Interstitial collagenase activity stimulates bone resorption by mouse marrow osteoclasts [1]. Her... more Interstitial collagenase activity stimulates bone resorption by mouse marrow osteoclasts [1]. Here, we show that collagenase activity promotes bone resorption by activating adherent osteoclasts to resorb bone. Inhibition of interstitial collagenase activity, either with peptidomimetic hydroxymates or with a specific anti-interstitial collagenase inhibiting antibody, reduced bone resorption by 73-92%. Equal numbers of osteoclasts adhered to bone in the presence of collagenase inhibitors and osteoclast survival was unaffected. In contrast, formation of actin rings and polarization of the vacuolar-H+-ATPase (V-ATPase) to ruffled membranes, two indicators of osteoclast activation, were decreased by inhibiting collagenase activity and stimulated in the presence of cleaved or heat-denatured type I collagen in proportion to increases and decreases of bone resorptive activity. Addition of excess recombinant osteoprotegerin-ligand to cultures did not restore bone resorption in the presence of interstitial collagenase inhibitors. These data support the hypothesis that cleaved collagen stimulates osteoclastic bone resorption by triggering cytoskeletal reorganization and transport of V-ATPase from cytoplasmic stores to ruffled membranes.
Vacuolar H + -ATPases (V-ATPases) are multisubunit enzymes that acidify compartments of the vacuo... more Vacuolar H + -ATPases (V-ATPases) are multisubunit enzymes that acidify compartments of the vacuolar system of all eukaryotic cells. In osteoclasts, the cells that degrade bone, V-ATPases, are recruited from intracellular membrane compartments to the ruffled ...
Osteoclasts differentiate from hematopoietic cells and resorb the bone in response to various sig... more Osteoclasts differentiate from hematopoietic cells and resorb the bone in response to various signals, some of which are received directly from noncellular elements of the bone. In vitro, adherence to the bone triggers the reduction of cell–cell fusion events between osteoclasts and the activation of osteoclasts to form unusual dynamic cytoskeletal and membrane structures that are required for degrading the bone. Integrins on the surface of osteoclasts are known to receive regulatory signals from the bone matrix. Regulation of the availability of these signals is accomplished by enzymatic alterations of the bone matrix by protease activity and phosphorylation/dephosphorylation events. Other membrane receptors are present in osteoclasts and may interact with as yet unidentified signals in the bone. Bone mineral has been shown to have regulatory effects on osteoclasts, and osteoclast activity is also directly modulated by mechanical stress. As understanding of how osteoclasts and othe...
Osteoclasts play a vital role in orthodontic tooth movement. Transactivation of nuclear factor ka... more Osteoclasts play a vital role in orthodontic tooth movement. Transactivation of nuclear factor kappaB (NFkappaB) by phosphorylation of the p65 component of NFkappaB at amino acid 536 (p65*(536)) plays a role in osteoclast differentiation stimulated by receptor activator of nuclear factor kappaB-ligand (RANK-L). We hypothesized that this transactivation pathway might be involved in the responses of alveolar bone cells during orthodontic tooth movement. We detected sharp increases in the levels of p65*(536) 3 and 12 hrs after the application of orthodontic stimuli in rats. In cell culture, osteoclast-like cells displayed no changes in p65*(536) in response to RANK-L, but levels rapidly increased after the cells were mechanically scraped. We conclude that p65*(536) is produced rapidly in response to orthodontic stimuli and mechanical insults, and may be important in bone remodeling associated with orthodontic tooth movement.
Objective: To evaluate a novel, yet simple appliance system for studying orthodontic tooth moveme... more Objective: To evaluate a novel, yet simple appliance system for studying orthodontic tooth movement in the rat that can be used to improve preclinical studies of tooth movement and be easily implemented into translational research, an important area for advancing orthodontics. Method: Male Sprague-Dawley rats (N=32) were lightly anesthetized (1-2.5% isoflurane, inhalation) and a prebent 0.014” Australian wire was bonded to the maxillary incisors and extended to the first molar and activated to produce a palatal tipping force. Light body impressions were taken and epoxy models generated prior to appliance bonding, then at weekly intervals. Digital images were captured and intermolar distances measured at each time point. Tooth movement was evaluated in alendronate (7mg/kg) and bis-enoxacin (25mg/kg/day) treated animals as compared to control animals. Cone beam CT images were generated to evaluate bony changes and animal weight was used as a surrogate measure of health. Result: Using ...
