Occlusal disharmony transiently decrease cognition via cognitive suppressor molecules and partially restores cognitive ability via clearance molecules.

Occlusal disharmony has been reported to be affected not only by cytokine and steroid hormone secretion and sympathetic activation in peripheral organs, but also by neurotransmitter release in the central nervous system. However, little is known about whether occlusal disharmony can decrease cognitive ability. We hypothesized that hyperocclusion decreases cognition via Alzheimer's disease-associated molecule expression in the brain. The present study is aimed to elucidate the relationships among occlusal disharmony, cytokine and cognitive-regulated molecule expression in the brain, and the impairment of learning and memory cognition. We examined the effect of hyperocclusion on the relationships among cytokine expression, cognitive suppressor molecules in the hippocampus, and cognition in behavior using a hyperocclusion mouse model. Hyperocclusion dramatically increased interleukin-1ß expression in the serum and hippocampus 1 week after hyperocclusal loading in 2-month-old mice, but no effects in 12-month-old mice. The social and long-term cognitive abilities of the 2-month-old mice were transiently downregulated close to the level of the 12-month-old mice 1 week after hyperocclusion and recovered to close to basal level via the expression of cognitive suppressor clearing proteins. The expression levels of amyloid-ß and phosphorylated tau were significantly upregulated 1 week after hyperocclusal loading in the hippocampus of 2-month-old mice but were constant in 12-month-old mice. Occlusal disharmony-induced interleukin-1ß expression may contribute to accumulation of cognitive suppressor molecules such as amyloid-ß and phosphorylated tau and activate their clearance proteins, resulting in protection against transient dementia in young but not older individuals.

Embedded Human Periodontal Ligament Stem Cells Spheroids Enhance Cementogenic Differentiation via Plasminogen Activator Inhibitor 1.

Spheroids reproduce the tissue structure that is found in vivo more accurately than classic two-dimensional (2D) monolayer cultures. We cultured human periodontal ligament stem cells (HPLSCs) as spheroids that were embedded in collagen gel to examine whether their cementogenic differentiation could be enhanced by treatment with recombinant human plasminogen activator inhibitor-1 (rhPAI-1). The upregulated expression of cementum protein 1 (CEMP1) and cementum attachment protein (CAP), established cementoblast markers, was observed in the 2D monolayer HPLSCs that were treated with rhPAI-1 for 3 weeks compared with that in the control and osteogenic-induction medium groups. In the embedded HPLSC spheroids, rhPAI-1 treatment induced interplay between the spheroids and collagenous extracellular matrix (ECM), indicating that disaggregated HPLSCs migrated and spread into the surrounding ECM 72 h after three-dimensional (3D) culture. Western blot and immunocytochemistry analyses showed that the CEMP1 expression levels were significantly upregulated in the rhPAI-1-treated embedded HPLSC spheroids compared with all the 2D monolayer HPLSCs groups and the 3D spheroid groups. Therefore, 3D collagen-embedded spheroid culture in combination with rhPAI-1 treatment may be useful for facilitating cementogenic differentiation of HPLSCs.

Enhancement of jaw bone regeneration via ERK1/2 activation using dedifferentiated fat cells.

BACKGROUND AIMS: Mesenchymal stem/stromal cells (MSCs) are multipotent and self-renewing cells that are extensively used in tissue engineering. Adipose tissues are known to be the source of two types of MSCs; namely, adipose tissue-derived MSCs (ASCs) and dedifferentiated fat (DFAT) cells. Although ASCs are sometimes transplanted for clinical cytotherapy, the effects of DFAT cell transplantation on mandibular bone healing remain unclear. METHODS: The authors assessed whether DFAT cells have osteogenerative potential compared with ASCs in rats in vitro. In addition, to elucidate the ability of DFAT cells to regenerate the jaw bone, the authors examined the effects of DFAT cells on new bone formation in a mandibular defect model in (i) 30-week-old rats and (ii) ovariectomy-induced osteoporotic rats in vivo. RESULTS: Osteoblast differentiation with bone morphogenetic protein 2 (BMP-2) or osteogenesis-induced medium upregulated the osteogenesis-related molecules in DFAT cells compared with those in ASCs. BMP-2 activated the phosphorylation signaling pathways of ERK1/2 and Smad2 in DFAT cells, but minor Smad1/5/9 activation was noted in ASCs. The transplantation of DFAT cells into normal or ovariectomy-induced osteoporotic rats with mandibular defects promoted new bone formation compared with that seen with ASCs. CONCLUSIONS: DFAT cells promoted osteoblast differentiation and new bone formation through ERK1/2 and Smad2 signaling pathways in vitro. The transplantation of DFAT cells promoted new mandibular bone formation in vivo compared with that seen with ASCs. These results suggest that transplantation of ERK1/2-activated DFAT cells shorten the mandibular bone healing process in cytotherapy.

