Enhanced immunomodulation and periodontal regeneration efficacy of subgingivally delivered progranulin-loaded hydrogel as an adjunct to non-surgical treatment for Class II furcation involvement in dogs.

AIM: To evaluate the effect of subgingival delivery of progranulin (PGRN)/gelatin methacryloyl (GelMA) complex as an adjunct to scaling and root planing (SRP) on an experimental periodontitis dog model with Class II furcation involvement (FI). MATERIALS AND METHODS: A Class II FI model was established, and the defects were divided into four treatment groups: (a) no treatment (control); (b) SRP; (c) SRP + GelMA; (d) SRP + PGRN/GelMA. Eight weeks after treatment, periodontal parameters were recorded, gingival crevicular fluid and gingival tissue were collected for ELISA and RT-qPCR, respectively, and mandibular tissue blocks were collected for micro computed tomography (micro-CT) scanning and hematoxylin and eosin (H&E) staining. RESULTS: The SRP + PGRN/GelMA group showed significant improvement in all periodontal parameters compared with those in the other groups. The expression of markers related to M1 macrophage and Th17 cell significantly decreased, and the expression of markers related to M2 macrophage and Treg cell significantly increased in the SRP + PGRN/GelMA group compared with those in the other groups. The volume, quality and area of new bone and the length of new cementum in the root furcation defects of the PGRN/GelMA group were significantly increased compared to those in the other groups. CONCLUSIONS: Subgingival delivery of the PGRN/GelMA complex could be a promising non-surgical adjunctive therapy for anti-inflammation, immunomodulation and periodontal regeneration.

Periodontal effect of augmented corticotomy-assisted orthodontics versus conventional orthodontics in treatment of adult patients with bialveolar protrusion.

BACKGROUND: The patients of bialveolar protrusion always demonstrate thin anterior alveoli which may aggravate subsequent gingival recession and bone loss during retraction. This study aimed to investigate the periodontal changes, including alveolar height, thickness, and area, and the width of keratinized gingiva, in mandibular anterior teeth after augmented corticotomy-assisted orthodontics (ACAO) compared with traditional orthodontics. METHODS: Twenty adult patients with skeletal class I bialveolar protrusion were selected from two groups: ACAO group (augmented corticotomy on the labial side of the anterior mandibular teeth, n = 10) and control group (conventional orthodontics, n = 10). In all patients, four first premolars were extracted and the incisors were retracted under the maximum anchorage. The measurements included the labial alveolar bone area, vertical alveolar bone height, alveolar bone thickness surrounding the mandibular anterior teeth, root length, gingival recession and width of keratinized gingiva after alignment (T0) and 3 months after space closure (T1). RESULTS: The labial alveolar height, area, and thicknesses all decreased after space closure in the control group but significantly increased in the ACAO group. The decrease in the lingual alveolar height was statistically less in the ACAO group than that in the control group. Besides, the width of keratinized gingiva increased in the ACAO group but decreased in the control group. There was no significant difference in the changes of root length between groups. The dentoalveolar changes between anterior teeth were consistent but with different scales. The lateral incisors gained the most labial bone height and area. CONCLUSION: Compared to conventional orthodontics, ACAO provided a more favorable effect of improving periodontal status surrounding the mandibular anterior teeth for Class I maxillary protrusion patients.

Progranulin inhibits LPS-induced macrophage M1 polarization via NF-кB and MAPK pathways.

