Photodynamic Therapy with Aminolevulinic Acid Enhances the Cellular Activity of Cells Cultured on Porcine Acellular Dermal Matrix Membranes Used in Periodontology

Abstract

This study aims to test a photodynamic protocol based on a gel containing aminolevulinic acid followed by red-LED (ALAD-PDT) irradiation on human gingival fibroblasts (hGFs) and osteoblasts (hOBs) cultured on a porcine acellular dermal matrix membrane (PADMM). In the previous literature, ALAD-PDT showed solid antibacterial activity and proliferative induction on HGFs cultured on plates and HOBs cultured on a cortical lamina. PADMMs are used in dentistry and periodontology to treat gingival recessions and to increase the tissue thickness in the case of a thin biotype without the risks or postoperative discomfort associated with connective tissue grafts. However, one of the possible complications in this type of surgery is represented by bacterial invasion and membrane exposition during the healing period. We hypothesized that the addition of ALAD-PDT to PADMMs could enhance more rapid healing and decrease the risks connected with bacterial invasion. In periodontal surgery, PADMMs are inserted after a full-thickness flap elevation between the bone and the flap. Consequently, all procedures were performed in parallel on hOBs and hGFs obtained by dental patients. The group control (CTRL) was represented by the unexposed cells cultured on the membranes, group LED (PDT) were the cells subjected to 7 min of red LED irradiation, and ALAD-PDT were the cells subjected to 45 min of ALAD incubation and then to 7 min of red LED irradiation. After treatments, all groups were analyzed for MTT assay and subjected to histological examination at 3 and 7 days and to the SEM observations at 3, 7, and 14 days. Different bone mineralization assays were performed to quantify the effects of ALAD-PDT on hOBs: ALP activity, ALP gene expression, osteocalcin, and alizarin red. The effects of ALAD-PDT on hGFs were evaluated by quantifying collagen 1, fibronectin, and MMP-8. Results showed that ALAD-PDT promoted cellular induction, forming a dense cellular network on hOBs and hGFs, and the assays performed showed statistically significantly higher values for ALAD-PDT with respect to LED alone and CTRLs. In conclusion, ALAD-PDT could represent a promising aid for enhancing the healing of gingival tissues after PADMM applications.

Keywords: aminolevulinic acid; dermal matrix membrane; gingival fibroblasts; oral osteoblasts; photodynamic therapy; red light.

Figure 1

Scanning electron microscopy (SEM) images of the membrane (PADMM) without cells. Magnification 390×.

Figure 2

Scanning electron microscopy (SEM) images of hGFs cultured on the PADMM and exposed to ALAD-PDT at 3, 7, and 14 days. (A) Magnification 390×; (B) Magnification 1000×; (C) Magnification 3000×; (D) Cell proliferation of hGFs cultured on the PADMM at 3, 7, and 14 days. (** p < 0.001; *** p < 0.0001).

Figure 3

Scanning electron microscopy (SEM) images of hOBs cultured on the PADMM and exposed to ALAD-PDT at 3, 7, and 14 days. (A) (Magnification = 390×; (B) Magnification, 1000×; (C) Magnification and 3000×; (D). Cell proliferation of hOBs cultured on the PADMM at 3, 7, and 14 days. (*** p < 0.0001).

Figure 4

hGFs interaction with PADMM. hGFs grew on the edges of the membrane at 3 days (A–C). At 7 days, they colonize the inside of the membrane (D–F). Magnification: 400×. The arrows pointed to the cells.

Figure 5

Interaction between hOBs and PADMM. At 3 days, osteoblast showed a round shape (A–C); at 7 days, they appeared more elongated (D–F). Magnification: 400×. The arrows pointed to the cells.

Figure 6

ALP activity of hOBs cultured for 7 days and exposed to ALAD-PDT protocol. Increased levels were observed after the treatment (*** p < 0.0001).

Figure 7

Mineralization was evaluated at 14 days in hOBs seeded on the membrane and exposed to ALAD-PDT. The qualitative analysis was performed by ARS (A), while the quantization was carried out by CPC (B). ALAD-PDT promoted the highest calcium deposits compared to CTRL. (*** p < 0.0001).

Figure 8

Real-time PCR of fibroblasts (HGFs) cultured on the matrix and treated with ALAD-PDT for Fibronectin 1 (FN1) (A), Collagen 1 (COL-1) (B), Metalloprotease 8 (MMP8) (C) at 3, 7, and 14 days post-seeding. ALAD-PDT induced increased expression of FN1, COL-1, and MMP8 at 14 days (* p < 0.05; ** p < 0.001; *** p < 0.0001).

Figure 9

Real-time PCR of osteoblasts (hOBs) seeded on the PADMM and treated with ALAD-PDT for genes encoding Alkaline Phosphatase (ALP) (A) and Osteocalcin (OCN) (B) at 3, 7, and 14 days post-seeding. ALP and OCN were more expressed in the ALAD-PDT group than in CTRL. (* p < 0.05; ** p < 0.001; *** p < 0.0001).

Figure 10

Human oral osteoblasts (hOBs) at optical microscopy at 5th passage. Magnification: 10×.