Experimental Tooth Movement Into New Bone Area Regenerated by Use of Bone Marrow-Derived Mesenchymal Stem Cells.

OBJECTIVE: The aim of this study was to examine experimental tooth movement into regenerated bone in alveolar cleft with mesenchymal stem cells and a granulated carbonated hydroxyapatite scaffold. DESIGN: An artificial bone defect was created bilaterally in upper incisor regions of beagle dogs to simulate alveolar clefts in patients with cleft palate. The mesenchymal stem cells derived from the iliac bone marrow were cultured and transplanted with carbonated hydroxyapatite into the bone defect area. Carbonated hydroxyapatite alone was transplanted on the control side. Six months after the transplantation, multi-bracket appliances were attached to the lateral incisors and canines on both sides of the maxilla to exert an orthodontic force of 100 × g using an elastic chain. The distance between lateral incisor and canine was measured, and standardized x-ray images were taken every month. The tissue after tooth movement was evaluated by histological observation. RESULTS: The experimental tooth movement, accompanied by resorption of regenerated bone and new bone formation, was achieved on the experimental and control sides. Although there was no difference in the amount of tooth movement obtained on the experimental and control sides during the 6-month experimental period, the rate of tooth movement varied on the control side; whereas, the rate was consistent on the experimental side. Root resorption of the tooth was observed on the control side in one dog. CONCLUSION: It is suggested that mesenchymal/carbonated hydroxyapatite transplantation therapy has great potential as a new treatment modality for bone regeneration in patients with cleft palate.

Synthesis of hyaluronan and superficial zone protein in synovial membrane cells modulated by fluid flow.

Hyaluronan (HA) and superficial zone protein (SZP) distribute in joint structures and play a crucial role in joint lubrication. The aim of this study was to examine the effect of fluid flow on the synthesis of both HA and SZP in synovial membrane cells. Shear stress was applied by fluid flow to the rabbit synovial membrane cell line, HIG-82. The mRNA levels of HA synthase 2 (HAS2) , HA synthase 3 (HAS3), and SZP were examined by real-time PCR. The levels of HA and SZP protein were determined by sandwich ELISA and western blotting, respectively. The expression of SZP protein was increased by the application of low-magnitude shear stress, whereas high-magnitude shear stress decreased expression of SZP protein. Meanwhile, the level of HA protein in culture was decreased when high-magnitude shear stress was applied. The levels of both HAS2 and HAS3 mRNAs were down-regulated by high-magnitude shear stress, resulting in a significant decrease in HA concentration. In conclusion, it is shown that the application of shear stress to synovial membrane cells substantially affects the synthesis of both HA and SZP, which are inhibited if excessive stress is applied.

Superficial zone protein affects boundary lubrication on the surface of mandibular condylar cartilage.

We examined the localization and boundary lubricating function of superficial zone protein (SZP) on the surface of mandibular condylar cartilage. Chondrocytes were separated from the surface layer of mandibular condylar cartilage of 6- to 9-month-old female pigs. A cyclic tensile strain of 7% or 21% cell elongation was applied to the cultured chondrocytes. Gene expression levels of cartilage matrix proteins and secretory phospholipase A(2) (sPLA(2)) were quantified by real-time polymerase chain reaction analysis. The friction coefficient of the mandibular condylar surface was measured by a friction tester before and after treatment with 0.1 U/ml sPLA(2). Significantly higher mRNA levels of SZP and type I collagen were found in chondrocytes from the superficial layer than in those in the other layers. The SZP mRNA level was up-regulated by cyclic tensile strain of 7% and 21% cell elongation. Cyclic tensile strain of 21% cell elongation up-regulated the sPLA(2) mRNA level. The friction coefficient of the condylar surface was increased significantly by treatment with sPLA(2). The removal of SZP from the surface layer of mandibular condylar cartilage by sPLA(2) resulted in a significant increase in the friction coefficient on the surface of articular cartilage.

ANGPTL2 Induces Synovial Inflammation via LILRB2.

