Periostin-integrin interaction regulates force-induced TGF-ß1 and α-SMA expression by hPDLSCs.

OBJECTIVE: Periostin (PN), a major matricellular periodontal ligament (PDL) protein, modulates the remodeling of the PDL and bone, especially under mechanical stress. This study investigated the requirement of PN-integrin signaling in force-induced expression of transforming growth factor-beta 1 (TGF-ß1) and alpha-smooth muscle actin (α-SMA) in human PDL stem cells (hPDLSCs). METHODS: Cells were stimulated with intermittent compressive force (ICF) using computerized controlled apparatus. Cell migration was examined using in vitro scratch assay. The mRNA expression was examined using real-time polymerase chain reaction. The protein expression was determined using immunofluorescent staining and western blot analysis. RESULTS: Stimulation with ICF for 24 h increased the expression of PN, TGF-ß1, and α-SMA, along with increased SMAD2/3 phosphorylation. Knockdown of POSTN (PN gene) decreased the protein levels of TGF-ß1 and pSMAD2/3 upon force stimulation. POSTN knockdown of hPDLSCs resulted in delayed cell migration, as determined by a scratch assay. However, migration improved after seeding these knockdown cells on pre-PN-coated surfaces. Further, the knockdown of αVß5 significantly attenuated the force-induced TGF-ß1 expression. CONCLUSION: Our findings indicate the importance of PN-αVß5 interactions in ICF-induced TGF-ß1 signaling and the expression of α-SMA. Findings support the critical role of PN in maintaining the PDL's tissue integrity and homeostasis.

Alteration of extracellular matrix proteins in atrophic periodontal ligament of hypofunctional rat molars.

OBJECTIVES: The aim of this study was to investigate the effect of mechanical force on possible dynamic changes of the matrix proteins deposition in the PDL upon in vitro mechanical and in vivo occlusal forces in a rat model with hypofunctional conditions. MATERIALS AND METHODS: Intermittent compressive force (ICF) and shear force (SF) were applied to human periodontal ligament stem cells (PDLSCs). Protein expression of collagen I and POSTN was analyzed by western blot technique. To establish an in vivo model, rat maxillary molars were extracted to facilitate hypofunction of the periodontal ligament (PDL) tissue of the opposing mandibular molar. The mandibles were collected after 4-, 8-, and 12-weeks post-extraction and used for micro-CT and immunohistochemical analysis. RESULTS: ICF and SF increased the synthesis of POSTN by human PDLSCs. Histological changes in the hypofunctional teeth revealed a narrowing of the PDL space, along with a decreased amount of collagen I, POSTN, and laminin in perivascular structures compared to the functional contralateral molars. CONCLUSION: Our results revealed that loss of occlusal force disrupts deposition of some major matrix proteins in the PDL, underscoring the relevance of mechanical forces in maintaining periodontal tissue homeostasis by modulating ECM composition.

Anti-inflammatory effects of the prostacyclin analogue iloprost in an in vitro model of inflamed human dental pulp cells.

Iloprost's anti-inflammatory effects on human dental pulp stem cells (HDPCs) are currently unknown. We hypothesized that iloprost could downregulate the expression of inflammatory-related genes and protein in an inflamed HDPC in vitro model. To induce inflammation, the HDPCs were treated with a cocktail of interleukin-1 beta, interferon-gamma, and tumour necrosis alpha, at a ratio of 1:10:100. Iloprost (10-6  M) was then added or not to the cultures. Interleukin-6 (IL-6) and interleukin-12 (IL-12) mRNA expression were assessed by real-time polymerase chain reaction. IL-6 protein expression was assessed by enzyme-linked immunosorbent assay. The results were analysed using one-way ANOVA or the Kruskal-Wallis test. The cytokine cocktail induced more robust IL-6 expression than LPS treatment. Iloprost slightly, yet significantly, downregulated IL-6 and IL-12 mRNA expression. These findings suggest that iloprost might be used as a beneficial component in vital pulp therapy.

Porphyromonas gingivalis Induces Bisphosphonate-Related Osteonecrosis of the Femur in Mice.