BACKGROUND: Macrophage M1 polarization plays a pivotal role in inflammatory diseases. Progranulin (PGRN) has potential anti-inflammation action, however, the effect of PGRN on macrophage M1 polarization has been poorly studied. Our study aimed to investigate the effect of PGRN on lipopolysaccharide (LPS)-induced macrophage M1 polarization and clarify the underlying mechanisms. METHODS: RAW264.7 cells were polarized to M1 macrophage by LPS with or without recombinant PGRN (rPGRN) and tumor necrosis factor alpha antibody (anti-TNF-α). A cell counting kit-8 assay (CCK-8), flow cytometry, Quantitative Real-Time PCR assay (q-PCR), Western blot assay and enzyme-linked immunosorbent assay (ELISA) were used to determine the effect of different treatments on cell proliferation, expression of surface phenotype marker and expressions and secretion of inflammatory cytokines. The activation of NF-κB/mitogen-activated protein kinase (MAPK) pathways and the nuclear translocation of NF-κB p65 were detected by Western blot and immunofluorescence respectively. THP-1 and primary bone marrow-derived monocytes (BMDMs) were also used to demonstrate effect of PGRN on expressions and secretion of inflammatory cytokines induced by LPS. RESULTS: In RAW264.7 cells, rPGRN at concentrations below 80 ng/ml significantly promoted cell proliferation in dose dependent fashion. rPGRN significantly inhibited LPS-induced change of phenotype (CD86/CD206 ratio) and function (tumor necrosis factor (TNF-α) and inducible nitric oxide synthase (iNOS) expressions). LPS-stimulated secretion of TNF-α and activated phosphorylation of IKKα/ß, IкBα, p65, JNK and p38 and the nucleus translocation of NF-кB p65 were also significantly downregulated by rPGRN. In addition, recombinant TNF-α (rTNF-α) significantly boosted TNF-α and iNOS expression vs the control group. Moreover, anti-TNF-α significantly inhibited LPS-induced TNF-α and iNOS expression. In THP-1 and BMDM cells, reversing effect of rPGRN on LPS-enhanced expressions of TNF-α and iNOS and secretion of TNF-α was further demonstrated. CONCLUSIONS: PGRN down-regulates LPS-induced macrophage M1 polarization in phenotype and function via NF-κB/MAPK signaling pathways.

Enhancement of periodontal tissue regeneration by conditioned media from gingiva-derived or periodontal ligament-derived mesenchymal stem cells: a comparative study in rats.

BACKGROUND: Evidence has demonstrated conditioned medium (CM) from periodontal ligament stem cells (PDLSCs) improved periodontal regeneration. Gingival mesenchymal stem cells (GMSCs) have been considered an alternative strategy for regenerative medicine. To determine whether GMSC-CM could promote periodontal wound healing, we compared the effects of GMSC-CM and PDLSC-CM on periodontal regeneration and the underlying mechanisms in rat periodontal defects. METHODS: Cell-free CMs were collected from PDLSCs, GMSCs, and gingival fibroblasts (GFs) using ultracentrifugation (100-fold concentration). Periodontal defects were created on the buccal side of the first molar in the left mandible of 90 rats by a surgical method. Collagen membranes loaded with concentrated CMs (α-MEM, GF-CM, GMSC-CM, PDLSC-CM) were transplanted into periodontal defects. After 1, 2, and 4 weeks, the animals were sacrificed and specimens including the first molar and the surrounding tissues were separated and decalcified. Hematoxylin-eosin and Masson's trichrome staining were performed to evaluate periodontal regeneration. Immunohistochemical staining for tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-10 was conducted to analyze inflammation. Immunohistochemistry of BSP-II and Runx2 was performed to analyze osteoblast differentiation. RESULTS: Histological analysis showed the amount of newly formed periodontal tissue was significantly higher in both the GMSC-CM and PDLSC-CM groups than in the other groups, with no significant difference between these two groups. At 1 and 2 weeks, the expression levels of TNF-α and IL-1ß were significantly lower in the GMSC-CM and PDLSC-CM groups than in the other three groups, while there was no significant difference between these two groups. IL-10 expression was significantly higher in the GMSC-CM group than in the PDLSC-CM group and the other three groups. At 1, 2, and 4 weeks, BSP-II and Runx2 expressions were significantly higher in the GMSC-CM and PDLSC-CM groups than in the other three groups, with no significant difference between the two groups. CONCLUSIONS: Our results demonstrate that GMSC-CM transplantation can significantly promote periodontal regeneration in rats and achieve the same effect as PDLSC-CM. The mechanism of periodontal regeneration may involve the regulation of inflammatory factors and the promotion of osteogenic differentiation of bone progenitor cells in the wound region by CMs from MSCs.

Proanthocyanidins Promote Osteogenic Differentiation of Human Periodontal Ligament Fibroblasts in Inflammatory Environment Via Suppressing NF-κB Signal Pathway.

