Impaired dental implant osseointegration in rat with streptozotocin-induced diabetes.

OBJECTIVE: Few studies have reported on the impact of oxidative stress on the dental implant failure. The aim of this study was to investigate the impact of hyperglycemia-induced oxidative stress on dental implant osseointegration in diabetes mellitus (DM). METHODS: Acid-treated titanium implants were bilaterally placed in the maxillary alveolar ridge of streptozotocin-induced diabetic (DM group) and control rats after extraction of first molars. Histological analysis and micro-push-out test were performed 4 weeks after surgery. Oxidative stress and osteogenic markers in the surrounding bone were quantified by real-time polymerase chain reaction. In the in vitro study, rat bone marrow-derived mesenchymal stem cells (BMMSCs) were cultured on acid-treated titanium discs in a high-glucose (HG) or normal environment. Intracellular reactive oxygen species (ROS), cell proliferation, alkaline phosphatase (ALP) activity, and extracellular calcification were evaluated following antioxidant treatment with N-acetyl-L-cysteine (NAC). RESULTS: The implant survival rate was 92.9% and 75.0% in control and DM group, respectively. Bone-implant contact and push-out loads were significantly lower in the DM group. Expression of superoxide dismutase 1 at the mRNA level and on immunohistochemistry was significantly lower in the DM group. In vitro experiments revealed that the HG condition significantly increased ROS expression and suppressed the proliferation and extracellular calcification of BMMSCs, while NAC treatment significantly restored ROS expression, cell proliferation, and calcification. The ALP activity of both groups was not significantly different. CONCLUSION: In diabetes, high-glucose-induced oxidative stress downregulates proliferation and calcification of BMMSCs, impairing osseointegration and leading to implant failure.

Possible Repositioning of an Oral Anti-Osteoporotic Drug, Ipriflavone, for Treatment of Inflammatory Arthritis via Inhibitory Activity of KIAA1199, a Novel Potent Hyaluronidase.

KIAA1199 has a strong hyaluronidase activity in inflammatory arthritis. This study aimed to identify a drug that could reduce KIAA1199 activity and clarify its effects on inflammatory arthritis. Rat chondrosarcoma (RCS) cells were strongly stained with Alcian blue (AB). Its stainability was reduced in RCS cells, which were over-expressed with the KIAA1199 gene (RCS-KIAA). We screened the drugs that restore the AB stainability in RCS-KIAA. The effects of the drug were evaluated by particle exclusion assay, HA ELISA, RT-PCR, and Western blotting. We further evaluated the HA accumulation and the MMP1 and three expressions in fibroblast-like synoviocytes (FLS). In vivo, the effects of the drug on symptoms and serum concentration of HA in a collagen-induced arthritis mouse were evaluated. Ipriflavone was identified to restore AB stainability at 23%. Extracellular matrix formation was significantly increased in a dose-dependent manner (p = 0.006). Ipriflavone increased the HA accumulation and suppressed the MMP1 and MMP3 expression on TNF-α stimulated FLS. In vivo, Ipriflavone significantly improved the symptoms and reduced the serum concentrations of HA. Conclusions: We identified Ipriflavone, which has inhibitory effects on KIAA1199 activity. Ipriflavone may be a therapeutic candidate based on its reduction of KIAA1199 activity in inflammatory arthritis.

Versican V1 in human endometrial epithelial cells promotes BeWo spheroid adhesion in vitro.

The endometrium extracellular matrix (ECM) is essential for embryo implantation. Versican, a large chondroitin sulfate proteoglycan that binds hyaluronan and forms large ECM aggregates, can influence fundamental physiological phenomena, such as cell proliferation, adhesion and migration. The present study investigated the possible role of versican in human embryo implantation. Versican V1 expression and secretion in human endometrial epithelial cells (EECs) was most prominent in the mid-secretory phase. Versican expression in EECs significantly increased after treatment with estrogen and progesterone, but not by estrogen alone. We also established versican V1-overexpressing Ishikawa (endometrial cancer cell line) cells (ISKW-V1), versican V3-overexpressing (ISKW-V3) and control GFP-overexpressing (ISKW-GFP) Ishikawa cells. By the in vitro implantation model, the attachment ratio of BeWo (choriocarcinoma cell line) spheroids to the monolayer of ISKW-V1, but not of ISKW-V3, was found significantly enhanced compared with attachment to the ISKW-GFP monolayer. The conditioned medium derived from ISKW-V1 (V1-CM) also promoted the attachment of BeWo spheroids to the ISKW monolayer. However, this attachment-promoting effect was abolished when V1-CM was pretreated with chondroitinase ABC, which degrades chondroitin sulfate. Therefore, out of the ECM components, versican V1 may facilitate human embryo implantation.

