Progranulin promotes osteogenic differentiation of human periodontal ligament stem cells via tumor necrosis factor receptors to inhibit TNF-α sensitized NF-kB and activate ERK/JNK signaling.

OBJECTIVE: To investigate the molecular mechanism of Progranulin (PGRN) in promoting osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in inflammatory environment. BACKGROUND: Progranulin is an antagonist of tumor necrosis factor (TNF) receptors (TNFRs) and is known to promote inflammatory periodontal bone defect regeneration. METHODS: TNFR1- and TNFR2-silenced hPDLSCs designed as hPDLSCs-sh-TNFR1 and hPDLSCs-sh-TNFR2 were cultured with osteoinductive medium containing TNF-α and (or) PGRN. Immunofluorescence, quantitative real-time PCR, and western blot were used to, respectively, detect expressions of TNFR1\TNFR2 and osteogenic differentiation markers as well as phosphorylation level in NF-κB\MAPK-related pathways. RESULTS: Immunofluorescence and real-time PCR showed that TNFR1 and TNFR2 positively expressed in hPDLSCs. TNF-α stimulation could significantly decrease the expressions of ALP and RUNX2 in hPDLSCs, whereas PGRN treatment could significantly enhance their expressions, and reverse TNF-α-mediated expression suppression of ALP and RUNX2 in hPDLSCs. In hPDLSCs-sh-TNFR1, TNF-α mediated osteogenic inhibition decreased, but both TNF-α + PGRN and alone PGRN significantly promoted expression of ALP and RUNX2. PGRN significantly enhanced expression of P-ERK1/2 and P-JNK, while corresponding inhibitors eliminated PGRN-stimulated osteogenic differentiation. In hPDLSCs-sh-TNFR2, no significant difference existed in osteogenic markers and P-JNK expression between the PGRN group and the control group. However, PGRN still activated P-ERK1/2 expression. Besides, PGRN antagonized TNF-α-enhanced NF-κB P65 expression. CONCLUSION: Progranulin promotes osteogenic differentiation of hPDLSCs via TNFR1 to inhibit TNF-α-sensitized NF-κB and via TNFR2 to activate JNK signaling. The mechanism by which PGRN activates ERK signaling remains to be explored.

Disturbed Expression of EphB4, but Not EphrinB2, Inhibited Bone Regeneration in an In Vivo Inflammatory Microenvironment.

The important role of ephrinB2-EphB4 signaling pathway in bone remodeling has been well established. However, it is still unclear whether this bidirectional signaling also has effects on the regenerative processes of bone defects created in an inflammatory microenvironment. In this study, an experimental animal model of bone defects treated with lentiviruses was prepared and an inflammatory microenvironment was established. Expression levels of bone marker genes were monitored in the newly formed bone tissue using quantitative reverse transcriptase polymerase chain reaction and western blot. Immunohistochemical (IHC) staining and histomorphometric analysis were also performed to evaluate bone healing processes. Compared with the pLenti6.3-ctrl group, the pLenti6.3-ephb4siRNA group exhibited lower expression levels of bone formation marker genes and a higher level of NFATc1 in the new bone tissue. In addition, the newly formed bone was thinner and the number of giant osteoclasts was higher in the pLenti6.3-ephb4siRNA group than that in the pLenti6.3-ctrl group. In contrast, there was no significant difference between the pLenti6.3-efnb2siRNA group and the pLenti6.3-ctrl group. In conclusion, EphB4 plays an irreplaceable role in bone regeneration in an inflammatory microenvironment, whereas the functional loss of ephrinB2 can be effectively compensated, most possibly by other ephrins with similar chemical structures.

Synthesis and discovery of novel pyrazole carboxamide derivatives as potential osteogenesis inducers.

A series of novel N-aryl-3-aryl-1-arylmethyl-1H-pyrazole-5-carboxamide derivatives 4a-l was synthesized by the reaction of 3-aryl-1-arylmethyl-1H-pyrazole-5-carbonyl chloride with substituted aniline in good to excellent yields. Structures of the compounds were determined by IR, (1) H NMR, and HR-MS spectroscopy. The molecular structure was confirmed by the X-ray crystal analysis of one compound (4j) that was prone to crystallization. These compounds were used to induce mouse osteoblast precursors MC3T3-E1 into osteoblasts and the induction was assessed by alkaline phosphatase (ALP) activity and the gene expression of bone sialoprotein (BSP). The results showed that the compounds 4a-d, 4g, 4h, and 4k could increase the ALP activity in comparison with the negative control group and compound 4h was the most effective one which could induce osteogenesis. Furthermore, mRNA expression of BSP which is a marker of osteogenesis was up-regulated by the compound 4h.

