Receptor activator of nuclear factor-kappa B is enriched in CD9-positive extracellular vesicles released by osteoclasts.

Aim: Receptor activator of nuclear factor-kappa B (RANK)-containing extracellular vesicles (EVs) bind RANK-Ligand (RANKL) on osteoblasts, and thereby simultaneously inhibit bone resorption and promote bone formation. Because of this, they are attractive candidates for therapeutic bone anabolic agents. Previously, RANK was detected in 1 in every 36 EVs from osteoclasts by immunogold electron microscopy. Here, we have sought to characterize the subpopulation of EVs from osteoclasts that contains RANK in more detail. Methods: The tetraspanins CD9 and CD81 were localized in osteoclasts by immunofluorescence. EVs were visualized by transmission electron microscopy. A Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) and immunoaffinity isolations examined whether RANK is enriched in specific types of EVs. Results: Immunofluorescence showed CD9 was mostly on or near the plasma membrane of osteoclasts. In contrast, CD81 was localized deeper in the osteoclast's cytosolic vesicular network. By interferometry, both CD9 and CD81 positive EVs from osteoclasts were small (56-83 nm in diameter), consistent with electron microscopy. The CD9 and CD81 EV populations were mostly distinct, and only 22% of the EVs contained both markers. RANK was detected by SP-IRIS in 2%-4% of the CD9-containing EVs, but not in CD81-positive EVs, from mature osteoclasts. Immunomagnetic isolation of CD9-containing EVs from conditioned media of osteoclasts removed most of the RANK. A trace amount of RANK was isolated with CD81. Conclusion: RANK was enriched in a subset of the CD9-positive EVs. The current study provides the first report of selective localization of RANK in subsets of EVs.

Metabolomic signatures distinguish extracellular vesicles from osteoclasts and odontoclasts.

AIMS: Pathological dental root resorption and alveolar bone loss are often detected only after irreversible damage. Biomarkers in the gingival crevicular fluid or saliva could provide a means for early detection; however, such biomarkers have proven elusive. We hypothesize that a multiomic approach might yield reliable diagnostic signatures for root resorption and alveolar bone loss. Previously, we showed that extracellular vesicles (EVs) from osteoclasts and odontoclasts differ in their protein composition. In this study, we investigated the metabolome of EVs from osteoclasts, odontoclasts and clasts (non-resorbing clastic cells). MATERIALS AND METHODS: Mouse haematopoietic precursors were cultured on dentine, bone or plastic, in the presence of recombinant RANKL and CSF-1 to trigger differentiation along the clastic line. On Day 7, the cells were fixed and the differentiation state and resorptive status of the clastic cells were confirmed. EVs were isolated from the conditioned media on Day 7 and characterized by nanoparticle tracking and electron microscopy to ensure quality. Global metabolomic profiling was performed using a Thermo Q-Exactive Orbitrap mass spectrometer with a Dionex UHPLC and autosampler. RESULTS: We identified 978 metabolites in clastic EVs. Of those, 79 are potential biomarkers with Variable Interdependent Parameters scores of 2 or greater. Known metabolites cytidine, isocytosine, thymine, succinate and citrulline were found at statistically higher levels in EVs from odontoclasts compared with osteoclasts. CONCLUSION: We conclude that numerous metabolites found in odontoclast EVs differ from those in osteoclast EVs, and thus represent potential biomarkers for root resorption and periodontal tissue destruction.

Genome Sequence of Arthrobacter Phage NathanVaag.

We report the genome sequence of bacteriophage NathanVaag, an actinobacteriophage isolated from soil in El Paso, Texas, that infects Arthrobacter sp. strain ATCC 21022. The 49,645-bp genome contains 73 predicted protein-coding genes. Based on gene content similarity to phages in the Actinobacteriophage Database, NathanVaag is assigned to phage cluster AO1.

Multivariate strategy for identifying and quantifying jet fuel contaminants by MCR-ALS/PLS models coupled to combined MIR/NIR spectra.

