Insensitive Effects of Inflammatory Cytokines on the Reference Genes of Synovial Fluid Resident-Mesenchymal Stem Cells Derived from Rheumatoid Arthritis Patients.

Mesenchymal stem cells derived from rheumatoid arthritis patients (RA-MSCs) provide an understanding of a variety of cellular and immunological responses within the inflammatory milieu. Sustained exposure of MSCs to inflammatory cytokines is likely to exert an influence on genetic variations, including reference genes (RGs). The sensitive effect of cytokines on the reference genes of RA-SF-MSCs may be a variation factor affecting patient-derived MSCs as well as the accuracy and reliability of data. Here, we comparatively evaluated the stability levels of nine RG candidates, namely GAPDH, ACTB, B2M, EEF1A1, TBP, RPLP0, PPIA, YWHAZ, and HPRT1, to find the most stable ones. Alteration of the RG expression was evaluated in MSCs derived from the SF of healthy donors (H-SF-MSCs) and in RA-SF-MSCs using the geNorm and NormFinder software programs. The results showed that TBP, PPIA, and YWHAZ were the most stable RGs for the normalization of H-SF-MSCs and RA-SF-MSCs using RT-qPCR, whereas ACTB, the most commonly used RG, was less stable and performed poorly. Additionally, the sensitivity of RG expression upon exposure to proinflammatory cytokines (TNF-α and IL-1ß) was evaluated. RG stability was sensitive in the H-SF-MSCs exposed to TNF-α and IL-1ß but insensitive in the RA-SF-MSCs. Furthermore, the normalization of IDO expression using ACTB falsely diminished the magnitude of biological significance, which was further confirmed with a functional analysis and an IDO activity assay. In conclusion, the results suggest that TBP, PPIA, and YWHAZ can be used in SF-MSCs, regardless of their exposure to inflammatory cytokines.

The influence of different hinge position on PTS during HTO: comparison between open-wedge and closed-wedge HTO.

PURPOSE: The purpose of this study was to determine the significance of hinge position through comparison between open-wedge and closed-wedge high tibial osteotomy (HTO) and to determine the ideal hinge position to minimize the effect of HTO on the posterior tibial slope (PTS) and medial proximal tibial angle (MPTA). METHODS: Procedures were performed on 32 cadaveric knees using open-wedge HTO with the standard hinge position or a low hinge position or closed-wedge HTO with the standard hinge position or a low hinge position. To define the standard hinge position in open wedge HTO, we drew a line 3-cm inferior to the medial tibial plateau toward the fibular head and located the intersection of this line with a longitudinal line 1-cm medial to fibular shaft. The low hinge position was then defined as the point 1-cm inferior to the standard position. For the standard hinge position for closed-wedge HTO, we drew a line parallel with joint line from 2-cm inferior to the lateral tibial plateau. The low hinge position was then defined as the point 1-cm inferior to the standard position. RESULTS: For the open-wedge procedure, osteotomy through the low hinge position resulted in a significantly greater PTS compared to osteotomy through the standard hinge position. MPTA was also significantly greater for the low hinge position compared to standard hinge position. In the closed-wedge HTO, neither the PTS nor MPTA was significantly different for the low and standard hinge positions. CONCLUSIONS: Hinge position significantly affects changes in the PTS and MPTA following open-wedge but not closed-wedge HTO. Understanding how to hinge position affects the PTS and MPTA is critical for surgeons performing open-wedge HTO procedures. Adopting an accurate hinge position is crucial for preventing complications, especially in open-wedge osteotomy, due to postoperative changes in the PTS and MPTA.

Tiron Has Negative Effects on Osteogenic Differentiation via Mitochondrial Dysfunction in Human Periosteum-Derived Cells.

Tiron is a potent antioxidant that counters the pathological effects of reactive oxygen species (ROS) production due to oxidative stress in various cell types. We examined the effects of tiron on mitochondrial function and osteoblastic differentiation in human periosteum-derived cells (hPDCs). Tiron increased mitochondrial activity and decreased senescence-associated ß-galactosidase activity in hPDCs; however, it had a detrimental effect on osteoblastic differentiation by reducing alkaline phosphatase (ALP) activity and alizarin red-positive mineralization, regardless of H2O2 treatment. Osteoblast-differentiating hPDCs displayed increased ROS production compared with non-differentiating hPDCs, and treatment with tiron reduced ROS production in the differentiating cells. Antioxidants decreased the rates of oxygen consumption and ATP production, which are increased in hPDCs during osteoblastic differentiation. In addition, treatment with tiron reduced the levels of most mitochondrial proteins, which are increased in hPDCs during culture in osteogenic induction medium. These results suggest that tiron exerts negative effects on the osteoblastic differentiation of hPDCs by causing mitochondrial dysfunction.