Combined effects of soluble vascular endothelial growth factor receptor FLT-1 gene therapy and cisplatin chemotherapy in human tongue carcinoma xenografts.

The aim of the present study was to assess the anti-tumor effect of a defective adenovirus that expresses soluble vascular endothelial growth factor (VEGF) receptor FLT-1 (AdsFLT-1) in combination with cisplatin (cis-diamminedichloroplatinum, DDP) on human tongue carcinoma Tca8113 cell xenografts that had been pre-established in nude mice. In vitro, Tca8113 cells secreted soluble FLT-1 (sFLT-1) after infection with AdsFLT-1, and the conditioned medium from AdsFLT-1-treated Tca8113 cells seemed to inhibit VEGF-induced proliferation of human umbilical vein endothelial cells. The combined effects of sFLT-1 gene therapy and DDP chemotherapy was then studied in well-established Tca8113 xenografts. The concentration of sFLT-1 in serum reached a peak 8 days after intratumoral injection of AdsFLT-1. In these tumors, AdsFLT-1 intratumoral injections had only a small effect. Interestingly, when the cells were also exposed to DDP chemotherapy, significantly higher (P<0.05), and possibly synergistic, anti-tumoral effects were observed that were highly correlated to a marked reduction in intratumoral vascularization and an increase in tumor-cell apoptosis. Together, these data emphasize the potential of combining an anti-angiogenic gene therapy strategy with a destructive approach directed against the tumor cells to fight human tongue carcinoma.

[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.

Tongue lipoma in an older male: A case report and literature review of patients with tongue lipoma reported in China.

Lipoma is the most common benign tumor that occurs at any region where adipose tissue is present. However, as the tongue is devoid of adipocyte it is an extremely rare site for a lipoma to develop, particularly in China. The present study reports the presence of a tongue lipoma in a 78-year-old man that measured 2.2×2.0×1.5 cm and was located on the left ventral region of the tongue. The tumor was completely excised, and subsequent to 4 years of follow-up, there was no recurrence of the lesion. In addition, the present study reviewed the literature concerning tongue lipomas in China and analyzed 18 other cases of patients with tongue lipomas in the past 30 years, between 1 January 1985 and 31 December 2014.

Clinical characteristics and analysis of familial oral lichen planus in eight Chinese families.

Oral lichen planus (OLP) is one of the most common oral mucosa diseases; however, familial OLP is uncommon. The present study reported and analyzed patients with familial OLP (n=18) in eight different Chinese families between January 1, 2012 and December 31, 2013. Parameters analyzed include gender, age at diagnosis, lesion distribution and lesion type. The follow-up period for each patient was a minimum of 1 year. In this survey, 18/88 individuals in the eight families were affected. Females were more frequently affected, and the female to male ratio for familial OLP (2.2:1) was higher compared with that previously reported for nonfamilial OLP (1.4:1). The age at diagnosis, lesion distribution and lesion type showed consistency with reports concerning nonfamilial OLP, with the exception of family VI, in which 4/5 children had OLP/LP lesions and were of an early age at diagnosis. There were two families in which three generations had been affected by OLP. In addition, it appeared that patients of the same generation in the same family were of a similar age at diagnosis. No malignant or premalignant lesion was identified in the 18 individuals diagnosed with OLP from the eight families. The present study supports the hypothesis that genetic predisposition may serve a role in the etiology of OLP.

Systemic BMSC homing in the regeneration of pulp-like tissue and the enhancing effect of stromal cell-derived factor-1 on BMSC homing.

