Faculty Bibliography

Oral cancer is notoriously painful. Activation of protease-activated receptor 2 (PAR₂, encoded by F2RL1) by proteases in the cancer microenvironment is implicated in oral cancer pain. PAR₂ is a G protein-coupled receptor (GPCR) expressed on neurons and cells in the cancer microenvironment. Sustained signaling of PAR₂ from endosomes of neurons mediates sensitization and nociception. We focused on the differential contribution of PAR₂ on oral cancer cells and neurons to oral cancer pain and whether encapsulation of a PAR₂ inhibitor, AZ3451 in nanoparticles (NP) more effectively reverses PAR₂ activation. We report that F2RL1 was overexpressed in human oral cancers and cancer cell lines. Deletion of F2RL1 on cancer cells reduced cancer-associated mechanical allodynia. A third-generation polyamidoamine dendrimer, functionalized with cholesterol was self-assembled into NPs encapsulating AZ3451. NP encapsulated AZ3451 (PAMAM-Chol-AZ NPs) more effectively reversed activation of PAR₂ at the plasma membrane and early endosomes than free drug. The PAMAM-Chol-AZ NPs showed greater efficacy in reversing nociception than free drug, with respect to both level and duration, in three preclinical mouse models of oral cancer pain. The antinociceptive efficacy was confirmed with an operant orofacial assay. Genetic deletion of F2RL1 on cancer cells or F2rl1 on neurons each partially reversed mechanical cancer allodynia. The remaining nociception could be effectively reversed by PAMAM-Chol-AZ NPs. These findings suggest that PAR₂ on oral cancer cells and neurons contribute to oral cancer nociception and NPs loaded with a PAR₂ antagonist provide increased antinociception and improved oral function compared to free drug.

BACKGROUND/PURPOSE/UNASSIGNED:Bone morphogenetic protein-9 (BMP-9) has demonstrated multiple advantages in promoting osteogenesis. Our previous findings have indicated that the use of an absorbable collagen membrane (ACM) as a carrier for growth factors is effective in stimulating bone regeneration. The objective of this study was to assess the synergistic impact of BMP-9 incorporated into ACM (ACM/BMP-9) on bone formation within rat mandibular bone defects. MATERIALS AND METHODS/UNASSIGNED:Circular bone defects of critical size were surgically induced on both sides of the rat mandibular bone, with subsequent random allocation into distinct groups: control, ACM alone, and ACM loaded with low (0.5 μg) or high (2.0 μg) concentrations of BMP-9. We conducted real-time in vivo micro-computerized tomography scans at the baseline and at 2, 4, and 6 weeks, and measured the volume of newly formed bone (NFB), bone mineral density (BMD) of NFB, and the closure percentage of the NFB area. Histological and histomorphometric analyses were performed at 6 weeks. RESULTS/UNASSIGNED:Real-time assessment revealed notably higher levels of bone volume, BMD, and closure percentage in the NFB area for the groups treated with ACM/BMP-9 compared to the control and ACM groups. Within the high concentration of BMP-9 group, the volume and BMD of NFB exhibited a significant increase at 6 weeks compared to baseline. Histological examination confirmed the existence of osteoblasts, osteocytes, and blood vessels within the NFB. CONCLUSION/UNASSIGNED:Considering the limitations of this research, the real-time evaluation finding indicates that ACM/BMP-9 effectively promotes bone formation in critical-size mandibular defects in rats.