No differences in mid- to long-term outcomes of computer-assisted navigation versus conventional total knee arthroplasty.

PURPOSE: Accurate implant position in total knee arthroplasty (TKA) can potentially lead to better long-term functional outcomes and implant survival. Recent studies on whether better clinical results could be obtained from computer-navigated or conventional TKA were inconclusive. In addition, recent reviews only included short-term follow-up studies without performing quantitative mid- to long-term follow-up analysis. Thus, the purpose of the present study was to perform a meta-analysis comparing mid- to long-term clinical outcomes (such as knee scoring and functional results) and radiological outcomes (such as normal alignment of the limb axis or component) between computer-navigated TKA and conventional TKA to determine which method of TKA could obtain better clinical and radiological results. METHODS: MEDLINE, EMBASE, the Cochrane Central Register of Controlled Trials (CENTRAL), Web of Science, and SCOPUS electronic databases were searched for relevant articles published through August 2018 that compared outcomes of computer-navigated TKA and conventional TKA. Data search, extraction, analysis, and quality assessment were performed according to the Cochrane Collaboration guidelines. Clinical and radiological outcomes of both techniques were evaluated using various outcome measures. RESULTS: Seven randomized controlled trials were included. Based on Knee Society Scores, the Western Ontario and McMaster Universities Osteoarthritis Index, pain, and range of motion, there were no significant differences in clinical outcomes between the two techniques. Based on outliers from the normal axis, outliers of femoral components in the coronal plane, and outliers of tibial components in the coronal plane, radiologic outcomes showed no significant differences between the two techniques either. CONCLUSIONS: The present study revealed that there were no significant differences in clinical or radiological outcomes between computer-navigated TKA and conventional TKA. It remains unclear which TKA technique yields better results in terms of mid- to long-term clinical and radiological outcomes. LEVEL OF EVIDENCE: I.

Parthenolide Has Negative Effects on In Vitro Enhanced Osteogenic Phenotypes by Inflammatory Cytokine TNF-α via Inhibiting JNK Signaling.

Nuclear factor kappa B (NF-κB) regulates inflammatory gene expression and represents a likely target for novel disease treatment approaches, including skeletal disorders. Several plant-derived sesquiterpene lactones can inhibit the activation of NF-κB. Parthenolide (PTL) is an abundant sesquiterpene lactone, found in Mexican Indian Asteraceae family plants, with reported anti-inflammatory activity, through the inhibition of a common step in the NF-κB activation pathway. This study examined the effects of PTL on the enhanced, in vitro, osteogenic phenotypes of human periosteum-derived cells (hPDCs), mediated by the inflammatory cytokine tumor necrosis factor (TNF)-α. PTL had no significant effects on hPDC viability or osteoblastic activities, whereas TNF-α had positive effects on the in vitro osteoblastic differentiation of hPDCs. c-Jun N-terminal kinase (JNK) signaling played an important role in the enhanced osteoblastic differentiation of TNF-α-treated hPDCs. Treatment with 1 µM PTL did not affect TNF-α-treated hPDCs; however, 5 and 10 µM PTL treatment decreased the histochemical detection and activity of alkaline phosphatase (ALP), alizarin red-positive mineralization, and the expression of ALP and osteocalcin mRNA. JNK phosphorylation decreased significantly in TNF-α-treated hPDCs pretreated with PTL. These results suggested that PTL exerts negative effects on the increased osteoblastic differentiation of TNF-α-treated hPDCs by inhibiting JNK signaling.

Upregulated TTYH2 expression is critical for the invasion and migration of U2OS human osteosarcoma cell lines.