Pulp regeneration caused by endogenous cells homing has become the new research spot in endodontics. However, the source of functional cells that are involved in and contributed to the reconstituting process has not been identified. In this study, the possible role of systemical BMSC in pulp regeneration and the effect of stromal cell-derived factor-1 (SDF-1) on stem cell recruitment and angiogenesis were evaluated. 54 mice were divided into three groups: SDF-1 group (subcutaneous pockets containing roots with SDF-1 absorbed neutralized collagen gel and the green fluorescent protein (GFP) positive BMSCs transplantation via the tail vein), SDF-1-free group (pockets containing roots with gel alone and GFP + BMSCs transplantation) and Control group (pockets containing roots with gel alone). The animals were sacrificed after the roots were implanted into subcutaneous pockets for 3 weeks. Histomorphometric analysis was performed to evaluate the regenerated tissue in the canal by hematoxylin and eosin (HE) staining. The homing of the transplanted BMSCs was monitored with a fluorescence microscope and immunohistochemical analysis. The expression of ALP in new formed tissue was detected immunohistochemically. Dental-pulp-like tissue and new vessels were regenerated and GFP-positive BMSCs and expression of ALP could be observed in both SDF-1 group and SDF-1-free group. Furthermore, more GFP+ cells, stronger expression of ALP and stronger angiogenesis were found in the SDF-1 group than in the SDF-1-free group. To conclude, systemic BMSC can home to the root canal and participate in dental-pulp-like tissue regeneration. Intracanal application of SDF-1 may enhance BMSC homing efficiency and angiogenesis.

Biomaterial strategies for stem cell maintenance during in vitro expansion.

Stem cells, having the potential for self-renewal and multilineage differentiation, are the building blocks for tissue/organ regeneration. Stem cells can be isolated from various sources but are, in general, available in too small numbers to be used directly for clinical purpose without intermediate expansion procedures in vitro. Although this in vitro expansion of undifferentiated stem cells is necessary, stem cells typically diminish their ability to self-renew and proliferate during passaging. Consequently, maintaining the stemness of stem cells has been recognized as a major challenge in stem cell-based research. This review focuses on the latest developments in maintaining the self-renewal ability of stem cells during in vitro expansion by biomaterial strategies. Further, this review highlights what should be the focus for future studies using stem cells for regenerative applications.

Human periodontal ligament derived progenitor cells: effect of STRO-1 cell sorting and Wnt3a treatment on cell behavior.

OBJECTIVES: STRO-1 positive periodontal ligament cells (PDLCs) and unsorted PDLCs have demonstrated potential for periodontal regeneration, but the comparison between unsorted cells and the expanded STRO-1 sorted cells has never been reported. Additionally, Wnt3a is involved in cell proliferation thus may benefit in vitro PDLC expansion. The aim was to evaluate the effect of STRO-1 cell sorting and Wnt3a treatment on cell behavior of human PDLCs (hPDLCs). MATERIALS AND METHODS: STRO-1 positive hPDLCs were sorted and the sorted cells were expanded and compared with their unsorted parental cells. Thereafter, hPDLCs were treated with or without Wnt3a and the cell proliferation, self-renewal, and osteogenic differentiation were evaluated. RESULTS: No differences were measured between the expanded STRO-1-sorted cells and unsorted parental cells in terms of proliferation, CFU, and mineralization capacity. Wnt3a enhanced the proliferation and self-renewal ability of hPDLCs significantly as displayed by higher DNA content values, a shorter cell population doubling time, and higher expression of the self-renewal gene Oct4. Moreover, Wnt3a promoted the expansion of hPDLCs for 5 passages without affecting cell proliferation, CFU, and osteogenic capacity. CONCLUSIONS: Expanded STRO-1-sorted hPDLCs showed no superiority compared to their unsorted parental cells. On the other hand, Wnt3a promotes the efficient hPDLC expansion and retains the self-renewal and osteogenic differentiation capacity.

Systemically transplanted human gingiva-derived mesenchymal stem cells contributing to bone tissue regeneration.

As novel postnatal stem cells, gingiva-derived mesenchymal stem cells (GMSCs) have been considered as an ideal candidate cell resource for tissue engineering and cell-based therapies. GMSCs implanted into sites of injury have been confirmed to promote the injury repair. However, no studies have demonstrated whether systemically transplanted GMSCs can home to the bone injuries and contribute to the new bone formation in vivo. In this study, we transplanted human GMSCs into C57BL/6J mice with defects in mandibular bone via the tail vein to explore the capacity of transplanted GMSCs to promote bone regeneration. Results showed that the transplanted GMSCs were detected in the bone defects and employed in new bone formation. And the newly formed bone area in mice with GMSCs transplantation was significantly higher than that in control mice. Our findings indicate that systemically transplanted GMSCs can not only home to the mandibular defect but also promote bone regeneration.

