Periodontal disease resulting from chronic dental infection can cause bone sequestration or, in the worst case, tooth loss. The current investigation evaluated anabolic treatment with a novel stem cell bone-targeting drug, LLP2A-Ale, with or without exogenous MSCs to promote bone formation and treat alveolar bone loss associated with PD.
Ligature-induced periodontitis is a well-established mouse model that recapitulates the pathogenesis of human periodontal disease, including bacteria film formation, host immune response stimulation, and alveolar bone loss. Ligand-induced bacterial load and peak periodontal bone loss were observed between days 5-8 post-ligature, which might sustain for 2-3 weeks, or resulted in tooth loss [13-15]. We started treatment on day 8, representing an intervention protocol. In this study, we provide radiographic and histologic evidence that PD induced alveolar bone loss and suppressed bone formation. Treatment with LLP2A-Ale, alone or in combination with MSCs, rescued alveolar bone loss by augmenting bone formation, supporting the use of an anabolic approach to sustain bone formation for the treatment of bone loss associated with PD. Importantly, PD was also associated with systemic changes in cytokines and chemokines and induced changes in peripheral bone formation and bone mass, resembling a systemic inflammatory disease. LLP2A-Ale monotherapy and LLP2A-Ale + MSCs were equally effective at attenuating the PD-induced suppression of bone formation, sustaining alveolar bone mass, and increasing systemic bone formation.
PD is characterized by both inflammation and bone loss [18]. Current medical treatments for PD include flap surgery/root planning, antibacterial oral rinses or antibiotics to control infection, and antiresorptive treatments to control periodontitis. Some non-surgical therapeutics, such as the adjunctive use of probiotics (Clinical study identifier NCT04069611) and Omega-3 polyunsaturated fatty acids (Clinical study identifier NCT04477395) are being evaluated for their efficacies in treating PD. Taurolidine gel was shown to have antimicrobial effects and useful for periodontal therapy [19]. Other experimental drugs, such as chlorhexidine, tetracycline/hydrochloride, locale application of statins, were used as adjunctive treatments to scaling and root planning and were found to have antimicrobial, anti-inflammatory, and modulated bone remodeling process in a rat model of PD [20-23].
Other medications for osteoporosis, such as bisphosphonates (BPs), are used to treat bone loss resulting from periodontitis. BPs were shown to inhibit alveolar bone resorption [24, 25]. However, chronic or high-dose BP treatment is associated with a higher risk for the development of osteonecrosis of the jaw in animals[4, 26-28] and humans [3, 4, 29, 30]. Other medications that affect bone turnover, such as a monoclonal antibody against receptor activator of NF-κB ligand (RANKL), inhibits bone resorption [31]. An antibody against high mobility group box 1 (HMGB1), a nonhistone DNA-binding protein that is secreted into the extracellular matrix in response to inflammation, suppresses the progression of periodontitis through an antiresorptive mechanism [32]. Antiresorptive agents such as BPs are also known for their antiangiogenic actions that potentially are a risk factor for the development of osteonecrosis [33, 34]. Anabolic treatments, such as teriparatide (human PTH (1-34), which effective against osteoporosis, may also help treat bone loss in both rat and mouse models of PD [35, 36]. Interestingly, these previous studies reported antiresorptive and anti-inflammatory mechanisms for daily PTH injections instead of the well-known bone growth effects usually observed for PTH [35, 36]. In one clinical trial, the application of PTH combined with periodontal surgery was superior to periodontal surgery alone at repairing localized bone defects [37]. PTH has also been shown to enhance alveolar bone formation in conjunction with bone grafts in animal studies [38, 39]. Biweekly injections of a new anabolic agent, a monoclonal antibody against sclerostin, for 6 weeks increased periodontal bone formation and alveolar bone in a rat model of PD [40]. Taken together, the data indicate that bone anabolic agents, rather than antiresorptive agents, may be more beneficial for the treatment of periodontal bone loss than anti-resorptive agents. On the other hand, our previous studies with LLP2A-Ale have found enhanced angiogenesis and blood vessel density in bone [7, 8]. The ability of LLP2A-Ale to stimulate both angiogenesis and bone formation suggests that this agent has considerable potential as a treatment of periodontal bone loss.