BACKGROUND:Hierarchical micro-nano structured topography along with surface chemistry modifications of dental implants have been suggested to positively contribute to the osseointegration process. However, the effect of such surface modifications on the molecular response as well as bone formation rate and quality are still unclear, especially in the early healing period. This study aimed to evaluate the effect of coating a double acid etched (DAE) implant surface with nano-sized (20 nm) hydroxyapatite (Nano) with respect to gene expression, histologic parameters, and nanomechanical properties when compared to DAE control at 1 and 2 weeks after implant placement in a rodent femur model. MATERIAL AND METHODS/METHODS:Expression of bone-related genes was determined by qRT-PCR (Col-I, Runx-2, Osx, Opn, Ocn, Alp). Histomorphometric evaluation of bone-to-implant contact (BIC) and bone area fraction occupancy (BAFO) within implant threads was performed using photomicrographs after histologic processing. Mechanical properties, reduced elastic modulus and hardness, were determined through nanoindentation. RESULTS:At 1 week, the Nano group demonstrated significantly higher expression of Col-I and Ocn compared to the DAE group, indicating upregulation of osteoprogenitor and osteoblast differentiation genes. At 2 weeks, Nano surface further exhibited enhanced gene expression of Col-I and Osx in comparison to the DAE surface, suggesting an increased mineralization of the newly formed bone. Nanoindentation analysis revealed that the Nano group presented no significant difference on the ranks of reduced elastic modulus and hardness compared to DAE for both timepoints. Histomorphometric analysis yielded no significant difference in the percentage of BIC and BAFO between the Nano and DAE surfaces at 1 and 2 weeks. However, Nano implants did present a higher mean value, ~50%, of BIC compared to DAE, ~30%, after 2 weeks in vivo. CONCLUSIONS:While no significant differences were observed in the amount and mechanical properties of newly formed bone, Nano surface positively and significantly increased the expression osteogenic genes compared to DAE surface at early healing periods.

The use of biomaterials and bioactive agents has shown promise in bone defect repair, leading to the development of strategies for bone regeneration. Various artificial membranes, especially collagen membranes (CMs) that are widely used for periodontal therapy and provide an extracellular matrix-simulating environment, play a significant role in promoting bone regeneration. In addition, numerous growth factors (GFs) have been used as clinical applications in regenerative therapy. However, it has been established that the unregulated administration of these factors may not work to their full regenerative potential and could also trigger unfavorable side effects. The utilization of these factors in clinical settings is still restricted due to the lack of effective delivery systems and biomaterial carriers. Hence, considering the efficiency of bone regeneration, both spaces maintained using CMs and GFs can synergistically create successful outcomes in bone tissue engineering. Therefore, recent studies have demonstrated a significant interest in the potential of combining CMs and GFs to effectively promote bone repair. This approach holds great promise and has become a focal point in our research. The purpose of this review is to highlight the role of CMs containing GFs in the regeneration of bone tissue, and to discuss their use in preclinical animal models of regeneration. Additionally, the review addresses potential concerns and suggests future research directions for growth factor therapy in the field of regenerative science.

Background and Aim: Periodontitis is an infectious disease caused by periodontal pathogens that are known to evade host immune responses. Recently, programmed death-ligand 1 (PD-L1) plays an important role in suppressing T cell function in cancer immunity. However, the relationship between PD-L1 and periodontal disease is unknown. The purpose of this study is to investigate the role of PD-L1 on regulation of T cells activation and osteoclast differentiation in periodontitis.
Method(s): PD-L1 mRNA expression was measured in Ca9-22 human gingival epithelial cells infected with/without Porphyromonas gingivalis. After Jurkat human T lymphocyte cells were co-cultured with Ca9-22 cells infected with/without P. gingivalis, expression of interferon gamma (IFN-gamma) as an activation marker of T cells was measured. After RAW 264.7 mouse monocyte/macrophage-like cells were treated with receptor activator of nuclear factor kappa-B ligand (RANKL) and PD-L1, mRNA expression of osteoclast differentiation markers (Cat-K and Cfms) was measured and numbers of osteoclast-like cells were counted. Using a mouse periodontitis model with silk threads ligated to the maxillary molars, Pd-l1 mRNA was measured in the gingival tissue.
Result(s): PD-L1 mRNA expression significantly induced in Ca9-22 cells infected with P. gingivalis compared to non-infected control (p < 0.05). IFN-gamma secretion was significantly inhibited in co-cultured with Ca9-22 cells infected P. gingivalis compared to non-infected control. In the periodontitis model, osteoclast-like cells were observed and Pd-l1 mRNA expression was significantly increased in the ligated side compared to the control side (p < 0.01). The number of osteoclast-like cells and mRNA expression of Cat-K and C-fms was significantly decreased in RAW 264.7 cells treated with RANKL and PD-L1 compared to non-treated control (p < 0.05).
Conclusion(s): These results suggest that PD-L1 may play an important role in periodontitis via attenuating T cell activation and osteoclast differentiation