Effect of umbilical cord mesenchymal stem cell in peri-implant bone defect after immediate implant: an experiment study in beagle dogs.

BACKGROUND: For the sake of reducing post extraction resorption, getting optimal positioning of the implant and shortening treatment time, immediate implant placement following tooth extraction has been proposed as a treatment option. However, the large bone defect peri-implant has a negative influence on the process of bone healing. In this study, umbilical cord mesenchymal stem cells (UCMSCs) were transplanted into the bone defect peri-implant inbeagle dogs and the effect of UCMSCs on bone regeneration in peri-implant were assessed. METHODS: The mandibular second, third and fourth premolars of 8 beagle dogs were extracted bilaterally. The defects in one side were filled with platelet-rich fibrin (PRF) and then UCMSCs were injected into the defect area, while the defects in the other side were filled with PRF only as control group. The titanium implant was placed into the distal root socket of each extracted tooth. The animals were sacrificed at week 2, 4 and 8 post operative. The bone defects adjacent to the implant which are 4 mm in height, 4 mm in the mesio-distal direction and 3.5 mm in the bucco-lingual direction were made after immediate implant. Histomorphometric analysis was performed using methylene blue-fuchsin acid staining and hematoxylin and eosin (HE) staining to evaluate bone regeneration. RESULTS: The direct bone-to-implant contact (BIC) in the experiment after 4 and 8 weeks was 56.47±1.18% and 76.23±2.08%; and in the control group was40.79±0.65% and 61.17±2.79%, respectively. The percentage of newly formed bone after 2, 4 and 8 weeks was 17.60±1.5%, 49.82±4.02% and 67.16±2.1% in experiment group; and in control group 14.30±1.25%, 37.04±2.29% and 58.83±3.36%, respectively. These results represented significant differences statistically. CONCLUSION: Intra-bone marrow injection of UCMSCs can promote new bone formation. UCMSCs can be used to as excellent seed cells to repair the large defect peri-implant after immediate implant.

Effect of umbilical cord mesenchymal stem cell in peri-implant bone defect after immediate implant: an experiment study in beagle dogs.

BACKGROUND: For the sake of reducing post extraction resorption, getting optimal positioning of the implant and shortening treatment time, immediate implant placement following tooth extraction has been proposed as a treatment option. However, the large bone defect peri-implant has a negative influence on the process of bone healing. In this study, umbilical cord mesenchymal stem cells (UCMSCs) were transplanted into the bone defect peri-implant in beagle dogs and the effect of UCMSCs on bone regeneration in peri-implant were assessed. METHODS: The mandibular second, third and fourth premolars of 8 beagle dogs were extracted bilaterally. The defects in one side were filled with platelet-rich fibrin (PRF) and then UCMSCs were injected into the defect area, while the defects in the other side were filled with PRF only as control group. The titanium implant was placed into the distal root socket of each extracted tooth. The animals were sacrificed at week 2, 4 and 8 post operation. The bone defects adjacent to the implant which are 4 mm in height, 4 mm in the mesio-distal direction and 3.5 mm in the bucco-lingual direction were made after immediate implant. Histomorphometric analysis was performed using methylene blue-fuchsin acid staining and hematoxylin and eosin (HE) staining to evaluate bone regeneration. RESULTS: The direct bone-to-implant contact (BIC) in the experiment after 4 and 8 weeks was 56.47 ± 1.18% and 76.23 ± 2.08%; and in the control group was40.79 ± 0.65% and 61.17 ± 2.79%, respectively. The percentage of newly formed bone after 2, 4 and 8 weeks was 17.60 ± 1.5%, 49.82 ± 4.02% and 67.16 ± 2.1% in experiment group; and in control group 14.30 ± 1.25%, 37.04 ± 2.29% and 58.83 ± 3.36%, respectively. These results represented significant differences statistically. CONCLUSION: Intra-bone marrow injection of UCMSCs can promote new bone formation. UCMSCs can be used to as excellent seed cells to repair the large defect peri-implant after immediate implant.

Hyperlipidemia compromises homing efficiency of systemically transplanted BMSCs and inhibits bone regeneration.