Angiopoietin-like proteins (ANGPTLs) are circulating proteins that are expressed in various cells and tissues and are thought to be involved in the repair and remodeling of damaged tissues; however, ANGPTL2 hyperfunction has been shown to cause chronic inflammation, leading to the progression of various diseases. ANGPTL2 is known to exert cellular effects via receptors such as integrin α5ß1 and leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2); however, their roles in ANGPTL2-induced inflammation remain unclear. In this study, we investigated the mechanisms underlying ANGPTL2-induced inflammation involving LILRB2 and various signaling pathways in human fibroblast-like synoviocytes (HFLS). The effects of ANGPTL2 and an anti-LILRB2 antibody on the gene expression of various inflammation-related factors were examined using real-time RT-PCR, while their effects on MAPK, NF-κB, and Akt phosphorylation were analyzed by western blotting. We found that the addition of ANGPTL2 enhanced the gene expression of inflammatory factors, whereas pretreatment with the anti-LILRB2 antibody for 12 h decreased the expression of these factors. Similarly, ANGPTL2 addition activated the phosphorylation of ERK, p38, JNK, NF-κB, and Akt in HFLS; however, this effect was significantly inhibited by pretreatment with the anti-LILRB2 antibody. Together, the findings of this study demonstrate that ANGPTL2 induces the expression of inflammatory factors via LILRB2 in synovial cells. Therefore, LILRB2 could be a potential therapeutic agent for treating matrix degradation in osteoarthritis.

Effects of human full-length amelogenin on the proliferation of human mesenchymal stem cells derived from bone marrow.

Amelogenins are enamel matrix proteins that play a crucial role in enamel formation. Recent studies have revealed that amelogenins also have cell signaling properties. Although amelogenins had been described as specific products of ameloblasts, recent research has demonstrated their expression in bone marrow stromal cells. In this study, we examined the effect of recombinant human full-length amelogenin (rh174) on the proliferation of human mesenchymal stem cells (MSCs) derived from bone marrow and characterized the associated changes in intracellular signaling pathways. MSCs were treated with rh174 ranging in dose from 0 to 1,000 ng/ml. Cell proliferative activity was analyzed by bromodeoxyuridine (BrdU) immunoassay. The expression of lysosomal-associated membrane protein 1 (LAMP1), a possible amelogenin receptor, in MSCs was analyzed. Anti-LAMP1 antibody was used to block the binding of rh174 to LAMP1. The MAPK-ERK pathway was examined by Cellular Activation of Signaling ELISA (CASE) kit and western blot analysis. A specific MAPK inhibitor, U0126, was used to block ERK activity. It was shown that rh174 increased the proliferation of MSCs and MAPK-ERK activity. The MSC proliferation and MAPK-ERK activity enhanced by rh174 were reduced by the addition of anti-LAMP1 antibody. Additionally, the increased proliferation of MSCs induced by rh174 was inhibited in the presence of U0126. In conclusion, it is demonstrated that rh174 increases the proliferation of MSCs by interaction with LAMP1 through the MAPK-ERK signaling pathway, indicating the possibility of MSC application to tissue regeneration in the orofacial region.

Applying an excessive mechanical stress alters the effect of subchondral osteoblasts on chondrocytes in a co-culture system.

Osteoarthritis (OA) sometimes occurs as a consequence of repeated microtrauma involved in parafunction, which may lead to microfracture in the subchondral bone. The aim of this in vitro study was to evaluate the effects of subchondral osteoblasts in loading with repeated excessive mechanical stress on the metabolism of overlying chondrocytes. A high-magnitude cyclic tensile stress of 15 kPa (30 cycles min(-1)) was applied to the cultured osteoblasts obtained from porcine mandibular condyles. The chondrocytes in alginate beads were then co-cultured with mechanically stressed or unstressed osteoblasts. Chondrocytes co-cultured with unstressed osteoblasts showed a phenotypic shift to hypertrophic chondrocytes, characterized by decreased expression of type II collagen, aggrecan, Sry-related HMG box (SOX-9), and cartilage oligomeric matrix protein (COMP) genes and increased expression of type X collagen and bone sialoprotein (BSP) genes, suggesting that the co-culture may change the chondrocyte differentiation to some extent. These changes were more distinct in chondrocytes co-cultured with excessively mechanically stressed osteoblasts. After co-culture with stressed osteoblasts, the expressions of matrix metalloproteinase (MMP)1, MMP3 and MMP13 genes were also enhanced and the synthesis of DNA, proteoglycan and collagen were significantly decreased in chondrocytes. These results demonstrate that alterations in cartilage metabolism can be induced by stressed osteoblasts, indicating a possible explanation for the onset and progression of OA.

