Therapeutic interaction of systemically-administered mesenchymal stem cells with peri-implant mucosa

Abstract

Objectives: The objective of this study was to investigate the effect of systemically transplanted mesenchymal stem cells (MSCs) on the peri-implant epithelial sealing around dental implants.

Materials and methods: MSCs were isolated from bone marrow of donor rats and expanded in culture. After recipient rats received experimental titanium dental implants in the bone sockets after extraction of maxillary right first molars, donor rat MSCs were intravenously transplanted into the recipient rats.

Results: The injected MSCs were found in the oral mucosa surrounding the dental implants at 24 hours post-transplantation. MSC transplantation accelerated the formation of the peri-implant epithelium (PIE)-mediated mucosa sealing around the implants at an early stage after implantation. Subsequently, enhanced deposition of laminin-332 was found along the PIE-implant interface at 4 weeks after the replacement. We also observed enhanced attachment and proliferation of oral mucous epithelial cells.

Conclusion: Systemically transplanted MSCs might play a critical role in reinforcing the epithelial sealing around dental implants.

Figure 1. Design of the in vivo and in vitro experiments.

(A) Photograph of the experimental implants (upper panel). Photograph of the implant in the rat oral cavity (lower panel). There is no apparent inflammation in the oral mucosa around the implant. (B) Experimental protocol for the in vivo study. Implantation was immediately performed after tooth extraction. Then, 24 hours after implantation, mesenchymal stem cells (MSCs) were injected via the tail vein. The structure of the epithelial tissue around the tooth or implant was observed after 1, 2, 3 and 4 weeks. (C) Experimental protocol for the in vitro study. Rat oral epithelial cells (OECs) were analyzed for changes in cell morphology 7 days after seeding OECs with MSCs under various culture conditions. (D) Experimental methods used for the in vitro study.

Figure 2. Multipotential differentiation of rat MSCs.

(A) Expression of stem cell markers in rat BMMSCs. Cells cultured in 100 mm culture dishes were fixed and immunostained with specific Abs for rat CD-44, CD-90, CD-105 or CD-11b. Cells were incubated with rhodamine- or FITC-conjugated secondary Abs. (A1) Under a fluorescence microscope, positive signals were quantified in five random fields and expressed as the percentage of total DAPI-positive cells (bar = 100 µm) (mean ± SD). (A2) Expression of cell surface markers on MSCs as determined by flow cytometry. (B) (B1) Osteogenic differentiation of MSCs. After culture under osteogenic differentiation conditions for 4 weeks, osteogenic differentiation was determined by Alizarin Red S staining and western blot analysis of specific proteins (ALP, Ranx-2, OCN). The graph shows the quantification of the Alizarin Red S dye content in differentiated osteocytes from independent experiments (mean ± SD). Scale bar, 50 µm. (B2) Adipogenic differentiation of MSCs. After culture under adipogenic differentiation conditions for 2 weeks, adipocyte differentiation was determined by Oil Red O staining and western blot analysis of specific proteins (PPAR-γ, LPL). The graph shows the quantification of the Oil Red O dye content in differentiated adipocytes from independent experiments (mean ± SD). *P<0.05 (C) Single colony-derived rat stem cells represented a putative MSC population with clonogenic renewal properties.

Figure 3. Accumulation of GFP-transgenic injected MSCs after tooth extraction or implantation.

(A) Around the experimental implants, injected MSCs selectively accumulated at the extraction site or peri-implant tissue. However, no double-positive MSCs were observed in gingival mucosa. Fibroblasts isolated from back skin as negative controls did not accumulate at any sites. Bar = 100 µm. (B) The accumulated MSCs remained around the extraction and implantation sites for approximately 1 to 2 weeks.

Figure 4. Formation of oral mucosae by injected rat MSCs after tooth extraction.

(1w) One week after extraction, a thin epithelial layer extended horizontally on the wound site in the MSC group (B), but not in the control group (A). (2, 3w) After 2 and 3 weeks, in both groups, the OE became mature in structure. In the MSC group, the wound site exhibited the same horizontal height as normal OE. (4w) After 4 weeks, both groups exhibited consolidated OE. Hematoxylin and eosin staining. Bar = 100 µm.

Figure 5. Laminin-332 distribution during the formation of peri-implant epithelium (PIE) following MSC injection.

(A) (1w) After 1 week, laminin-332 was expressed in the BM of the new epithelium and in the FC, but not in the epithelial layer facing the implant. After 2 weeks, laminin-332 was intensely expressed in the CT, and the innermost cells of the PIE facing the implant were positive for laminin-332 in the MSC group. (3w) After 3 weeks, a weak positive reaction for laminin-332 was observed in the PIE as a thin band along the implant-PIE interface in the MSC group only. (4w) After 4 weeks, the PIE was completely formed in both groups. Laminin-332 was scarcely expressed along the upper portion of the implant-PIE interface. Laminin-332 was weakly expressed along the BM. Hematoxylin staining. Bar = 100 µm. (B) Lower panels show schematics of these tissue arrangements in the gingiva around the implant. Green lines show laminin-332-positive areas.

Figure 6. Relationship between MSCs and OECs in co-culture.

(A) Rate of proliferation as determined by Brd-U assay. The rate of OEC proliferation in the trans-well condition was significantly lower than in cells grown with C-medium, but the proliferation rate was markedly increased by indirect culture with MSCs. (bar = 100 µm) (B) Apoptosis analysis by FACS. Apoptosis of OECs was detected and quantified by FACS after annexin V and 7AAD staining. The lower table was arranged from the upper data. The rate of apoptosis in OECs in the Co-cul condition was much higher than in the trans-well condition. Each data point represents the mean ± SD of two parallel experiments. #; P<0.05 versus trans-well.