Proanthocyanidins (PA) have been proven to suppress inflammation and promote osteogenic differentiation. However, whether PA could promote osteogenic differentiation of human periodontal ligament fibroblasts (HPDLFs) in inflammatory environment is unclear. Here, HPDLFs were stimulated by tumor necrosis factor-α (TNF-α), PA, or their combination, and osteogenic differentiation- and mineralization-associated markers were detected by quantitative real-time polymerase chain reaction (qRT-PCR), alizarin red S staining, and alkaline phosphatase (ALP) activity assay. The result showed that PA significantly upregulated expression of osteogenesis-related genes and proteins and ALP activity in HPDLFs compared with the control in non-inflammatory environment. Moreover, PA significantly reversed inhibition of osteogenesis-related gene and protein expression, ALP activity, and mineralization caused by TNF-α. The underlying mechanism was that PA could regulate osteogenesis of HPDLFs via suppressing nuclear factor-kappa beta (NF-κB) signal pathway. These findings suggest that PA may contribute to bone generation in inflammatory microenvironment via suppressing NF-κB signal pathway. Thus, PA may be a potential treatment agent for periodontal bone regeneration.

N-WASP knockdown upregulates inflammatory cytokines expression in human gingival fibroblasts.

OBJECTIVE: The neuronal wiskott-aldrich syndrome protein (N-WASP) is a member of the wiskott-aldrich syndrome protein (WASP) family. N-WASP plays a vital role in promoting cell migration, receptor signaling and immune inflammatory responses. This study aimed to observe the changes in the expression of inflammatory factors and involving pathways after N-WASP knockdown in human gingival fibroblasts (HGFs). DESIGN: Gingival inflammatory condition of N-WASP knockout mice was evaluated by H&E staining. N-WASP in HGFs was knockdown by siRNA and the best knockdown efficiency was determined by qRT-PCR and immunofluorescence. The mRNA levels of interleukin (IL)-6, IL-8, C-C motif ligand 2 (CCL2), superoxide dismutase 2 (SOD2) and prostaglandin endoperoxide synthase 2 (PTGS2) were evaluated by qRT-PCR after N-WASP knockdown with or without mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) inhibitors. The protein levels of IL-6, IL-8 and CCL2 were assessed by ELISA. Western blotting was used to detect the activation of NF-κB and MAPK signaling pathways. RESULTS: Gingival tissue from N-WASP knockout mice exhibited an inflammatory reaction. The expression of IL-6, IL-8, CCL2, SOD2 and PTGS2 was significantly upregulated after N-WASP knockdown in HGFs for 6, 24 and 48 h, except for the SOD2 at 6 h. N-WASP knockdown significantly activated the signaling pathways of NF-κB and MAPK. The inhibitors of p65, p38, ERK and JNK clearly decreased IL-6, IL-8, CCL2, SOD2 and PTGS2 expression after N-WASP knockdown. CONCLUSION: These data indicated that N-WASP deficiency in HGFs increases the production of inflammatory cytokine and is regulated via NF-κB and MAPK signaling pathways.

One-Year Results Evaluating the Effects of Concentrated Growth Factors on the Healing of Intrabony Defects Treated with or without Bone Substitute in Chronic Periodontitis.

BACKGROUND The restoration of damaged periodontium, especially one-wall intrabony defects, is a major challenge for clinicians. Concentrated growth factors (CGF) are a 100% autologous fibrin with multiple concentrated growth factors. The rigid fibrin structure of CGF makes it possible to preserve or reconstruct the initial bone volume. The aim of this study was to evaluate the clinical healing patterns after surgical application of CGF with and without a Bio-Oss graft in one-wall infrabony defects. MATERIAL AND METHODS We randomly divided 120 one-wall intrabony defects in 54 patients into 4 groups: flap surgery alone (Group 1), flap surgery with autologous CGF (Group 2), flap surgery with Bio-Oss (Group 3), and flap surgery with CGF+Bio-Oss (Group 4). Clinical parameters such as probing depth (PD) and clinical attachment level (CAL) change were recorded at baseline and at 6 and 12 months postoperatively. RESULTS At 12 months postoperatively, Group 2 showed significant improvement in clinical parameters over Group 1 (P<0.05) and the results were significantly greater in Groups 3 and 4 compared to the other groups (P<0.05). Although no significant difference was noted between Groups 3 and 4 in clinical parameters (P>0.05) compared to Group 3, the mean change of CAL at 6-12 months in Group 4 was not significant (P>0.05). CONCLUSIONS CGF reduced periodontal intrabony defects depth and, when mixed with Bio-Oss, CGF showed better results in the early period and the effect was more stable.

The biological behavior optimization of human periodontal ligament stem cells via preconditioning by the combined application of fibroblast growth factor-2 and A83-01 in in vitro culture expansion.

