Characterization of the soft-tissue wall lining residual periodontal pockets and implications in periodontal wound healing

Periodontitis is initiated by hyper-inflammatory responses within the tissues which in turn generate dysbiotic ecological changes within the microbial communities [1, 2]. Four distinct but overlapping phases (i.e., hemostasis, inflammation, proliferation, and maturation) characterize periodontal wound healing. In the presence of residual inflammation, granulation tissue (GT) is formed in an attempt to reconstruct the site, which is characterized by a highly dense network of blood arteries and capillaries, increased cellular density of fibroblasts and macrophages, and irregularly organized collagen fibers [3]. During regenerative periodontal surgery, GT is routinely excised [4, 5] aiming to promote wound healing and new attachment [6, 7], as it does no longer occupy the space that would be otherwise populated by regenerative cells. From a clinical standpoint, GT excision helps to control local inflammation and bleeding and thus, facilitates the debridement of a site, while it creates space for biomaterial grafting [8]. It should be noted that lower densities of immunocompetent cells were found in regenerated gingival units following resection of the pocket soft-tissue wall, compared to sites that this lining was left intact in situ [9].

However, there is controversy in the literature regarding the clinical benefits of resecting the pocket soft-tissue lining. Gingival curettage and GT removal in adjunction to scaling and root planing (SRP) failed to demonstrate additional clinical improvements over SRP alone [10]. Gingival curettage as a stand-alone procedure or when combined with SRP is not considered as a method of treatment [11] having little—if any—effect on gain in clinical attachment levels (CAL) [12]. Interestingly, it has been recently demonstrated that cells isolated from periodontal GT express embryonic stem cell markers [13] and appear to have osteogenic potential [14]. Remaining periodontal pockets following subgingival instrumentation, albeit inflamed, possess MSC-like cells with similar immunophenotypic characteristics to those isolated from healthy gingival crevices [15]. A pro-inflammatory milieu is found to suppress pluripotency, viability, and migration of human periodontal ligament stem cells (hPDLSCs), while these cell properties are promoted when inflammatory processes are regulated restoring the regenerative capacity of the cells [16].

Hence, a recent clinical trial studied the treatment outcomes of the Minimal Access Flap by retaining the soft-tissue wall of intrabony defects and thus, the multipotent stem cells that reside within it aiming to potentially promote tissue healing while minimizing gingival recession [17]. The current study aimed to characterize the soft-tissue wall lining residual periodontal pockets following step 1 and step 2 periodontal therapy, regarding wound healing-related parameters versus healthy gingival crevices in the same individuals.

During inflammation, lymphatic vessels sprout and proliferate within the infiltrated tissues [23], to facilitate the homing of leukocytes, eliminate the antigens, and drain the interstitial fluid from the inflammatory site [24]. However, there is a void in the literature regarding the contribution of lymphangiogenesis in periodontal wound healing and bone loss [25]. The lymphangiogenesis-related genes PROX1 [26], LYVE1 [27], and PDPN [28] are mainly used to distinguish lymphatic vessels from blood vessels. Current findings demonstrated that these genes were expressed in diseased and healthy gingival biopsies indicating a lymphangiogenesis potential in both types of tissue. However, the significantly greater expression of PROX1 found in diseased tissues implies the presence of residual inflammation that was also clinically evident. The lymphatic response is known to be long-lasting, even after the inflammatory challenge has been removed [23], and if inflammation reoccurs, the remaining expanded vessel network contributes to the initiation of adaptive immune responses [29]. Although an increased vessel network is associated with inflammation resolution, lymphangiogenesis has also been demonstrated to play a role in perpetuating chronic inflammatory disorders [30]. Thus, the responses of lymphatic endothelial cells within the chronically inflamed gingivae may shed light into periodontal pathogenesis and this remains to be further studied.