Localization and expression pattern of amelotin, odontogenic ameloblast-associated protein and follicular dendritic cell-secreted protein in the junctional epithelium of inflamed gingiva.

The purpose of this study is to elucidate the localization of amelotin (AMTN), odontogenic ameloblast-associated protein (ODAM) and follicular dendritic cell-secreted protein (FDC-SP) at the junctional epithelium (JE) in Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans infected mice and inflamed and non-inflamed human gingiva. We performed immunostaining to determine the localization and expression pattern of AMTN, ODAM and FDC-SP. AMTN, ODAM and FDC-SP in A. actinomycetemcomitans infected mice did not change dramatically compared with non-infected mice. AMTN and FDC-SP expressions were observed stronger in P. gingivalis infected mice at early stage. However, at the following stage, the coronal part of the AMTN expression disappeared from the JE, and FDC-SP expression decreased due to severe inflammation by P. gingivalis. ODAM expressed internal and external basal lamina, and the expression increased not only at early stage but also at the following stage in the inflammatory JE induced by P. gingivalis. In the human gingival tissues, AMTN was detected at the surface of the sulcular epithelium and JE in the non-inflamed and inflamed gingiva, and the localization did not change the process of inflammation. ODAM and FDC-SP were more widely detected at the sulcular epithelium and JE in the non-inflamed gingiva. In the inflamed gingiva, localization of ODAM and FDC-SP was spread into the gingival epithelium, compared to AMTN. These studies demonstrated that the expression pattern of AMTN, ODAM and FDC-SP at the JE were changed during inflammation process and these three proteins might play an important role in the resistance to inflammation.

Versican/PG-M is essential for ventricular septal formation subsequent to cardiac atrioventricular cushion development.

Versican (Vcan)/proteoglycan (PG)-M is a large chondroitin sulfate proteoglycan which forms a proteoglycan/hyaluronan (HA) aggregate in the extracellular matrix (ECM). We tried to generate the Vcan knockout mice by a conventional method, which resulted in mutant mice Vcan(Δ3/Δ3) whose Vcan lacks the A subdomain of the G1 domain. The Vcan knockout embryos died during the early development stage due to heart defects, but some Vcan(Δ3/Δ3) embryos survived through to the neonatal period. The hearts in Vcan(Δ3/Δ3) newborn mice showed normal cardiac looping, but had ventricular septal defects. Their atrioventricular canal (AVC) cushion was much smaller than those of wild-type (WT) embryos, and the extracellular space for cardiac jelly was narrow. The Vcan deposition in the Vcan(Δ3/Δ3) AVC cushion had decreased, whereas the HA deposition was maintained and condensed. In the tip of ventricular septa, both Vcan and HA had decreased. The cell proliferation based on the number of Ki67-positive cells had remarkably increased in both the AVC cushion and ventricular septa, compared with that of WT embryos. Vcan(Δ3/Δ3) seemed to have endocardial and mesenchymal mixed characteristics. When the ex vivo explant culture of these regions was performed on the collagen gel, hardly any migration to make sufficient space for the ECM construction was apparent. Our results suggest that the proteoglycan aggregates are necessary in both the AVC cushion and ventricular septa to fuse interventricular septa, and the Vcan A subdomain plays an essential role for the interventricular septal formation by constituting the proteoglycan aggregates.

Versican V1 isoform regulates cell-associated matrix formation and cell behavior differentially from aggrecan in Swarm rat chondrosarcoma cells.