Cisplatin-induced programmed cell death ligand-2 expression is associated with metastasis ability in oral squamous cell carcinoma.

Programmed cell death ligands (PD-Ls) are expressed in tumor cells where they bind to programmed cell death-1, an immunocyte co-receptor, resulting in tumor cell evasion from the immune system. Chemotherapeutic drugs have been recently reported to induce the expression of PD-L, such as PD-L1, in some cancer cells. However, little is known regarding PD-L2 expression and its role in oral squamous cell carcinoma (OSCC). In this study, we examined the effect of cisplatin on the expression and regulation of PD-L2 in OSCC cell lines and analyzed malignant behavior in PD-L2-expressing cells using colony, transwell and transformation assays. In addition, we examined PD-L2 expression in the tumor tissues of OSCC patients using cytology and tissue microarray methods. In OSCC cell lines, cisplatin treatment upregulated PD-L2 expression, along with that of the drug efflux transporter ABCG2, via signal transducers and activator of transcription (STAT) 1/3 activation. Moreover, PD-L2-positive or PD-L2-overexpressing cells demonstrated upregulation in both invasion and transformation ability but not in proliferation compared with PD-L2-negative or PD-L2-silencing cells. PD-L2 expression was also observed in OSCC cells of cytology samples and tissue from OSCC patients. The intensity of PD-L2 expression was correlated with more malignant morphological features in the histological appearance and an invasive pattern. Our findings indicate that cisplatin-upregulated PD-L2 expression in OSCC via STAT1/3 activation and the expression of PD-L2 are likely to be associated with malignancy in OSCC. The PD-L2 expression in cisplatin-resistant OSCC cells may be a critical factor in prognosis of advanced OSCC patients.

Three-dimensional spheroids of mesenchymal stem/stromal cells promote osteogenesis by activating stemness and Wnt/ß-catenin.

Mesenchymal stem/stromal cells (MSCs) are multipotent and self-renewal cells that are widely used in regenerative medicine. The culture of three-dimensional (3D) spheroid MSCs more accurately mimics the biological microenvironment. However, it is unclear which key molecules are responsible for the cell fate control of MSCs during 3D spheroid formation and their impact on the functional characteristics of these stem cells. Furthermore, it remains unclear what effects 3D spheroid MSC transplantation has on new bone formation compared with that of 2D monolayer MSCs. We assessed whether the osteogenerative potential of 3D spheroid MSCs is greater than that of 2D monolayer MSCs in vitro. In addition, to elucidate the ability of 3D spheroid MSCs to regenerate bone, we examined the effects of transplanting wild-type (WT) or knockout (KO) spheroid MSCs on new bone formation in mice calvarial defect model in vitro. The 3D spheroid MSC culture dramatically upregulated into stemness markers compared with the 2D monolayer MSC culture. In contrast, BMP-2 significantly increased the osteogenesis-related molecules in the 3D spheroid MSCs but, in turn, downregulated the stemness markers. BMP-2 activated Smad1/5 together with Wnt/ß-catenin in 3D spheroid MSCs. Transplantation of these MSCs into aged mice with calvarial defects promoted new bone formation compared with that of 2D monolayer MSCs. In contrast, transplantation of 3D or 2D ß-catenin knockout MSCs induced little new bone formation. The 3D spheroid MSC culture had higher stemness compared with the 2D monolayer MSC culture. The culture of 3D spheroid MSCs rapidly promoted osteoblastogenesis and bone formation through synergistic activation of the Wnt/ß-catenin pathway in vitro. The transformation of 3D spheroid, but not 2D monolayer, MSCs promoted new bone regeneration in vivo. These results indicate that transplantation of 3D spheroid MSCs in regeneration therapy contributes to a shorter regenerative healing process, including new bone formation.