One of the most prominent characteristics of bisphosphonate-related osteonecrosis of the jaw(BRONJ) is its site-specificity. Osteonecrosis tends to occur specifically in maxillofacial bones, in spite of a systemic administration of the medicine. Previous studies suggested rich blood supply and fast bone turnover might be reasons for BRONJ. Yet, a sound scientific basis explaining its occurrence is still lacking. The present study aimed to explore the role of Porphyromonas gingivalis (P. gingivalis), an important oral pathogen, on the site-specificity of bisphosphonate-induced osteonecrosis and to elucidate its underlying mechanism. Mice were intraperitoneally injected with zoledronic acid (ZA) or saline for 3 weeks. In the third week, the right mandibular first molars were extracted and circular bone defects with a diameter of 1 mm were created in right femurs. After the operation, drug administration was continued, and P. gingivalis suspension was applied to the oral cavities and femur defects. The mice were killed after four or eight weeks postoperatively. The right mandibles and femurs were harvested for micro-CT and histological analyses. A poor healing of bone defects of both jaws and femurs was noted in mice injected with both ZA and P. gingivalis. Micro-CT analysis showed a decreased bone volume, and histological staining showed an increased number of empty osteocyte lacunae, a decreased collagen regeneration, an increased inflammatory infiltration and a decreased number of osteoclasts. In addition, the left femurs were collected for isolation of osteoclast precursors (OCPs). The osteoclastogenesis potential of OCPs was analyzed in vitro. OCPs extracted from mice of ZA-treated groups were shown to have a lower osteoclast differentiation potential and the expression level of related genes and proteins was declined. In conclusion, we established a mouse model of bisphosphonate-related osteonecrosis of both the jaw and femur. P. gingivalis could inhibit the healing of femur defects under the administration of ZA. These findings suggest that P. gingivalis in the oral cavity might be one of the steering compounds for BRONJ to occur.

The Prostacyclin Analog Iloprost Promotes Cementum Formation and Collagen Reattachment of Replanted Molars and Up-regulates Mineralization by Human Periodontal Ligament Cells.

INTRODUCTION: This study evaluated the use of the prostacyclin analog iloprost as a root surface treatment agent in promoting acellular cementum (AC) formation and collagen reattachment after tooth replantation in vivo. In addition, its effect on human periodontal ligament cell (hPDLC) mineralization was assessed in vitro. METHODS: First molars of 8-week-old Wistar rats were extracted. In 1 group, the root surfaces were treated with Hank's balanced salt solution (HBSS), and the other group's root surfaces were treated with 10-6 mol/L iloprost before replantation. At day 30, maxillae were prepared for micro-computed tomographic imaging and histomorphometric analysis. The effect of iloprost on mineralization by hPDLCs was analyzed by mineralized nodule formation and quantitative polymerase chain reaction at 7 and 14 days. RESULTS: Micro-computed tomographic imaging demonstrated a significant higher bone volume in the iloprost groups, whereas the HBSS groups had extensive bone and root resorption. Histologic analysis revealed deposition of a thick AC layer along the root in the iloprost group with well-organized periodontal ligament fibers inserted into the cementum. The HBSS group demonstrated more osteoclasts than the iloprost group. In vitro, iloprost-treated hPDLCs had a significantly increased RUNX2, OSX, BSP, and ALP gene expression that coincided with an increased deposition of mineralized nodules. These effects were abrogated by a PGI2 receptor inhibitor. CONCLUSIONS: Our results revealed that iloprost promoted PDL regeneration in replanted molars. Furthermore, resorption of the roots was decreased, whereas AC deposition was stimulated. Iloprost-treatment increased hPDLC mineralization and was mediated by PGI2 receptor activation. These observations indicate that iloprost may be a promising root surface treatment agent.

Mutation in the CCAL1 locus accounts for bidirectional process of human subchondral bone turnover and cartilage mineralization.

OBJECTIVES: To study the mechanism by which the readthrough mutation in TNFRSF11B, encoding osteoprotegerin (OPG) with additional 19 amino acids at its C-terminus (OPG-XL), causes the characteristic bidirectional phenotype of subchondral bone turnover accompanied by cartilage mineralization in chondrocalcinosis patients. METHODS: OPG-XL was studied by human induced pluripotent stem cells expressing OPG-XL and two isogenic CRISPR/Cas9-corrected controls in cartilage and bone organoids. Osteoclastogenesis was studied with monocytes from OPG-XL carriers and matched healthy controls followed by gene expression characterization. Dual energy X-ray absorptiometry scans and MRI analyses were used to characterize the phenotype of carriers and non-carriers of the mutation. RESULTS: Human OPG-XL carriers relative to sex- and age-matched controls showed, after an initial delay, large active osteoclasts with high number of nuclei. By employing hiPSCs expressing OPG-XL and isogenic CRISPR/Cas9-corrected controls to established cartilage and bone organoids, we demonstrated that expression of OPG-XL resulted in excessive fibrosis in cartilage and high mineralization in bone accompanied by marked downregulation of MGP, encoding matrix Gla protein, and upregulation of DIO2, encoding type 2 deiodinase, gene expression, respectively. CONCLUSIONS: The readthrough mutation at CCAL1 locus in TNFRSF11B identifies an unknown role for OPG-XL in subchondral bone turnover and cartilage mineralization in humans via DIO2 and MGP functions. Previously, OPG-XL was shown to affect binding between RANKL and heparan sulphate (HS) resulting in loss of immobilized OPG-XL. Therefore, effects may be triggered by deficiency in the immobilization of OPG-XL Since the characteristic bidirectional pathophysiology of articular cartilage calcification accompanied by low subchondral bone mineralization is also a hallmark of OA pathophysiology, our results are likely extrapolated to common arthropathies.

Osteopontin induces osteogenic differentiation by human periodontal ligament cells via calcium binding domain-ALK-1 interaction.