Ion channels have recently emerged as stable biomarkers and anticancer targets particularly when the applications of the currently available therapeutic regimens are limited, as in case of osteosarcoma, a malignant bone tumor. Here, we evaluated the expression of TTYH2, a presumably calcium-activated chloride channel, in a human osteosarcoma cell line U2OS. We used small-interfering RNA (siRNA)-mediated gene silencing to demonstrate the downregulation in the expression of TTYH2 that resulted in the decrease in the invasion and migration, but not proliferation, of U2OS cells. The expression levels of Slug and ZEB1, the transcription factors involved in epithelial-mesenchymal transition, significantly reduced after TTYH2 silencing. Based on these results, we suggest that TTYH2 may serve as a novel target for the treatment of osteosarcoma.

Gait analysis after total hip arthroplasty using direct anterior approach versus anterolateral approach: a systematic review and meta-analysis.

BACKGROUND: Comparative studies of total hip arthroplasty using the direct anterior approach (DAA) compared with the anterolateral approach (ALA) by gait analysis compared the results of the two groups, the damage to the abductor muscle, with objective and detailed kinematic as well as kinetic data of actual gait. The purpose of this systematic review was to analyze the differences in gait such as time-dependent parameters, kinetics, and kinematics after THA using the DAA compared with ALA. METHODS: PubMed Central, OVID Medline, Cochrane Collaboration Library, Web of Science, EMBASE and AHRQ carried out a comprehensive search for all relevant randomized controlled trials and comparative studies, up to December 2018. Based on the following criteria, studies were selected: 1) study design: randomized controlled trials or non-randomized comparative studies; 2) study population: patients with primary osteoarthritis or avascular necrosis; 3) intervention: total hip arthroplasty by DAA or ALA; 4) Kinetic and kinematic data after gait analysis in the plains during postoperative follow-up. RESULTS: Of the 148 studies, 7 randomized controlled trials and 5 comparative studies were finally included in this systematic review. The peak hip flexion within 3 months after surgery was described in two studies and was significantly higher in the DAA group. (OR = 1.90; 95% CI [1.67,2.13]; P < 0.01, Z = 16.18). The gait speed within 3 months after surgery was reported in 3 studies and was significantly higher in the DAA group than in the ALA group. (SMD = 0.17; 95% CI [0.12,0.22]; P < 0.01, Z = 6.62) There was no difference between the two groups in stride length, step length, and hip range of motion in sagittal plane. CONCLUSIONS: In this meta-analysis, gait speed and peak hip flexion within 3 months after surgery were significantly higher in the DAA group than in the ALA group. Despite a few significant differences between two approaches, determining whether the reported differences in terms of postoperative gait values are clinically meaningful remains a substantial challenge.

The Role of Human Umbilical Vein Endothelial Cells in Osteogenic Differentiation of Dental Follicle-Derived Stem Cells in In Vitro Co-cultures.

Angiogenesis and vascularization are essential for the growth and survival of most tissues. Engineered bone tissue requires an active blood vessel network for survival and integration with mature host tissue. Angiogenesis also has an effect on cell growth and differentiation in vitro. However, the effect of angiogenic factors on osteoprogenitor cell differentiation remains unclear. We studied the effects of human umbilical vein endothelial cells (HUVECs) on osteogenic differentiation of dental follicle-derived stem cells (DFSCs) in vitro by co-culturing DFSCs and HUVECs. Cell viability, based on metabolic activity and DNA content, was highest for co-cultures with a DFSC/HUVEC ratio of 50:50 in a 1:1 mixture of mesenchymal stem cell growth medium and endothelial cell growth medium. Osteoblastic and angiogenic phenotypes were enhanced in co-cultures with a DFSC/HUVEC ratio of 50:50 compared with DFSC monocultures. Increased expression of angiogenic phenotypes and vascular endothelial growth factor (VEGF) levels were observed over time in both 50:50 DFSC/HUVEC co-cultures and DFSC monocultures during culture period. Our results showed that increased angiogenic activity in DFSC/HUVEC co-cultures may stimulate osteoblast maturation of DFSCs. Therefore, the secretion of angiogenic factors from HUVECs may play a role in the osteogenic differentiation of DFSCs.

Which Technique Is Better for Treating Patellar Dislocation? A Systematic Review and Meta-analysis.