Lipopolysaccharide induces bacterial autophagy in epithelial keratinocytes of the gingival sulcus.

BACKGROUND: Interactions of resident bacteria and/or their producing lipopolysaccharide (LPS) with sulcular epithelial keratinocytes may be regulated by autophagy in the gingival sulcus. In this study, we investigated an induction of bacterial autophagy in exfoliative sulcular keratinocytes of the gingival sulcus and cultured keratinocytes treated with Porphyromonas gingivalis-originated LPS (PgLPS). RESULTS: Exfoliative sulcular keratinocytes showed an induction of autophagy, in addition to increased expression of LPS-mediated factors including lipopolysaccharide-binding protein and toll-like receptors (TLRs), leading to co-localization of bacteria with autophagosomes. In contrast, exfoliative keratinocytes from the free gingiva did not show similar autophagy. Autophagy activity in human cultured keratinocyte cells (HaCaT) was induced by PgLPS, which was dependent partially on the AMP-activated protein kinase (AMPK) pathway via increased intracellular reactive oxygen species (ROS) and was in association with an activation of TLR4 signaling. After incubation of cultured keratinocytes with E.coli BioParticles following PgLPS stimulation, co-localization of bioparticles with autophagosomes was enhanced. Conversely, blockage of autophagy with 3-methyladenin and LPS-binding with polymyxin B led to significant reduction of co-localization of particles with autophagosomes. CONCLUSION: These findings indicate that PgLPS-induced autophagy is at least partially responsible for interaction between bacteria and sulcular keratinocytes in the gingival sulcus.

Impact of glycemic control with sitagliptin on the 2-year progression of arterial stiffness: a sub-analysis of the PROLOGUE study.

BACKGROUND: No conclusive evidence has been obtained yet on the significance of the effects of dipeptidyl peptidase-4 (DPP-4 inhibitor) treatment on the arterial stiffness in clinical settings. In addition, the effects of good glycemic control on the arterial stiffness have also not been clarified yet. As a sub-analysis of the PROLOGUE study, we examined the effect of a DPP-4 inhibitor (sitagliptin) on the 2-year progression of the arterial stiffness and also to determine the effect of good glycemic control on the rate of progression of the arterial stiffness. METHODS: In the PROLOGUE study, the study participants were either allocated to add-on sitagliptin treatment or to continued treatment with conventional anti-diabetic agents. Among the 463 participants of the PROLOGUE study, we succeeded in measuring the brachial-ankle pulse wave velocity (baPWV) at least two times during the 2-year study period in 96 subjects. RESULTS: The changes in the baPWV during the study period were similar between the both groups (i.e., with/without staglipitin), overall. On the other hand, when the study subjects were divided into two groups according to the glycemic control status during the study period {good glycemic control group (GC) = hemoglobin (Hb)A1c <7.0 at both 12 and 24 months after the treatment randomization; poor glycemic control group (PC) = HbA1c ≥7.0 at either 12 months, 24 months, or both}, the 2-year increase of the baPWV was marginally significantly larger in the PC group (144 ± 235 cm/s) as compared to that the GC group (-10 ± 282 cm/s) (p = 0.036). CONCLUSION: While the present study could not confirm the beneficial effect of sitagliptin per se on the arterial stiffness, the results suggested that good glycemic control appears to be beneficial for delaying the annual progression of the arterial stiffness. Trial registration University Hospital Medical Information Network Clinical Trials Registry UMIN000004490.

Long-term effect of sitagliptin on endothelial function in type 2 diabetes: a sub-analysis of the PROLOGUE study.

