Because MSC treatment is being introduced more widely as a clinically available therapy, the method of administration must be considered to better mitigate risk. Although for a number of other factors also need consideration, including cell source, cell donor condition, cell population, and timing of MSC administration, this study only focused on comparison between systemic and local injection of MSCs into a rat oral implant model.
Sealing and defense at the PIE–implant interface are very important because dental implants in the oral mucosa are at high risk of inflammation. However, sealing between the PIE and implant is much weaker than that between the junctional epithelium (JE) and teeth [3], possibly owing to an inferiority of adhesion structures at the PIE-implant interface [15]. We therefore aimed to assess the influence of MSCs during implant treatment. Our previous report showed a positive effect of systemically injected MSCs for the improvement of peri-implant tissue sealing and acceleration of tissue healing [11].
HRP penetration on implant surface
In the systemic group, a strong HRP reaction was seen only in the coronal portion of the PIE on the implant surface (Fig. 2a). In the control and local groups, HRP reaction was not only found in the coronal PIE region on the surface of the PIE but also in the connective tissue. Furthermore, in the middle and apical PIE regions of these latter groups, the deep layers of PIE cells exhibited the strongest HRP reaction. This result meant that the PIE with these groups had only a weak epithelial sealing, and had been penetration of the external factors to the surrounding tissue of implant.
The systemic group exhibited a significant improvement in blocking HRP penetration (Fig. 2b) compared with both the local and control groups, which were comparable.
Distribution of Ln-332 in the peri-implant oral mucosa
In the systemic group, immunohistochemical staining of Ln-332 showed a positive reaction along the whole implant-PIE interface at 4 weeks (Fig. 3a). In the local group, the Ln-332 deposition pattern in the PIE was comparable to that of the control group. In the oral mucosa around both local and control group implants, Ln-332-positive staining was apparent at the apical portion of the implant-PIE interface, but the upper portion of the interface did not exhibit Ln-332 detection. Only in the control group was the PIE-connective tissue interface intensely stained at the end of the PIE. Absence of Ln-332 staining was noted in the buccal mucosa underlying the OSE or OE in all groups.
As shown in Fig. 3b, expression of adhesion proteins on the interface between PIE-implant was significantly lower in the control and local groups compared with the systemic group.
Ln-332 is the major adhesive ligand for integrin α6β4, which interacts with the cytoskeletal elements, and is a component of the hemidesmosomes, epithelial adhesion plaques that tack the plasma membrane of the epithelial cells [21,22,23]. Moreover, Ln is expressed at the interface between the JE and natural tooth [24, 25] and is thought to be critical for the attachment of gingival epithelial cells to substrates [26, 27]. In our previous study, Ln was implicated in the adhesion of the PIE to the dental implant [20, 28]. Therefore, we observed the distribution of Ln during PIE formation around the implant to eliminate the influence of transplanted MSCs on the OE.
Connecting Ln and α6β4 integrin activates intracellular signaling pathways, such as the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling pathways, which control cell migration, adhesion, and survival [29,30,31]. Our previous study showed that insulin-like growth factor-1 (IGF-1)-activated PI3K signaling promoted epithelial adhesion via HD activation of PI3K signaling and improved epithelial sealing around the implant [32]. Some studies indicate that MSCs activate intrinsic MSCs or various other cells through paracrine expression of IGF-1, epidermal growth factor (EGF), or platelet-derived growth factor (PDGF). Therefore, we highlight the importance of direct contact between MSCs and epithelial cells in order to change cell characteristics or activate cell differentiation.
Whole body MSC accumulation
GFP/CD-90 double-positive cells were detected and counted in various tissues, including the mucosa around the experimental implants (Fig. 4a, b). Although few double-positive MSCs were observed in the liver and heart 1 day after MSC injection, double-positive MSCs were observed in the lung and peri-implant tissue after both systemic and local injection. Figure 4c shows changes in MSC numbers in the rat blood over time. In the local group, intravascular MSC number peaked at day 5, while in the systemic group, MSC number declined quickly at the early time points 3 days after the administration.
Subcutaneously administrated cells or drugs are reported to take a few days to be delivered into the body through vessel bloods [33, 34]. This may be owed to difficulty of the cells in securing vascular accesses to the target site because of a lack of blood vessels at the buccinators, while systemic MSC homing occurs more readily through the bloodstream [35].
The effects of MSC treatment on levels of serum inflammatory cytokines IL-2, IL-4, and IL-10 in the implant model rat are shown (Fig. 4d). Systemic MSC injection resulted in lower IL-2 and IL-4 levels and higher IL-10 levels compared with local MSC injection and the control.
Accumulation of MSCs at the peri-implant tissue
An interesting study disclosed that intraperitoneal MSCs migrated and engrafted at the inflamed colon and passed through the whole intestinal wall reaching the luminal side [36]. Although we were unable to trace the exact migration of our locally administrated MSCs by observed fragmentary, in vivo imaging or tracking with superparamagnetic iron oxide might enable this using a series of flow [14, 37].