The investigation and control of jet fuel contamination for private aircrafts has gained attention due to the softer monitoring in comparison to commercial aviation. The possible contamination with kerosene solvent (KS) makes this investigation more challenging, since it has physicochemical similarities with jet fuel. To help solve this problem, a chemometric methodology was applied in this research combining multivariate curve resolution with alternating least squares (MCR-ALS) and partial least squares (PLS) models coupled to near- and mid-infrared spectroscopies (MIR/NIR) in order to detect and quantify KS in blends with JET-A1 using 23 samples (5-60% v/v). Additionally, 98 samples were stored for 60 days, and principal component analysis, genetic algorithm, and successive projections algorithm were coupled to linear discriminant analysis (PCA-LDA, GA-LDA, and SPA-LDA) in order to classify the blends according to the bands assigned to oxidation products, such as phenols and carboxylic acids. GA-LDA and SPA-LDA models were accurate and reached 100% sensitivity and specificity. Physicochemical analysis was not able to detect the presence of KS in contaminated jet fuel samples, even in high concentrations. The use of MIR-NIR combined spectra improved the quantification results, thus decreasing the experimental error from 5.22% (using only NIR) to 1.64%. PLS regression quantified the content of KS with high accuracy (RMSEP < 1.64%, R2 > 0.995). The MCR-ALS model stood out for recovering the spectral profile of kerosene solvent by segregating it from jet fuel spectra. The development of models using chemometric tools contributed to a fast, low-cost, and efficient process for quality control that can be applied in the fuel industry.

Otitis Media and Hearing Loss in Patients With Nonsyndromic Craniosynostosis: A Multicenter Study.

OBJECTIVES: The objectives of this study were to: (1) determine the prevalence of otitis media with effusion in patients with nonsyndromic craniosynostosis; (2) determine the prevalence of hearing loss in patients with nonsyndromic craniosynostosis; and (3) identify potential patterns and outcomes in patients with nonsyndromic craniosynostosis. METHODS: A retrospective chart review was conducted at 2 academic institutions, St Christopher's Hospital for Children and SUNY Upstate Medical University, from January 2015 through August 2018, to identify patients having nonsyndromic craniosynostosis with a concurrent diagnosis of otitis media and/or hearing loss. The demographic data and categorical variables were analyzed using descriptive statistics and chi-square testing, respectively. RESULTS: In the entire cohort of patients (N = 113, age range 0-123 months), 36% had otitis media with effusion on either history, physical examination, tympanometry, and/or imaging. Half (50%) of patients with coronal synostosis had otitis media with effusion compared to sagittal (40.7%), metopic (26.3%), multiple (25%), and lambdoid (0%). However, these differences were not statistically significant (P = .190). Most patients had normal hearing (91%), while a minority had either conductive (7%) or sensorineural (2%) hearing loss. CONCLUSION: The presence of otitis media in our cohort of patients with nonsyndromic craniosynostosis appears to be at the upper limit of normal when compared to historical rates in normocephalic children. Synostosis subtype did not appear to predict the presence of otitis media. Only 9% of patients with nonsyndromic craniosynostosis were found to have a hearing loss.

RANKL and RANK in extracellular vesicles: surprising new players in bone remodeling.

Receptor activator of nuclear factor kappa B-ligand (RANKL), its receptor RANK, and osteoprotegerin which binds RANKL and acts as a soluble decoy receptor, are essential controllers of bone remodeling. They also play important roles in establishing immune tolerance and in the development of the lymphatic system and mammary glands. In bone, RANKL stimulates osteoclast formation by binding RANK on osteoclast precursors and osteoclasts. This is required for bone resorption. Recently, RANKL and RANK have been shown to be functional components of extracellular vesicles (EVs). Data linking RANKL and RANK in EVs to biological regulatory roles are reviewed, and crucial unanswered questions are examined. RANKL and RANK are transmembrane proteins and their presence in EVs allows them to act at a distance from their cell of origin. Because RANKL-bearing osteocytes and osteoblasts are often spatially distant from RANK-containing osteoclasts in vivo, this may be crucial for the stimulation of osteoclast formation and bone resorption. RANK in EVs from osteoclasts has the capacity to stimulate a RANKL reverse signaling pathway in osteoblasts that promotes bone formation. This serves to couple bone resorption with bone formation and has inspired novel bifunctional therapeutic agents. RANKL- and RANK- containing EVs in serum may serve as biomarkers for bone and immune pathologies. In summary, EVs containing RANKL and RANK have been identified as intercellular regulators in bone biology. They add complexity to the central signaling network responsible for maintaining bone. RANKL- and RANK-containing EVs are attractive as drug targets and as biomarkers.

Activation of (pro)renin by (pro)renin receptor in extracellular vesicles from osteoclasts.