Inhibition of the proliferation of human periodontal ligament fibroblasts by hyaluronidase.

Hyaluronan (HA) exists in various living tissues as one of the major matrix macromolecules, and is well known to play an integral role in cell differentiation and proliferation. The present study was conducted to elucidate whether or not the proliferation of periodontal ligament (PDL) cells are affected specifically by the degradation of HA by hyaluronidasze (HAase). Human PDL fibroblasts were isolated and cultured with and without 15-150U/ml bovine testicular HAase from 1 to 11 days after seeding. The cells were also cultured with anti-CD44 antibody of 2 microg/ml. For the control against the anti-CD44 antibody treatment, 2 microg/ml IgG was used. The HA-dependent pericellular matrix was visualized by particle-exclusion assay. The number of cells was counted by MTT assay during the proliferation. The mRNA levels of HA synthases (HASs), HAases (HYALs) and CD44s were examined by a quantitative real-time PCR analysis. The cell proliferation was inhibited by the treatment with HAase and anti-CD44 antibody in cultured PDL fibroblasts. HASs mRNAs were down-regulated, whereas HYALs mRNAs were up-regulated significantly by the treatment with HAase and anti-CD44 antibody. The CD44s mRNA level exhibited no significant changes. These results suggest that HA may contribute to modulate the proliferation of cultured human PDL cells through a CD44-mediated mechanism.

An adolescent patient with multiple impacted teeth.

Multiple impacted permanent teeth is uncommon and rarely reported in the literature. This article reports the treatment of an adolescent patient with multiple impacted teeth without systemic disease. A 9-year 2-month-old boy complained of a delay of eruption of the first molars. All first molars were unerupted, and the left deciduous second molar was a submerged tooth. The panoramic radiograph showed all permanent teeth except the incisors were unerupted and, especially for the first molars, spontaneous eruption was not expected. His medical history was uneventful. A lingual arch appliance and a segmental arch were placed on the mandibular and maxillary dentitions, respectively, to guide eruption of the impacted first molars. After traction of the first molars, eruption of the impacted lower premolars was induced. Furthermore, at 15 years the impacted mandibular second molars were also positioned properly by use of the lingual arch with auxiliary wires. After achieving traction of the impacted teeth, tooth alignment was initiated using multibracket appliances after the bilateral extraction of the second premolars. After 22 months of treatment with multibracket appliances, an acceptable occlusion was achieved with a Class I molar relationship. After 2 years of retention an acceptable occlusion was maintained without any relapse in the occlusion. Since a delay in the treatment of impacted teeth may induce secondary problems such as root dilacerations and ankylosis, it is highly recommended to perform early treatment of multiple impacted teeth during adolescence.

The C-terminus of the amelogenin peptide influences the proliferation of human bone marrow mesenchymal stem cells.

BACKGROUND: Amelogenins are a family of enamel matrix proteins that are important for formation of enamel. Amelogenins may induce division of mesenchymal stem cells (MSCs), among others. Recently, the C-terminus of the amelogenin peptide (AMG-CP) has been shown to enhance the proliferation of cementoblast lineage cells. The role of the amelogenin peptide on the proliferation of human MSCs and related alterations in the intracellular signaling pathway were studied. METHODS: MSCs were exposed to AMG-CP in vitro. The MTS and 5-bromo-2'-deoxyuridine (BrdU) assays were used to determine proliferation. Expression of the amelogenin receptor, lysosomal-associated membrane protein 1 (LAMP1), was examined in MSCs with western blotting. Binding of AMG-CP to LAMP1 was inhibited with anti-LAMP1 antibody. Components of the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) pathway were studied with western blotting and enzyme-linked immunosorbent assay, and U0126, an MAPK inhibitor, was used to inhibit ERK activity. RESULTS: MSC proliferation was significantly increased in the presence of AMG-CP and significantly inhibited by anti-LAMP1 antibody or U0126. Increased phosphorylated ERK1/2 was observed in the presence of AMG-CP, and decreased phosphorylated ERK1/2 was seen in the presence of anti-LAMP1 antibody or U0126. CONCLUSION: A C-terminal amelogenin variant increased the proliferation of MSCs via an interaction with LAMP1 and the MAPK-ERK signaling pathway, indicating the possibility of using MSCs for tissue regeneration in the craniofacial region.