PURPOSE: To clarify the discrepancy in surgical options and present evidence to treat patellar dislocation by evaluating which of the techniques yields better improvement in stability and functional recovery for patellar dislocation. METHODS: The MEDLINE, Embase, Cochrane Central Register of Controlled Trials, Web of Science, and Scopus electronic databases were searched for relevant articles comparing the outcomes of medial patellofemoral ligament (MPFL) treatment published up until August 2017. Data searching, extraction, analysis, and quality assessment were performed based on The Cochrane Collaboration guidelines. Clinical outcomes were evaluated using various outcome values in various techniques. For results with high heterogeneity, 95% prediction intervals (PIs) were also investigated. RESULTS: Eleven clinical studies were investigated. In patients with primary patellar dislocation, there were no significant differences in all evaluated outcomes between the conservative and surgical treatment groups. For patients with recurrent patellar dislocation, MPFL reconstruction was associated with a favorable Kujala score (mean difference, -8.91; 95% confidence interval, -14.05 to -3.77; I2 = 94%; 95% PI, -9.64 to -8.1) and Lysholm score (mean difference, -13.51; 95% confidence interval, -21.35 to -5.68; I2 = 96%; 95% PI, -14.86 to -12.16) when compared with soft tissue realignment surgery. CONCLUSIONS: Although surgical treatment of the MPFL for primary patellar dislocation is not superior to conservative treatment in restoring knee function and clinical outcomes, MPFL reconstruction is associated with more favorable clinical outcomes compared with medial soft tissue realignment surgery in patients with recurrent patellar dislocation. Double-bundle MPFL reconstruction seems to provide more favorable outcomes than single-bundle MPFL reconstruction, but this finding should be interpreted with caution because the evidence levels were low and were from only a few studies. LEVEL OF EVIDENCE: Level III, meta-analysis.

The effects of different hinge positions on posterior tibial slope in medial open-wedge high tibial osteotomy.

PURPOSE: The purpose of this study was to determine the standard hinge position to minimize effects from medial open-wedge high tibial osteotomy (HTO) on the posterior tibial slope. METHODS: Sixteen cadaveric knees underwent medial open-wedge osteotomy using either the standard or the low hinge position. To define the standard hinge position, a line 3 cm inferior to the medial tibial plateau towards the fibular head and located its intersection with a longitudinal line 1 cm medial to the fibular shaft was drawn. Low hinge position was defined as the point 1 cm inferior to the standard position. After tibial osteotomy, computed tomography scans of each knee were taken and three-dimensional models were constructed to characterize hinge position orientation and measure the osteotomy site effects on posterior tibial slope, medial proximal tibial angle, and gap ratio (the ratio of the anterior to posterior gap in the opened wedge). RESULTS: In two low hinge position specimens, the tibial lateral cortex hinge fracture occurred. Osteotomy through the low hinge position resulted in significantly greater posterior tibial slope compared to the standard hinge position (mean ± standard deviation) (11.2 ± 3.0° and 5.6 ± 2.5°, respectively; p < 0.001). Medial proximal tibial angle was also significantly greater for low compared to standard hinge position (95.4 ± 3.5° and 88.0 ± 3.5°, respectively; p < 0.001). Gap ratio was not significantly different between the two groups. CONCLUSION: Hinge position significantly affects the posterior tibial slope and medial proximal tibial angle following medial open-wedge HTO. Accurate hinge position is crucial to prevent complications from changes in posterior tibial slope and medial proximal tibial angle after surgery.

The role of isolated posterior cruciate ligament reconstruction in knees with combined posterior cruciate ligament and posterolateral complex injury.

PURPOSE: This is a meta-analysis comparing biomechanical outcomes to determine whether an isolated posterior cruciate ligament (PCL) reconstruction can restore normal knee kinematics in a combined PCL/posterolateral complex (PLC) injury and whether double-bundle (DB) PCL reconstruction is superior in controlling posterior and rotational laxity compared with single-bundle (SB) PCL reconstruction in a PCL/PLC-deficient knee. METHODS: A number of electronic databases were searched for relevant articles published through August 2016 that compared biomechanical outcomes of PCL reconstruction in patients who underwent reconstruction for combined PCL/PLC deficiencies. Data were searched, extracted, analysed, and assessed for quality according to Cochrane Collaboration guidelines, and biomechanical outcomes were evaluated using various outcome values. The results are presented as relative ratios for binary outcomes and standard mean differences for continuous outcomes with 95% confidence intervals. RESULTS: Five biomechanical studies were included in this meta-analysis. There were significant differences in laxities such as posterior tibial translation (PTT), external rotation, varus rotation, and PTT coupled with external rotation in the isolated PCL reconstruction group compared with the native PCL group. Furthermore, there were no significant differences in laxities such as PTT, external rotation, or varus rotation between the SB and DB PCL reconstruction groups. CONCLUSION: Isolated PCL reconstruction, whether SB or DB, could not restore normal knee kinematics in the PCL/PLC-deficient knee. In such cases, residual laxity after isolated PCL reconstruction can be controlled successfully with PLC reconstruction. Therefore, simultaneous PCL and PLC reconstruction is recommended for patients with combined PCL/PLC injury.