Objectives: By using computational chemistry and in vitro screening, we identified enoxacin as an... more Objectives: By using computational chemistry and in vitro screening, we identified enoxacin as an inhibitor of the interaction between the B2-subunit of vacuolar H+-ATPase (V-ATPase) and F-actin. Enoxacin reduced osteoclast formation and bone resorption in mouse marrow cultures in vitro without affecting osteoblast function [1]. Our goals were to identify additional small molecules with this activity, and to identify the mechanisms by which bone resorption is inhibited. Methods: New inhibitors were identified by computation chemistry (Binhib16), or by constructing a chemical derivative of enoxacin by adding a bisphosphonate backbone (Bis-enoxacin). Marrow osteoclasts were differentiated by treatment with calcitriol and loaded onto bone slices, then treated with inhibitors. Raw 264.7 cells were stimulated with recombinant RANKL in the presence or absence of inhibitors. Apoptotic cells were identified using a colorimetric TUNEL assay. Osteoclasts were identified by staining for tartra...
Objectives: Several lines of evidence suggest that binding between the B2-subunit of vacuolar H+-... more Objectives: Several lines of evidence suggest that binding between the B2-subunit of vacuolar H+-ATPase (V-ATPase) and microfilaments is vital for osteoclast function. Recent data show that enoxacin, a small molecule inhibitor of this interaction, inhibits osteoclast bone resorption in vitro, and both cancer growth and metastasis and horizontal alveolar bone loss triggered by polymicrobial periodontal infections in rodent model systems. Binding between V-ATPase and microfilaments has been shown to be vital for vesicle sorting controlled by the WASH complex. Here we tested whether the WASH complex is involved in osteoclast function. Methods: WASH complex proteins were detected by Western blot, real time quantitative PCR and immunofluorescence microscopy, and knocked down from osteoclasts by RNA interference. Osteoclasts were monitored by staining of actin rings with phalloidin and V-ATPase with anti-E subunit antibody. Results: Elements of the WASH complex, WASH, FAM21 and CapZβ, wer...
Objective: Exosomes are 30-120 nm vesicles that are secreted from many types of cells. We hypothe... more Objective: Exosomes are 30-120 nm vesicles that are secreted from many types of cells. We hypothesized that osteoclast-derived exosomes might be involved in the regulation of bone remodeling. As an initial test of this idea, we isolated exosomes from osteoclast-like cells in culture and then applied them to calcitriol-stimulated mouse marrow cultures to determine their effects on osteoclast-differentiation. Method: Osteoclast-like cells were produced by stimulating RAW 264.7 cells with recombinant RANKL. Exosomes were isolated using ExoQuickTM. Exosomes were negative-stained with uranyl acetate and visualized by transmission electron microscopy. Exosomes (10 µg/ml) were applied to calcitriol-stimulated mouse marrow cultures. After 6 days, osteoclasts were detected by staining for tartrate-resistant acid phosphatase (TRAP) activity. ANOVA and T-tests were used to analyze data. Result: Transmission electron microscopy revealed that vesicles isolated using ExoQuickTM were predominantly...
Objectives: Enoxacin, a fluoroquinolone antibiotic, possesses anti-osteoclastic properties and st... more Objectives: Enoxacin, a fluoroquinolone antibiotic, possesses anti-osteoclastic properties and stimulates miRNA expression. Similarly, enoxacin-linked bisphosphonate, bis-enoxacin, inhibits osteoclast formation and bone resorption. Periodontal diseases are complex and multifactorial caused by polymicrobial subgingival pathogens including Porphyromonas gingivalis (Pg), Treponema denticola (Td), and Tannerella forsythia (Tf). This study was designed to test the efficacy of bis-enoxacin, enoxacin, and alendronate in inhibiting osteoclast alveolar bone resorption (ABR) in a polymicrobial periodontal disease rat model. Methods: Sprague-Dawley rats were orally infected with Pg FDC 381, Td ATCC 35404, and Tf ATCC 43037 strains for 4 days consecutively every other week for 12 weeks. A mixture of 109 cells (Pg 3.3X108+Td 3.3108+Tf 3.3X108) mixed with 4% carboxymethylcellulose was administered orally. Bis-enoxacin (5, 25mg/kg), enoxacin (25 mg/kg), alendronate (1, 2.5mg/kg), and doxycycline (...