The (pro)renin receptor (PRR) is a multifunctional integral membrane protein that serves as a component of the vacuolar H+-ATPase (V-ATPase) and also activates (pro)renin. We recently showed that full-length PRR, found as part of a V-ATPase sub-complex, is abundant in extracellular vesicles shed by osteoclasts. Here, we tested whether these extracellular vesicles stimulate (pro)renin. Extracellular vesicles isolated from the conditioned media of RAW 264.7 osteoclast-like cells or primary osteoclasts were characterized and counted by nanoparticle tracking. Immunoblotting confirmed that full-length PRR was present. Extracellular vesicles from osteoclasts dose-dependently stimulated (pro)renin activity, while extracellular vesicles from 4T1 cancer cells, in which we did not detect PRR, did not activate (pro)renin. To confirm that the ability of extracellular vesicles from osteoclasts to stimulate (pro)renin activity was due to the PRR, the "handle region peptide" from the PRR, a competitive inhibitor of PRR activity, was tested. It dose-dependently blocked the ability of extracellular vesicles to stimulate the enzymatic activity of (pro)renin. In summary, the PRR, an abundant component of extracellular vesicles shed by osteoclasts, stimulates (pro)renin activity. This represents a novel mechanism by which extracellular vesicles can function in intercellular regulation, with direct implications for bone biology.

The management of paediatric burns with Burns and Wounds ointment and burdock leaves: a case series.

OBJECTIVE: In the Amish community, natural therapies, such as Burns and Wounds (B&W) ointment and burdock leaves, are preferred over modern medicine when treating burn wounds. The primary aim of this case series is to highlight the use and clinical outcomes of this treatment for paediatric Amish patients. METHOD: At the a paediatric burn centre, two patients were treated with B&W ointment and burdock leaves. The first patient was 11 months old with 17% total body surface area (TBSA) partial and full-thickness scald burns to her lower extremities. The second patient was 24 months old with 20% TBSA partial-thickness scald burns to the torso, bilateral upper extremities, neck and chin. RESULTS: Soon after presentation to the hospital, both patients developed positive wound cultures and required cessation of ointment and burdock leaf therapy. Both patients ultimately underwent surgical interventions. CONCLUSION: Managing burn wounds with B&W ointment and burdock leaves should be considered as an additional option for wound care in select cases. However, the efficacy of this therapy is limited and standard-of-care modern medical burn treatments should remain an option for these patients. It is critically important to build a mutually respectful relationship with Amish patients' community leaders, as this allows open communication and collaboration in patient care and increases the likelihood that Amish guardians will bring their children to a hospital when necessary.

Craniofacial and neck burns in the pediatric population.

INTRODUCTION: Burn injuries can present with catastrophic physical and psychiatric harm with extensive, long-term sequelae. The pediatric population may especially be at-risk given this population's early neurocognitive and behavioral state of development. Innovations in treatment modalities and the development of evidence-based guidelines have helped mitigate burn morbidity and mortality in the pediatric population. Unfortunately, a surprising dearth of literature identifies risk-factors, epidemiological data, injury mechanisms, and prognostic factors within the pediatric population in the setting of craniofacial burns. METHODS: An analysis of emergency department visits under the National Electronic Injury Surveillance System was conducted for the most recent 5-year period available (2014-2018). Available information includes demographical data, such as age and sex, mechanism of injury, visit circumstances, as well as visit disposition. Additionally, details surrounding the injury, including type of burn and anatomical location of injury, were compared. RESULTS: After a review of results, a total of 2599 patients were included for analysis. Our study shows that infants and young children are at increased risk for grave injury 27.3% and 13% of infants and toddlers transferred or admitted, respectively, p < 0.05). 59.8% of infant burns in particular were caused by liquid or kitchen products, while 44.5% of burns in toddlers were caused by chemical products (p < 0.05 for both). Conversely, adolescents are at greater risk of burns in the setting of occupational and hobby-based activities (20.4% of adolescent burns). CONCLUSIONS: Craniofacial burns in the pediatric population may present with complex pathology and sometimes necessitate advanced care. Presentations and prognoses are different dependent upon age and injury mechanism. These findings may serve as important framework in the establishment of guidelines for medical and legislative reform.

The Use of an Autologous Cell Harvesting and Processing Device to Decrease Surgical Procedures and Expedite Healing in Two Pediatric Burn Patients.