Background/purpose/UNASSIGNED:Fibroblast growth factor-2 (FGF-2) regulates bone formation. The concept of guided bone regeneration using a resorbable collagen membrane (RCM) is generally accepted in implant dentistry. This study aimed to investigate the bone healing pattern in rat mandibular bone defects in real-time with and without RCM containing FGF-2 (RCM/FGF-2). Materials and methods/UNASSIGNED:Critical-size circular bone defects (4.0 mm diameter) were created on both sides of the rat mandibular bone. The defects were randomly divided into the following groups: control, RCM alone, RCM containing low (0.5 μg) or high (2.0 μg) concentration of FGF-2. We performed real-time in vivo micro-computerized tomography scans at the baseline and at 2, 4, and 6 weeks, and measured the volume of newly formed bone (NFB), bone mineral density (BMD) of NFB, and the closure percentage of the NFB area. At 6 weeks, the mandibular specimens were assessed histologically and histomorphometrically to evaluate the area of new bone regeneration. Results/UNASSIGNED:Real-time assessment revealed a significant increase in the volume, BMD, and closure percentage of the NFB area in the RCM/FGF-2-treated groups than that in the control and RCM groups. In the H-FGF-2 group, the volume and BMD of NFB exhibited a significant increase at 6 weeks than that at the baseline. Histological evaluation revealed the presence of osteoblasts, osteocytes, and blood vessels within the NFB. Conclusion/UNASSIGNED:The real-time in vivo experiment demonstrated that RCM/FGF-2 effectively promoted bone regeneration within the critical-size mandibular defects in rats and verified new bone formation starting in the early postoperative phase.

BACKGROUND:We developed a non-viral vector, a combination of HIV-1 Tat peptide modified with histidine and cysteine (mTat) and polyethylenimine, jetPEI (PEI), displaying the high efficiency of plasmid DNA transfection with little toxicity. Since the highest efficiency of INTERFERin (INT), a cationic amphiphilic lipid-based reagent, for small interfering RNA (siRNA) transfection among six commercial reagents was shown, we hypothesized that combining mTat/PEI with INT would improve transfection efficiency of siRNA delivery. To elucidate the efficacy of the hybrid vector for siRNA silencing, β-actin expression was measured after siRNA β-actin was transfected with mTat/PEI/INT or other vectors in HSC-3 human oral squamous carcinoma cells. RESULTS:mTat/PEI/INT/siRNA produced significant improvement in transfection efficiency with little cytotoxicity compared to other vectors and achieved ≈ 100% knockdown of β-actin expression compared to non-treated cells. The electric charge of mTat/PEI/INT/siRNA was significantly higher than INT/siRNA. The particle size of mTat/PEI/INT/siRNA was significantly smaller than INT/siRNA. Filipin III and β-cyclodextrin, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/PEI/INT/siRNA transfection, while chlorpromazine, an inhibitor of clathrin-mediated endocytosis, did not inhibit mTat/PEI/INT/siRNA transfection. Furthermore, the transfection efficiency of mTat/PEI/INT at 4 °C was significantly lower than 37 °C. CONCLUSIONS:These findings demonstrated the feasibility of using mTat/PEI/INT as a potentially attractive non-viral vector for siRNA delivery.

