent TGF-β1 (LTGF-β1) via a specific methionine residue (at position 253 on LAP). Laser-activated TGF-β1 was capable of differentiating human dental stem cells in vitro. Further, an in vivo pulp capping model in rat teeth demonstrated significant increase in dentin regeneration after LPL treatment. These in vivo effects were abrogated in TGF-β receptor II (TGF-βRII) conditional knockout (DSPP(Cre)TGF-βRII(fl/fl)) mice or when wild-type mice were given a TGF-βRI inhibitor. These findings indicate a pivotal role for TGF-β in mediating LPL-induced dental tissue regeneration. More broadly, this work outlines a mechanistic basis for harnessing resident stem cells with a light-activated endogenous cue for clinical regenerative applications.

Conflict of interest statement

Competing interests: The authors declare that they have no competing interests. D.J.M. and P.R.A. are inventors on a patent application filed on this work.

Figures

Fig. 1
Fig. 1. LPL induces tertiary dentin in a rodent model
(A) An occlusal defect (arrow, top panel) exposed the pulp in the maxillary first molar in rats. These defects were subsequently irradiated with LPL, packed with microspheres or Ca(OH)2 dressing, and sealed with a restoration (bottom panel). (B) Image and quantification of inflammatory response with a myeloperoxidase probe at 24 hours after pulpal exposure and restoration (Control) versus pulpal exposure followed by LPL treatment (Laser). Data are means ± SD (n = 3). P > 0.05, paired two-tailed t test. (C) Dentin repair imaged by high-resolution μCT. Red arrows in top panel indicate cement filling, and yellow arrows in lower panel indicate tertiary dentin deposition along walls of pulp chamber. (D) Quantification of mineralized tissue formation in defects after 12 weeks using high-resolution μCT imaging after pulp exposure alone (Control) and LPL treatment (Laser). Tissue formation defined by the ratio of bone volume (BV) to total tooth volume (TV). Data are individual animals represented by unique symbols (n = 7). Red lines denote means; P value determined by Wilcoxon matched-pairs signed rank test. (E) Histological analysis of decalcified teeth by hematoxylin and eosin (H&E) and toluidine blue staining, with polarized illumination. Sections were taken from tissue adjacent to the defect. # indicates regions of tertiary dentin. Scale bar, 200 μm. (F) Mineral content of non-decalcified sections by scanning electron microscopy (SEM)–EDS. Data are means ± SD (n ≥ 3). Inset image depicts pseudocolored SEM image of regions assessed for analyses. TD, tertiary dentin. (G) LPL-treated non-decalcified tooth samples (n = 2) were imaged using Raman microscopy to CH. Scale bar, to see compositional map of ratio of phosphate (PO43−) 100 μm. Inset shows quantitative histograph depicting spatial distribution of intrapulpal islands (arrow) of tertiary dentin induction. # indicates regions of tertiary dentin within the pulp.
Fig. 2
Fig. 2. LPL treatment generates ROS in mammalian cells in vitro
(A) Mv1Lu cells were treated with LPL at various fluences and assessed for superoxide generation with MitoSOX Red. Mitochondria were counterstained with MitoTracker Green. Red and green overlays are shown representative of three independent experiments. Some cells were preincubated with NAC (1 mM) before LPL (3 J/cm2) treatment or antimycin A treatment as a positive control. Scale bars, 20 μm. (B) Superoxide generation after LPL (3 J/cm2) treatment of Mv1Lu cells. Data are means ± SD (n = 3). P value determined by two-tailed t test. (C) H2O2 generation in Mv1Lu cells (revealed by CM-H2DCFDA) in response to LPL irradiation, with or without NAC (1 mM). Mitochondria were counterstained with MitoTracker Red. Red and green overlays are shown. Scale bar, 100 μm. (D) Griess assay for NO generation after LPL (3 J/cm2) treatment of Mv1Lu cells and mouse dental papilla cells (MDPC-23). (E) H2O2 generation assessed with Amplex UltraRed after LPL treatment of FBS at various fluences. Some samples were preincubated with NAC (1 mM) before LPL treatment. (F) LPL (3 J/cm2)–induced generation of hydroxyl radicals in cell-free serum, as assessed with proxylhydroxylamine. In (D) to (F), data are means ± SD (n = 3). P values determined by two-tailed t test. These findings indicate that LPL treatment induces tertiary dentin formation within the rat tooth pulp.
Fig. 3
Fig. 3. LPL-generated ROS activates LTGF-β1 in vitro