BACKGROUND: As a sub-analysis of the PROLOGUE study, we evaluated the long-term effect of sitagliptin, a dipeptidyl peptidase 4 inhibitor, on endothelial function in the conduit brachial artery in patients with type 2 diabetes. METHODS: In the PROLOGUE study, patients were randomly assigned to either add-on sitagliptin treatment (sitagliptin group) or continued conventional antihyperglycemic treatment (conventional group). Among the 463 participants in the PROLOGUE study, FMD was measured in 17 patients in the sitagliptin group and 18 patients in the conventional group at the beginning and after 12 and 24 months of treatment. RESULTS: HbA1c levels were significantly decreased after 12 and 24 months of treatment compared to baseline values in both groups (7.0 ± 0.4 vs. 6.6 ± 0.3 and 6.6 ± 0.4 % in the sitagliptin group; 7.0 ± 0.6 vs. 6.6 ± 0.7 and 6.6 ± 0.7 % in the conventional group; P < 0.05, respectively). There was no significant difference between FMD values at baseline and after 12 and 24 months in the sitagliptin group (4.3 ± 2.6 vs. 4.4 ± 2.1 and 4.4 ± 2.3 %, P = 1.0, respectively). Although FMD had a tendency to increase from 4.3 ± 2.4 % at baseline to 5.2 ± 1.9 % after 12 months and 5.1 ± 2.2 % after 24 months in the conventional group, there was no significant difference between FMD values at baseline and after 12 and 24 months (P = 0.36 and 0.33, respectively). CONCLUSIONS: Add-on sitagliptin to conventional antihyperglycemic drugs in patients with type 2 diabetes did not alter endothelial function in the conduit brachial artery measured by FMD during a 2-year study period. Sitagliptin may be used without concern for an adverse effect on endothelial function in patients with type 2 diabetes. TRIAL REGISTRATION: University hospital Medical Information Network (UMIN) Center: ID UMIN000004490.

Mesenchymal stem cell spheroids exhibit enhanced in-vitro and in-vivo osteoregenerative potential.

BACKGROUND: Mesenchymal stem cells (MSCs) are a favored cell source for regenerative medicine because of their multilinage potential. However, the conventional monolayer technique used to culture MSCs, inadequately overcomes their low differentiation capacity. Culture of MSCs in multicellular spheroids, more accurately mimics the in-vivo microenvironment; thus, resolving this problem. In this study, we assessed whether the osteoregenerative potential of MSC spheroids is greater than that of monolayer MSCs. RESULTS: MSC spheroids were generated from rat MSCs (rMSCs) using low-binding plates. Real-time reverse transcription-polymerase chain reaction and immunocytochemical analysis indicated that osteogenic properties were accelerated in MSC spheroids compared with monolayer rMSCs when treated with an osteoblast-inducer reagent for 7 days. Moreover, increased calcium deposition was visualized in MSC spheroids using Alizarin red staining. In a rat calvarial defect model, micro-computed tomography and histological assays showed that MSC spheroid-engrafted defects experienced enhanced bone regeneration. CONCLUSIONS: Our in-vitro and in-vivo results reveal that MSCs in the spheroid culture exhibit enhanced osteoregenerative efficiency compared with monolayer MSCs.

Dynamic changes in cell-surface expression of mannose in the oral epithelium during the development of graft-versus-host disease of the oral mucosa in rats.

BACKGROUND: The role of cell-surface glycoconjugates in oral mucosal graft-versus-host disease (GVHD) is still unclear, even though molecular changes in the oral epithelium are essential for the pathogenesis of these lesions. In this study, we investigated changes in the binding of mannose (Man)-specific Lens culinaris lectin (LCA) in the oral mucosa of rats with GVHD. METHODS: Lewis rat spleen cells were injected into (Lewis x Brown Norway) F1 rats to induce systemic GVHD, including oral mucosal lesions. Tongue and spleen samples were evaluated using lectin histochemistry, immunohistochemistry, Western blotting, transwell migration assays and Stamper-Woodruff binding assays. RESULTS: Binding of Man-specific LCA expanded to the epithelial layers of the tongue in GVHD-rats. An expansion of LCA binding was related to the increased expression of mannosyltransferase in the oral mucosa. CD8+ cells, effector cells of oral mucosal GVHD, expressed mannose-binding protein (MBP) and migrated to the medium containing Man in the transwell migration assay. Adherence of CD8+ cells to the oral epithelium could be inhibited by pretreating CD8+ cells with MBP antibody and/or by pretreating sections with Man-specific LCA. CONCLUSIONS: Increased expression of Man on keratinocytes leads to the migration and/or adhesion of CD8+ cells in the surface epithelium, which is mediated in part by the MBP/Man-binding pathway during the development of oral mucosal GVHD.

