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.

Increase in the Number of Bone Marrow Osteoclast Precursors at Different Skeletal Sites, Particularly in Long Bone and Jaw Marrow in Mice Lacking IL-1RA.

Recently, it was shown that interleukin-1ß (IL-1ß) has diverse stimulatory effects on different murine long bone marrow osteoclast precursors (OCPs) in vitro. In this study, interleukin-1 receptor antagonist deficient (Il1rn-/-) and wild-type (WT) mice were compared to investigate the effects of enhanced IL-1 signaling on the composition of OCPs in long bone, calvaria, vertebra, and jaw. Bone marrow cells were isolated from these sites and the percentage of early blast (CD31hi Ly-6C-), myeloid blast (CD31+ Ly-6C+), and monocyte (CD31- Ly-6Chi) OCPs was assessed by flow cytometry. At the time-point of cell isolation, Il1rn-/- mice showed no inflammation or bone destruction yet as determined by histology and microcomputed tomography. However, Il1rn-/- mice had an approximately two-fold higher percentage of OCPs in long bone and jaw marrow compared to WT. Conversely, vertebrae and calvaria marrow contained a similar composition of OCPs in both strains. Bone marrow cells were cultured with macrophage colony stimulating factor (M-CSF) and receptor of NfκB ligand (RANKL) on bone slices to assess osteoclastogenesis and on calcium phosphate-coated plates to analyze mineral dissolution. Deletion of Il1rn increased osteoclastogenesis from long bone, calvaria, and jaw marrows, and all Il1rn-/- cultures showed increased mineral dissolution compared to WT. However, osteoclast markers increased exclusively in Il1rn-/- osteoclasts from long bone and jaw. Collectively, these findings indicate that a lack of IL-1RA increases the numbers of OCPs in vivo, particularly in long bone and jaw, where rheumatoid arthritis and periodontitis develop. Thus, increased bone loss at these sites may be triggered by a larger pool of OCPs due to the disruption of IL-1 inhibitors.

TLR3 activation modulates immunomodulatory properties of human periodontal ligament cells.

BACKGROUND: Toll-like receptors (TLR) are a group of receptors that play roles in the innate immune system. Human periodontal ligament cells (hPDL cells) express several TLRs, including TLR3, a nucleotide sensing receptor that recognizes double-stranded RNA from viral infection. However, its role in hPDL cells is unclear. The aim of this study was to investigate the responses of hPDL cells in terms of immunomodulation after TLR3 engagement. METHODS: HPDL cells were treated with various doses of poly I:C, a TLR3 activator. The expression of interferon-gamma (IFNγ), indoleamine 2,3 dioxygenase (IDO), and human leukocyte antigen G (HLA-G) was determined. Chemical inhibitors and small interfering RNA (siRNA) were used to confirm the role of TLR3. Coculture with human peripheral blood mononuclear cells (PBMCs) with poly I:C-activated hPDL cells was performed. RESULTS: Endosomal TLR3 in hPDL cells was observed by immunocytochemistry. Addition of poly I:C significantly enhanced the expression and secretion of IFNγ, IDO, and HLA-G. Knockdown of TLR3 using siRNA decreased the poly I:C-induced expression of these three molecules. Bafilomycin-A, an inhibitor of auto-phagosome and lysosome fusion, inhibited poly I:C-induced IDO and HLA-G expression, whereas cycloheximide and a TLR3-neutralizing antibody had no effect. In co-culture experiments, poly I:C-activated hPDL cells inhibited PBMCs proliferation and increased mRNA expression of forkhead box P3 (FOXP3), a transcription factor which is a marker of regulatory T cells. CONCLUSION: Our findings indicated that TLR3 engagement of hPDL cells induced immunosuppressive properties of these cells. Because immunosuppressive properties play an important role in tissue healing and regeneration, activation of TLR3 may help to attenuate tissue destruction by limiting the inflammatory process and perhaps initiate the healing and regeneration process of the periodontium.

In Vivo Bone Regeneration Induced by a Scaffold of Chitosan/Dicarboxylic Acid Seeded with Human Periodontal Ligament Cells.

Chitosan/dicarboxylic acid (CS/DA) scaffold has been developed as a bone tissue engineering material. This study evaluated a CS/DA scaffold with and without seeded primary human periodontal ligament cells (hPDLCs) in its capacity to regenerate bone in calvarial defects of mice. The osteogenic differentiation of hPDLCs was analyzed by bone nodule formation and gene expression. In vivo bone regeneration was analyzed in mice calvarial defects. Eighteen mice were divided into 3 groups: one group with empty defects, one group with defects with CS/DA scaffold, and a group with defects with CS/DA scaffold and with hPDLCs. After 6 and 12 weeks, new bone formation was assessed using microcomputed tomography (Micro-CT) and histology. CS/DA scaffold significantly promoted in vitro osteoblast-related gene expression (RUNX2, OSX, COL1, ALP, and OPN) by hPDLCs. Micro-CT revealed that CS/DA scaffolds significantly promoted in vivo bone regeneration both after 6 and 12 weeks (p < 0.05). Histological examination confirmed these findings. New bone formation was observed in defects with CS/DA scaffold; being similar with and without hPDLCs. CS/DA scaffolds can be used as a bone regenerative material with good osteoinductive/osteoconductive properties.