BACKGROUND: As the optimal source of seed cells in periodontal tissue engineering, periodontal ligament stem cells (PDLSCs) have always been researched to improve cell expansion due to their limited resource and spontaneous differentiation in vitro cultivation. Fibroblast growth factor-2 (FGF-2) has been proven to stimulate bone marrow mesenchymal stem cells (BMMSCs) proliferation and maintain their pluripotency when being added to the culture medium. As a small molecule inhibitor of transforming growth factor-beta receptors (TGF-ßRs), A83-01 can also promote cell proliferation. Therefore, the aim of this study was to verify whether the combined application of FGF-2 and A83-01 could augment cell quantity and quality during in vitro culture. METHODS: PDLSCs were preconditioned with A83-01, FGF-2, or their combination. A cell counting kit-8 (CCK8) assay, cell apoptosis assay, ALP activity assay, Alizarin Red S staining assay, RT-PCR assay, Western blot assay and ELISA were used to determine the sustained effects of different preconditioning strategies on the proliferation, apoptosis, stemness, osteogenic differentiation and paracrine action of PDLSCs. RESULTS: The combined application of FGF-2 and A83-01 significantly augmented cell expansion, reduced cell apoptosis, magnified stemness expression, promoted later osteogenic differentiation and mineralization and increased paracrine action of PDLSCs compared with the control. Moreover, the combination presented significant advantages in enhancing proliferation, stemness expression and paracrine action over FGF-2 alone. CONCLUSIONS: The combined application of A83-01 and FGF-2 may be an improved strategy for PDLSCs biological behavior optimization in culture expansion and advantageous for reinforcing proliferation, stemness expression and cytokine secretion over FGF-2 alone.

Progranulin Promotes Regeneration of Inflammatory Periodontal Bone Defect in Rats via Anti-inflammation, Osteoclastogenic Inhibition, and Osteogenic Promotion.

Progranulin (PGRN) has been proved to play a crucial role in anti-inflammation and osteogenesis promotion; thus, it was hypothesized that PGRN could promote bone regeneration in periodontal disease. In this experiment, the periodontal bone defects were established in periodontitis rats; recombinant human progranulin (rhPGRN), tumor necrosis factor alpha inhibitor (anti-TNF-α), or phosphate buffer saline (PBS)-loaded collagen membrane scaffolds were implanted within defects and the rats were sacrificed at scheduled time points. Volume of new bone was assessed by radiological and histomorphometric analyses. Expression of osteogenesis-related markers and tumor necrosis factor-α (TNF-α) was evaluated using immunohistochemistry. Tartrate-resistant acid phosphatase (TRAP) staining was also performed to determine the number of osteoclasts. Immunofluorescence (IF) staining was performed to explore the interaction between rhPGRN and tumor necrosis factor receptors (TNFRs). The results showed that the rhPGRN group had significantly superior quantity and quality of newly formed bone, higher expression of alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and TNFR2 compared with the PBS group and the anti-TNF-α group. Similarly to the anti-TNF-α group, the rhPGRN group also exhibited the significant inhibitory effect on the expression of TNF-α and the number of TRAP-positive cells compared with the PBS group. Hence, our experiment suggests that PGRN promotes regeneration of inflammatory periodontal bone defect in rats via anti-inflammation, osteoclastogenic inhibition, and osteogenic promotion. Local administration of PGRN may provide a new therapeutic strategy for periodontal bone regeneration.

Periodontitis May Restrain the Mandibular Bone Healing via Disturbing Osteogenic and Osteoclastic Balance.

Periodontitis has been advocated as a systematic chronic low-grade infection burden. However, the relationship between periodontitis and bone defect healing has not been elucidated. One hundred and eight male Wister rats were randomly assigned into three groups: control (healthy) group, periodontitis group, and periodontitis plus human tumor necrosis factor receptorII:IgG Fc fusion protein (rhTNFR:Fc) group. The experimental periodontitis model was established by ligaturing with orthodontic wire and silk suture plus local administration of 20 µl of lipopolysaccharide (LPS). Mandibular bone defects in size of 4 × 2 × 1 mm were created for all the rats and rhTNFR:Fc subcutaneously injected at neck at a dose of 2.5 mg/kg every 3 days for the periodontitis plus rhTNFR:Fc group. The gene and protein expressions of bone-related markers in the healing tissue were monitored and new bone formation was histologically evaluated. Tartrate-resistant acid phosphatase (TRAP) staining was performed to determine the number of osteoclasts. The results showed that the mRNA and protein expressions of osteogenesis-related markers were significantly lower while nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) gene expression was significantly higher in the periodontitis group. The periodontitis group showed decreased new bone formation and increased number of osteoclasts when compared to the control group. However, there was no significant difference between the periodontitis plus rhTNFR:Fc group and the control group. These results demonstrated that periodontitis may restrain the mandibular bone healing via disturbing osteogenic and osteoclastic balance in which tumor necrosis factor-α (TNF-α) could act as a leverage.