AMPK activation enhances osteoblast differentiation on a titanium disc via autophagy.

PURPOSE: The acquisition of osseointegration during implant therapy is slower and poorer in patients with diabetes compared with healthy persons. The serum concentration of adiponectin in patients with type II diabetes is lower than that of healthy persons via the suppression of AMP-activated protein kinase (AMPK). Therefore, we hypothesized that the AMPK activation enhances bone formation around implants, resulting in the improved acquisition of osseointegration. The purpose of this study was to evaluate the impact of AMPK activation on osteoblast differentiation and its mechanism of downstream signaling on titanium disc (Ti). METHODS: Confluent mouse pre-osteoblasts (MC3T3-E1) cells (1 × 105 cells/well) were cultured with BMP-2 for osteoblast differentiation, in the presence or absence AICAR, an AMPK activator. We examined the effects of AMPK activation on osteoblast differentiation and the underlying mechanism on a Ti using a CCK8 assay, a luciferase assay, quantitative RT-PCR, and western blotting. RESULTS: Although the proliferation rate of osteoblasts was not different between a Ti and a tissue culture polystyrene dish, the addition of AICAR, AMPK activator slightly enhanced osteoblast proliferation on the Ti. AICAR enhanced the BMP-2-dependent transcriptional activity on the Ti, leading to upregulation in the expression of osteogenesis-associated molecules. AICAR simultaneously upregulated the expression of autophagy-associated molecules on the Ti, especially LC3-II. AdipoRon, an adiponectin receptor type1/type2 activator activated AMPK, and upregulated osteogenesis-associated molecules on Ti. CONCLUSIONS: AMPK activation enhances osteoblast differentiation on a Ti via autophagy, suggesting that it promotes the acquisition of osseointegration during implant therapy.

Lupus-like oral mucosal lesions in mercury-induced autoimmune response in Brown Norway rats.

BACKGROUND: Administration of mercury at nontoxic doses induces systemic autoimmune disease in Brown Norway (BN) rats. The pathogenesis of lupus-like oral mucosal lesion by mercury-induced autoimmunity is still unclear, even though the oral mucosa is observed to be commonly affected in mercury-treated BN rats. In this study, we investigated the immunopathology of lupus-like oral mucosal lesions in a model of mercury-induced systemic autoimmunity. METHODS: Brown Norway male rats were injected subcutaneously with either phosphate-buffered saline (control) or mercury at a dose of 1.0 mg per kilogram of body weight on days 0, 3, 5, and 7. Blood, kidney, and tongue samples were taken at various timepoints for evaluation by immunohistochemistry, RT-PCR, and lupus band test (LBT). RESULTS: Oral mucosal lesions were classified according to three consecutive temporal phases on the basis of infiltration of immunocompetent cells as follows: (phase I) infiltration of MHC class II+ dendritic cells (DC) and macrophages; (phase II) addition of ED1+ macrophage infiltrates; and (phase III) focal infiltration of pan T cells following increased infiltration of DC and macrophages. Dense infiltration of DC and macrophages was observed in the basement membrane (BM) zone of the oral epithelium. Tissue expression of IL-4 mRNA was detected in early lesions (phase I), suggesting that locally produced IL-4 may be responsible for Th2-mediated immune response. A linear and continuous smooth pattern of fluorescence was observed in the oral epithelial BM in addition to renal glomeruli, indicating immune complex deposits. CONCLUSIONS: Local autoimmune responses are involved in the pathogenesis of mercury-induced lupus-like lesions of the oral mucosa.