Cyclic Tensile Strain Upregulates Pro-Inflammatory Cytokine Expression Via FAK-MAPK Signaling in Chondrocytes.

Excessive mechanical stimulation is considered an important factor in the destruction of chondrocytes. Focal adhesion kinase (FAK) is non-receptor tyrosine kinase related to a number of different signaling proteins. Little is known about the function of FAK in chondrocytes under mechanical stimulation. In the present study, we investigated the function of FAK in mechanical signal transduction and the mechanism through which cyclic tensile strain (CTS) induces expression of inflammation-related factors. Mouse ATDC5 chondrogenic cells were subjected to CTS of 0.5 Hz to 10% cell elongation with an FAK inhibitor. The expression of genes encoding COX-2, IL-1ß, and TNF-α was examined using real-time RT-PCR after CTS application with FAK inhibitor. Phosphorylation of p-38, ERK, and JNK was analyzed by Western blotting. Differences in COX-2 expression following pretreatment with FAK, p-38, ERK, and JNK inhibitors were compared by Western blotting. We found that CTS increased the expression of genes encoding COX-2, IL-1ß, and TNF-α and activated the phosphorylation of FAK, p-38, ERK, and JNK. Pretreatment with an FAK inhibitor for 2 h reduced the expression of genes encoding COX-2, IL-1ß, and TNF-α induced by CTS-associated inflammation and decreased phosphorylation of FAK, p-38, ERK, and JNK. Pretreatment with FAK, p-38, ERK, and JNK inhibitors markedly suppressed COX-2 and IL-1ß protein expression. In conclusion, FAK appears to regulate inflammation in chondrocytes under CTS via MAPK pathways.

Education of dentists in China.

China is geographically located in the east of Asia and its population exceeds 1.3 billion. An understanding of dental education in China is thus of interest. However, as there is little published information on this topic, this paper provides information about China regarding its dental history, dental school system including curriculum and dental licensure. High school graduates take a nationwide entrance examination to apply for dental school, of which there are more than 50 in China. A five year dental education leads to the BDS degree. Dental school graduates must then pass the nationwide licensure examination to practise dentistry. Currently, there are not adequate numbers of dentists to provide the necessary oral health care for people living outside metropolitan areas.

Effects of low-intensity pulsed ultrasound on the expression of cyclooxygenase-2 in mandibular condylar chondrocytes.

AIMS: To determine the effect of low-intensity pulsed ultrasound (LIPUS) on cyclooxygenase-2 (COX-2) expression and related mechanisms by using cultured articular chondrocytes derived from porcine mandibular condyles after treatment with interleukin-1 beta (IL-1ß). METHODS: Chondrocytes were derived from porcine mandibular condylar cartilage and cultured. The cells were treated with or without 10 ng/mL IL-1ß. At the same time, the cells were exposed to LIPUS for 20 minutes. After LIPUS exposure, the conditioned medium was changed to a fresh one without IL-1ß, and the cells were incubated for 0 to 24 hours. The effects of LIPUS on IL-1ß-treated chondrocytes were examined in terms of the expression of p-integrin ß1, COX-2, and phosphorylated extracellular signal-related kinase (p-ERK) 1/2 by real-time polymerase chain reaction (PCR) and Western blot analyses. Differences in the means among multiple groups were examined by one-way analysis of variance (ANOVA) for all groups at each time point, followed by a Scheffé multiple comparison test as a post-hoc test; Student t test was also used. RESULTS: COX-2 mRNA level was upregulated by the treatment with IL-1ß and was suppressed significantly (P < .01) by LIPUS exposure. Furthermore, LIPUS enhanced gene expression and phosphorylation of integrin ß, and it inhibited the expression of p-ERK 1/2. CONCLUSION: LIPUS exposure inhibited IL-1ß-induced COX-2 expression through the integrin ß1 receptor followed by the phosphorylation of ERK 1/2. Despite the restricted duration of its effect, LIPUS is suggested to be a potential candidate as a preventive and auxiliary treatment to suppress the degradation of articular chondrocytes in temporomandibular joint osteoarthritis.

