Expression of SARS-CoV-2 entry factors in human oral tissue.

The distribution of cells expressing SARS-CoV-2 entry factor angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) in human oral tissues were tested. The investigation was conducted with normal flesh tissue and paraffin-embedded specimens. The ACE2 and TMPRSS2 expression was detected with all subjects in the normal mucosa of the keratinized stratified squamous epithelia of the tongue and non-keratinized stratified squamous epithelia of the lip and cheek. It was found that ACE2 is expressed in the cytoplasm and on the cell membrane mainly in the stratum granulosum of the epithelia while the TMPRSS2 is strongly expressed on the cell membrane mainly in the stratum granulosum and stratum spinosum, but not in the stratum basale. Antibodies' reactions for ACE2 and TMPRSS2 were not observed in the nuclei or keratin layer. The expression of ACE2 and TMPRSS2 in the oral epithelia appears to be general, and the expression was also observed in the mucous and serous acini of the labial glands. The SARS-CoV-2 may transiently attach to the oral mucosa and the minor salivary glands which are present under all of the oral mucosa. The oral cavity can be considered an important organ for SARS-CoV-2 attachment and may provide a preventive medical avenue to guard against COVID-19 by preventing saliva from scattering.

Intramedullary injury combined with osteoporosis therapeutics regulates targeted local osteogenesis.

Bone marrow ablation prompts transient bone formation in nearly the entire medullary cavity before marrow regeneration occurs. Here, we establish a procedure to direct bone formation in a desired particular site within the medullary cavity for support of biomedical devices. Local intramedullary injury was performed in the tibiae of rats and parathyroid hormone (PTH), alendronate, or saline was administered. Newly generated bone in the medulla was assessed by micro-CT and histology. To evaluate the function of newly generated bone, animals received intramedullary injury in tibiae followed by daily PTH. At day-14, implants were placed in the endocortical bone and the bone response to the implants was assessed. The fate of newly generated bone was compared with and without implants. We found that neither intramedullary injury nor medication alone resulted in bone formation. However, when combined, substantial bone was generated locally inside the diaphyseal medulla. Newly formed bone disappeared without implant placement but was retained with implants. Bone was especially retained around and between the implants. This study found that local bone marrow disruption followed by PTH or alendronate generated substantial cancellous bone locally in the diaphyseal medulla. This approach offers promise as a tissue engineering tool in medicine and dentistry.

Mouse anti-RANKL antibody delays oral wound healing and increases TRAP-positive mononuclear cells in bone marrow.

OBJECTIVES: Denosumab, a human monoclonal antibody (mAb) that neutralizes receptor activator for nuclear factor κB ligand (RANKL), is associated with osteonecrosis of the jaw. However, the effect of denosumab on oral wounds is unclear. The aim was to determine the effect of anti-RANKL mAb on oral wounds and bone marrow. MATERIALS AND METHODS: The direct effect of the mAb on fibroblasts, macrophages, and osteoclasts were assessed in vitro. In vivo, mouse anti-RANKL mAb was administered to mice for 9 weeks prior to palatal bone denudation surgery. Mice were euthanized 3 weeks post-surgery, and wound healing was histomorphometrically analyzed. Long bones were assessed using micro-computed tomography, quantitative real-time polymerase chain reaction, and flow cytometry. RESULTS: The mAb had no effect on macrophages and fibroblasts but significantly suppressed osteoclast proliferation in vitro. The mAb treatment significantly increased bone mass by suppressing osteoclasts in vivo. The expression of pro-osteoclastic genes was promoted in the bone marrow of the mAb-administered animals. Consistently, the mAb significantly induced the development of tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells (MNCs) but not osteoclasts in bone marrow. The mAb treatment had no effect on gross healing of the palatal wounds. However, significant inflammation was retained in the connective tissue facing the once denuded bone surface. CONCLUSIONS: Repair of the damaged palate was delayed, and significant inflammation was sustained in the connective tissue by anti-RANKL mAb treatment. CLINICAL RELEVANCE: Denosumab impairs osteoclastic bone repair. Care should be exercised to minimize osseous trauma when invasive procedures are performed on patients taking denosumab.

Chemotherapeutic and antiresorptive combination therapy suppressed lymphangiogenesis and induced osteonecrosis of the jaw-like lesions in mice.