Objective: To carry out an immunoassay analysis of biomarkers expressed in gingival crevicular f... more 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 ...
To effectively inhibit bone resorption and prevent failure of endodontically treated teeth, an id... more To effectively inhibit bone resorption and prevent failure of endodontically treated teeth, an ideal intracanal medicament would decrease inflammation along with osteoclast-mediated bone resorption. Bacterial components such as lipopolysaccharides (LPS) are implicated in the development of pulpal and periapical inflammation as well as induce osteoclastogenesis. Propolis is a natural, non-toxic substance collected from bee's wax that has been used for many years in folk medicine. Propolis has been demonstrated to have antibacterial properities along with anti-inflammatory affects on macrophages. In addition, our previous studies have shown that propolis inhibits osteoclast maturation. However, the effect of propolis on the inflammatory response of pulp cells and osteoclasts function has yet to be explored. Objectives: The purpose of this study was to evaluate whether propolis alters the inflammatory response of four endodontically-relevant cell lines: mouse odontoblast-like cells...
The purpose of this study was to determine the direct effects of lipopolysaccharide (LPS) on oste... more The purpose of this study was to determine the direct effects of lipopolysaccharide (LPS) on osteoclastogenesis and to assess the ability of calcium hydroxide-Ca(OH)(2)-to inhibit the osteoclast formation stimulated by LPS. RAW 264.7 cells were cultured with 50 ng/mL recombinant receptor activator of NF-kappaB ligand (RANKL) for 72 hours. RANKL was then removed, and the cells were treated with 0, 1, 10, or 100 ng/mL of LPS, Ca(OH)(2)-treated LPS, or 50 ng/mL of RANKL as a positive control for an additional 48 hours. Cells were fixed and stained with fluorescein isothiocyanate-conjugated phalloidin to detect actin ring formation, and histochemistry was performed to detect multinucleated cells expressing tartrate-resistant acid phosphatase activity. LPS induced osteoclast-like cell (OCL) formation in a dose-dependent manner when osteoclast precursor RAW 264.7 cells were pretreated for 72 hours with RANKL. Ca(OH)(2) significantly inhibited the ability of LPS to stimulate OCL formation....
Interstitial collagenase activity stimulates bone resorption by mouse marrow osteoclasts [1]. Her... more Interstitial collagenase activity stimulates bone resorption by mouse marrow osteoclasts [1]. Here, we show that collagenase activity promotes bone resorption by activating adherent osteoclasts to resorb bone. Inhibition of interstitial collagenase activity, either with peptidomimetic hydroxymates or with a specific anti-interstitial collagenase inhibiting antibody, reduced bone resorption by 73-92%. Equal numbers of osteoclasts adhered to bone in the presence of collagenase inhibitors and osteoclast survival was unaffected. In contrast, formation of actin rings and polarization of the vacuolar-H+-ATPase (V-ATPase) to ruffled membranes, two indicators of osteoclast activation, were decreased by inhibiting collagenase activity and stimulated in the presence of cleaved or heat-denatured type I collagen in proportion to increases and decreases of bone resorptive activity. Addition of excess recombinant osteoprotegerin-ligand to cultures did not restore bone resorption in the presence of interstitial collagenase inhibitors. These data support the hypothesis that cleaved collagen stimulates osteoclastic bone resorption by triggering cytoskeletal reorganization and transport of V-ATPase from cytoplasmic stores to ruffled membranes.
Vacuolar H + -ATPases (V-ATPases) are multisubunit enzymes that acidify compartments of the vacuo... more Vacuolar H + -ATPases (V-ATPases) are multisubunit enzymes that acidify compartments of the vacuolar system of all eukaryotic cells. In osteoclasts, the cells that degrade bone, V-ATPases, are recruited from intracellular membrane compartments to the ruffled ...
Osteoclasts differentiate from hematopoietic cells and resorb the bone in response to various sig... more Osteoclasts differentiate from hematopoietic cells and resorb the bone in response to various signals, some of which are received directly from noncellular elements of the bone. In vitro, adherence to the bone triggers the reduction of cell–cell fusion events between osteoclasts and the activation of osteoclasts to form unusual dynamic cytoskeletal and membrane structures that are required for degrading the bone. Integrins on the surface of osteoclasts are known to receive regulatory signals from the bone matrix. Regulation of the availability of these signals is accomplished by enzymatic alterations of the bone matrix by protease activity and phosphorylation/dephosphorylation events. Other membrane receptors are present in osteoclasts and may interact with as yet unidentified signals in the bone. Bone mineral has been shown to have regulatory effects on osteoclasts, and osteoclast activity is also directly modulated by mechanical stress. As understanding of how osteoclasts and othe...