The dynamic mechanical viscoelastic properties of the temporomandibular joint disc: The role of collagen and elastin fibers from a perspective of polymer dynamics.

The temporomandibular joint disc is a structure, characterized as heterogeneous fibrocartilage, and is composed of macromolecular biopolymers. Despite a large body of characterization studies, the contribution of matrix biopolymers on the dynamic viscoelastic behavior of the disc is poorly understood. Given the high permeability and low concentration of glycosaminoglycans in the disc, it has been suggested that poro-elastic behavior can be neglected and that the intrinsic viscoelastic nature of solid matrix plays a dominant role in governing its time-dependent behavior. This study attempts to quantify the contribution of collagen and elastin fibers to the viscoelastic properties of the disc. Using collagenase and elastase, we perturbed the collagen and elastin fibrillar network in porcine temporomandibular joint discs and investigated the changes of dynamic viscoelastic properties in five different regions of the disc. Following both treatments, the storage and loss moduli of these regions were reduced dramatically up to the point that the tissue was no longer mechanically heterogeneous. However, the proportion of changes in storage and loss moduli were different for each treatment, reflected in the decrease and increase of the loss tangent for collagenase and elastase treated discs, respectively. The reduction of storage and loss moduli of the disc correlated with a decrease of biopolymer length. The present study indicates that the compositional and structural changes of collagen and elastin fibers alter the viscoelastic properties of the disc consistent with polymer dynamics.

Cyclic tensile force-upregulated IL6 increases MMP3 expression by human periodontal ligament cells.

OBJECTIVE: Cyclic tensile force (CTF) modulates physiological responses of periodontal ligament (PDL) cells. PDL cells are mechanosensitive and are able to maintain tissue homeostasis; a process mediated by the expression of particular cytokines including interleukin 6 (IL6). It is unknown whether CTF-induced IL6 regulates the expression of MMPs, enzymes needed for tissue remodeling. DESIGN: Human PDL cells were subjected to 10% elongation strain of CTF at a frequency of 60 rpm continuously for 6 h. RNA and proteins were extracted and analyzed for IL6 and MMP expression by quantitative real-time PCR and ELISA, respectively. Using a neutralizing anti-IL6 antibody and addition of recombinant human IL6 at concentrations of 0.1, 1, 10 ng.mL-1 were performed to clarify whether CTF-upregulated IL6 increased MMP expression. Inhibitors of intracellular signaling molecules were employed to reveal possible pathway(s) of IL6-induced MMP expression. RESULTS: CTF-induced IL6 expression coincided with an increased MMP3 expression. A neutralizing anti-IL6 antibody attenuated the CTF-increased MMP3 expression, whereas stimulating the cells with recombinant human IL6 increased MMP3 expression. Both PI3K and MAPK pathways were essential in the IL6 induced expression of MMP3. CONCLUSION: Our findings suggest a role of CTF in the modulation of expression of IL6 and MMP3 and thus in the regulation of homeostasis and remodeling of the periodontal ligament.

IL-1ß Damages Fibrocartilage and Upregulates MMP-13 Expression in Fibrochondrocytes in the Condyle of the Temporomandibular Joint.

The temporomandibular joint (TMJ), which differs anatomically and biochemically from hyaline cartilage-covered joints, is an under-recognized joint in arthritic disease, even though TMJ damage can have deleterious effects on physical appearance, pain and function. Here, we analyzed the effect of IL-1ß, a cytokine highly expressed in arthritic joints, on TMJ fibrocartilage-derived cells, and we investigated the modulatory effect of mechanical loading on IL-1ß-induced expression of catabolic enzymes. TMJ cartilage degradation was analyzed in 8-11-week-old mice deficient for IL-1 receptor antagonist (IL-1RA-/-) and wild-type controls. Cells were isolated from the juvenile porcine condyle, fossa, and disc, grown in agarose gels, and subjected to IL-1ß (0.1-10 ng/mL) for 6 or 24 h. Expression of catabolic enzymes (ADAMTS and MMPs) was quantified by RT-qPCR and immunohistochemistry. Porcine condylar cells were stimulated with IL-1ß for 12 h with IL-1ß, followed by 8 h of 6% dynamic mechanical (tensile) strain, and gene expression of MMPs was quantified. Early signs of condylar cartilage damage were apparent in IL-1RA-/- mice. In porcine cells, IL-1ß strongly increased expression of the aggrecanases ADAMTS4 and ADAMTS5 by fibrochondrocytes from the fossa (13-fold and 7-fold) and enhanced the number of MMP-13 protein-expressing condylar cells (8-fold). Mechanical loading significantly lowered (3-fold) IL-1ß-induced MMP-13 gene expression by condylar fibrochondrocytes. IL-1ß induces TMJ condylar cartilage damage, possibly by enhancing MMP-13 production. Mechanical loading reduces IL-1ß-induced MMP-13 gene expression, suggesting that mechanical stimuli may prevent cartilage damage of the TMJ in arthritic patients.