Osteonecrosis of the jaw (ONJ) is a serious adverse event that occurs predominantly in patients on both antiresorptive and antineoplastic therapies. However, how these combination therapies are connected to the high frequency of ONJ in this particular patient population is unclear. This study's aim was to determine a mechanism of ONJ associated with the combination therapy of antiresorptives and chemotherapeutics. Mice received zoledronic acid (ZA) in conjunction with melphalan or dexamethasone. The maxillary first molars were extracted 3 weeks after the initiation of treatment and wound healing assessed at 4 weeks post-extractions using microcomputed tomography and immunohistochemistry. Mice receiving the combination treatment of ZA and melphalan developed ONJ-like lesions, while ONJ-like lesions were not found in mice on ZA or melphalan monotherapy, or the combination treatment of ZA and dexamethasone. ONJ lesions were characterized by a lack of epithelium, exposed necrotic bone, severe inflammatory cell infiltration, and minimal bone formation. Fluorescent immunohistochemistry showed that lymphatic vessel formation was significantly suppressed in ONJ-like lesions with a concomitant decrease in F4/80(+) macrophages expressing vascular endothelial growth factor C (VEGFC). Interestingly, significantly suppressed lymphatics were also found in the draining lymph nodes of mice on the combination treatment of ZA and melphalan. Thus, suppressed lymphangiogenesis was strongly associated with the development of ONJ-like lesions in the current study. Since lymphangiogenesis is critical in the resolution of inflammation during wound healing, inflammation control may serve as a potential strategy to prevent ONJ.

Antiresorptives and osteonecrosis of the jaw.

Osteonecrosis of the jaw (ONJ) is an uncommon condition noted to occur in patients who are receiving osteoclast-targeted antiresorptive therapy. The incidence of ONJ in patients taking oral antiresorptives for the management of osteoporosis is low (approximately 1:100,000), whereas it is higher (∼10%) in patients taking intravenous bisphosphonates for the treatment of metastatic bone diseases. The etiology and pathophysiology of ONJ is unclear. No established preventive or treatment modalities are currently available. Although ONJ is a rare condition, it is imperative for oral care providers to have updated knowledge, as a large number of patients on antiresorptives are seeking oral care. In this comprehensive review, we focus on ONJ and bisphosphonate therapy and dissect the currently available evidence to establish a clinical approach to assess risk, preventive measures, and management of ONJ.

Increased numbers of nonattached osteoclasts after long-term zoledronic acid therapy in mice.

Osteoclasts are key players in the maintenance of bone, which is an endocrine target and organ. Bisphosphonates, used for the management of metastatic bone diseases and osteoporosis, suppress osteoclasts. However, the impact of continuously suppressed osteoclasts is unknown. In this study, mice received zoledronic acid (ZA) for 13 months, nearly half the lifespan of mice, and the effects of continual osteoclast suppression on the bone environment and oral wound healing were determined. ZA therapy suppressed osteoclasts, resulting in significantly more bone mass compared with control. Despite continuous and intense suppression of bone loss in mice receiving ZA, serum calcium levels were maintained in the normal range. No differences were noted in serum tartrate-resistant acid phosphatase (TRAP) 5b levels between ZA-treated and control mice. Histomorphometric analyses of bones revealed that ZA therapy significantly decreased osteoclasts on the bone surface but, instead, substantially increased TRAP(+) mononuclear cells and osteoclasts that were not on the bone surface. When oral trauma was induced, such TRAP(+) mononuclear and nonattached osteoclasts increased considerably with increased inflammatory cell infiltration in the wounds. As a result, oral wound healing was hindered at the connective tissue level. Healing of the epithelium was unaffected. These findings indicate that the continual suppression of osteoclasts does not affect serum calcium levels and that long-term ZA therapy stimulates nonattached osteoclast and TRAP(+) mononuclear cell formation that are expanded rapidly in response to oral trauma. Caution should be exercised when using the serum TRAcP5b to estimate the efficacy of antiresorptive therapy.

Effect of zoledronate on oral wound healing in rats.