Versican, a large chondroitin sulfate proteoglycan that binds hyaluronan and is composed of large extracellular matrix aggregates, has been shown to correlate with tumor progression. No studies have examined the roles of versican in chondrosarcoma nor compared them to those of aggrecan. In clinical specimens of human chondromatous tumors, versican expression was significantly increased in malignant tumors, moreover, as the tumor grade increased. To clarify the roles of versican in chondrosarcoma, versican splicing variant 1, variant 3 or only GFP was stably transfected to Swarm rat chondrosarcoma cells with Trap-In System. Forced expression of versican V1 isoform in Swarm rat chondrosarcoma cells induced a marked increase of cell-associated matrix compared to V3-, GFP- transfected or RCS cells. Versican was immunolocalized in a fashion similar to that of hyaluronan and more diffusively than aggrecan. Anchor-dependent and -independent growth was not affected by versican isoform expression, whereas cell motility and migration were significantly enhanced by V1 isoform transfection. Tumors formed in vivo with V1-transfected cells exhibited more myxomatous area and included more spindle shaped cells. These results support the concept that versican has the capacity to form more extensive cell-associated matrix than aggrecan, and the prominent matrix formation alters the cell behavior of chondrosarcoma more aggressively. These observations suggest that versican expression may serve as a marker of tumor grade determination in chondrosarcoma and possibly help to decide on therapeutic targets in higher grades of chondrosarcoma.

Gene expression of type II collagen is regulated by direct interaction with Kruppel-like factor 4 and AT-rich interactive domain 5B.

We have previously found and characterized two pairs of enhancer elements, E1 and E2, in the type II collagen alpha 1 chain (COL2A1) gene. Subsequent studies have suggested that these enhancers function differently in the regulation of gene expression. For example, histone deacetylase 10 modifies only the E2 enhancer region to affect gene expression. Therefore, in this study, we aimed to clarify the transcriptional complex formed at each enhancer region by identifying transcription factors that specifically bind to each enhancer element. To this end, we used chondrocytic cell lines established using our unique silent reporter system and overexpressed candidate transcription factors in these cells. We found two transcription factors, other than the SOX trio, that directly bound to COL2A1 and regulated its expression. The first was Kruppel-like factor-4 (KLF4), which bound to the promoter proximal region, and the second was AT-rich interactive domain 5B (ARID5B) which bound to the E1 enhancer element. Further studies are needed to identify factors that specifically bind to the E2 enhancer element. In any case, our findings provide an important insight into the molecular mechanisms underlying the regulation of COL2A1. In this paper, we reevaluated the previous analysis of transcription factors involved in the regulation of COL2A1 expression.

Forced expression of KIAA1199, a novel hyaluronidase, inhibits tumorigenicity of low-grade chondrosarcoma.

Hyaluronan (HA) has been shown to play crucial roles in the tumorigenicity of malignant tumors. Chondrosarcoma, particularly when low-grade, is characterized by the formation of an extracellular matrix (ECM) containing abundant HA, and its drug/radiation resistance has become a clinically relevant problem. This study aimed to evaluate the effects of a novel hyaluronidase, KIAA1199, on ECM formation as well as antitumor effects on chondrosarcoma. To clarify the roles of KIAA1199 in chondrosarcoma, mouse KIAA1199 was stably transfected to Swarm rat chondrosarcoma (RCS) cells (histologically grade 1). We investigated the effects of KIAA1199 on RCS cells in vitro and an autografted model in vivo. HA binding protein (HABP) stainability and ECM formation in KIAA1199-RCS was markedly suppressed compared with that of control cells. No significant changes in messenger RNA expression of Has1, Has2, Has3, Hyal1, or Hyal2 were observed. KIAA1199 expression did not affect proliferation or apoptosis but inhibited migration and invasion of RCS cells. In contrast, the expression of KIAA1199 significantly inhibited the growth of grafted tumors and suppressed the stainability of alcian blue in tumor tissues. Although there was no direct inhibitory effect on proliferation in vitro, induction of KIAA1199 showed the antitumor effects in grafted tumor growth in vivo possibly due to changes in the tumor microenvironment such as inhibition of ECM formation. Forced expression of KIAA1199 exhibits antitumor effects on low-grade chondrosarcoma, which has chemo- and radio-therapy resistant features. Together, KIAA1199 could be a novel promising therapeutic tool for low-grade chondrosarcoma, mediated by the degradation of HA.