PURPOSE: Mesenchymal stem cells (MSCs) can selectively home to bone defects and play an essential role in promoting bone regeneration. As an adverse effect factor for bone metabolism, hyperlipidemia significantly impairs bone regeneration. In this study, bone marrow stromal cells (BMSCs) were systemically transplanted into a hyperlipidemic mouse model to explore the effect of hyperlipidemia on stem cell recruitment and bone regeneration. METHODS: Hyperlipidemia was established in ApoE-/- mice (on C57BL/6J background) fed with a high fat diet (HFD) for five weeks. C57BL/6 mice fed with the same diet served as controls. BMSCs labeled with the green fluorescent protein (GFP) were then injected via the tail vein and bone defects were created in the mandibles. The animals were sacrificed at weeks 1, 2 and 4 after surgery, and the fate of the transplanted BMSCs was monitored with a fluorescence microscope and immunohistochemical analysis. After hematoxylin and eosin (HE) staining and Masson's Trichrome (MT) staining, histomorphometric analysis was performed to evaluate bone regeneration. RESULTS: In both groups transplanted with BMSCs, the number of GFP-positive BMSCs detected in the bone defects reached its peak at 1 week after surgery and was decreased thereafter. However, at all time points, less GFP+ cells were detected in the ApoE-/- mice than in the corresponding control mice. BMSCs transplantation significantly enhanced new bone formation, but to a lesser degree in the ApoE-/- mice when compared with the control mice. CONCLUSIONS: Hyperlipidemia compromises homing efficiency of systemically transplanted BMSCs and inhibits bone regeneration.

The role of small molecules in bone regeneration.

Although several methods have been used in bone regeneration medicine, current methods still have many limitations. The tissue used for autogenous bone graft is limited and allograft has weak osteoinductive activity. Tissue engineering provides a good choice for bone regeneration. However, the growth factors needed have a high price and short half-life. Recently, a number of small molecules have been confirmed to have osteoinductive activity and some have been clinically used. Natural small molecules including decalpenic acid, flavonoids, quinones can be extracted from plants and others can be synthesized according to the structure designed or mimicking the structure of natural small molecules. Small molecules can act as co-activator of BMP2 pathway or activate Wnt pathway; others can be the inhibitors of NF-κB signaling pathway. This review gives an overview on the small molecules with osteoinductive activity and discusses the mechanism of the small molecules.

[Construction and identification of a recombinant lentiviral expression plasmid pLentiTrident1-hBMP2-Neo-hNGF].

PURPOSE: To construct and confirm a recombinant lentiviral vector containing human bone morphogenetic protein 2 (hBMP2) and human nerve growth factor (hNGF). METHODS: The Neomycin gene was digested from pLentiTrident1-EGFP-Neo and then was subcloned into lentiviral vector. The hBMP2 and hNGF genes were amplified by polymerase chain reaction (PCR), and then the PCR product was inserted to proper sites of the vector. Finally, the recombinant vector pLentiTrident1-hBMP2-Neo-hNGF was confirmed by agarose gel electrophoresis and DNA sequence analysis. RESULTS: The construction of recombinant lentiviral vector pLentiTrident-hBMP2 -Neo-hNGF was confirmed through restriction enzyme maping analysis and DNA sequencing. CONCLUSIONS: The recombinant lentiviral vector which can coexpress hBMP2 and hNGF is successfully constructed,which lays a solid foundation of studying the effect of neuro factors on bone regeneration.

Osteopromotive activity of a novel pyrazole carboxamide derivative.

AIMS: To investigate the effect of 1-(4-(tert-butyl)benzyl)-N-(4-methoxyphenyl)-3-phenyl-1H-pyrazole-5-carboxamide (Pyr-C) on the proliferation and osteogenic differentiation of MC3T3-E1 cells. MATERIALS & METHODS: MTT and BrdU incorporation assay were used to determine cell survival and proliferation. The gene expression levels of osteogenic markers were determined using real-time PCR and ALP activity was detected. Western-blot analysis was used to determine the protein expression of BSP and OPN. The long-term effect of Pyr-C on mineralization deposition was measured by Alizarin Red Staining. RESULTS: Pyr-C inhibited cell proliferation and increased ALP activity. Gene expression of ALP, BSP, OCN, Runx2, and Osterix was up-regulated in Pyr-C-induced group. Pyr-C increased the protein expression of BSP at day 7, 14 and 21, and OPN at day 14, 21 and 28. Meanwhile, Pyr-C enhanced the mineral deposition. CONCLUSION: Pyr-C inhibits proliferation and stimulates osteogenic differentiation of MC3T3-E1 cells.