Identification of SAMT family proteins as substrates of MARCH11 in mouse spermatids.

MARCH11, a RING-finger transmembrane ubiquitin ligase, is predominantly expressed in spermatids and localized to the trans-Golgi network (TGN) and multivesicular bodies (MVBs). Because ubiquitination acts as a sorting signal of cargo proteins, MARCH11 has been postulated to mediate selective protein sorting via the TGN-MVB pathway. However, the physiological substrate of MARCH11 has not been identified. In this study, we have identified and characterized SAMT1, a member of a novel 4-transmembrane protein family, which consists of four members. Samt1 mRNA and its expression product were found to be specific to the testis and were first detected in germ cells 25 days after birth in mice. Immunohistochemical analysis further revealed that SAMT1 was specifically expressed in haploid spermatids during the cap and acrosome phases. Confocal microscopic analysis showed that SAMT1 co-localized with MARCH11 as well as with fucose-containing glycoproteins, another TGN/MVB marker, and LAPM2, a late endosome/lysosome marker. Furthermore, we found that MARCH11 could increase the ubiquitination of SAMT1 and enhance its lysosomal delivery and degradation in an E3 ligase activity-dependent manner. In addition, the C-terminal region of SAMT1 was indispensable for its ubiquitination and proper localization. The other member proteins of the SAMT family also showed similar expression profile, intracellular localization, and biochemical properties, including ubiquitination by MARCH11. These results suggest that SAMT family proteins are physiological substrates of MARCH11 and are delivered to lysosomes through the TGN-MVB pathway by a ubiquitin-dependent sorting system in mouse spermatids.

Endovascular retrieval of an elongated Supera stent.

Stenting is used to achieve artery patency, and the Supera stent, a self-expanding interwoven nitinol stent, has produced good clinical outcomes. A 70-year-old woman with peripheral artery disease had experienced intermittent claudication (Fontaine stage IIb). Endovascular treatment was performed for a chronic total occlusion TransAtlantic InterSociety Consensus class II type B lesion. A Supera stent (Abbott Vascular, Santa Clara, CA) was used. However, it had become severely elongated to the proximal end in the superficial femoral artery and was removed using a balloon inserted from the side and trapped to the guide sheath with the distal end of the stent outside the sheath. After this bailout, an alternate stent could be placed through an antegrade approach to the contralateral common femoral artery.

Epipharyngeal Abrasive Therapy Down-regulates the Expression of SARS-CoV-2 Entry Factors ACE2 and TMPRSS2.

BACKGROUND: The epipharynx, with its high expression of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) entry factors angiotensin-converting enzyme 2 (ACE2) and transmembrane protease, serine 2 (TMPRSS2), is a primary target for SARS-CoV-2 replication in the early stage of Coronavirus Disease 19 (COVID-19). Epipharyngeal abrasive therapy (EAT) is a treatment for epipharyngitis in Japan which involves applying zinc chloride to the epipharyngeal mucosa. In this study, we evaluated the expression patterns of ACE2 and TMPRSS2 in tissue samples from patients before and after EAT. PATIENTS AND METHODS: The study subjects were seven patients that had not been treated with EAT and 11 patients that had. For immunohistochemical assessment of the epipharyngeal mucosa, the staining intensity of ACE2 and TMPRSS2 was described as an immunohistochemical score (IHC score). RESULTS: The IHC scores for ACE2 and TEMPRSS2 in the EAT-treated group were 3.40-fold and 1.81-fold lower, respectively, than those in the non-treated group (p=0.0208 and p=0.0244, respectively). CONCLUSION: EAT down-regulates the expression of SARS-CoV-2 entry factors ACE2 and TMPRSS2. Thus, EAT has potential as a novel COVID-19 preventative method.

Application of the sliding window method and Mask-RCNN method to nuclear recognition in oral cytology.