Osteoclasts play a vital role in orthodontic tooth movement. Transactivation of nuclear factor ka... more Osteoclasts play a vital role in orthodontic tooth movement. Transactivation of nuclear factor kappaB (NFkappaB) by phosphorylation of the p65 component of NFkappaB at amino acid 536 (p65*(536)) plays a role in osteoclast differentiation stimulated by receptor activator of nuclear factor kappaB-ligand (RANK-L). We hypothesized that this transactivation pathway might be involved in the responses of alveolar bone cells during orthodontic tooth movement. We detected sharp increases in the levels of p65*(536) 3 and 12 hrs after the application of orthodontic stimuli in rats. In cell culture, osteoclast-like cells displayed no changes in p65*(536) in response to RANK-L, but levels rapidly increased after the cells were mechanically scraped. We conclude that p65*(536) is produced rapidly in response to orthodontic stimuli and mechanical insults, and may be important in bone remodeling associated with orthodontic tooth movement.
Objective: To evaluate a novel, yet simple appliance system for studying orthodontic tooth moveme... more Objective: To evaluate a novel, yet simple appliance system for studying orthodontic tooth movement in the rat that can be used to improve preclinical studies of tooth movement and be easily implemented into translational research, an important area for advancing orthodontics. Method: Male Sprague-Dawley rats (N=32) were lightly anesthetized (1-2.5% isoflurane, inhalation) and a prebent 0.014” Australian wire was bonded to the maxillary incisors and extended to the first molar and activated to produce a palatal tipping force. Light body impressions were taken and epoxy models generated prior to appliance bonding, then at weekly intervals. Digital images were captured and intermolar distances measured at each time point. Tooth movement was evaluated in alendronate (7mg/kg) and bis-enoxacin (25mg/kg/day) treated animals as compared to control animals. Cone beam CT images were generated to evaluate bony changes and animal weight was used as a surrogate measure of health. Result: Using ...
Objectives: By using computational chemistry and in vitro screening, we identified enoxacin as an... more Objectives: By using computational chemistry and in vitro screening, we identified enoxacin as an inhibitor of the interaction between the B2-subunit of vacuolar H+-ATPase (V-ATPase) and F-actin. Enoxacin reduced osteoclast formation and bone resorption in mouse marrow cultures in vitro without affecting osteoblast function [1]. Our goals were to identify additional small molecules with this activity, and to identify the mechanisms by which bone resorption is inhibited. Methods: New inhibitors were identified by computation chemistry (Binhib16), or by constructing a chemical derivative of enoxacin by adding a bisphosphonate backbone (Bis-enoxacin). Marrow osteoclasts were differentiated by treatment with calcitriol and loaded onto bone slices, then treated with inhibitors. Raw 264.7 cells were stimulated with recombinant RANKL in the presence or absence of inhibitors. Apoptotic cells were identified using a colorimetric TUNEL assay. Osteoclasts were identified by staining for tartra...
Objectives: Several lines of evidence suggest that binding between the B2-subunit of vacuolar H+-... more Objectives: Several lines of evidence suggest that binding between the B2-subunit of vacuolar H+-ATPase (V-ATPase) and microfilaments is vital for osteoclast function. Recent data show that enoxacin, a small molecule inhibitor of this interaction, inhibits osteoclast bone resorption in vitro, and both cancer growth and metastasis and horizontal alveolar bone loss triggered by polymicrobial periodontal infections in rodent model systems. Binding between V-ATPase and microfilaments has been shown to be vital for vesicle sorting controlled by the WASH complex. Here we tested whether the WASH complex is involved in osteoclast function. Methods: WASH complex proteins were detected by Western blot, real time quantitative PCR and immunofluorescence microscopy, and knocked down from osteoclasts by RNA interference. Osteoclasts were monitored by staining of actin rings with phalloidin and V-ATPase with anti-E subunit antibody. Results: Elements of the WASH complex, WASH, FAM21 and CapZβ, wer...