[Establishment of the inferior alveolar nerve transection combined experimental periodontitis animal modal in rats].

PURPOSE: To establish an animal model of the inferior alveolar nerve (IAN) transected combined experimental periodontitis in rats, in order to provide a foundation for exploring the function of nerve factor in the occurrence and development of periodontitis and periodontal tissue regeneration in vivo. METHODS: Thirty-six adult SPF Wistar rats were divided into 3 groups randomly. The IAN-transection and periodontitis group (P1): steel-wire was used to ligate to the dental cervix of the dual first lower molars, the gingiva was lacerated using a dental probe, and the surgical transection of the IAN-transection was applied to the left side of the experimental rats, the animals were provided with high-carbonhydrate diet after surgery;the IAN-transection group (P2): the surgical transection of the IAN-transection was applied to the left side of the experimental group, the animals were fed on in routine way after surgery; the sham surgical group(N): no surgery was performed, the animals were fed on in routine way after surgery.6 weeks after the operation, specimens were collected by dissecting the operation areas after internal fixation and observed by clinical examination, X-ray, and histological examination. SPSS13.0 software package were used for statistical analysis. RESULTS: The IAN-transection operation was successfully conducted in the study and there was no significant necrosis or ulceration observed in the nerve dissected rats. All experimental periodontitis models were established successfully at the time point; there was severe destruction of the periodontal tissues especially in the P1 group. There was bone losses in the specimens of IAN-transection side than that in non IAN-transection side according to X-ray examination 6 weeks after operation both in the P1 and P2 group, especially in the P1 group. Periodontal tissues on both sides of the P2 group had no obvious inflammation and periodontal destruction. CONCLUSIONS: The experimental periodontitis animal model in various nerve conditions can be successfully conducted. The inferior alveolar nerve transection can result in severe destruction of the periodontal tissues, but it can not result in inflammation of normal periodontal tissue.

[Expression of periodontal ligament-associated protein-1 in normal periodontal tissues and cells in rat].

OBJECTIVE: To examine the expression of periodontal ligament-associated protein-1 (PLAP-1) in the periodontal tissues and periodontal ligament cells (PDLC). METHODS: The PLAP-1 expression in normal periodontal tissue was examined by immunohistochemistry. The protein expression and mRNA transcription of PLAP-1 in PDLC were investigated by immunocytochemistry and reverse transcription-polymerase chain reaction. RESULTS: PLAP-1 was expressed in periodontium but not in cementum, alveolar bone and gingival tissues. PLAP-1 expression was observed in cell plasma, but not in nuclei. There was a 350 bp electrophoresis band representing PLAP-1 mRNA. CONCLUSIONS: PLAP-1 may play a role in physiology of periodontal tissues and cells in normal adult rats.

[Quantitative detection of human cytomegalovirus in aggressive and chronic periodontitis lesions].

OBJECTIVE: To establish a quantitative real-time PCR assay for the detection of human cytomegalovirus (HCMV) DNA load in subgingival specimens from the patients with aggressive and chronic periodontitis, and to investigate the relationship between HCMV infection and the periodontal status. METHODS: A total of 114 subgingival plaque specimens were taken from 18 subjects with aggressive priodontiti (AgP), 24 subjects with chronic periodontitis (CP) and 15 healthy control subjects. Standard quantification was performed with recombinant plasmid containing a conserved fragment of HCMV. The SYBR Green I fluorescent quantitative real-time PCR assay was established based on positive plasmid. HCMV DNA load in the specimens were detected with quantitative real-time PCR based on SYBR Green I fluorescence. RESULTS: HCMV were detected in 58.3% of AgP sites and 41.7% of CP sites, however, only 6.7% of periodontally-healthy sites were HCMV positive. The detection rate of HCMV in periodontitis lesions was significantly higher than in periodontal health (P < 0.01). High copy-counts more than 10(4) of HCMV were detected in 33.3% of AgP sites, which were significantly higher than in CP sites (10.4%) (P < 0.05). CONCLUSION: Subgingival infection with HCMV is closely associated with periodontitis. Active HCMV infection may be related to the rapid tissue destruction of AgP.