Cytokines play an essential role in the pathogenesis of PD. The host response can attenuate periodontal bone loss, mostly through anti-resorptive mechanisms [41, 42]. In PD tissue, abundant neutrophils are localized in connective tissue [43, 44]. In the initial steps of periodontal disease, gingival epithelial cells defend against bacterial infection by secreting cytokines such as interleukin (IL)-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), and monocyte chemotactic protein (MCP-1) from gingival tissues to induce the migration of immune cells into inflammation sites [45-47]. These various inflammatory cytokines, while defend against bacterial infections, may indirectly stimulate osteoclastogenesis and bone resorption. Other chemokines, such as CXCL10, CXCL12, CXCL13, and CCL5, may affect osteoblast precursors or osteoblasts and therefore bone formation [48-52]. When the inflammation proceeds to the subepithelial space, it induces damage to the underlying bone through recruitment of osteoclast precursors and induce bone resorption, resulting in no net bone loss [53, 54]. If the inflammatory infiltrate persists near the bone, the bone formation will be uncoupled due to the inhibitory effects of cytokines on osteoblasts [18]. However, inflammation is complicated for PD; while low-level inflammation may be protective against the constant bacterial insult, high levels of key inflammatory factors, such as IL-1, TNF-α, and IL-17R, can result in more severe disease [55-57], and IL-10 may protect against periodontal bone loss [58]. Alendronate was used in LLP2A-Ale to achieve bone-targeted effects [5, 6], however, we have not observed anti-resorptive effects in other published studies [5-8]. Additionally, we initiated treatment with LLP2A-Ale with and without MSCs when the periodontal bone loss was established [13, 14], and we chose to focus on evaluating the ability of LLP2A-Ale to augment new bone formation. Three weeks after the ligature was placed to induce PD, the surrounding alveolar bone had reduced the mineral apposition rate, corresponding to reduced osteoblast activity, and this may have contributed to the observed alveolar bone loss.
It is among the shortcoming of our report that we did not measure pocket formation, a surrogate for inflammation and bone loss, and the local inflammatory cells infiltration in the periodontal tissues. We measured the systemic serum cytokine levels and found that PD is not localized to the dental area but was associated with increased levels of interferon-gamma inducible protein 10 (IP-10), macrophage inflammatory protein-1alpha (MIP-1α), RANKL, G-CSF, and macrophage inflammatory protein 2 (MIP2, CXCL2). Higher expression levels of IP-10, RANTE, MCP-1, and MIP2α were found in gingival biopsies from PD patients [59, 60], and a sustained increase in the concentration of both MIP-1a and MCP-1 was associated with increasing severity of PD [61]. Moreover, investigators have reported that PD may trigger a systemic immune response associated with chronic disorders, including cardiovascular disease, obesity/type 2 diabetes, and rheumatoid arthritis [62]. In particular, we found that PD increased the circulating levels of G-CSF and MIP2 (CXCL2), both of which attenuate osteoblast differentiation [63-65]; this result was consistent with our observation of a reduced mineral apposition rate locally in the oral region and systemically, which ultimately resulted in localized and generalized bone loss. Both the elevated serum levels of G-CSF and MIP2 levels were suppressed by LLP2A-Ale treatment, thus maintaining periodontal bone formation and bone mass. LLP2A-Ale + MSCs increased systemic bone formation and bone mass, which was consistent with our previous observations in other models of bone disease [5-8]. However, in this PD model of inflammation and bone loss, we did not see an additive effect of MSC transplantation for alveolar bone formation during the treatment of periodontal bone loss despite the observation that transplanted MSCs homed to the periodontal regions. MSCs are known to have paracrine effects that support anti-inflammatory activity, angiogenesis, and osteogenesis within the bone microenvironment [8], but we have yet to evaluate the fate of these transplanted MSCs engrafted in alveolar bone and ascertain whether they have an anti-inflammatory or pro-angiogenetic effect in the longer term. Rather than using bone-marrow-derived MSCs, other sources of MSCs, especially the oral stem cells that are derived from the periodontal ligament, dental pulp, exfoliated deciduous teeth, dental follicle, and gingival et al., may offer superior regenerative effects to support periodontal repair or regeneration [66-68]. Periodontal ligament -derived stem cells were used to treat PD in humans (clinical trial identifier: NCT02523651). Apart from oral stem cells, other regenerative approaches such as the use of biomaterials, by themselves or used as scaffolds to load cells or drugs, extracellular vesicles, hold promising therapeutic future to repair and grow bone [69, 70]. Nevertheless, additional studies are needed to determine the beneficial effects of using dental-derived stem cells and LLP2A-Ale for PD-induced bone loss. Perhaps combination treatments of dental-derived MSC and an anabolic agent would be ideal to treat more a challenge degenerative dental diseases, such as repairing osteonecrosis of the jaw, to reduce inflammation, restore angiogenesis, and induce bone regeneration.
In conclusion, LLP2A-Ale treatment stimulated alveolar bone formation and reversed alveolar bone loss induced by ligature induced PD. LLP2A-Ale also reduces PD-induced circulating inflammatory proteins, especially, G-CSF and MIP2. Treatment with the combination of MSCs and LLP2A further increased peripheral skeletal bone formation and bone mass but has no additive effect in terms of further increasing alveolar bone formation. Monotherapy with weekly injections of LLP2A-Ale is a novel therapeutic option for the treatment of bone loss associated with periodontitis.