Effects of Osteogenic-Conditioned Medium from Human Periosteum-Derived Cells on Osteoclast Differentiation.

Stem/progenitor cell-based regenerative medicine using the osteoblast differentiation of mesenchymal stem cells (MSCs) is regarded as a promising approach for the therapeutic treatment of various bone defects. The effects of the osteogenic differentiation of stem/progenitor cells on osteoclast differentiation may have important implications for use in therapy. However, there is little data regarding the expression of osteoclastogenic proteins during osteoblastic differentiation of human periosteum-derived cells (hPDCs) and whether factors expressed during this process can modulate osteoclastogenesis. In the present study, we measured expression of RANKL in hPDCs undergoing osteoblastic differentiation and found that expression of RANKL mRNA was markedly increased in these cells in a time-dependent manner. RANKL protein expression was also significantly enhanced in osteogenic-conditioned media from hPDCs undergoing osteoblastic differentiation. We then isolated and cultured CD34+ hematopoietic stem cells (HSCs) from umbilical cord blood (UCB) mononuclear cells (MNCs) and found that these cells were well differentiated into several hematopoietic lineages. Finally, we co-cultured human trabecular bone osteoblasts (hOBs) with CD34+ HSCs and used the conditioned medium, collected from hPDCs during osteoblastic differentiation, to investigate whether factors produced during osteoblast maturation can affect osteoclast differentiation. Specifically, we measured the effect of this osteogenic-conditioned media on expression of osteoclastogenic markers and osteoclast cell number. We found that osteoclastic marker gene expression was highest in co-cultures incubated with the conditioned medium collected from hPDCs with the greatest level of osteogenic maturation. Although further study will be needed to clarify the precise mechanisms that underlie osteogenic-conditioned medium-regulated osteoclastogenesis, our results suggest that the osteogenic maturation of hPDCs could promote osteoclastic potential.

Lin28a enhances in vitro osteoblastic differentiation of human periosteum-derived cells.

Despite a capacity for proliferation and an ability to differentiate into multiple cell types, in long-term culture and with ageing, stem cells show a reduction in growth, display a decrease in differentiation potential, and enter senescence without evidence of transformation. The Lin28a gene encodes an RNA-binding protein that plays a role in regulating stem cell activity, including self-renewal and differentiation propensity. However, the effect of the Lin28a gene on cultured human osteoprecursor cells is poorly understood. In the present study, alkaline phosphatase activity, alizarin red-positive mineralization, and calcium content, positive indicators of osteogenic differentiation, were significantly higher in cultured human periosteum-derived cells (hPDCs) with Lin28a overexpression compared with cells without Lin28a overexpression. Lin28a overexpression by hPDCs also increased mitochondrial activity, which is essential for cellular proliferation, as suggested by a reduced presence of reactive oxygen species and significantly enhanced lactate levels and ATP production. Our results suggest that, in hPDCs, the Lin28a gene enhances osteoblastic differentiation and increases mitochondrial activity. Although Lin28a is known as a marker of undifferentiated human embryogenic stem cell, there is limited evidence regarding the influence of Lin28a on osteoblastic differentiation of cultured osteoprecursor cells. This study was to examine the impact of Lin28a on osteogenic phenotypes of human periosteum-derived cells. Their phenotypes can be similar to those of mesenchymal stem cells. Our results suggest that the Lin28a gene enhances the osteoblastic differentiation of human periosteum-derived cells. In addition, the Lin28a gene increases mitochondrial activity in human periosteum-derived cells.

The involvement of histone methylation in osteoblastic differentiation of human periosteum-derived cells cultured in vitro under hypoxic conditions.

