Therapeutic delivery of CCL2 modulates immune response and restores host-microbe homeostasis.

Many chronic inflammatory diseases are attributed to disturbances in host-microbe interactions, which drive immune-mediated tissue damage. Depending on the anatomic setting, a chronic inflammatory disease can exert unique local and systemic influences, which provide an exceptional opportunity for understanding disease mechanism and testing therapeutic interventions. The oral cavity is an easily accessible environment that allows for protective interventions aiming at modulating the immune response to control disease processes driven by a breakdown of host-microbe homeostasis. Periodontal disease (PD) is a prevalent condition in which quantitative and qualitative changes of the oral microbiota (dysbiosis) trigger nonresolving chronic inflammation, progressive bone loss, and ultimately tooth loss. Here, we demonstrate the therapeutic benefit of local sustained delivery of the myeloid-recruiting chemokine (C-C motif) ligand 2 (CCL2) in murine ligature-induced PD using clinically relevant models as a preventive, interventional, or reparative therapy. Local delivery of CCL2 into the periodontium inhibited bone loss and accelerated bone gain that could be ascribed to reduced osteoclasts numbers. CCL2 treatment up-regulated M2-macrophage and downregulated proinflammatory and pro-osteoclastic markers. Furthermore, single-cell ribonucleic acid (RNA) sequencing indicated that CCL2 therapy reversed disease-associated transcriptomic profiles of murine gingival macrophages via inhibiting the triggering receptor expressed on myeloid cells-1 (TREM-1) signaling in classically activated macrophages and inducing protein kinase A (PKA) signaling in infiltrating macrophages. Finally, 16S ribosomal ribonucleic acid (rRNA) sequencing showed mitigation of microbial dysbiosis in the periodontium that correlated with a reduction in microbial load in CCL2-treated mice. This study reveals a novel protective effect of CCL2 local delivery in PD as a model for chronic inflammatory diseases caused by a disturbance in host-microbe homeostasis.

Ultrasound-based jawbone surface quality evaluation after alveolar ridge preservation.

BACKGROUND: Bone readiness for implant placement is typically evaluated by bone quality/density on 2-dimensional radiographs and cone beam computed tomography at an arbitrary time between 3 and 6 months after tooth extraction and alveolar ridge preservation (ARP). The aim of this study is to investigate if high-frequency ultrasound (US) can classify bone readiness in humans, using micro-CT as a reference standard to obtain bone mineral density (BMD) and bone volume fraction (BVTV) of healed sockets receiving ARP in humans. METHODS: A total of 27 bone cores were harvested during the implant surgery from 24 patients who received prior extraction with ARP. US images were taken immediately before the implant surgery at a site co-registered with the tissue biopsy collection location, made possible with a specially designed guide, and then classified into 3 tiers using B-mode image criteria (1) favorable, (2) questionable, and (3) unfavorable. Bone mineral density (hydroxyapatite) and BVTV were obtained from micro-CT as the gold standard. RESULTS: Hydroxyapatite and BVTV were evaluated within the projected US slice plane and thresholded to favorable (>2200 mg/cm3; >0.45 mm3/mm3), questionable (1500-2200 mg/cm3; 0.4-0.45 mm3/mm3), and unfavorable (<1500 mg/cm3; <0.4 mm3/mm3). The present US B-mode classification inversely scales with BMD. Regression analysis showed a significant relation between US classification and BMD as well as BVTV. T-test analysis demonstrated a significant correlation between US reader scores and the gold standard. When comparing Tier 1 with the combination of Tier 2 and 3, US achieved a significant group differentiation relative to mean BMD (p = 0.004, true positive 66.7%, false positive 0%, true negative 100%, false negative 33.3%, specificity 100%, sensitivity 66.7%, receiver operating characteristics area under the curve 0.86). Similar results were found between US-derived tiers and BVTV. CONCLUSION: Preliminary data suggest US could classify jawbone surface quality that correlates with BMD/BVTV and serve as the basis for future development of US-based socket healing evaluation after ARP.

Intrasocket reactive tissue: The state of current knowledge.

This review focuses on intrasocket reactive tissue and its impact on extraction socket healing. It summarises the current knowledge about intrasocket reactive tissue from a histopathological and biological perspective and discusses the mechanisms by which residual intrasocket reactive tissue can have a positive or negative effect on healing. Additionally, it provides an overview of the various hand and rotary instruments that are currently used for intrasocket reactive tissue debridement. The review also discusses preserving intrasocket reactive tissue as a socket sealing material and the benefits this may offer. It presents clinical cases where either removal or preservation of intrasocket reactive tissue was adopted following extraction and prior to alveolar ridge preservation. Future studies are needed to investigate the suggested beneficial effects of intrasocket reactive tissue on socket healing outcomes.

Local induction of regulatory T cells prevents inflammatory bone loss in ligature-induced experimental periodontitis in mice.

Periodontitis (periodontal disease) is a highly prevalent disease, affecting over 65 million adults in the United States alone. Characterized by an overburden of invasive bacteria, gum inflammation and plaque buildup, over time, these symptoms can result in severe loss of gingival tissue attachment, bone resorption and even tooth loss. Although current treatments (local antibiotics and scaling and root planing procedures) target the bacterial dysbiosis, they do not address the underlying inflammatory imbalance in the periodontium. In the healthy steady state, the body naturally combats destructive, imbalanced inflammatory responses through regulatory pathways mediated by cells such as regulatory T cells (Tregs). Consequently, we hypothesized that local enrichment of regulatory lymphocytes (Tregs) could restore local, immunological homeostasis and prevent the main outcome of bone loss. Accordingly, we locally delivered a combination of TGFß, Rapamycin, and IL2 microspheres in a ligature-induced murine periodontitis model. Herein, we have demonstrated this preventative treatment decreases alveolar bone loss, increases the local ratio of Tregs to T effector cells and changes the local microenvironment's expression of inflammatory and regenerative markers. Ultimately, these Treg-inducing microspheres appear promising as a method to improve periodontitis outcomes and may be able to serve as a platform delivery system to treat other inflammatory diseases.

