Associations of LRP5 Gene With Bone Mineral Density, Bone Turnover Markers, and Fractures in the Elderly With Osteoporosis.

Objective: The study aimed to explore the associations of rs4988300 and rs634008 in the low-density lipoprotein receptor-related protein 5 (LRP5) gene with bone mineral density (BMD), bone turnover markers (BTM), and fractures in elderly patients with osteoporosis (OP). Methods: Our study included 328 unrelated OP patients with or without fractures. Genomic DNA was extracted for genotyping. BTM levels were assessed by electrochemiluminescence (ECL). Dual-energy X-ray absorptiometry (DXA) was employed to measure BMD in the lumbar spine (LS) and proximal femur. Basic features between the OP and fracture groups were analyzed using the t-test. The Chi-square test was performed to analyze the differences in allele and genotype frequencies. The associations of single-nucleotide polymorphisms (SNPs) with BMD and BTM in the subgroups were investigated by the analysis of covariance (ANCOVA) adjusted for confounding factors. Results: In both females and males, individuals with fractures exhibited higher BTM levels and lower BMD values than those with OP (P < 0.05). The allele and genotype frequencies of rs4988300 in the subgroups were significantly different (P < 0.05). In both females and males suffering from OP, participants with rs4988300 GG or rs634008 TT presented lower procollagen I N-terminal propeptide (PINP) levels (P < 0.05). Women with OP carrying rs4988300 GG exhibited lower BMD values at FN and TH (P < 0.05). In both females and males with fractures, individuals carrying rs4988300 GG genotype or rs634008 TT genotype exhibited lower PINP levels and BMD values at FN and TH than those with other genotypes (P < 0.05). Conclusions: Rs4988300 and rs634008 polymorphisms in the LRP5 gene are associated with bone phenotypes in the elderly with OP or fractures.

In vitro Flow Perfusion Maintaining Long-term Viability of the Rat Groin Fat Flap: A Novel Model for Research on Large-scale Engineered Tissues.

BACKGROUND: Large-scale muscle tissue engineering remains a major challenge. An axial vascular pedicle and perfusion bioreactor are necessary for the development and maintenance of large-scale engineered muscle to ensure circulation within the construct. We aimed to develop a novel experimental model of a large-scale engineered muscle flap from an existing rat groin fat flap. METHODS: A fat flap based on the superficial inferior epigastric vascular pedicle was excised from rats and placed into a perfusion bioreactor. The flaps were kept in the bioreactor for up to 7 weeks, and transdifferentiation of adipose to muscle tissue could have taken place. This system enabled myogenic-differentiation medium flow through the bioreactor at constant pH and oxygen concentration. Assessment of viability was performed by an immunofluorescence assay, histological staining, a calcein-based live/dead test, and through determination of RNA quantity and quality after 1, 3, 5, and 7 weeks. RESULTS: Immunofluorescence staining showed that smooth muscle around vessels was still intact without signs of necrosis or atrophy. The visual assessment of viability by the calcein-based live/dead test revealed viability of the rat adipose tissue preserved in the bioreactor system with permanent perfusion. RNA samples from different experimental conditions were quantified by spectrophotometry, and intact bands of 18S and 28S rRNA were detected by gel electrophoresis, indicating that degradation of RNA was minimal. CONCLUSIONS: Flow perfusion maintains the long-term viability of a rat groin engineered muscle flap in vitro, and a large-scale vascularized muscle could be engineered in a perfusion bioreactor.

[Variations in expressions of periostin and related factors in early stage of wound healing and scar remodeling in rats].

OBJECTIVE: To investigate the expressions of periostin (PN), angiopoietin-1 (Ang-1), vascular epithelial growth factor (VEGF) and fetal liver kinase-1 (Flk-1) during the processes of scar formation and modulation in rat cutaneous wounds and probe into their roles in wound healing and scaring. METHODS: Eighty-two male Sprague-Dawley (SD) rats were randomly divided into 10 groups with 8-9 rats in each group. Two 2 cm×2 cm full-thickness excisional wounds in the back were created in each rat. The wound surface was observed, and the healing area was measured. The pathological change was observed after hematoxylin and eosin (HE) staining. The expressions of PN, Ang-1, VEGF and Flk-1 in wound surface scar at 4-8 weeks were determined with immunohistochemistry. The expressions of PN, Ang-1 and VEGF were determined by Western blotting. The normal skin was served as control. RESULTS: HE staining showed that the wound surface tissue had healed with epithelization at 4-8 weeks. Immunohistochemistry results showed that there was no significant difference in Flk-1 expression between wound surface tissue and normal skin. The PN expression (A value/µm(2)) in wound surface tissue was significantly lower than that in normal skin at 5 weeks (2.43±0.44 vs. 4.24±0.50, P<0.05), and the expression of Ang-1 and VEGF (A value/µm(2)) at 4, 5, 6, 8 weeks was significantly lower than that in normal skin (Ang-1: 3.51±0.93, 3.10±0.57, 2.77±0.59, 2.77±1.26 vs. 4.89±0.48; VEGF: 1.76±0.68, 1.75±0.49, 1.99±0.42, 1.94±0.86 vs. 4.86±1.63, all P<0.05). In wound surface scar, PN and Flk-1 positive signal was found in cell, and the Ang-1 and VEGF positive signal in extracellular matrix. Western blotting data demonstrated that the expressions of PN, Ang-1 and VEGF peaked at the 10th day after excision with increases to 7.90-22.56 folds compared with normal skin (PN: 2.45±1.51 vs. 0.31±0.19, Ang-1: 18.43±15.20 vs. 1.53±1.42, VEGF: 6.09±4.66 vs. 0.27±0.13, P<0.05 or P<0.01), and then followed with a decrease. CONCLUSIONS: PN, Ang-1, VEGF and Flk-1 are transiently overexpressed in early stage of full-thickness cutaneous wound healing in rats. Their expressions vary in wounds and scars. They participate in the healing of full-thickness cutaneous wounds together and may be essential for the proliferation stage during wound healing.