Although oxygen concentrations affect the growth and function of mesenchymal stem cells (MSCs), the impact of hypoxia on osteoblastic differentiation is not understood. Likewise, the effect of hypoxia-induced epigenetic changes on osteoblastic differentiation of MSCs is unknown. The aim of this study was to examine the in vitro hypoxic response of human periosteum-derived cells (hPDCs). Hypoxia resulted in greater proliferation of hPDCs as compared with those cultured in normoxia. Further, hypoxic conditions yielded decreased expression of apoptosis- and senescence-associated genes by hPDCs. Osteoblast phenotypes of hPDCS were suppressed by hypoxia, as suggested by alkaline phosphatase activity, alizarin red-S-positive mineralization, and mRNA expression of osteoblast-related genes. Chromatin immunoprecipitation assays showed an increased presence of H3K27me3, trimethylation of lysine 27 on histone H3, on the promoter region of bone morphogenetic protein-2. In addition, mRNA expression of histone lysine demethylase 6B (KDM6B) by hPDCs was significantly decreased in hypoxic conditions. Our results suggest that an increased level of H3K27me3 on the promoter region of bone morphogenetic protein-2, in combination with downregulation of KDM6B activity, is involved in the suppression of osteogenic phenotypes of hPDCs cultured in hypoxic conditions. Although oxygen tension plays an important role in the viability and maintenance of MSCs in an undifferentiated state, the effect of hypoxia on osteoblastic differentiation of MSCs remains controversial. In addition, evidence regarding the importance of epigenetics in regulating MSCs has been limited. This study was to examine the role hypoxia on osteoblastic differentiation of hPDCs, and we examined whether histone methylation is involved in the observed effect of hypoxia on osteogenic differentiation of hPDCs.

Effect of oleanolic acid on the activity, secretion and gene expression of matrix metalloproteinase-3 in articular chondrocytes in vitro and the production of matrix metalloproteinase-3 in vivo.

In the present study, we tried to examine whether oleanolic acid regulates the activity, secretion and gene expression of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as the production of MMP-3 in the knee joint of rat to evaluate the potential chondroprotective effect of oleanolic acid. Rabbit articular chondrocytes were cultured in a monolayer, and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure interleukin-1ß (IL-1ß)-induced gene expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), ADAMTS-5 and type II collagen. In rabbit articular chondrocytes, the effects of oleanolic acid on IL-1ß-induced secretion and proteolytic activity of MMP-3 were investigated using western blot analysis and casein zymography, respectively. The effect of oleanolic acid on in vivo MMP-3 protein production was also examined, after intra-articular injection to the knee joint of rat. The results were as follows: (1) oleanolic acid inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5, but increased the gene expression of type II collagen; (2) oleanolic acid reduced the secretion and proteolytic activity of MMP-3; (3) oleanolic acid suppressed the production of MMP-3 protein in vivo. These results suggest that oleanolic acid can regulate the activity, secretion and gene expression of MMP-3, by directly acting on articular chondrocytes.

Erratum to: Betulin suppressed interleukin-1b-induced gene expression, secretion and proteolytic activity of matrix metalloproteinase in cultured articular chondrocytes and production of matrix metalloproteinase in the knee joint of rat.

[This corrects the article on p. 19 in vol. 21, PMID: 28066137.].

Betulin suppressed interleukin-1ß-induced gene expression, secretion and proteolytic activity of matrix metalloproteinase in cultured articular chondrocytes and production of matrix metalloproteinase in the knee joint of rat.

We investigated whether betulin affects the gene expression, secretion and proteolytic activity of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as in vivo production of MMP-3 in the rat knee joint to evaluate the potential chondroprotective effect of betulin. Rabbit articular chondrocytes were cultured and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure interleukin-1ß (IL-1ß)-induced gene expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), ADAMTS-5 and type II collagen. Effect of betulin on IL-1ß-induced secretion and proteolytic activity of MMP-3 was investigated using western blot analysis and casein zymography, respectively. Effect of betulin on MMP-3 protein production was also examined in vivo. The results were as follows: (1) betulin inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5, but increased the gene expression of type II collagen; (2) betulin inhibited the secretion and proteolytic activity of MMP-3; (3) betulin suppressed the production of MMP-3 protein in vivo. These results suggest that betulin can regulate the gene expression, secretion, and proteolytic activity of MMP-3, by directly acting on articular chondrocytes.