INTRODUCTION: Autologous cell harvesting and processing devices are designed to facilitate the harvesting of cells using enzymatic and physical disruption techniques to immediately apply non-cultured autologous cell suspension (ACS) to the wound area. OBJECTIVE: This case report evaluates clinical outcomes following application of cellular suspension with split-thickness skin grafts (STSGs) as an adjunct for definitive closure of burn injuries and donor sites in 2 pediatric patients. MATERIALS AND METHODS: The cases were performed under a humanitarian use protocol following institutional review board approval at St. Christopher's Hospital for Children (Philadelphia, PA). RESULTS: The first patient was a 4-year-old girl with partial- and full-thickness (32% total body surface area) burn injuries of her head, trunk, flank, arms, thighs, and feet. The patient was discharged 19 days following ACS treatment. The second patient was an 18-month-old girl with partial- and full-thickness (21% total body surface area) burns involving the bilateral lower extremities. She was discharged 22 days after ACS treatment with widely meshed autograft. Neither patient required additional surgical interventions. All treatment and donor areas for both patients remained uninfected and neither patient experienced any unexpected treatment-related adverse events. CONCLUSIONS: These cases are the first of their kind reported in the pediatric population and suggest ACS in conjunction with STSGs can help decrease surgical procedures and expedite healing in pediatric patients with large surface burns.

Actin and Actin-Associated Proteins in Extracellular Vesicles Shed by Osteoclasts.

Extracellular vesicles (EVs) are shed by all eukaryotic cells and have emerged as important intercellular regulators. EVs released by osteoclasts were recently identified as important coupling factors in bone remodeling. They are shed as osteoclasts resorb bone and stimulate osteoblasts to form bone to replace the bone resorbed. We reported the proteomic content of osteoclast EVs with data from two-dimensional, high resolution liquid chromatography/mass spectrometry. In this article, we examine in detail the actin and actin-associated proteins found in osteoclast EVs. Like EVs from other cell types, actin and various actin-associated proteins were abundant. These include components of the polymerization machinery, myosin mechanoenzymes, proteins that stabilize or depolymerize microfilaments, and actin-associated proteins that are involved in regulating integrins. The selective incorporation of actin-associated proteins into osteoclast EVs suggests that they have roles in the formation of EVs and/or the regulatory signaling functions of the EVs. Regulating integrins so that they bind extracellular matrix tightly, in order to attach EVs to the extracellular matrix at specific locations in organs and tissues, is one potential active role for actin-associated proteins in EVs.

The proteome of extracellular vesicles released by clastic cells differs based on their substrate.

Extracellular vesicles (EVs) from osteoclasts are important regulators in intercellular communication. Here, we investigated the proteome of EVs from clastic cells plated on plastic (clasts), bone (osteoclasts) and dentin (odontoclasts) by two-dimensional high performance liquid chromatography mass spectrometry seeking differences attributable to distinct mineralized matrices. A total of 1,952 proteins were identified. Of the 500 most abundant proteins in EVs, osteoclast and odontoclast EVs were 83.3% identical, while clasts shared 70.7% of the proteins with osteoclasts and 74.2% of proteins with odontoclasts. For each protein, the differences between the total ion count values were mapped to an expression ratio histogram (Z-score) in order to detect proteins differentially expressed. Stabilin-1 and macrophage mannose receptor-1 were significantly-enriched in EVs from odontoclasts compared with osteoclasts (Z = 2.45, Z = 3.34) and clasts (Z = 13.86, Z = 1.81) and were abundant in odontoclast EVs. Numerous less abundant proteins were differentially-enriched. Subunits of known protein complexes were abundant in clastic EVs, and were present at levels consistent with them being in assembled protein complexes. These included the proteasome, COP1, COP9, the T complex and a novel sub-complex of vacuolar H+-ATPase (V-ATPase), which included the (pro) renin receptor. The (pro) renin receptor was immunoprecipitated using an anti-E-subunit antibody from detergent-solubilized EVs, supporting the idea that the V-ATPase subunits present were in the same protein complex. We conclude that the protein composition of EVs released by clastic cells changes based on the substrate. Clastic EVs are enriched in various protein complexes including a previously undescribed V-ATPase sub-complex.

Clastic cells in orthodontic treatment: Translational challenges and recent advances.