Spatio-temporal expression of activating transcription factor 5 in the skeletal development of mouse limb.

There are no data about the expression pattern of activating transcription factor 5 (Atf5) during limb development. The aim of the present study was to describe the expression of Atf5 throughout mouse limb development. Some specimens were examined for the expression of type II collagen alpha1 (Col2a1), sex-determining region Y-related high-mobility-group-box 9 (Sox9), and activating transcription factor 2 (Atf2) by in situ hybridization. ATDC5 cells, mouse limb buds, and epiphyseal cartilage were examined for the expression of Atf5alpha and Atf5beta by quantitative real-time PCR. Atf5 transcripts were detected in condensed mesenchymal cells, cartilage primordia, proliferative, prehypertrophic, and articular chondrocytes, but not in hypertrophic chondrocytes. Atf5 was predominantly expressed by osteoblasts in the primary spongiosa. At the postnatal stage, Atf5 was expressed in epiphyseal chondrocytes and osteoblasts lining the bone trabeculae. Spatial distribution of Col2a1, Sox9, and Atf2 transcripts closely corresponded to that of Atf5 within developing limbs, except for bony tissues. In cartilage, Atf5alpha is the dominant form in the embryonic stage as well as in the postnatal stage.

Expression of type II collagen and aggrecan genes is regulated through distinct epigenetic modifications of their multiple enhancer elements.

To maintain normal function of cartilage tissue normally, the presence of a sufficient amount of type II collagen and aggrecan is essential, and their synthesis is tightly regulated. Therefore, understanding the mechanisms that control the expression of type II collagen and aggrecan would be useful for understanding gene expression changes in diseases such as osteoarthritis. Recently, we have identified two pairs of enhancer elements, termed E1 and E2 in the type II collagen gene and Ea and Eb in the aggrecan gene. However, their different mechanisms of action remained unclear. Thus, the central aim of this study was to clarify the different transcriptional regulation mediated through each enhancer element. To this end, we established different stable reporter cell lines that express a reporter gene under the control of different enhancer elements using a silent reporter system we previously constructed. Using these cell lines, we found that dexamethasone, forskolin, and trichostatin A affect the gene expression of type II collagen and aggrecan via different enhancer elements. Moreover, we clarified that E1 and E2 enhancer activities are regulated through distinct epigenetic modifications by histone deacetylase 10 and sirtuin 6.

Suppression of hyaluronan synthesis attenuates the tumorigenicity of low-grade chondrosarcoma.

Hyaluronan (HA) has been shown to play crucial roles in the tumorigenicity of malignant tumors. Chondrosarcoma, particularly when low-grade, is characterized by the formation of an extracellular matrix (ECM) containing abundant HA, and its drug/radiation resistance has become a clinically relevant problem. This study aimed to evaluate the effects of an HA synthesis inhibitor, 4-methylumbelliferone (MU), on ECM formation as well as antitumor effects in chondrosarcoma. We investigated the effects of MU on rat chondrosarcoma (RCS) cells with a grade I histological malignancy in vitro and in vivo grafted model. HA binding protein (HABP) stainability on and around the RCS cells was effectively reduced with treatment of MU. ECM formation was markedly suppressed by MU at a dose of 1.0 mM. Cell proliferation was significantly reduced by MU at 24 h. Cell motility and invasion were suppressed in a dose-dependent manner by MU. No significant changes in mRNA expression of Has1-3 were observed. Furthermore, MU inhibited the growth of grafted tumors in vivo. Histologically, chondrosarcoma cells of control tumors showed a cell-clustering structure. HABP stainability was markedly decreased in the MU-treated group. These results suggest that MU exhibits antitumor effects on low-grade chondrosarcoma, via inhibition of HA accumulation and ECM formation. MU, which is an approved drug in bile therapy, could be a new off-label medication for chondrosarcomas. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1573-1580, 2018.