Transglutaminases factor XIII-A and TG2 regulate resorption, adipogenesis and plasma fibronectin homeostasis in bone and bone marrow.

Appropriate bone mass is maintained by bone-forming osteoblast and bone-resorbing osteoclasts. Mesenchymal stem cell (MSC) lineage cells control osteoclastogenesis via expression of RANKL and OPG (receptor activator of nuclear factor κB ligand and osteoprotegerin), which promote and inhibit bone resorption, respectively. Protein crosslinking enzymes transglutaminase 2 (TG2) and Factor XIII-A (FXIII-A) have been linked to activity of myeloid and MSC lineage cells; however, in vivo evidence has been lacking to support their function. In this study, we show in mice that TG2 and FXIII-A control monocyte-macrophage cell differentiation into osteoclasts as well as RANKL production in MSCs and in adipocytes. Long bones of mice lacking TG2 and FXIII-A transglutaminases, show compromised biomechanical properties and trabecular bone loss in axial and appendicular skeleton. This was caused by increased osteoclastogenesis, a cellular phenotype that persists in vitro. The increased potential of TG2 and FXIII-A deficient monocytes to form osteoclasts was reversed by chemical inhibition of TG activity, which revealed the presence of TG1 in osteoclasts and assigned different roles for the TGs as regulators of osteoclastogenesis. TG2- and FXIII-A-deficient mice had normal osteoblast activity, but increased bone marrow adipogenesis, MSCs lacking TG2 and FXIII-A showed high adipogenic potential and significantly increased RANKL expression as well as upregulated TG1 expression. Chemical inhibition of TG activity in the null cells further increased adipogenic potential and RANKL production. Altered differentiation of TG2 and FXIII-A null MSCs was associated with plasma fibronectin (FN) assembly defect in cultures and FN retention in serum and marrow in vivo instead of assembly into bone. Our findings provide new functions for TG2, FXIII-A and TG1 in bone cells and identify them as novel regulators of bone mass, plasma FN homeostasis, RANKL production and myeloid and MSC cell differentiation.

[Short-term effect of ibuprofen on clinical indexes and cytokines in the gingival crevicular fluid of patients with severe chronic periodontitis].

PURPOSE: To explore the short-term effects of ibuprofen on clinical indexes and cytokines in the gingival crevicular fluid of patients with severe chronic periodontitis. METHODS: Twenty subjects with severe chronic periodontitis but otherwise healthy participated in the study and they were divided into two groups randomly. The patients in the experimental group took ibuprofen 300mg, bid for 5 days after scaling and root planing (SRP), while patients in the control group only underwent SRP. Clinical indexes were recorded at baseline, 1 w, 2 w, 4 w, respectively. Meanwhile, the levels of tumor necrosis factor alpha (TNF-α), osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL) in the gingival crevicular fluid were detected. SPSS 17.0 software package was used for statistical analysis. RESULTS: At each time point, both the clinical data and the levels of TNF-α, RANKL, OPG and RANKL/OPG between the experimental group and the control group were not statistically significant (P>0.05). CONCLUSIONS: We can't disclose the positive effect of ibuprofen's short-term oral administration on the treatment of severe chronic periodontitis.

A case of multiple verruciform xanthoma in gingiva.

We present an unusual case of multiple verruciform xanthomas in the gingiva of a patient with no systemic diseases.

Role of serum IL-23/IL-17 axis in the relationship between periodontitis and coronary heart disease.

To explore the role of the IL-23/IL-17 axis in the relationship between periodontitis and coronary heart disease (CHD), 97 subjects were recruited and divided into four groups: (1) CHD + periodontitis, (2) CHD, (3) periodontitus alone, and (4) healthy. The demographic characteristics and periodontal status of all subjects were recorded, and the serum levels of IL-23/IL-17 were detected by enzyme-linked immunoabsorbent assay. Results showed that the serum levels of IL-23/IL-17 in groups 1, 2, and 3 were higher compared with group 4. Group 1 manifested the highest level of serum IL-23/IL-17. A significant positive correlation between IL-23 and IL-17 levels was seen in the three patients groups; groups 1 and 3 also had significant positive correlations with probing depth and attachment loss. The results indicate that there may be an association between periodontitis and CHD, and the IL-23/IL-17 axis may play an important role in the pathologic process of both diseases.