CCL2 loaded microparticles promote acute patency in silk-based vascular grafts implanted in rat aortae.

Cardiovascular disease is the leading cause of death worldwide, often associated with coronary artery occlusion. A common intervention for arterial blockage utilizes a vascular graft to bypass the diseased artery and restore downstream blood flow; however, current clinical options exhibit high long-term failure rates. Our goal was to develop an off-the-shelf tissue-engineered vascular graft capable of delivering a biological payload based on the monocyte recruitment factor C-C motif chemokine ligand 2 (CCL2) to induce remodeling. Bi-layered silk scaffolds consisting of an inner porous and outer electrospun layer were fabricated using a custom blend of Antherea Assama and Bombyx Mori silk (lyogel). Lyogel silk scaffolds alone (LG), and lyogel silk scaffolds containing microparticles (LGMP) were tested. The microparticles (MPs) were loaded with either CCL2 (LGMP+) or water (LGMP-). Scaffolds were implanted as abdominal aortic interposition grafts in Lewis rats for 1 and 8 weeks. 1-week implants exhibited patency rates of 50% (7/14), 100% (10/10), and 100% (5/5) in the LGMP-, LGMP+, and LG groups, respectively. The significantly higher patency rate for the LGMP+ group compared to the LGMP- group (p = 0.0188) suggests that CCL2 can prevent acute occlusion. Immunostaining of the explants revealed a significantly higher density of macrophages (CD68+ cells) within the outer vs. inner layer of LGMP- and LGMP+ constructs but not in LG constructs. After 8 weeks, there were no significant differences in patency rates between groups. All patent scaffolds at 8 weeks showed signs of remodeling; however, stenosis was observed within the majority of explants. This study demonstrated the successful fabrication of a custom blended silk scaffold functionalized with cell-mimicking microparticles to facilitate controlled delivery of a biological payload improving their in vivo performance. STATEMENT OF SIGNIFICANCE: This study outlines the development of a custom blended silk-based tissue-engineered vascular graft (TEVG) for use in arterial bypass or replacement surgery. A custom mixture of silk was formulated to improve biocompatibility and cellular binding to the tubular scaffold. Many current approaches to TEVGs include cells that encourage graft cellularization and remodeling; however, our technology incorporates a microparticle based delivery platform capable of delivering bioactive molecules that can mimic the function of seeded cells. In this study, we load the TEVGs with microparticles containing a monocyte attractant and demonstrate improved performance in terms of unobstructed blood flow versus blank microparticles. The acellular nature of this technology potentially reduces risk, increases reproducibility, and results in a more cost-effective graft when compared to cell-based options.

Local Sustained Delivery of Anti-IL-17A Antibodies Limits Inflammatory Bone Loss in Murine Experimental Periodontitis.

Periodontal disease (PD) is a chronic destructive inflammatory disease of the tooth-supporting structures that leads to tooth loss at its advanced stages. Although the disease is initiated by a complex organization of oral microorganisms in the form of a plaque biofilm, it is the uncontrolled immune response to periodontal pathogens that fuels periodontal tissue destruction. IL-17A has been identified as a key cytokine in the pathogenesis of PD. Despite its well documented role in host defense against invading pathogens at oral barrier sites, IL-17A-mediated signaling can also lead to a detrimental inflammatory response, causing periodontal bone destruction. In this study, we developed a local sustained delivery system that restrains IL-17A hyperactivity in periodontal tissues by incorporating neutralizing anti-IL-17A Abs in poly(lactic-coglycolic) acid microparticles (MP). This formulation allowed for controlled release of anti-IL-17A in the periodontium of mice with ligature-induced PD. Local delivery of anti-IL-17A MP after murine PD induction inhibited alveolar bone loss and osteoclastic activity. The anti-IL-17A MP formulation also decreased expression of IL-6, an IL-17A target gene known to induce bone resorption in periodontal tissues. This study demonstrates proof of concept that local and sustained release of IL-17A Abs constitutes a promising therapeutic strategy for PD and may be applicable to other osteolytic bone diseases mediated by IL-17A-driven inflammation.

Biorelevant and screening dissolution methods for minocycline hydrochloride microspheres intended for periodontal administration.

Currently, there is no compendial-level method to assess dissolution of particulate systems administered in the periodontal pocket. This work seeks to develop dissolution methods for extended release poly(lactic-co-glycolic acid) (PLGA) microspheres applied in the periodontal pocket. Arestin®, PLGA microspheres containing minocycline hydrochloride (MIN), is indicated for reduction of pocket depth in adult periodontitis. Utilizing Arestin® as a model product, two dissolution methods were developed: a dialysis set-up using USP apparatus 4 and a novel apparatus fabricated to simulate in vivo environment of the periodontal pocket. In the biorelevant method, the microspheres were dispersed in 250 µL of simulated gingival crevicular fluid (sGCF) which was enclosed in a custom-made dialysis enclosure. sGCF was continuously delivered to the device at a biorelevant flow rate and was collected daily for drug content analysis using UPLC. Both methods could discriminate release characteristics of a panel of MIN-loaded PLGA microspheres that differed in composition and process conditions. A mechanistic model was developed, which satisfactorily explained the release profiles observed using both dissolution methods. The developed methods may have the potential to be used as routine quality control tools to ensure batch-to-batch consistency and to support evaluation of bioequivalence for periodontal microspheres.