[Chondrogenic differentiation and transcription factors].

Molecular understanding of cartilage differentiation has been improved during the past decade. The finding of Sox9 in particular has improved our understanding of the molecular events during cartilage formation. However, a relatively few transcription factors have been proven to be involved in chondrogenesis. Therefore, the majority of transcription factors and their networks for the differentiation and maturation of chondrocytes may well remain to be elucidated. Here, we summarize the present state of research on transcription factors involved in the differentiation of chondrocytes.

Development and application of a new Silent reporter system to quantitate the activity of enhancer elements in the type II Collagen Gene.

Type II collagen is a major component of cartilage, which provide structural stiffness to the tissue. As a sufficient amount of type II collagen is critical for maintaining the biomechanical properties of cartilage, its expression is tightly regulated in chondrocytes. Therefore, it is essential to elucidate in detail the transcriptional mechanism that controls expression of type II collagen, in particular by two enhancer elements we recently discovered. To systematically analyze and compare enhancer activities, we developed a novel reporter assay system that exploits site-specific integration of promoter and enhancer elements to activate a transcriptionally silent reporter gene. Using this system, we found that the enhancer elements have distinct characteristics, with one exhibiting additive effects and the other exhibiting synergistic effects when repeated in tandem.

Role of Versican in the Pathogenesis of Peritoneal Endometriosis.

CONTEXT: Sampson's theory cannot explain why only some cycling women develop peritoneal endometriosis. Few studies have focused on the pelvic peritoneum, which receives regurgitated endometrial tissues. We hypothesized that molecular alterations in the peritoneum are involved in the development of peritoneal endometriosis and conducted a microarray analysis to compare macroscopically normal peritoneum sampled from women with peritoneal endometriosis (endometriotic peritoneum) and those without (non-endometriotic peritoneum). Versican, a major proteoglycan component of the extracellular matrix, is one of the molecules up-regulated in endometriotic peritoneum. OBJECTIVE: To investigate the role of versican in peritoneal endometriosis. Design, Patients, and Main Outcome Measure: Endometriotic peritoneum and non-endometriotic peritoneum were subjected to RT-PCR, immunostaining, and Western blotting. The versican V1 isoform was stably transfected into Chinese hamster ovary cells (CHO-V1), and the effects of CHO-V1-derived conditioned medium (V1-CM) on primary human endometrial stromal cells were investigated with attachment, invasion, and proliferation assays. The effects of peritoneal fluid collected from endometriotic women (endometriotic PF) or cytokines/growth factors, which were shown to be elevated in endometriotic PF, on versican expression in a human peritoneal cell line (HMrSV5) were also examined. RESULTS: Versican V1 expression levels were significantly higher in endometriotic peritoneum. In vitro, V1-CM promoted attachment to the HMrSV5 cell monolayer as well as the Matrigel invasion of endometrial stromal cells. Although versican V1 expression was up-regulated by TGF-ß1 in HMrSV5 cells, it remained unchanged in endometriotic PF. CONCLUSIONS: Our results suggest the involvement of peritoneal versican in the development of peritoneal endometriosis.

Gene trap screening for cell surface and extracellular matrix molecules produced by chondrocytes.

We have developed a simple and unique strategy for identifying cell surface and extracellular matrix molecules produced by chondrocytes. Our strategy comprises two methods, retrovirus-based signal sequence gene trapping and culturing of Swarm rat chondrosarcoma chondrocytes. After infection with a retrovirus vector and isolation of hygromycin-resistant clones, trapped genes could be easily identified by the 5' rapid amplification of cDNA ends (5' RACE) method. Furthermore, the treatment of isolated clones with gadolinium chloride enabled us to determine whether the trapped gene expression was dependent on the state of chondrogenic differentiation.

Identification of genes preferentially expressed in articular cartilage by suppression subtractive hybridization.