Bone repair by periodontal ligament stem cellseeded nanohydroxyapatite-chitosan scaffold.

BACKGROUND: A nanohydroxyapatite-coated chitosan scaffold has been developed in recent years, but the effect of this composite scaffold on the viability and differentiation of periodontal ligament stem cells (PDLSCs) and bone repair is still unknown. This study explored the behavior of PDLSCs on a new nanohydroxyapatite-coated genipin-chitosan conjunction scaffold (HGCCS) in vitro as compared with an uncoated genipin-chitosan framework, and evaluated the effect of PDLSC-seeded HGCCS on bone repair in vivo. METHODS: Human PDLSCs were cultured and identified, seeded on a HGCCS and on a genipin-chitosan framework, and assessed by scanning electron microscopy, confocal laser scanning microscopy, MTT, alkaline phosphatase activity, and quantitative real-time polymerase chain reaction at different time intervals. Moreover, PDLSC-seeded scaffolds were used in a rat calvarial defect model, and new bone formation was assessed by hematoxylin and eosin staining at 12 weeks postoperatively. RESULTS: PDLSCs were clonogenic and positive for STRO-1. They had the capacity to undergo osteogenic and adipogenic differentiation in vitro. When seeded on HGCCS, PDLSCs exhibited significantly greater viability, alkaline phosphatase activity, and upregulated the bone-related markers, bone sialoprotein, osteopontin, and osteocalcin to a greater extent compared with PDLSCs seeded on the genipin-chitosan framework. The use of PDLSC-seeded HGCCS promoted calvarial bone repair. CONCLUSION: This study demonstrates the potential of HGCCS combined with PDLSCs as a promising tool for bone regeneration.

[Effect of hypoxia on the expression of matrix metalloproteinase and tissue inhibitors of matrix metalloproteinase mRNA in human periodontal ligament fibroblasts in vitro].

OBJECTIVE: To investigate the effect of hypoxia on the expression of matrix metalloproteinase (MMP) and tissue inhibitors of matrix metalloproteinase (TIMP) in human periodontal ligament fibroblasts (HPDLF). METHODS: HPDLF were cultured in α-minima essential medium (α-MEM) and subcultured at confluence. In the hypoxic groups, cells were incubated in a humidified atmosphere of 1%O(2), 5%CO(2), 94%N(2) at 37°C for 12, 24 and 48 h, respectively. In the normoxic control group, cells were incubated under normoxic conditions of 20%O(2), 5%CO(2), 75%N(2). The mRNA expression of MMP and TIMP was measured using reverse transcription-polymerase chain reaction (RT-PCR). The data was analyzed by Student's t test, one-way ANOVA and LSD test with SPSS 13.0 software package. RESULTS: The expression of MMP-2, TIMP-1 and TIMP-2 mRNA in the hypoxia groups was higher than that in control. The expression of MMP-2 mRNA in hypoxic groups showed a significantly increasing trend. There was significant difference between the hypoxic group and the normoxic control group in the expression of MMP-2 mRNA in HPDLF (P < 0.01). The expression of TIMP-1, TIMP-2 mRNA in hypoxic groups of 12 h was momentarily increased. There was significant difference between the hypoxic 12 h group and the normoxic control group in the expression of TIMP-1, TIMP-2 mRNA in HPDLF (P < 0.05). However, with prolonged hypoxia time, the expression of TIMP-1, TIMP-2 mRNA in hypoxic groups showed a significantly declining trend, there were significant differences between the hypoxic 12, 24 and 48 h group and the normoxic control group in the expression of TIMP-2 mRNA in HPDLF (P < 0.05). The expression of MMP-1 mRNA in hypoxic groups of 12 h was momentarily decreased and then increased after 24 h of hypoxia. There were significant differences between the hypoxic 48 h group and the normoxic control group in the expression of MMP-1 mRNA in HPDLF (P < 0.05). There were significant differences between the hypoxic 12 h group and the normoxic control group in the ratio of MMP-1/TIMP-1 mRNA (P < 0.05). The ratio of MMP-2/TIMP-2 mRNA in the hypoxia group significantly increased compared with normoxic group. There were significant differences between the hypoxic group and the normoxic control group in the ratio of MMP-2/TIMP-2 mRNA (P < 0.05). CONCLUSIONS: Hypoxia could change the expression of MMP and TIMP mRNA and other relevant growth factors and also lead to the imbalance of MMP-2/TIMP-2 mRNA expression. It is suggested that the imbalance of MMP-2/TIMP-2 expression may be closely correlated with the occurrence and development of periodontal disease and play an important role in the process of periodontal tissue destruction in periodontitis.