Prostacyclin Analog Promotes Human Dental Pulp Cell Migration via a Matrix Metalloproteinase 9-related Pathway.

INTRODUCTION: During dental pulp healing, progenitor cells migrate to the injured site. This study investigated the effect of iloprost (an exogenous prostacyclin [PGI2]) on enhancing human dental pulp cell (HDPC) migration and its underlying mechanism. METHODS: HDPC migration was analyzed using a wound scratch assay. HDPCs were obtained from extracted teeth and cultured in the presence of iloprost for 24 and 72 hours. Immunofluorescent staining for matrix metalloproteinase 9 (MMP-9), quantitative polymerase chain reaction gene expression analysis, gelatin zymography, and enzyme-linked immunosorbent assay of MMP-9 expression were performed. A PGI2 (IP) antagonist, protein kinase A (PKA) inhibitor, and MMP-9 inhibitor were used to inhibit the IP receptor, PKA signaling pathway, and MMP-9 activity, respectively. RESULTS: A mechanically applied scratch in HDPC cultures closed more rapidly in the presence of iloprost. This result coincided with increased MMP-9 messenger RNA and protein expression and higher gelatinase activity. These iloprost-enhanced effects were inhibited by an IP receptor antagonist or a PKA inhibitor. Forskolin, a PKA activator, increased MMP-9 expression concomitant with increased migration. The application of a selective MMP-9 inhibitor resulted in decreased iloprost-induced migration. CONCLUSIONS: MMPs play an important role in cell migration by degrading components of the extracellular matrix. In this study, iloprost accelerated HDPC migration in a wound scratch assay. MMP-9 expression was increased concomitantly by iloprost and appeared to be mediated by the IP-PKA pathway. These observations suggest that iloprost may enhance dental pulp tissue healing by up-regulating MMP-9. The PGI2 analog might be a promising biomolecule in dental pulp regenerative treatment.

Transmission Electron Microscopy of Bone.

Electron microscopic analysis of mineralized tissues like bone and dentin is essential for understanding of cell-cell/cell-matrix interactions, and the three-dimensional organization of these tissues. This chapter describes a few methods to process mineralized tissues obtained from different sources for ultrastructural analysis by transmission electron microscopy.

Molecular pathways of cell-mediated degradation of fibrillar collagen.

Fibrillar collagens are the most abundant components of the extracellular matrix and provide stability to connective tissues, such as bone, cartilage and skin. An imbalance in collagen turnover inevitably affects the function of these tissues. Therefore, the molecular and cellular mechanisms involved in the synthesis and degradation of collagen have received increasing attention. This short review attempts to summarize our present understanding of how different pathways of collagen degradation are used by different cell types.

Cyclic tensile force stimulates BMP9 synthesis and in vitro mineralization by human periodontal ligament cells.

Periodontal ligament (PDL) cells are mechanosensitive and have the potential to differentiate into osteoblast-like cells under the influence of cyclic tensile force (CTF). CTF modulates the expression of regulatory proteins including bone morphogenetic proteins (BMPs), which are essential for the homeostasis of the periodontium. Among the BMPs, BMP9 is one of the most potent osteogenic BMPs. It is yet unknown whether CTF affects the expression of BMP9 and mineralization. Here, we demonstrated that continuously applied CTF for only the first 6 hr stimulated the synthesis of BMP9 and induced mineral deposition within 14 days by human PDL cells. Stimulation of BMP9 expression depended on ATP and P2Y 1 receptors. Apyrase, an ecto-ATPase, inhibited CTF-mediated ATP-induced BMP9 expression. The addition of ATP increased the expression of BMP9. Loss of function experiments using suramin (a broad-spectrum P2Y antagonist), MRS2179 (a specific P2Y 1 receptor antagonist), MRS 2365 (a specific P2Y 1 agonist), U-73122 (a phospholipase C [PLC] inhibitor), and thapsigargin (enhancer of intracytosolic calcium) revealed the participation of P2Y 1 in regulating the expression of BMP9. This was mediated by an increased level of intracellular Ca 2+ through the PLC pathway. A neutralizing anti-BMP9 antibody decreased mineral deposition, which was stimulated by CTF for almost 45% indicating a role of BMP9 in an in vitro mineralization. Collectively, our findings suggest an essential modulatory role of CTF in the homeostasis and regeneration of the periodontium.