BACKGROUND: We aimed to develop an artificial intelligence (AI)-assisted oral cytology method, similar to cervical cytology. We focused on the detection of cell nuclei because the ratio of cell nuclei to cytoplasm increases with increasing cell malignancy. As an initial step in the development of AI-assisted cytology, we investigated two methods for the automatic detection of cell nuclei in blue-stained cells in cytopreparation images. METHODS: We evaluated the usefulness of the sliding window method (SWM) and mask region-based convolutional neural network (Mask-RCNN) in identifying the cell nuclei in oral cytopreparation images. Thirty cases of liquid-based oral cytology were analyzed. First, we performed the SWM by dividing each image into 96 × 96 pixels. Overall, 591 images with or without blue-stained cell nuclei were prepared as the training data and 197 as the test data (total: 1,576 images). Next, we performed the Mask-RCNN by preparing 130 images of Class II and III lesions and creating mask images showing cell regions based on these images. RESULTS: Using the SWM method, the highest detection rate for blue-stained cells in the evaluation group was 0.9314. For Mask-RCNN, 37 cell nuclei were identified, and 1 cell nucleus was identified as a non-nucleus after 40 epochs (error rate:0.027). CONCLUSIONS: Mask-RCNN is more accurate than SWM in identifying the cell nuclei. If the blue-stained cell nuclei can be correctly identified automatically, the entire cell morphology can be grasped faster, and the diagnostic performance of cytology can be improved.

DNA/protamine complex paste for an injectable dental material.

A DNA/protamine complex powder was prepared by reaction between DNA and protamine sulfate solution with stirring in order to develop a new injectable biomaterials for dental therapy. The powder of DNA/protamine complex became paste by kneading the complex powder and distilled water. Complex formation was confirmed by FT-IR measurement. The complex paste had a porous structure and its viscosity was approximately 280.1 Pas. The paste could easily pass through a needle of 0.25 mm internal diameter. It seemed that DNA/protamine complex paste has suitable viscosity for clinical use as an injectable biomaterial. Although, the complex paste delayed the growth speed of Staphylococcus aureus, Pseudomonas aeruginosa, Porphyromonas gingivalis and Prevotella intermedia for limited periods, it cannot kill and inhibit growing bacteria. The complex paste disk showed a mild tissue response and gradually degraded after the implantation into the soft tissue of rats. These results suggested that this DNA/protamine complex paste could be a useful material for a biodegradable biomaterial. In particular, this paste will be applicable as an injectable biomaterial using syringe for the repair of defects of living tissue, GBR treatment and/or GTR treatment in dentistry.

Antibody-Conjugated Gel-Coated Single-Walled Carbon Nanotubes as Photothermal Agents.

Photothermal therapy (PTT) using near-infrared (NIR) light is an attractive treatment modality for cancer, in which photothermal agents absorb energy from photons and convert it into thermal energy to lead to cancer cell death. Among the various organic and inorganic materials, single-walled carbon nanotubes (SWCNTs) are promising candidates for NIR photothermal agents due to their strong absorption in this region as well as their high photothermal conversion efficiency. In the development of the SWCNT-based PTT materials, modifications of SWCNTs to offer a stable dispersion for biocompatibility as well as to target the tumor of choice while maintaining their NIR absorption have been required. While modification of SWCNTs through noncovalent methods can be achieved, these modifications can be easily reversed in the body. Contrarily, modifications through covalent attachments, while more desirable, may compromise the NIR absorption characteristics of the SWCNTs. Previously, we reported the development of a synthetic strategy to coat SWCNTs with a cross-linked polymer (i.e., a gel) through a process called CNT Micelle Polymerization and successfully introduced maleimide groups that allowed for postmodification through the ene-thiol reaction without deteriorating the NIR absorption. In this report, we postmodify thiol-containing antibodies (anti-TRP-1, a melanoma specific protein) using maleimide chemistry and find that the SWCNTs conjugated with anti-TRP-1 maintain the characteristic NIR absorption as SWCNTs with dispersion stability. It is estimated that 50 maleimide groups are incorporated in one SWCNT (ca. 280 nm long) and they are modified with 32 TRP-1 fragments. Finally, we successfully use these targeted SWCNTs for the PTT of the melanoma cell line using NIR light (1064 nm; 2 W, 5 min). Our method can be extended to a vast array of specific antibodies as well as other targeting agents.