Effects of bFGF on the Modulation of Apoptosis in Gingival Fibroblasts with Different Host Ages.

The purpose of this study was to investigate the effects of basic fibroblast growth factor (bFGF) treatment on the proliferation and apoptosis of cultured gingival fibroblasts (GFs). Human GFs were isolated from the palatal gingival tissues of 16 healthy volunteers ranging in the age from 9 to 35 years old. Cultured GFs were subjected to the analyses for cell proliferation by ELISA assay, gene expression by RT-PCR analysis, and apoptosis potency by caspase-3 assay. The cell proliferation activity and gene expression of type-I collagen and caspase-3 activity were enhanced significantly by the treatment with bFGF in cultured GFs. Furthermore, the activity of caspase-3 in cultured GFs from young subjects was significantly higher than that in GFs from adults. It is shown that bFGF significantly enhances the gene expression of type-I collagen in cultured fibroblasts from human gingival tissues. It also demonstrated that bFGF modulates the apoptosis of periodontal fibroblasts, and the effect is higher in young subjects, indicating a significant role of bFGF in the prevention of scar formation during wound healing.

Differential effects of amelogenin on mineralization of cementoblasts and periodontal ligament cells.

BACKGROUND: Amelogenin is a major component of developing extracellular enamel matrix proteins and plays a crucial role during the formation of tooth enamel. In addition, amelogenins are suggested to exert biologic functions as signaling molecules through cell-surface receptors. The purpose of this study is to examine the effect of recombinant human full-length amelogenin (rh174) on the mineralization of human cementoblasts (HCEMs) and human periodontal ligament cells (HPDLs). METHODS: HCEMs, namely, a cell line immortalized by transfection of human telomerase reverse transcription gene, and HPDLs isolated from human first premolars were cultured and treated with 0 to 1,000 ng/mL rh174. The messenger ribonucleic acid (mRNA) levels of alkaline phosphatase (ALP), osteocalcin (OCN), and bone sialoprotein (BSP) were examined by real-time polymerase chain reaction analysis. The protein levels of OCN and BSP were examined by Western blot analysis. ALP activity and calcium deposition of cell cultures were also determined. Mineralization of cells was evaluated by red dye staining. RESULTS: The treatment of HCEMs with rh174 upregulated the ALP, OCN, and BSP mRNA levels. In addition, the protein levels of OCN and BSP, ALP activity, and calcium deposition were enhanced, resulting in enhanced mineralization. Conversely, there were no significant effects of rh174 on the mineralization of HPDLs. CONCLUSION: The present study shows that rh174 enhances mineralization accompanied by upregulation of mineralization markers in HCEMs, whereas it has no effect on that in HPDLs, suggesting different effects of amelogenin on PDL and cementum.

Bone regeneration in artificial jaw cleft by use of carbonated hydroxyapatite particles and mesenchymal stem cells derived from iliac bone.

Objectives of the Study. Cleft lip and palate (CLP) is a prevalent congenital anomaly in the orofacial region. Autogenous iliac bone grafting has been frequently employed for the closure of bone defects at the jaw cleft site. Since the related surgical procedures are quite invasive for patients, it is of great importance to develop a new less invasive technique. The aim of this study was to examine bone regeneration with mesenchyme stem cells (MSCs) for the treatment of bone defect in artificially created jaw cleft in dogs. Materials and Methods. A bone defect was prepared bilaterally in the upper incisor regions of beagle dogs. MSCs derived from iliac bone marrow were cultured and transplanted with carbonated hydroxyapatite (CAP) particles into the bone defect area. The bone regeneration was evaluated by standardized occlusal X-ray examination and histological observation. Results. Six months after the transplantation, perfect closure of the jaw cleft was achieved on the experimental side. The X-ray and histological examination revealed that the regenerated bone on the experimental side was almost equivalent to the original bone adjoining the jaw cleft. Conclusion. It was suggested that the application of MSCs with CAP particles can become a new treatment modality for bone regeneration for CLP patients.