BACKGROUND: Recently we have generated recombinant human osteopontin (rhOPN) using a plant platform (Nicotiana benthamiana) and demonstrated, when coated on culture plates, its osteogenic induction capacity of human periodontal ligament (PDL) cells. The aim of this study is to elucidate the molecular mechanism underlying the rhOPN-induced osteogenic differentiation of human PDL cells. METHODS: Full length rhOPN (FL-OPN) and three constructs of OPN containing integrin binding domain (N142), calcium binding domain (C122) and mutated calcium-binding domain (C122δ) were generated from N. benthamiana. Human PDL cells were isolated from extracted third molars and cultured on FL-OPN, N142, C122, or C122δ-coated surfaces. Real-time PCR and Western blot analyses were used to determine mRNA and protein expression. In vitro calcification was determined by Alizarin red staining. A chemical inhibitor and RNAi silencing were used to elucidate signaling pathways. In silico analyses were performed to predict the protein-protein interaction. In vivo analysis was performed using a rat calvaria defect model. RESULTS: Human PDL cells seeded on FL-OPN and C122-coated surfaces significantly increased both mRNA and protein expression of osterix (OSX) and enhanced in vitro calcification. Soluble FL-OPN as well as a surface coated with N142 did not affect OSX expression. Inhibition of activin receptor-like kinase (ALK-1) abolished the induction of osterix expression. In silico analysis suggested a possible interaction between the calcium binding domain (CaBD) of OPN and ALK-1 receptor. C122, but not C122δ coated surfaces, induced the expression of p-Smad-1 and this induction was inhibited by an ALK-1 inhibitor and RNAi against ALK-1. In vivo data showed that 3D porous scaffold containing C-122 enhanced new bone formation as compared to scaffold alone. CONCLUSION: The results suggest that next to full length OPN, the CaBD of OPN, if coated to a surface, induces osteogenic differentiation via interaction with ALK-1 receptor.

LAMP-2 Is Involved in Surface Expression of RANKL of Osteoblasts In Vitro.

Lysosome associated membrane proteins (LAMPs) are involved in several processes, among which is fusion of lysosomes with phagosomes. For the formation of multinucleated osteoclasts, the interaction between receptor activator of nuclear kappa ß (RANK) and its ligand RANKL is essential. Osteoclast precursors express RANK on their membrane and RANKL is expressed by cells of the osteoblast lineage. Recently it has been suggested that the transport of RANKL to the plasma membrane is mediated by lysosomal organelles. We wondered whether LAMP-2 might play a role in transportation of RANKL to the plasma membrane of osteoblasts. To elucidate the possible function of LAMP-2 herein and in the formation of osteoclasts, we analyzed these processes in vivo and in vitro using LAMP-2-deficient mice. We found that, in the presence of macrophage colony stimulating factor (M-CSF) and RANKL, active osteoclasts were formed using bone marrow cells from calvaria and long bone mouse bone marrow. Surprisingly, an almost complete absence of osteoclast formation was found when osteoclast precursors were co-cultured with LAMP-2 deficient osteoblasts. Fluorescence-activated cell sorting FACS analysis revealed that plasma membrane-bound RANKL was strongly decreased on LAMP-2 deficient osteoblasts. These results suggest that osteoblastic LAMP-2 is required for osteoblast-induced osteoclast formation in vitro.

Alteration of structural and mechanical properties of the temporomandibular joint disc following elastase digestion.

The temporomandibular joint disc is a fibrocartilaginous structure, composed of collagen fibers, elastin fibers, and proteoglycans. Despite the crucial role of elastin fibers in load-bearing properties of connective tissues, its contribution in temporomandibular joint disc biomechanics has been disregarded. This study attempts to characterize the structural-functional contribution of elastin in the temporomandibular joint disc. Using elastase, we selectively perturbed the elastin fiber network in porcine temporomandibular joint discs and investigated the structural, compositional, and mechanical regional changes through: (a) analysis of collagen and elastin fibers by immunolabeling and transmission electron microscopy; (b) quantitative analysis of collagen tortuosity, cell shape, and disc volume; (c) biochemical quantification of collagen, glycosaminoglycan and elastin content; and (d) cyclic compression test. Following elastase treatment, microscopic examination revealed fragmentation of elastin fibers across the temporomandibular joint disc, with a more pronounced effect in the intermediate regions. Also, biochemical analyses of the intermediate regions showed significant depletion of elastin (50%), and substantial decrease in collagen (20%) and glycosaminoglycan (49%) content, likely due to non-specific activity of elastase. Degradation of elastin fibers affected the homeostatic configuration of the disc, reflected in its significant volume enlargement accompanied by remarkable reduction of collagen tortuosity and cell elongation. Mechanically, elastase treatment nearly doubled the maximal energy dissipation across the intermediate regions while the instantaneous modulus was not significantly affected. We conclude that elastin fibers contribute to the restoration and maintenance of the disc resting shape and actively interact with collagen fibers to provide mechanical resilience to the temporomandibular joint disc.