(A) Free cysteines in serum and in a solution of rhLTGF-β1 after LPL treatment (3 J/cm2). Data are means ± SD (n = 3). P values determined by two-tailed t test. (B) Control and LPL-treated serum samples were labeled with IAEDANS and then separated by gel electrophoresis; specific bands were visualized by ultraviolet, indicating the labeling of LPL-induced conformational changes in putative candidates. Right panel shows the same samples, transferred onto a nitrocellulose membrane and subjected to immunoblotting against TGF-β1. Asterisk indicates serum albumin. (C) TGF-β1 activation in serum after treatment with LPL at increasing fluences assessed with ELISA. Data are means ± SD (n > 3). P value was determined by two-tailed t test with Bonferroni correction compared to control (no laser treatment). (D) Phosphorylated Smad2 after LPL treatment of wild-type MEFs or MEFs transfected with mutated (M253A) LTGF-β1. Mutated MEFs were also treated with recombinant TGF-β1 to ensure signaling competency (without laser treatment).
Fig. 4
Fig. 4. LPL-induced odontoblastic differentiation of human dental stem cells (hDSCs)
(A) Phospho-Smad2 expression in hDSCs after LPL treatment. TGF-β1 treatment served as a positive control. Some cells were pretreated with TGF-βRI inhibitor, SB431542, before LPL treatment. Actin served as a loading control. (B) The TGF-β responsiveness of hDSCs was assessed by nuclear translocation of p-Smad2/3 after LPL treatment. Counterstaining for cytoskeletal actin (phalloidin) and nuclei [4′,6-diamidino-2-phenylindole (DAPI)] was performed. Tricolor overlays are shown. Some samples were preincubated with NAC or SB431542 before LPL treatments. Scale bar, 20 μm. (C) Immunofluorescent labeling of stem cell markers Stro-1 and CD90 in hDSCs 7 days after LPL or TGF-β1 treatment. Controls are untreated hDSCs. Nuclei were counterstained with DAPI. Inset shows red channel only. Scale bar, 50 μm. (D) Western blots for stem cell markers CD44, CD106, and CD117 in hDSCs 7 days after LPL or TGF-β1 treatment. Actin served as a loading control. (E) Dentin matrix expression was assessed by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) in hDSCs, 7 days after LPL treatment. Gel images were inverted (black bands) for clarity of presentation.
Fig. 5
Fig. 5. LPL directs dentin differentiation in 2D and 3D mouse pre-odontoblast cultures
(A) The kinetics of TGF-β1 responsiveness of MDPC-23 after LPL or TGF-β1 treatment was assessed with phospho-Smad2 Western blot. (B) ALP activity 3 days after LPL treatment of mouse MDPC-23 cells. Some cells were preincubated with NAC or SB431542 before LPL treatments. (C) SEM image of PLG macroporous scaffolds used for 3D culture (left). Polychromatic SEM technique shows cells seeded within scaffold pores (right). Scale bars, 100 μm (left) and 50 μm (right). (D) Left panel shows quantification of luciferase activity from TGF-β reporter (p3TP luc) Mv1Lu cells at 24 hours after LPL or TGF-β1 treatment. Some scaffolds were preincubated with NAC before LPL treatment. Panel on the right shows hypercolored image of in situ luciferase imaging of reporter cells within 3D scaffolds. (E) LPL induction of mineralizing phenotype assessed by ALP activity in mouse odontoblast-like cells (MDPC-23) cultured in 3D scaffold cultures at 21 days. (F) Western blots for DMP1 and OPN after LPL treatment of MDPC-23 cells cultured in 3D PLG scaffolds for 21 days. In (B), (D), and (E), data are means ± SD (n = 3). P values determined by two-tailed t test.
Fig. 6
Fig. 6. TGF-β mediates the effect of LPL on dentin formation in vivo
(A) Quantitation of induced tertiary dentin volume, as indicated by the ratio of bone volume (BV) to total tooth volume (TV), of rats treated with SB431542 alone or with LPL treatment assessed by μCT at 12 weeks. Means (red lines) are presented; each data point represents individual animal (n = 4). P value determined by Wilcoxon matched-pairs signed rank test. (B) H&E and toluidine blue staining, with polarized illumination, of tooth sections obtained from rats treated with SB431542 (TGF-βRI inhibitor) with or without LPL. Scale bar, 200 μm. Asterisk denotes tertiary dentin noted along the pulp walls. (C) Pulp cells from DSPPCreTGF-βRIIfl/fl teeth were treated with TGF-β1 and BMP4 and assessed for ALP activity. Data are means ± SD (n = 4). P values determined by two-tailed t test. (D) Quantitation of dentin volume in teeth of DSPPCreTGF-βRIIf/f mice with no treatment (Control) or after LPL treatment (Laser) at 8 weeks. Means (red lines) are presented; each data point represents an individual animal (n = 3). P value determined by Wilcoxon matched-pairs signed rank test. (E) H&E and toluidine blue staining, with polarized illumination, of tooth sections obtained from DSPPCreTGF-βRIIlfl/fl mice that were either untreated (Control) or LPL-irradiated (Laser). Scale bar, 200 μm.

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