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, 9 (4), 519-26

Engineering of Tooth-Supporting Structures by Delivery of PDGF Gene Therapy Vectors

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Engineering of Tooth-Supporting Structures by Delivery of PDGF Gene Therapy Vectors

Qiming Jin et al. Mol Ther.

Abstract

Platelet-derived growth factor (PDGF) exerts potent effects on wound healing including the regeneration of tooth-supporting structures. Limitations of topical protein delivery to periodontal osseous defects include transient biological activity and the bioavailability of PDGF at the wound site. The objective of this investigation was to determine the feasibility of in vivo PDGF-B gene transfer to stimulate periodontal tissue regeneration in large tooth-associated alveolar bone defects in rats. Periodontal lesions (0.3 x 0.2 cm in size) were treated with a 2.6% collagen matrix alone or a matrix containing adenoviruses encoding luciferase (control), a dominant negative mutant of PDGF-A (PDGF-1308), or PDGF-B. Block biopsies were harvested at 3, 7, and 14 days post-gene delivery and descriptive histology and histomorphometric analyses were performed. The defects treated with Ad-PDGF-B demonstrated greater proliferating cell nuclear antigen positively stained cells and strong evidence of bone and cementum regeneration beyond that of Ad-luciferase and Ad-PDGF-1308 groups. Quantitative image analysis showed a nearly fourfold increase in bridging bone and sixfold increase in tooth-lining cemental repair in the Ad-PDGF-B-treated sites compared to lesions treated with Ad-luciferase or collagen matrix alone, which showed limited hard tissue neogenesis. In addition, the Xenogen In Vivo Imaging System revealed sustained and localized gene expression of the luciferase reporter at the periodontal lesions for up to 21 days after gene transfer. These results indicate that in vivo direct gene transfer of PDGF-B stimulates alveolar bone and cementum regeneration in large periodontal defects. Gene therapy utilizing PDGF-B may offer the potential for periodontal tissue engineering applications.

Figures

FIG. 1
FIG. 1
Adenovirus transduction efficiency in vitro and in vivo. (A) The transduction efficiency of Ad-PDGF-B in rat dermal fibroblasts was evaluated by Northern blotting and compared to Ad-luciferase, Ad-PDGF-1308, and no treatment (NT). Forty-eight hours after transduction, a high intensity of PDGF-B gene expression was noted following Ad-PDGF-B gene transfer, while other groups failed to exhibit PDGF-B gene expression. (B) Images of a representative rat receiving Ad-luciferase to the mandible depict the kinetics of luciferase expression for a period of 28 days. The alveolar bone of the mandibulae received 20 μl of 2.5 × 1011 PN/ml Ad-luciferase contained in a 2.6% collagen matrix. Evaluation of luciferase expression was then assessed by the use of a Xenogen In Vivo Imaging System. (C) Quantitative intensity of luciferase expression was measured as relative light units at days 1 –28 post-gene transfer. The highest level of luciferase expression occurred at day 1 post-gene transfer and rapidly decreased to 20% by day 14 compared with the expression at day 1 post-gene transfer. Luciferase expression continued to decline over time and was undetectable 28 days following gene delivery. Bars represent standard deviation (n = 3).
FIG. 2
FIG. 2
Photomicrographs of PCNA immunohistochemistry at days 3 and 7 postsurgery and gene delivery. Positively stained cells can be visualized by the brown nuclei. At 3 days postsurgery, most of the positively stained cells were seen along the defect periphery. However, more positive cells were detected along the defect and the carrier peripheries following Ad-PDGF-B treatment compared with the other treatment groups. At day 7, a larger number of positively stained cells were associated with the defects treated with Ad-PDGF-B compared to other groups (100× original magnification). Arrows indicate positively stained cells. Arrowheads indicate the edges of defects.
FIG. 3
FIG. 3
Histological microphotographs of periodontal alveolar bone defects treated for 14 days after gene delivery of Ad-PDGF-B, Ad-PDGF-1308, or Ad-luciferase or collagen matrix only (40× original magnification, left; 200× original magnification, right). Arrows on left side indicate alveolar bone wound edges. Limited alveolar bone formation occurred in the collagen, Ad-luc, and Ad-PDGF-1308 defects, while significant bone bridging was noted most extensively in sites treated with Ad-PDGF-B (dashed line). A thin layer of newly formed cementum (arrowheads) was observed only in the Ad-PDGF-B-treated defects. In addition, more vascularization (blue arrows) was seen in the periodontal ligament region of the Ad-PDGF-B-treated lesions. Asterisks indicate the collagen carrier.
FIG. 4
FIG. 4
Histomorphometric analysis of the specimens 14 days postsurgery and gene delivery. Ad-PDGF-B treatment increased cementum formation, compared to Ad-luciferase treatment (*P < 0.05). Furthermore, Ad-PDGF-B treatment not only improved the bridging length of newly formed alveolar bone compared to Ad-luciferase (**P < 0.01) and collagen matrix alone (*P < 0.05) groups, but also enhanced the percentage of alveolar bone fill compared to Ad-luciferase and Ad-PDGF-1308 treatments (*P < 0.05) (n = 6–8 specimens/group).

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