In this study, GFP-MSCs took several days to be observed at the target organ after local injection (Fig. 5B (b, c)). Some cells were observed in the mass of the injected area (Fig. 5B(a)), while others were observed indirectly circulating within the whole body or were slightly accumulating at the wound area (Fig. 5B (d)). Specially, these results showed that the most of injected MSCs in the local group got delayed to accumulate around the implant. In the systemic group, GFP/CD-90 double-positive cells were observed around the apical portion of the PIE-like epithelial structure at days 3 and 5 (Fig. 5B (b, c)), after which positive staining declined over time. In the local group, MSC location was limited to the buccal mucosa near the experimental implant at early stage; however, MSC accumulation was observed at the mucosa around the implant from day 5 onwards (Fig 5B). On the contrary, the MSCs did not accumulate on the implant surface, unlike in the systemic group, and they remained around the implantation site for approximately 1 week.
Detection of apoptotic GFP-MSCs
Due to the existence of muscles, connective tissue, dermal layer, and basement membrane, cells within the mass of the injected area encounter these barriers, inhibiting the distance of migration between the application region and inflammatory site, which has an estimated diameter of 20–30 μm (Fig. 6). High-density cell injection at the topical region is also an obstacle for homing, thus using a vasodilator like heparin, culturing the cells under hypoxic condition, maintaining a lower confluence, or the addition of IL-3, IL-6, IGF-1, tumor necrosis factor alpha (TNF-α), or interferon-gamma (IFN-γ), can be used to increase C-X-C chemokine receptor type 4 (CXCR-4) [38], which is a specific receptor for stromal-derived-factor-1 (SDF-1, also called CXCL12), an MSC chemotactic factor that could improve homing efficiency [39].
Locally injected MSCs decreased sharply in number from the buccal site within a week (Fig. 6a). One day after injection, some 7-AAD-positive cells were detected within the MSC mass at the subcutaneous tissue. From days 3 and 5, MSCs began to undergo apoptosis within the local administration region. At day 7 after injection, the level of MSC apoptosis was approximately 90% (Fig. 6b).
Relationship between cell density and differentiation
Isolated MSCs were seeded at four concentrations (5 × 102, 5 × 103, 5 × 104, 5 × 105 MSCs/ml) in adipogenic and osteogenic induction conditions. When the medium was switched to adipogenic and osteogenic differentiation medium, the MSC concentrations were approximately 30, 60, 80, and 100%, respectively. MSCs differentiation into adipocytes and osteoblasts was determined by PPARγ and Runx2 immunofluorescence staining, respectively (Fig. 7 A, B). At a density of 5 × 104 MSCs/ml, adipogenic and osteogenic differentiation were confirmed by increased expression of specific adipogenic markers and Runx2, respectively, using western blotting. Therefore, cells at a suitable cell density within the MSC mass were induced to undergo osteogenic or adipogenic differentiation. However, 3 days after injection, the MSCs began to undergo apoptosis over time in the local administration region. A high ratio of apoptosis occurred immediately after local administration of MSCs, which reduced the amount of viable cells at early stage.
MSC migration from the local mass
Figure 7C and D showed the suitable amount of MSCs had much better positive effect for the migration and adhesion of OECs to titanium surface. MSC attachment within the upper Transwell chamber was determined 24 h after seeding by fluorescence microscopy, as shown in Fig. 7D (a). The majority of MSCs appeared flattened with numerous cytoplasmic extensions and lamellipodia. The majority of MSCs passed through the transwell pores when seeded at a density of 5×104. However, at a density of 5 × 104 the MSCs more readily passed through the 8 µm pores compared with cells at other densities.
OEC and MSC coculture at various seeding densities.
MSCs were seeded at a range of densities (5 × 102, 5 × 103, 5 × 104, 5 × 105 cells/ml) within the upper Transwell chamber, and OECs were cultured on titanium plates like as titanium implant surface, as shown in Fig. 7D (b, c). Only at a density of 5 × 103 did the MSCs activate OEC migration and adhesion.
Furthermore, ELISA indicated that this may not have regulated inflammation because there was no significant difference in expression of IL-10 detected at 24-hour post injection compared with the control (data not shown). The blood stream is thought to be a suitable environment for MSCs, since their survival is higher in this source than within inflamed tissues [37]. To therefore ensure a constant number of viable cells, repeat doses with smaller cell numbers or scatter injection points may benefit local MSC administration. This may permit more cells to be intravasated into the blood vessels, and offer an antiphlogistic effect to the inflammation sites. In terms of MSC administration timing, an earlier response is believed to be more effective to clinical outcomes [36, 40], although similar results have been obtained following delayed administration in some studies. Above all, investing research efforts in identifying the most efficacious route for MSC delivery is a critical matter because there is currently no consensus.