Differential regulation of collagen, lysyl oxidase and MMP-2 in human periodontal ligament cells by low- and high-level mechanical stretching.

BACKGROUND AND OBJECTIVE: Mechanical stretching modulates extracellular matrix (ECM) protein synthesis by periodontal ligament (PDL) cells. However, the mechanoregulation of lysyl oxidase (LOX), a key enzyme for collagen cross-linking, is not fully understood. In the present study, we hypothesized that low-level and high-level mechanical stretching differentially regulates collagen deposition and the expression of LOX and the enzymes responsible for ECM degradation, such as MMP-2 in PDL cells. MATERIAL AND METHODS: Human PDL cells were cultured on flexible-bottom culture plates and subjected to cyclic mechanical stretching (3% and 10% elongation at 0.1 Hz) for 24 and 48 h in a Flexercell FX-4000 strain unit. The levels of expression of type I collagen alpha 1 (COL1A1), type III collagen alpha 1 (COL3A1), lysyl oxidase (LOX), MMP2 and TIMP2 mRNAs were analyzed using an RT-PCR technique. The cell layer and the culture medium were separately collected and processed for detection of the following ECM-related molecules: (i) total collagen content using a Sircol dye-binding method; (ii) LOX protein expression by western blotting; (iii) LOX activity using a fluorometric assay; and (iv) MMP-2 enzyme activity by gelatin zymography. RESULTS: Low-level (3%) mechanical stretching of PDL cells upregulated the expression of COL1A1, COL3A1 and LOX mRNAs, enhanced the production of collagen and increased the LOX activity but did not change the level of expression of MMP2 or TIMP2 mRNA. The collagen content and LOX activity showed obvious elevation in the medium, but not in the cell layer. High-level (10%) mechanical stretching downregulated COL1A1 mRNA but upregulated COL3A1 mRNA; however, the effect on COL3A1 was smaller, and occurred earlier, compared with the effect on the COL1A1 gene. High-level mechanical stretching upregulated the expression of MMP2 and TIMP2 mRNAs but did not change collagen production or LOX activity. Moreover, high-level mechanical stretching increased the level of pro-MMP-2, especially in the cell layer. CONCLUSIONS: This study substantiates the mechanoregulation of the expression of ECM-related molecules in PDL cells. High-level mechanical stretching upregulated the expression of MMP2 and TIMP2 mRNAs, but did not affect collagen production or LOX activity. In addition to increasing the transcription of COL1A1, COL3A1 and LOX genes, low-level mechanical stretching enhanced total collagen production and LOX activity, which should favor ECM stabilization. As an effective regulator of ECM remodeling, mechanical stretching can be exploited in periodontal regeneration and ligament tissue engineering via application of appropriate mechanical stimulation.

Human opioid µ-receptor A118G polymorphism may protect against central pruritus by epidural morphine for post-cesarean analgesia.

BACKGROUND: Intrathecal or epidural morphine used for post-operative analgesia frequently induces central type pruritus. The purpose of this study was to investigate the association between the severity of central type pruritus induced by epidural morphine for post-cesarean analgesia and the A118G polymorphism of the human µ-opioid receptor gene (OPRM1). METHODS: Pregnant women (212) received pure epidural morphine (2 mg) twice per day for post-cesarean analgesia. Blood samples were collected and sequenced with high-resolution melting analysis to detect three different genotypes of OPRM1 (AA, AG and GG). We interviewed all candidates 24 h post-operatively to record the clinical phenotype with subjective complaints and objective observations. RESULTS: The genotyping revealed that 99 women (46.7%) were AA, 88 (41.5%) were AG and 25 (11.8%) were GG. Sixty-two of 212 women suffered from significant pruritus (29.2%), and 150 of 212 women had non-significant pruritus (70.8%). In genotype AA, 33 patients (53.2%) experienced significant pruritus, 26 (41.9%) in genotype AG and 3 (4.8%) in genotype GG. The G allele was a statistically independent protective factor for individuals developing pruritus, and the multivariate-adjusted odds ratio was 0.27. There was a trend for progressively decreasing severity scores among the three groups, with the lowest severity score (0.72) for pruritus in the GG group. CONCLUSIONS: The incidence of significant pruritus in the recessive type (GG) was significantly lower compared with the dominant types (AA+AG). The recessive G allele in the A118G polymorphism may have protective effects against significant pruritus after epidural morphine for post-cesarean analgesia.