[Effects of enamel matrix proteins on the proliferation of human gingival epithelial cells in vitro].

PURPOSE: To evaluate the effect of enamel matrix proteins (EMPs) on the proliferation of human gingival epithelial cells in vitro. METHODS: EMPs were extracted from pig tooth germ by acetic acid. The gingival tissues cut off during gingivectomy were separated into two parts through Dispase II digestion and the epithelium part was cultured to acquire the gingival epithelial cells. The first passage epithelial cells were inoculated into 96-well plate, 22500 cells per well, and exposed to different concentrations of EMPs (50, 100, 200 microg/ml respectively). The control was epithelial cells cultured in the same medium except without EMPs. The proliferation rates were carried out over a 5-day period and assessed by an MTT assay and the data were analysed by one-way variance. RESULTS: It was shown that gingival epithelial cells well attached and spread on EMPs-coated substrata. There were no significant differences between the control group and various concentrations of EMPs groups at the initial stage, however, EMPs at a concentration of 200 microg/ml significantly inhibited gingival epithelial cells proliferation from day 3 over the experiment. CONCLUSIONS: The proliferation of gingival epithelial cells was significantly inhibited by EMPs in a dose- and time-dependent manner, which provides some evidence for the mechanism of EMPs in promoting the periodontal tissue regeneration.

[Preliminary study on adult odontoblast culture in situ].

PURPOSE: To establish an in situ culture model of adult odontoblasts. METHODS: Thirty intact and healthy third molars freshly prepared from 20-30 year old individuals were randomly divided into three groups. Each group had 10 molars. Group 1 was pulp tissue extraction group. Group 2 was serum-containing culture group. Group 3 was serum-free culture group. The root was dissected from the crown and the pulp was pulled out to make odontoblasts remaining in the crown. The odontoblasts were cultured in situ either in medium containing serum or serum-free medium for up to 7 days. The growth status of the cells was examined by light microscopy and cell morphology and distribution was analyzed by scanning electron microscopy. Cell viability was determined by trypan blue staining. RESULTS: After pulp removal at room temperature, odontoblasts remained in the wall of the pulp chamber, and kept viable and good morphology during the 7-day culture. CONCLUSION: We have successfully established an in situ culture model of adult primary odontoblasts in either serum-containing or serum-free medium.

[Effects of EMPs on growth and attachment of human BMSCs].

PURPOSE: The present study is to evaluate the effects of enamel matrix proteins(EMPs) on the attachment, spreading and proliferation of human bone marrow stromal cells(hBMSCs) in vitro. METHODS: Human BMSCs were obtained from human bone marrow aspiration and cultured in DMEM medium with 10% fetal bovine serum (FBS). EMPs was added into medium in several concentrations (50,100, 200, 300 microg/ml) as experimental groups. BMSCs were cultured without EMPs as control group. Attachment ability of hBMSCs was detected by counting cell number. Cell spreading rates were performed at various culture times by analysis of micrographs taken at predetermined sites of each wells. Cell proliferation rates were assessed by MTT assay. Data was statistically analyzed with SAS6.12 software for one-way ANOVA. RESULTS: It was shown that BMSCs were cultured successfully in vitro. There was no significant change between the control group and experimental groups in cell attachment and cell spreading rate. However, the proliferation of BMSCs was significantly stimulated by EMPs in a dose- and time-dependent manner. EMPs at a concentration of 200 microg/ml significantly enhanced BMSCs proliferation (P < 0.05). CONCLUSION: EMPs could promote the proliferating ability of human BMSCs, but have no effects on its attachment and spreading.Supported by National "863" Project (Grant No. 2002AA205013), Shanghai Municipal Education Development Fund(Grant No.2002-02) and Research Fund of Science and Technology Committee of Shanghai Municipality (Grant No.04dz05601).