The gene profiling in the inflamed and non-inflamed gingival tissues demonstrated that out of the 84 study key genes that are central to wound healing, 65 were expressed in both inflamed and healthy gingival tissues. However, gene expression between the pair-matched tissue specimens significantly differed for six genes; TGF-α, EGFR, TGF-β1, COL5A3, MIF, and ITGA3. The TGF-α and TGF-β1 are important growth factors in periodontal healing but they have distinct biological roles. Transforming growth factor-alpha acts as a ligand for the epidermal growth factor receptor (EGFR), triggering cell proliferation, differentiation, and development [31]. Transforming growth factor-beta-1 promotes the production of GT by enhancing the expression of extracellular matrix (ECM)-related genes such as fibronectin, fibronectin receptor, collagen, and protease inhibitors [32]. It is also implicated in the recruitment of inflammatory cells and enhancement of tissue debridement by macrophages [32, 33]. In line with current findings, TGF-β1 mRNA expression has been found higher in gingival tissue obtained from patients with periodontitis when compared to tissue harvested from subjects free of periodontitis [34]. In the current study, macrophage migration inhibitory factor (MIF) was significantly upregulated in GT samples. Macrophage inhibitory factor is a pro-inflammatory mediator [35] and has been shown to contribute to both the inflammatory and migration/proliferation phases of wound healing [36]. In the inflammatory phase, chronic expression of MIF would serve to attenuate wound healing progression due to its potent inflammatory potential. Therapeutic targeting of MIF might be promising in diseases with impaired/unbalanced wound healing [36]. However, due to its pro-proliferative activity, MIF expression may contribute to improving wound healing by exerting its effects on fibroblasts [36], and this is central to periodontal regeneration. The current study indicated that COL5A3 gene was significantly upregulated in GT samples. Collagen-V, a protein produced by the COL5A gene, is the connector between the basement membrane and stroma and promotes cell attachment and migration [37]. It has been shown to regulate the initiation of collagen fibril assembly, making it an essential regulator of fibril formation and matrix organization [38]. A recent study has demonstrated that matrix containing COL5A3 may be required for the initiation of wound healing [39]. In addition, ITGA3 was significantly upregulated in GT samples. Integrins are heterodimeric transmembrane proteins that play significant roles in cell proliferation, differentiation, cell-cell attachment, adhesion, and signal transduction between the cell and the ECM during developmental and pathological processes [40]. They can also bind to other ECM proteins such as vitronectin, fibronectin, laminin, and collagen because of their adhesive nature [41]. In conclusion, the upregulated genes in the diseased tissues were involved in the proliferation phases of wound healing by collagen deposition, matrix remodeling, and integrin signaling indicating that despite the remaining inflammatory stimulus, the infiltrated tissues are in a process of restoration, possibly for prolonged time.

This study also investigated the properties of isolated MSCs in diseased versus healthy tissue biopsies. The scratch migration assay comprises a semi-quantitative measurement of migration of monolayer cultured MSCs, indicating their capacity to proliferate and bridge the generated gap in vitro. It should be noted that due to the manual technique employed to generate the scratch, there might be variation in the depth, size, and borders of the scratch. This limitation was overcome by the transwell migration assay which sought to reveal the actual potential of the cells to migrate towards a chemoattractant agent. Current data demonstrated a lower proliferation rate of MSCs isolated from the diseased gingival tissues over the healthy tissues and a tendency for lower migration rate of the former than the latter cells. This finding is in partial agreement with previous data [42], which demonstrated that the population doubling ability of healthy gingival MSCs was greater than that of inflamed gingival MSCs, indicating that the inflammatory state of gingival tissues has an impact on the clonal expansion of residing stem/progenitor cells.

Of interest is the interpretation of current findings from the angle of collagen apposition. In line with the gene profiling analysis which demonstrated the upregulation of genes related to collagen synthesis, greater production of collagen was found by the MSCs isolated in the diseased biopsy over the healthy gingival tissue. It might well be that MSCs in GT are committed to collagen production and matrix synthesis rather than migration and/or proliferation. During wound healing, the fibroblasts proliferate to produce ECM and restore the damaged tissues. This role is seemingly “turned on” in inflamed tissues as an attempt to restore the tissues; however, with GT formation, the site remains unresolved in the long-term unless treatment reforms the site and successfully controls inflammation. However, consistent with the current study, other studies in animal models [43‐45] and in humans [46] have shown that GT has higher levels of collagen than non-inflamed tissues in the early stages of wound healing. These data do not reflect on the organization of the collagen bundles within the tissues and possibly indicate an effort of the host to repair the damaged tissue by increased production of collagen.

Interestingly, a clinical study demonstrated that non-resective surgical approaches that retained the soft-tissue lining of the pocket led to greater residual lesions with greater densities of immunocompetent cells in the regenerated gingival tissues at 6 months [9], implying an inferior healing outcome. However, a relatively short time frame for re-evaluation should be considered in that study design [47]. The current study sheds light into the wound healing process of gingival tissues with remaining inflammation and follows up previous studies on this topic which investigated the outcomes of preserving the pocket gingival lining [15, 17]. This in vitro study was conducted on tissue biopsies retrieved from six non-smoking donors, and despite a relatively small sample size of biopsies (three pairs of biopsies for each arm of the experiment), there was homogeneity in the donors in terms of a similar periodontal clinical status. Because of the small quantities of specimens, three donors were used for the gene profiling analysis and the remaining three for the cell culture assays. However, despite the different specimens used in the two experimental arms of the study, there was a consistent trend across all donors. It should be noted that the samples of health versus disease were pair-matched, to exclude any biological variability across donors. Further research is suggested to determine the role that the upregulated genes related to wound healing might play in a successful treatment outcome and whether the different components of the ECM might play a role in inflammation control and maturation of the GT into connective tissue.