We have developed a unique delivery system of growth factors using collagen membranes (CMs) to induce bone regeneration. We hypothesized that fibroblast growth factor18 (FGF-18), a pleiotropic protein that stimulates proliferation in several tissues, can be a good candidate to use our delivery system for bone regeneration. Cell viability, cell proliferation, alkaline phosphatase activity, mineralization, and marker gene expression of osteoblastic differentiation were evaluated after mouse preosteoblasts were cultured with a CM containing FGF-18, a CM containing platelet-derived growth factor, or a CM alone. Furthermore, expression of microRNA, especially miR-133a and miR-135a involving inhibition of osteogenic factors, was measured in preosteoblasts with CM/FGF-18 or CM alone. A sustained release of FGF-18 from the CM was observed over 21 days. CM/FGF-18 significantly promoted cell proliferation, alkaline phosphatase activity, and mineralization compared to CM alone. Gene expression of type I collagen, runt-related transcription factor 2, osteocalcin, Smad5, and osteopontin was significantly upregulated in CM/FGF-18 compared to CM alone, and similar to CM/platelet-derived growth factor. Additionally, CM/FGF-18 downregulated expression of miR-133a and miR-135a. These results suggested that released FGF-18 from a CM promotes osteoblastic activity involved with downregulation of miR-133a and miR-135a.

OBJECTIVES: Osteogenic protein-1 (OP-1) has shown osteoinductive activities and is useful for clinical treatments, including bone regeneration. Regenerative procedures using a bioabsorbable collagen membrane (BCM) are well established in periodontal and implant dentistry. We evaluated the subsequent effects of the BCM in combination with OP-1 on bone regeneration in a rat mandibular circular critical-sized bone defect in vivo. DESIGN: We used 8 rats that received surgery in both sides of the mandible, and created the total 16 defects which were divided into 4 groups: Group 1; no treatment, as a control, Group 2; BCM alone, Group 3; BCM containing low dose 0.5mug of OP-1 (L-OP-1), and Group 4; BCM containing high dose 2.0mug of OP-1 (H-OP-1). Newly formed bone was evaluated by micro computed tomography (micro-CT) and histological analyses at 8 weeks postoperatively. In quantitative and qualitative micro-CT analyses of the volume of new bone formation, bone density, and percentage of new bone area was evaluated. RESULTS: BCM with rhOP-1 significantly increased and accelerated bone volume, bone mineral density, and percentage of new bone area compared to control and BCM alone at 8 weeks after surgery; these enhancements in bone regeneration in the OP-1-treated groups were dose-dependent. CONCLUSIONS: OP-1 delivered with a BCM may have effective osteoinductive potency and be a good combination for bone regeneration. The use of such a combination device for osteogenesis may result in safer and more predictable bone regenerative outcomes in the future.

Virus-mediated gene delivery shows promise for the treatment of chronic pain. However, viral vectors have cytotoxicity. To avoid toxicities and limitations of virus-mediated gene delivery, we developed a novel nonviral hybrid vector: HIV-1 Tat peptide sequence modified with histidine and cysteine residues combined with a cationic lipid. The vector has high transfection efficiency with little cytotoxicity in cancer cell lines including HSC-3 (human tongue squamous cell carcinoma) and exhibits differential expression in HSC-3 ( approximately 45-fold) relative to HGF-1 (human gingival fibroblasts) cells. We used the nonviral vector to transfect cancer with OPRM1, the mu-opioid receptor gene, as a novel method for treating cancer-induced pain. After HSC-3 cells were transfected with OPRM1, a cancer mouse model was created by inoculating the transfected HSC-3 cells into the hind paw or tongue of athymic mice to determine the analgesic potential of OPRM1 transfection. Mice with HSC-3 tumors expressing OPRM1 demonstrated significant antinociception compared with control mice. The effect was reversible with local naloxone administration. We quantified beta-endorphin secretion from HSC-3 cells and showed that HSC-3 cells transfected with OPRM1 secreted significantly more beta-endorphin than control HSC-3 cells. These findings indicate that nonviral delivery of the OPRM1 gene targeted to the cancer microenvironment has an analgesic effect in a preclinical cancer model, and nonviral gene delivery is a potential treatment for cancer pain.