Biological characteristics of human adipose-derived stem cells and their response to periostin in vitro.

BACKGROUND: Many studies on periostin have focused on its role in tumors and vascular reconstruction. However, the effect of periostin on stem cell function remains unclear. The aim of this study was to enhance vitality in adipose-derived stem cells (ADSCs), the effect of periostin on the function of ADSCs was observed. METHODS: Human ADSCs (hADSCs) were isolated from human adipose tissue by collagenase I digestion and collected in multi-periods for in vitro culture. CD29, CD34, CD44, CD45 and CD105 were detected by flow cytometry. In addition, directed differentiation of hADSCs was induced using adipogenic, osteogenic and chondrogenic induction mediums. The induced morphological changes were observed using oil red O, Alizarin red and alcian blue staining. Periostin was administered to hADSCs in an acidic environment. The treatments of cells were divided into three groups: a periostin group (P); an acidic control group (A); a normal group (N). Then the resulting cell proliferation and migration were detected using a Cell Counting Kit-8 (CCK-8) and a transwell chamber assay, respectively. RESULTS: The detection rates of CD29, CD44, CD105, CD34 and CD45 were 98.89%, 93.73%, 86.99%, 0.19% and 0.16%. The specific staining of cells was positive after induction culture. The mean absorbance of the cells in group P and A at 12 hours were 16.67% and 22.22% greater than group N, respectively (P < 0.01). The mean absorbance of cells from group P was 20.00% greater than that of group A at 48 hours (P < 0.05). The mean number of migratory cells per visual field in group A was 50.38% lower than that in group N (P < 0.05). The migratory cell number in group P was 119.98% greater than that in group A (P < 0.05). CONCLUSIONS: The acidic environment impacted hADSC proliferation and inhibited cell migration. However, periostin was able to promote the proliferation and migration of hADSCs despite the acidic environment.

[The expressions of periostin and the related factors during healing process of full-thickness cutaneous wound in rat].

OBJECTIVE: To observe the expression of osteoblast-specific factor 2 (periostin, PN), angiopoietin-1 (Ang-1), vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 [VEGFR-2/fetal liver kinase-1 (FLK-1)] in wound surface and its peripheral skin, and their effects on wound healing in rats. METHODS: Forty-eight Sprague-Dawley (SD) rats were randomly divided into six groups, with 8 rats in each group. An area of 2 cm×2 cm full-thickness skin was excised on both sides of the back of rats. Specimens from wounds were obtained on 1, 4, 7, 10, 14, 21 days after operation, and histological evaluation and immunohistochemical staining of PN, Ang-1, VEGF and FLK-1 were made to determine their expression levels. Normal skin specimens were obtained as normal controls. RESULTS: The expressions of PN, Ang-1, VEGF and FLK-1 were significantly increased in wound surface after operation. Compared with the skin of normal controls, the expression of PN in the tissues of wound increased by 234.4% on the 1st day, and then increased continuously up to 597.9% on the 7th day (reaching the peak) after operation, followed by a decrease, the increase rate was 280.9% on the 21st day, and still remained at a high level (all P < 0.05). The expression of Ang-1 in the tissue of wound increased by 128.1% on the 1st day and 327.5% on the 4th day (reaching the peak), and then, it was gradually decreased. The increase rate was only 80.5% on the 14th day and it rose slightly later (all P < 0.05). The expression of VEGF in the tissues of wound reached the peak (165.8%) on the 7th day. Then it decreased with a slight fluctuation (all P < 0.05). The expression of FLK-1 in the tissues of wound was increased by 56.1% on the 1st day, and the level remained. It reached the peak by an increase of 70.1% on the 7th day (both P < 0.05). Then, it was lowered after the 10th day (all P > 0.05). CONCLUSIONS: The expressions of PN, Ang-1, VEGF, FLK-1 were obviously increased during healing of skin wound, with different peaking time and expressing rates. The increase in expression of PN showed the longest duration and highest peak value. The PN, Ang-1, VEGF, FLK-1 all play a role in the wound healing process, and PN might play an important role during the healing process of a full-thickness cutaneous wound.