OBJECTIVES: Orthodontic treatment consists of numerous appliance activations that rely on stimulation of osteoclasts at alveolar bone sites. However, the action of osteoclast-like cells on dentin ("odontoclasts") is a pathological side effect of orthodontic treatment. The aim of this article is twofold: (a) To report preliminary results from ongoing cell culture experiments to identify unique markers of dentin resorption, and (b) To discuss our work using nanoparticle tracking analysis (NTA) and exosomes for developing biological fluid-based biopsies to monitor clastic cell activity. SETTING AND SAMPLE POPULATION: Twelve healthy volunteers in permanent dentition. MATERIAL AND METHODS: For the in vitro experiments, murine clastic cell precursors were cultured on dentin or bone slices for 7 days and phage-display biopanning was used to identify molecular surface differences between osteoclasts and odontoclasts. In the human study, gingival crevicular fluid (GCF) samples were collected using different tools and analysed for protein and exosome recovery. RESULTS: Biopanning generated antibody fragments that were uniquely reactive to odontoclasts. Numerous nanoparticles in the size range of exosomes were detected in all of the human GCF samples. CONCLUSIONS: Our results support that there are molecular differences between osteoclasts and odontoclasts. Emerging technologies may allow the use of exosomes in GCF as a clinical tool to detect markers of root resorption.

Extracellular vesicle identification in tooth movement models.

Extracellular vesicles (EVs) are 30-150 nm in diameter vesicles released by cells that serve important intercellular regulatory functions. EVs include exosomes and microvesicles. Exosomes form in multivesicular bodies and are released extracellularly as the multivesicular bodies fuse with the plasma membrane. Microvesicles bud directly from the plasma membrane. Here, we examine methods that are available or emerging to detect and study EVs during orthodontic tooth movement (OTM). EV's involvement in regulating bone remodelling associated with OTM may be demonstrated by adding isolated EVs to an animal model to change the rate of tooth movement. Exosomes in multivesicular bodies might be detected by immunogold labelling of markers in sections from the tooth and jaw and detection by electron microscopy. Gingival crevicular fluid (GCF) is enriched in EVs. Detection and characterization of EVs released by osteoclasts during resorption have been described, and this information could be used to analyse EVs in OTM models. Regulatory EVs may be enriched in the GCF from teeth that are being moved or are undergoing root resorption. Emerging approaches, including nanoparticle tracking, ExoView and micro- and nanofluidics, show promise for studying EVs in the GCF. Techniques that amplify signal, including polymerase chain reaction (PCR), provide the sensitivity necessary to utilize EVs from GCF as biomarkers. Studies of the role of EVs in OTM will provide fresh insight that may identify means for enhancing OTM procedures. EVs in GCF may include biomarkers for bone remodelling during OTM, orthodontic-associated root resorption, and other dental pathologies.

Enoxacin and bis-enoxacin stimulate 4T1 murine breast cancer cells to release extracellular vesicles that inhibit osteoclastogenesis.

Enoxacin and its bone-seeking bisphosphonate derivative, bis-enoxacin, have recently captured attention as potential therapeutic agents for the treatment of cancer and bone disease. No differences in growth or survival of 4T1 murine breast cancer cells were detected at a concentration of 50 µM of enoxacin or bis-enoxacin. Growth was perturbed at higher concentrations. Both 50 µM enoxacin and bis-enoxacin stimulated increases in the number of GW/Processing bodies, but there were minimal changes in microRNA levels. Extracellular vesicles (EVs) released from 4T1 cells treated with 50 µM enoxacin or 50 µM bis-enoxacin stimulated proliferation of RAW 264.7 cells, and both significantly inhibited osteoclastogenesis in calcitriol-stimulated mouse marrow. EVs from 4T1 cells treated with enoxacin and bis-enoxacin displayed small reductions in the amount of microRNA (miR)-146a-5p and let-7b-5p. In marked contrast, miR-214-3p, which has been shown to regulate bone remodeling, was increased 22-fold and 30-fold respectively. We conclude that enoxacin and bis-enoxacin trigger the release of EVs from 4T1 cancer cells that inhibit osteoclastogenesis.

Clinical Characteristics of Localized Aggressive Periodontitis in Primary Dentition.

