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Targeting and Immobilization of Bioactive Peptides on Dentin Matrix

¹ Department of Endodontics and
² Department of Anatomy and Cell Biology, School of Dental Medicine, University of Pennsylvania, 240 South 40th Street, Levy Bldg, Rm 423, Philadelphia, PA 19104, USA; and
³ Department of Conservative Dentistry, University of Ulsan, Asan Medical Center, Seoul, Korea

View larger version (24K): [in this window] [in a new window]   Figure 1. Poly-glutamate targeting and transglutaminase–crosslinking result in stable retention of synthetic peptide to mineralized dentin matrix. (A) Streptavidin-HRP-DAB staining reaction within the dentin chambers pre-treated with test solutions. Note the intense staining within the chamber that was pre-treated with biotinylated fully functional peptide [*(Btn)-TG-MB-RGD] and transglutaminase, lesser staining within the chamber that was pre-treated with peptide [*(Btn)-TG-MB-RGD] alone, transglutaminase alone, or the background PBS (control) groups. (B) The average pixel density of the staining in the dentin chambers was measured and shown as the difference between the experimental groups and the PBS group (control).

View larger version (71K): [in this window] [in a new window]   Figure 2a. Attachment of HCDC to dentin in vitro. Cell attachment to the dentin matrix (Coomassie blue staining). (A) Poly-glutamate targeting and transglutaminase-crosslinking of the peptide (TG-MB-RGD and transglutaminase) to mineralized dentin matrix results in a significant increase in cell attachment compared with that to the dentin surfaces that were treated with (B) transglutaminase alone and (C) PBS alone. The cells also spread better on the surfaces pre-treated with both the fully functional peptide and transglutaminase compared with the other groups. Original magnifications at 100x and 200x (inset). Scale bar represents 20 µm.

View larger version (16K): [in this window] [in a new window]   Figure 2b. Quantitative analysis of HCDC cell attachment to dentin. *Statistically significant (by ANOVA). In the presence of transglutaminase, the highest number of cells was attached to the dentin matrix surface that was pre-treated with the fully functional peptide (TG-MB-RGD), followed by the TG-null peptide and the MB-null peptide, indicating that both of the active domains (i.e., both TG and MB domains) in a synthetic peptide are required for peptide retention and the greatest biological effect.

View larger version (103K): [in this window] [in a new window]   Figure 3a. Cementogenesis in vivo. Morphology of transplants showing newly formed cementum matrix (CMtx) deposition against the dentin vehicle (Vc). Original magnification, 250x. Dentin powder was pre-treated with (A) PBS, (B) *(Btn)-TG-MB-RGD peptide, (C) transglutaminase (TGase), (D) *(Btn)-TG-MB-RGD peptide and transglutaminase, (E) TG-MB-RGD peptide and transglutaminase, (F) TG-null peptide and transglutaminase, (G) MB-null peptide and transglutaminase, and (H) RGD-null peptide and transglutaminase. Sequestering the peptides via the mineral-binding domain on the surface of mineralized matrix, followed by the transglutaminase-catalyzed immobilization of such peptides to the organic part of root matrix via the transglutaminase substrate sequence, increased cementum formation in vivo (D,E). Also note that the newly formed cementum matrix abuts dentin vehicle, creating a tight dentin-cementum interface that does not separate, especially evident when dentin powder was pre-treated with fully functional peptides (TG-MB-RGD) and transglutaminase (D,E), indicating that a stable binding of the peptide is critical for creating a tight dentin-cementum interface. White arrows indicate the "splitting" of newly formed cementum from the vehicle (A,F,H).

View larger version (26K): [in this window] [in a new window]   Figure 3b. Morphometric analysis of the in vivo cementogenesis on modified dentin substrate. Note the significant increase in the cementum formation on the dentin surface that was treated with biotinylated fully functional peptide [*(Btn)-TG-MB-RGD] or fully functional peptide (TG-MB-RGD) and transglutaminase (D,E). *Statistically significant (by non-paired Student’s t test).

Journal of Dental Research, Vol. 86, No. 10, 968-973 (2007)
DOI: 10.1177/154405910708601010


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