Interleukin-1 beta affects cyclooxygenase-2 expression and cartilage metabolism in mandibular condyle.

Extracellular matrix degradation in mandibular condylar cartilage is mediated by various cytokines in the temporomandibular joint (TMJ). Interleukin-1 beta (IL-1ß) is detected in joint structures with pathologic status, and participates in catabolic action in the extracellular matrix. The purpose of this study was to investigate the effects of IL-1ß on cyclooxygenase-2 (COX-2) expression and cartilage metabolism using cultured chondrocytes from mandibular condyle. Articular chondrocytes from the porcine mandibular condylar cartilage around the surface were cultured and treated with 0-10 ng/ml IL-1ß or 0-1000 ng/ml prostaglandin (PGE(2)) for 0-24h. The mRNA levels of COX-2, MMP-1, -3, and -13 were evaluated by real-time PCR analysis. The protein levels of PGE(2) and MMPs were examined by ELISA and Western blot analysis, respectively. The expression levels of COX-2 and PGE(2) were enhanced by exogenous IL-1ß in chondrocytes. The mRNA levels of MMP-1, -3, and -13 were up-regulated by PGE(2) treatment dose-dependently. It is shown that the expression of COX-2/PGE(2) was enhanced by IL-1ß in articular chondrocytes from mandibular condyle, and that MMP-1, -3, and -13 were induced by PGE(2), suggesting that IL-1ß-induced COX-2/PGE(2) play a crucial role in catabolic processes of mandibular condylar cartilage under inflammatory conditions.

Amelogenin enhances the proliferation of cementoblast lineage cells.

BACKGROUND: It is well known that enamel matrix proteins play a crucial role in tooth root formation and amelogenesis. Because amelogenin is a major enamel matrix protein, it is assumed that amelogenin also affects the metabolism of cementum. However, the biologic functions of amelogenin in cementoblasts remain unclear. The purpose of this study is to examine the effect of recombinant human full-length amelogenin (rh174) on the proliferation of cultured human cementoblast-like (HCEM) and human periodontal ligament (HPDL) cells. METHODS: HCEM and HPDL cells were cultured and treated with 100 ng/mL rh174 in the presence or absence of an anti-cluster of differentiation (CD) 63 blocking antibody. Cell proliferation was evaluated using a cell proliferation enzyme-linked immunosorbent assay 5-bromo-2-deoxyuridine kit and quantification of the cell number by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium-inner salt assay. The phosphorylation of extracellular signal-regulated kinases (ERK) 1/2 was measured by enzyme-linked immunosorbent assay and Western blot analysis. RESULTS: The proliferation of HCEM and HPDL cells was enhanced significantly (P <0.05) by treatment with rh174, and inhibited significantly (P <0.05) by the addition of anti-CD63 blocking antibody. In addition, the ratio of phosphorylated ERK1/2 to total ERK1/2 became significantly larger (P <0.05) by treatment with rh174, and was reduced significantly by the addition of anti-CD63 blocking antibody in both HCEM and HPDL cells. CONCLUSION: The results show that rh174 interacts with CD63, and rh174/CD63 interaction activates the ERK1/2 signaling pathway, enhancing the proliferation activities of HCEM and HPDL cells.

Modulation of hyaluronan catabolism in chondrocytes by mechanical stimuli.