OBJECTIVES: Due to the low prevalence of localized aggressive periodontitis (LAP), clinical characteristics of LAP in primary dentition are derived from a few case reports/series in the literature. The goal of this study was to determine common clinical characteristics such as bone and root resorption patterns, in a series of cases with LAP in primary dentition. We hypothesize these cases present aggressive periodontal bone destruction starting mostly around first primary molars and atypical root resorption patterns. STUDY DESIGN: We have evaluated 33 LAP cases in primary dentition for pattern of bone destruction, root resorption and early exfoliation. RESULTS: Cases evaluated were aged 5-12 (mean=8.7 years). Thirty cases presented more severe bone loss on first than second molars, with relatively fast progression to second molars, altered pattern of root resorption, mostly external (n=16) and early exfoliation of primary teeth due to periodontal bone loss, rather than physiologic root resorption (n=11). CONCLUSIONS: This study showed common clinical characteristics found in LAP in primary molars, including possible initiation on first primary molars and abnormal root resorption patterns. These characteristics are important to be early identified and treated in order to prevent possible progression into the permanent dentition.

Acid-base machines: electrical work from neutralization reactions.

We have developed an electrochemical system that performs electrical work due to changes in alkaline ion and proton activities associated with acidic solution neutralization. This system can be used to treat wastewater, contributing to sustainable growth. The system includes an electrochemical machine that operates between an acidic and a basic reservoir to produce work in cycles comprising four stages: two isothermal ionic insertion/de-insertion steps and two steps involving acid and base injection. On the basis of the mixing free energy associated with the reaction free energy, we have developed the thermodynamic formalism by considering reversible electrochemical processes to determine the maximum work performed by this acid-base machine and the efficiency. Electrochemical methods in the time and frequency domains helped in investigating the kinetics of sodium ions and proton insertion in host matrices consisting of copper hexacyanoferrate and phosphomolybdic acid, respectively, to improve our understanding of the factors underlying dissipation as a function of pH and pNa. The full cell composed of these insertion electrodes was used as a proof of concept. It performed a maximum work of 26.4 kJ per mol of electro-inserted ion from HCl solution neutralization with the addition of NaOH, to simulate acidic wastewater treatment in a profitable and sustainable way.

A Pediatric Patient With an Orbital Respiratory Epithelial Cyst.

Respiratory epithelial cysts are rare orbital cysts that can arise secondary to choristomatous rests of respiratory epithelium. Approximately 15 congenital cases have been described in the literature, making it a rare disease entity. We present a case of a 14-month-old Middle Eastern male with a right infraorbital respiratory epithelial cyst. Magnetic resonance imaging of the brain and orbits revealed a right infraorbital cyst hyperintense on T1-weighted images and followed fluid density on T2-weighted images. This cyst was noted to displace the globe superiorly and inferior rectus muscle laterally. This cyst was excised using a transconjunctival approach. Histologically, the cyst wall was lined by ciliated columnar cells with interspersed mucus-containing cells and ciliated transitional epithelium was present, establishing the diagnosis of respiratory epithelial cyst. To our knowledge, this is the youngest patient with a respiratory epithelial cyst of the orbit reported in the literature.

Immunoassay analysis of proteins in gingival crevicular fluid samples from resorbing teeth.

OBJECTIVE: To carry out an immunoassay analysis of biomarkers expressed in gingival crevicular fluid (GCF) with the main goal of finding a useful diagnostic pattern to distinguish between resorbing deciduous teeth and nonresorbing controls. MATERIALS AND METHODS: A split-mouth design was used in this study with a total of 22 GCF samples collected from 11 patients in the mixed dentition. For each child, one deciduous molar with radiographic evidence of root resorption was used as the test tooth whereas the contralateral first permanent molar with formed roots was used as the control tooth. Samples were processed with immunoassays using a panel of selected biomarkers including interleukin-1 beta (IL-1b), interleukin-1 receptor antagonist (IL-1RA), nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG), matrix metalloproteinase-9 (MMP-9), and dentin sialoprotein (DSP). RESULTS: There were no statistically significant differences in levels of IL-1b, OPG, and MMP-9 between test and control sites (P > .05). IL-1RA was the only biomarker to show a significant down-regulation (P  =  .04) in GCF samples collected from resorbing teeth. RANKL data showed a heavily skewed distribution and was deemed unreliable. Only one deciduous GCF sample had detectable levels of DSP; therefore, no further statistical calculation was applicable because of the limited amount of data for this biomarker. CONCLUSIONS: This study indicated that IL1-RA is down-regulated in GCF from resorbing primary molars, thus suggesting this cytokine as a potential analyte to be included in a panel that can discriminate between resorbing and nonresorbing teeth.