Autophagy Has a Beneficial Role in Relieving Cigarette Smoke-Induced Apoptotic Death in Human Gingival Fibroblasts.

The deleterious role of cigarette smoke has long been documented in various human diseases including periodontal complications. In this report, we examined this adverse effect of cigarette smoke on human gingival fibroblasts (HGFs) which are critical not only in maintaining gingival tissue architecture but also in mediating immune responses. As well documented in other cell types, we also observed that cigarette smoke promoted cellular reactive oxygen species in HGFs. And we found that this cigarette smoke-induced oxidative stress reduced HGF viability through inducing apoptosis. Our results indicated that an increased Bax/Bcl-xL ratio and resulting caspase activation underlie the apoptotic death in HGFs exposed to cigarette smoke. Furthermore, we detected that cigarette smoke also triggered autophagy, an integrated cellular stress response. Interesting, a pharmacological suppression of the cigarette smoke-induced autophagy led to a further reduction in HGF viability while a pharmacological promotion of autophagy increased the viability of HGFs with cigarette smoke exposures. These findings suggest a protective role for autophagy in HGFs stressed with cigarette smoke, highlighting that modulation of autophagy can be a novel therapeutic target in periodontal complications with cigarette smoke.

Cultured Human Periosteum-Derived Cells Can Differentiate into Osteoblasts in a Perioxisome Proliferator-Activated Receptor Gamma-Mediated Fashion via Bone Morphogenetic Protein signaling.

The differentiation of mesenchymal stem cells towards an osteoblastic fate depends on numerous signaling pathways, including activation of bone morphogenetic protein (BMP) signaling components. Commitment to osteogenesis is associated with activation of osteoblast-related signal transduction, whereas inactivation of this signal transduction favors adipogenesis. BMP signaling also has a critical role in the processes by which mesenchymal stem cells undergo commitment to the adipocyte lineage. In our previous study, we demonstrated that an agonist of the perioxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipocyte differentiation, stimulates osteoblastic differentiation of cultured human periosteum-derived cells. In this study, we used dorsomorphin, a selective small molecule inhibitor of BMP signaling, to investigate whether BMP signaling is involved in the positive effects of PPARγ agonists on osteogenic phenotypes of cultured human periosteum-derived cells. Both histochemical detection and bioactivity of ALP were clearly increased in the periosteum-derived cells treated with the PPARγ agonist at day 10 of culture. Treatment with the PPARγ agonist also caused an increase in alizarin red S staining and calcium content in the periosteum-derived osteoblasts at 2 and 3 weeks of culture. In contrast, dorsomorphin markedly decreased ALP activity, alizarin red S staining and calcium content in both the cells treated with PPARγ agonist and the cells cultured in osteogenic induction media without PPARγ agonist during the culture period. In addition, the PPARγ agonist clearly increased osteogenic differentiation medium-induced BMP-2 upregulation in the periosteum-derived osteoblastic cells at 2 weeks of culture as determined by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), immunoblotting, and immunocytochemical analyses. Although further study will be needed to clarify the mechanisms of PPARγ-regulated osteogenesis, our results suggest that the positive effects of a PPARγ agonist on the osteogenic phenotypes of cultured human periosteum-derived cells seem to be dependent on BMP signaling.

Effects of prunetin on the proteolytic activity, secretion and gene expression of MMP-3 in vitro and production of MMP-3 in vivo.

We investigated whether prunetin affects the proteolytic activity, secretion, and gene expression of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as in vivo production of MMP-3 in the rat knee joint to evaluate the potential chondroprotective eff ect of prunetin. Rabbit articular chondrocytes were cultured in a monolayer, and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure interleukin-1ß (IL-1ß)-induced expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), and ADAMTS-5. In rabbit articular chondrocytes, the effects of prunetin on IL-1ß-induced secretion and proteolytic activity of MMP-3 were investigated using western blot analysis and casein zymography, respectively. The eff ect of prunetin on MMP-3 protein production was also examined in vivo. The results were as follows: (1) prunetin inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5; (2) prunetin inhibited the secretion and proteolytic activity of MMP-3; (3) prunetin suppressed the production of MMP-3 protein in vivo. These results suggest that prunetin can regulate the gene expression, secretion, and proteolytic activity of MMP-3, by directly acting on articular chondrocytes.