PURPOSE: Osteonecrosis of the jaw (ONJ) is a growing concern in patients who receive bisphosphonates that target osteoclasts. As osteoclasts play multifunctional roles in the bone marrow, their suppression likely affects bone homeostasis and alters wound healing of the jaw. The objective was to delineate the impact of osteoclast suppression in the bone marrow and wound healing of the jaw. EXPERIMENTAL DESIGN: Zoledronate was administered to senile rats for 14 weeks. A portion of the gingiva was removed to denude the palatal bone. Gene expression in the bone marrow was assessed and histologic sections were analyzed to determine the wound healing status. RESULTS: Angiogenesis-related genes, CD31 and VEGF-A, were not altered by zoledronate. VEGF-C, which plays a role in lymphangiogenesis, was suppressed. There was a decrease in gene expression of Tcirg1 and MMP-13. Bone denudation caused extensive osteocyte death indicative of bone necrosis. In zoledronate-treated rats, the necrotic bone was retained in the wound while, in controls, osteoclastic resorption of the necrotic bone was prominent. Even though large necrotic bone areas existed in zoledronate-treated rats, overlaying soft tissue healed clinically. Immunohistochemical staining showed rich vascularity in the overlaying soft tissue. CONCLUSIONS: Zoledronate therapy impacts bone marrow by suppressing genes associated with lymphangiogenesis and tissue remodeling, such as VEGF-C and MMP-13. Zoledronate was associated with impaired osseous wound healing but had no effect on angiogenic markers in the bone marrow or soft tissue wound healing. Zoledronate selectively blunts healing in bone but does not affect soft tissue healing in the oral cavity.

Updates on osteonecrosis of the jaw.

PURPOSE OF REVIEW: Osteonecrosis of the jaw (ONJ) is an uncommon condition noted to occur in patients with cancer who are receiving intravenous bisphosphonates. The cause of ONJ remains unknown. The leading hypotheses addressing the mechanism of ONJ are reviewed here. RECENT FINDINGS: The present clinical data suggest that ONJ may occur in approximately 5% of patients with metastatic bone disease. The ability to predict an individual's risk of developing ONJ remains elusive. It is likely that an altered bone microenvironment and/or host defense mechanisms effected by medications used to treat patients with metastatic bone disease contributes to the development of ONJ. Medications that significantly reduce osteoclastic activity are associated with ONJ. Preclinical models of ONJ are being developed but to establish such an intricate systemic condition in animals is challenging. SUMMARY: The ONJ field has progressed via knowledge gained by case reports, population-based studies, and emerging animal models. Still, there are myths that need to be resolved and important clues that need to be investigated. Understanding the pathophysiology of this condition will be critical to improve patient care. Communications between oncologists, dentists, basic scientists, and patients are central to effective treatment and research for this condition.

Significance of the epithelial collar on the subepithelial connective tissue graft.

BACKGROUND: Most clinicians adopt two versions of the subepithelial connective tissue graft (SCTG) procedure, SCTG with or without the epithelial collar on the graft combined with a coronally advanced flap (CAF). However, limited evidence is available to determine whether a retained epithelial collar on an SCTG is needed for a better outcome. The goal of this study was to compare the clinical outcomes of the two SCTG techniques (i.e., SCTG with or without an epithelial collar). METHODS: Twenty patients with Miller Class I or II gingival defects >/=2.0 mm were recruited for the study. The patients were randomly assigned to receive an SCTG with a retained epithelial collar + CAF (SCTGE; n = 10) or an SCTG without an epithelial collar + CAF (SCTGN; n = 10). Clinical parameters, including recession depth, recession width (RW), width of keratinized gingiva (KW), clinical attachment level (CAL), probing depth (PD), gingival index (GI), and plaque index (PI), were assessed at baseline and 3 and 6 months after surgery. RESULTS: SCTGE and SCTGN groups exhibited significant root coverage at 3 and 6 months compared to baseline (P <0.05). The SCTGE group had mean root coverage of 97.50% +/- 7.90% at 6 months compared to 89.10% +/- 25.93% in the SCTGN group, with no significant difference between the groups. At 6 months, complete root coverage was seen in nine of 10 and seven of 10 subjects from SCTGE and SCTGN groups, respectively. Mean KW at 3 months for the SCTGE group was 4.10 +/- 1.10 mm, whereas in the SCTGN group it was 2.75 +/- 0.68 mm. Mean RW was 0 mm and 1.20 +/- 1.60 mm for SCTGE and SCTGN groups, respectively. KW and RW were statistically significantly different between the two groups at 3 months; however, this significance was not seen at 6 months. Other clinical parameters (CAL, PD, thickness of the recipient gingival tissue, PI, GI, and the wound healing index) showed no significant differences between the groups at any time point. CONCLUSIONS: Both SCTG techniques (with or without the epithelial collar) provided predictable and successful root coverage (>/=89%). This study suggests that a retained epithelial collar on the SCTG may not provide a significant benefit with regard to clinical parameters.

