Soft tissue augmentation using silk gels: an in vitro and in vivo study

Abstract

Background: Restoration of a three-dimensional shape with soft tissue augmentation is a challenge for surgical reconstruction and esthetic improvement of intraoral mucosa and perioral skin tissues. A connective tissue graft or free gingival graft, classically used for such indications, requires a donor site, which may lead to various clinical complications.

Methods: In this article, a new three-dimensional scaffold made of silk fibroin that could be of great interest for these indications was studied. Mechanical tests were conducted to characterize the physical properties of the materials. The biocompatibility of such scaffolds was positively assessed in vitro using a combination of immunostaining, 5-bromo-2'-deoxyuridine proliferation assays, and histologic staining. Finally, the shaped material was grafted subcutaneously in nude mice for a long-time implantation study.

Results: Human fibroblasts embedded in this material had a survival rate up to 68.4% and were able to proliferate and synthesize proteins. One month after subcutaneous implantation, the three-dimensional soft tissue augmentation was stable, and histologic analysis revealed revascularization of the area through the biomaterial. A mild inflammatory reaction disappeared after 12 weeks.

Conclusion: The results indicate that silk-gel material was able to create a lasting three-dimensional soft tissue augmentation and is a promising biomaterial for periodontal and maxillofacial therapies, either as a scaffold for cells or alone as a biomaterial.

Figures

Figure 1

In vivo cylinder-shaped sample (8 × 6 mm) (A) and microstructural image of a liquid nitrogen frozen/freeze-dried gel observed under a field emission scanning electron microscope (B; bar = 1μm). Comparison of the mechanical properties of the silk and collagen gels using strain-to-failure and stress-relaxation tests. C) For each experiment, four samples were tested. Error bars represent the SD.

Figure 2

In vitro evaluation of fibroblasts inside the silk gels. A) Survival rates after 1 and 6 DIV were measured using the MTT assay for cell viability. Each experiment was performed in triplicate. Error bars represent the SD. B) Inside the silk scaffold, fibroblasts presented a rounded morphology (hematoxylin and eosin stain). Cells were able to proliferate, as confirmed by BrdU staining (C), and synthesizing proteins, as shown by the presence of procollagen, a precursor of collagen, deposited around the fibroblasts (D) (procollagen immunostain). E) No apoptotic cells could be detected (TUNEL stain). Bar = 50 μm.

Figure 3

In vivo experiment. Macroscopic observations and short-term histologic analyses. Mice grafted with silk-gel cylinder-shaped samples on their back as observed on day 7 (A), day 14 (B), and week 12 (C). The augmentation created by the silk gels was clearly visible at each stage (black arrows). D) Visual inspection of a biopsy specimen showed healthy tissues surrounding the graft after 1 month. Histologic analysis with hematoxylin and eosin staining (E through H) showed infiltration by fibroblasts inside the two different gels on day 7 (E and G). A high-inflammation process (black arrow) was observed around collagen gels (F) with inflammatory cells, mainly neutrophils, eosinophils, and histiocytes. H) On day 14, a mild inflammation process with the presence of only histiocytes (white arrow) is observed at the periphery of silk gels. Bar = 50 μm (E and G) and 25 μm (F and H).

Figure 4

In vivo experiment: long-term histologic analyses. A through C) Histologic analyses with hematoxylin and eosin staining showed a complete disappearance of the inflammation process around silk gels at 12 weeks after grafting, with a partial fragmentation of the material. A) At this stage, a large piece of the silk material was detected. B and C) Fibroblasts and stromal tissues were found in the interstitial spaces between the silk-gel fragments (black arrow in C) associated with a complete vascularization (white arrow in C) of the area. Bar = 500 μm (A) and 50 μm (B and C).