Suppression subtractive hybridization is very effective to enrich differentially expressed genes in two different tissues or cells. We therefore used the technique to identify characteristic genes expressed in rat knee joint articular cartilage as compared to rat costal cartilage. In this study, we revealed that several genes were enriched in a subtracted articular cartilage cDNA library. The most enriched gene is lubricin that is a putative key molecule for joint lubrication. The second gene is milk fat globule epidermal growth factor (EGF) factor 8, MFG-E8 whose expression has never been observed in cartilage. Other enriched genes are known to be expressed in cartilage, however their differential expressions in cartilages have not been necessarily common. The preferential expression of characteristic genes in articular cartilage would provide unique properties to the tissue. Our findings will provide a new view of articular cartilage.

Characterization of an anti-decorin monoclonal antibody, and its utility.

6B6 is a monoclonal antibody raised against a purified small dermatan sulfate proteoglycan from human ovarian fibroma capsule, has Although it been widely used as an anti-decorin monoclonal antibody, its epitope has not yet been characterized at the molecular level. Here, we show that 6B6 is specific to decorin. The antibody recognized human, mouse, and bovine decorin core protein, but not biglycan. Using recombinant decorin domains, we determined that the epitope lies within the region of amino acid residues 50-65, termed the cysteine cluster region. Cross-reactivity among species further narrowed it down to a primary sequence of residues 57-65. We also established the conditions for immunostaining. 6B6 stained both frozen and fixed sections. Whereas the glycosaminoglycan chain of decorin inhibited access of the antibody in immunoblotting, pretreatment of tissue sections with chondrotinase ABC did not affect the intensity of staining, suggesting that the glycosaminoglycan chain is integrated and the Cys cluster region oriented outside of the collagen fibrils in the tissue. When 6B6 was applied to enzyme-linked immunosorbent assay, a concentration as low as 0.5 microg/ml of decorin was detectable by either direct or sandwich ELISA. 6B6 is thus a sensitive and reliable antibody to study functions of decorin from various aspects.

[Proteoglycan knock-out mice and osteoarthritis].

There are several types of small leucine-rich proteoglycans expressed in articular cartilage and its surrounding tissues. Many of these proteoglycans are functioning as a modulator of collagen fibrillogenesis via binding to the surface of collagen fibers. Therefore, deficiencies of their expression in knock-out mice often result in the formation of abnormal collagen fibers leading to the instability of the join, and finally trigger osteoarthritis.

A candidate enhancer element responsible for high-level expression of the aggrecan gene in chondrocytes.

Aggrecan is the most abundant proteoglycan in cartilage. It contains a lot of negatively charged glycosaminoglycan chains along the core protein, providing a large osmotic swelling pressure within the cartilage. Therefore, the biomechanical properties of cartilage, such as its compressive load-bearing capacity, are highly dependent on the presence of abundant aggrecan in the cartilage matrix. To elucidate the transcriptional mechanism that leads to abundant expression of aggrecan by chondrocytes, we screened for enhancer elements in 130 kb of the aggrecan gene, Agc1, using a reporter assay system that we previously developed. The system is based on co-transfection of candidate enhancer elements and reporter constructs into Swarm rat chondrosarcoma chondrocytes that retain a high level of aggrecan expression. We found an element that might be involved in high-level expression of Agc1 gene in chondrocytes in vivo. The element is located 30 kb upstream of the Agc1 transcription start site.

A newly identified enhancer element responsible for type II collagen gene expression.

Type II collagen is a major component of cartilage where it is present at a high concentration, which is essential for the functional maintenance of the tissue. Therefore, any fundamental understanding of the physiology of cartilage tissue must include an understanding of the mechanism that allows the high level of expression of type II collagen gene, Col2a1, by chondrocytes. To this end, we developed a new reporter assay system based on the co-transfection of candidate enhancer elements and reporter construct into Swarm rat chondrosarcoma chondrocytes that allowed their stable expression. Using this system, we screened more than 70 kb of the Col2a1 gene and found an enhancer domain that is responsible for the regulation of its expression level. The domain is localized in intron 7, and consists of an 800-bp region that contains within it a previously unidentified domain, ∼140 bp in size.