Objective: Exosomes are 30-120 nm vesicles that are secreted from many types of cells. We hypothe... more Objective: Exosomes are 30-120 nm vesicles that are secreted from many types of cells. We hypothesized that osteoclast-derived exosomes might be involved in the regulation of bone remodeling. As an initial test of this idea, we isolated exosomes from osteoclast-like cells in culture and then applied them to calcitriol-stimulated mouse marrow cultures to determine their effects on osteoclast-differentiation. Method: Osteoclast-like cells were produced by stimulating RAW 264.7 cells with recombinant RANKL. Exosomes were isolated using ExoQuickTM. Exosomes were negative-stained with uranyl acetate and visualized by transmission electron microscopy. Exosomes (10 µg/ml) were applied to calcitriol-stimulated mouse marrow cultures. After 6 days, osteoclasts were detected by staining for tartrate-resistant acid phosphatase (TRAP) activity. ANOVA and T-tests were used to analyze data. Result: Transmission electron microscopy revealed that vesicles isolated using ExoQuickTM were predominantly...
Objectives: Enoxacin, a fluoroquinolone antibiotic, possesses anti-osteoclastic properties and st... more Objectives: Enoxacin, a fluoroquinolone antibiotic, possesses anti-osteoclastic properties and stimulates miRNA expression. Similarly, enoxacin-linked bisphosphonate, bis-enoxacin, inhibits osteoclast formation and bone resorption. Periodontal diseases are complex and multifactorial caused by polymicrobial subgingival pathogens including Porphyromonas gingivalis (Pg), Treponema denticola (Td), and Tannerella forsythia (Tf). This study was designed to test the efficacy of bis-enoxacin, enoxacin, and alendronate in inhibiting osteoclast alveolar bone resorption (ABR) in a polymicrobial periodontal disease rat model. Methods: Sprague-Dawley rats were orally infected with Pg FDC 381, Td ATCC 35404, and Tf ATCC 43037 strains for 4 days consecutively every other week for 12 weeks. A mixture of 109 cells (Pg 3.3X108+Td 3.3108+Tf 3.3X108) mixed with 4% carboxymethylcellulose was administered orally. Bis-enoxacin (5, 25mg/kg), enoxacin (25 mg/kg), alendronate (1, 2.5mg/kg), and doxycycline (...
Objective: To carry out an immunoassay analysis of biomarkers expressed in gingival crevicular f... more 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 ...
To effectively inhibit bone resorption and prevent failure of endodontically treated teeth, an id... more To effectively inhibit bone resorption and prevent failure of endodontically treated teeth, an ideal intracanal medicament would decrease inflammation along with osteoclast-mediated bone resorption. Bacterial components such as lipopolysaccharides (LPS) are implicated in the development of pulpal and periapical inflammation as well as induce osteoclastogenesis. Propolis is a natural, non-toxic substance collected from bee's wax that has been used for many years in folk medicine. Propolis has been demonstrated to have antibacterial properities along with anti-inflammatory affects on macrophages. In addition, our previous studies have shown that propolis inhibits osteoclast maturation. However, the effect of propolis on the inflammatory response of pulp cells and osteoclasts function has yet to be explored. Objectives: The purpose of this study was to evaluate whether propolis alters the inflammatory response of four endodontically-relevant cell lines: mouse odontoblast-like cells...
The purpose of this study was to determine the direct effects of lipopolysaccharide (LPS) on oste... more The purpose of this study was to determine the direct effects of lipopolysaccharide (LPS) on osteoclastogenesis and to assess the ability of calcium hydroxide-Ca(OH)(2)-to inhibit the osteoclast formation stimulated by LPS. RAW 264.7 cells were cultured with 50 ng/mL recombinant receptor activator of NF-kappaB ligand (RANKL) for 72 hours. RANKL was then removed, and the cells were treated with 0, 1, 10, or 100 ng/mL of LPS, Ca(OH)(2)-treated LPS, or 50 ng/mL of RANKL as a positive control for an additional 48 hours. Cells were fixed and stained with fluorescein isothiocyanate-conjugated phalloidin to detect actin ring formation, and histochemistry was performed to detect multinucleated cells expressing tartrate-resistant acid phosphatase activity. LPS induced osteoclast-like cell (OCL) formation in a dose-dependent manner when osteoclast precursor RAW 264.7 cells were pretreated for 72 hours with RANKL. Ca(OH)(2) significantly inhibited the ability of LPS to stimulate OCL formation....
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Papers by L. Holliday