Role of Bcl2 in osteoclastogenesis and PTH anabolic actions in bone.

INTRODUCTION: B-cell leukemia/lymphoma 2 (Bcl2) is a proto-oncogene best known for its ability to suppress cell death. However, the role of Bcl2 in the skeletal system is unknown. Bcl2 has been hypothesized to play an important anti-apoptotic role in osteoblasts during anabolic actions of PTH. Although rational, this has not been validated in vivo; hence, the impact of Bcl2 in bone remains unknown. MATERIALS AND METHODS: The bone phenotype of Bcl2 homozygous mutant (Bcl2(-/-)) mice was analyzed with histomorphometry and muCT. Calvarial osteoblasts were isolated and evaluated for their cellular activity. Osteoclastogenesis was induced from bone marrow cells using RANKL and macrophage-colony stimulating factor (M-CSF), and their differentiation was analyzed. PTH(1-34) (50 microg/kg) or vehicle was administered daily to Bcl2(+/+) and Bcl2(-/-) mice (4 days old) for 9 days to clarify the influence of Bcl2 ablation on PTH anabolic actions. Western blotting and real-time PCR were performed to detect Bcl2 expression in calvarial osteoblasts in response to PTH ex vivo. RESULTS: There were reduced numbers of osteoclasts in Bcl2(-/-) mice, with a resultant increase in bone mass. Bcl2(-/-) bone marrow-derived osteoclasts ex vivo were significantly larger in size and short-lived compared with wildtype, suggesting a pro-apoptotic nature of Bcl2(-/-) osteoclasts. In contrast, osteoblasts were entirely normal in their proliferation, differentiation, and mineralization. Intermittent administration of PTH increased bone mass similarly in Bcl2(+/+) and Bcl2(-/-) mice. Finally, Western blotting and real-time PCR showed that Bcl2 levels were not induced in response to PTH in calvarial osteoblasts. CONCLUSIONS: Bcl2 is critical in osteoclasts but not osteoblasts. Osteoclast suppression is at least in part responsible for increased bone mass of Bcl2(-/-) mice, and Bcl2 is dispensable in PTH anabolic actions during bone growth.

The zinc finger mutation C417R of I-kappa B kinase gamma impairs lipopolysaccharide- and TNF-mediated NF-kappa B activation through inhibiting phosphorylation of the I-kappa B kinase beta activation loop.

The activation of the I-kappaB kinase (IKK) complex by TNF or LPS stimulates phosphorylation and degradation of I-kappaBalpha, leading to the nuclear translocation of NF-kappaB. The IKK complex is mainly composed of two catalytic subunits, IKKalpha and IKKbeta, and a chaperon subunit IKKgamma. Although IKKgamma does not have catalytic activity, it is essential for IKK activation induced by multiple stimuli. Importantly, the key residue cysteine 417 at the zinc finger domain of IKKgamma has been found to be mutated to arginine (IKKgammaC417R) in a human genetic disorder called the anhydrotic ectodermal dysplasia with immunodeficiency. To understand the underlying mechanisms of immunodeficiency, we examined whether the IKKgammaC417R mutant modified IKK activation and NF-kappaB transcription stimulated by LPS or TNF in human monocytes. We found that overexpression of IKKgammaC417R severely impaired LPS- and TNF-induced I-kappaBalpha phosphorylation and degradation in a dominant-negative fashion. Also, LPS- and TNF-induced NF-kappaB transcription was inhibited by IKKgammaC417R. The reconstitution of IKKgamma, but not IKKgammaC417R, in IKKgamma-deficient cells restored NF-kappaB signaling, indicating the zinc finger structure of IKKgamma plays a key role in IKK activation. Moreover, C417R mutation in IKKgamma abolished both LPS- and TNF-induced phosphorylation of the activation loop of IKKbeta. Collectively, our results indicated that the zinc finger structure of IKKgamma plays a key role in LPS- and TNF-induced NF-kappaB activation. The anhydrotic ectodermal dysplasia with immunodeficiency patients' immunodeficiency may be associated with NF-kappaB defect in response to bacterial stimulation.