Hyaluronan (HA) is a component of the extracellular matrices of cartilage contributing to the structural and functional integrity. HA metabolism is regulated by both anabolic and catabolic processes; however, a great deal more of the detail has been unknown yet. The purpose of this study was to clarify the effect of excessive mechanical load on the expression and activity of hyaluronidase (HYAL) in chondrocytes with a special reference to the expressions of IL-1beta and tumor necrosis factor (TNF)-alpha. A cyclic tensile load of 22.8% cell elongation, regarded as an excessive mechanical stimulus, was applied to cultured rabbit knee articular chondrocytes. HYAL1, HYAL2, IL-1beta, and TNF-alpha mRNA levels were examined by quantitative real-time PCR analysis. The HYAL activity in culture medium was examined by HA zymography. Both HYAL1 and HYAL2 mRNA levels were upregulated significantly by the loading in cultured chondrocytes. HYAL activity was also enhanced as compared with unloaded controls. The IL-1beta mRNA level was upregulated significantly by the loading, and TNF-alpha mRNA level was slightly upregulated. HYAL1 and HYAL2 mRNA levels were upregulated significantly by IL-1beta treatment, resulting in a slight increase in HYAL activity. These results show that the expression of HYAL1 and HYAL2 in articular chondrocytes is enhanced by excessive mechanical stimuli and affected in part by induction of IL-1beta, leading to HA catabolism in articular cartilage.

Effects of mechanical load on the expression and activity of hyaluronidase in cultured synovial membrane cells.

Hyaluronan (HA) has lubricating and buffering functions in joints. Mechanical load is a regulatory factor of HA metabolism in joints, and HA synthesis by synovial membrane cells is modulated by mechanical load. However, the effects of mechanical load on HA catabolism by hyaluronidase (HYAL) in synovial membrane cells remain unclear. The purpose of this study was to evaluate the effects of cyclic tensile load on the expression and activity of HYAL in synovial membrane cells. A cyclic tensile load of 22.8% cell elongation was applied to cultured rabbit synovial membrane cells for 3 to 48 h with or without cycloheximide. HYAL1 and HYAL2 mRNA levels were evaluated by means of real-time polymerase chain reaction (PCR) analysis. HYAL activity in the cell culture was analyzed by means of HA zymography with or without HYAL2-small interfering (si) RNA. Levels of both HYAL1 and HYAL2 mRNA were up-regulated significantly (p < 0.01) by the cyclic tensile load with or without cycloheximide. HYAL activity was detected in the loaded cell cultures and was suppressed substantially by HYAL2-siRNA. HYAL activity was undetectable in unloaded cell cultures. These results show that a cyclic tensile load induces the expression and activity of HYAL in synovial membrane cells, suggesting that increased HYAL by mechanical load affects HA catabolism in synovial fluid.

Effects of hyaluronan oligosaccharide on the expression of MMP-1 in periodontal ligament cells.

It is well known that low-molecular weight hyaluronan (HA) is detected in human periodontal tissue under inflammatory conditions. HA oligosaccharide (HAoligo) has been demonstrated to induce matrix metalloproteinase (MMP)-1 expression in dendritic cells and chondrocytes, however, the bioactivities of HAoligo in periodontal ligament (PDL) cells remain unclear. In this study, we investigated the effect of HAoligo on MMP-1 expression in human PDL (HPDL) cells and the mechanisms in terms of the signal transmission. HAoligo was generated and purified from commercial human umbilical cord HA. HPDL cells were isolated from healthy ligaments, and cultured with HAoligo for 0-24h. The expression of MMP-1, tissue inhibitor of MMPs (TIMP)-1 and TIMP-2 was analyzed by real-time PCR and Western blot analyses. Effects of specific inhibitors of p38 mitogen-activated protein kinase (MAPK) activity, MAPK kinase activity and NFkB activity on HAoligo-induced MMP-1 expression in HPDL cells were also investigated by real-time PCR and Western blot analyses. HAoligo remarkably enhanced MMP-1 expression in both mRNA and protein levels, but no effect was shown on the expression of TIMP-1 and TIMP-2 mRNAs. Inhibition of p38MAPK activity decreased the MMP-1 expression. Neither inhibition of NFkB nor MAPKK activity affected the MMP-1 expression. It was suggested that HAoligo induces MMP-1 expression in HPDL cells, and p38MAPK plays a crucial role in signal transduction for MMP-1 inducted by HAoligo.