[Effect of periostin on the function of human umbilical vein endothelial cells in acidic environment].

OBJECTIVE: To observe the effect of recombinant periostin protein on the proliferation, apoptosis and migration of human umbilical vein endothelial cells (HUVECs) in acidic environment. METHODS: HUVECs were divided into three groups: normal control (pH 7.31±0.03), acidic control (pH 6.95±0.05) and experimental groups [(periostin 100 µg/L, (pH 6.95±0.05)]. The cellular proliferation,apoptosis and migration were measured by Cell Counting Kit-8(CCK-8) assay, Annexin V-FITC with Flow Cytometer(at 60 h after periostin was given) and Transwell chamber assay (at 20 h after periostin was given). RESULTS: In CCK-8 assay, the optical density values D450 nm (at 72 h after periostin was given) of normal control, acidic control and experimental groups were 0.71± 0.05, 0.62±0.04, 0.69±0.02, respectively. In the same order, the total apoptotic rate (%) of the three groups were 13.06±1.35, 16.95±0.46, 12.97±1.60, respectively; the numbers of migrated cells of the three groups were 67.45±11.88,44.89±8.67,64.60±9.63, respectively. Compared with normal control, the D(450 nm) value and the number of migrated cells of acidic control reduced 12.68% (P<0.01) and 33.44% (P<0.01) respectively. Otherwise, the total apoptotic rate (%) increased by 29.79% (P<0.05). Compared with acidic control, the D(450 nm) value and the number of migrated cells of experimental group increased by 11.29% (P<0.01) and 43.90% (P<0.01), respectively. Meantime, the total apoptotic rate (%) reduced 23.48% (P<0.05). CONCLUSION: In acidic environment, the HUVECs' abilities of proliferation and migration were lower; while, the apoptotic value was raised. The recombinant periostin protein could counteract the acidic influence by enhancing the cellular abilities of proliferation and migration. Periostin could make endothelial cells reisist acidosis and keep growing to form blood vessels so that the local histiocytes could get enough nutrients for survival.

[Literature mining and bioinformatic analysis of dysregulated genes in hypertrophic scar].

OBJECTIVE: To explore the pathogenesis mechanism of hypertrophic scar (HS) and the effective means for its clinical treatment, the difference of the gene expressions between HS and normal skin was compared. METHODS: The differentially expressed genes between HS and normal skin were obtained by mining PubMed. The dysregulated genes in HS were analyzed by a series of bioinformatics methods, including protein-protein interaction networks, pathways, Gene Ontology and functional annotation clustering analysis. RESULTS: A total of 55 dysregulated genes in HS was identified (46 up-regulated genes and 9 down-regulated genes). Fifty-one genes were found to encode proteins with interaction network, including up-regulated genes TGFB1, FN1, JUN, COL1A1, CTGF, VEGFA, FOS, COL3A1, IGF1, IL4, PELO, SMAD2, TIMP1, PCNA, and ITGA4 and down-regulated genes ITGB1 and DCN as the central nodes for this network. The dysregulated genes in HS involved in a variety of biological pathways, such as focal adhesion formation, integrin signal transduction, and tumor formation. Furthermore, the dysregulated genes in HS played the important roles in biological processes of cell surface receptor linked signal transduction, tissue development, cell proliferation and apoptosis, and macromolecule biosynthetic process, as well as in molecular function of calcium ion binding, double-stranded DNA binding, heparin binding, promoter binding and MAP kinase activity. The results of functional annotation clustering analysis revealed that the dysregulated genes in HS involved in epidermis development, angiogenesis, and apoptosis. CONCLUSION: Such key genes as TGFB1, FN1, and JUN, along with the pathways, biological processes and molecular functions involving epidermis development, angiogenesis, and extracellular matrix-integrin-focal adhesion signal transduction may play the important roles in the development of HS. The investigations of the dysregulated genes in HS could provide the new targets for clinical treatment.

[Construction of periostin shRNA vectors and their effects on the expression of periostin in fibroblasts].

OBJECTIVE: For studying the functions of periostin (POSTN) further, the recombinant plasmid and lentiviral vector for human periostin were constructed. The mRNA and protein expressions of POSTN were tested after the infection with the shRNA plasmid and lentivirus of POSTN in primary keloid fibroblasts (KFb). METHODS: We designed a specific sequence of small hair RNA targeting POSTN gene which containing both sense and antisense oligo DNA of the targeting sequence. It was cloned into the vectors to construct a plasmid and a lentiviral vector. The plasmid was converted into the competent DH5α E.coli, which confirmed by PCR and sequencing. Then the viral vector and the packed systemic vector were co-transfected 293T cells to get the lentivirus. KFb was infected with the plasmid and lentivirus. The expressions of POSTN in KFb were detected by RT-PCR and Western blotting. MTT was used to examine the proliferation of KFb and SFb. RESULTS: The expression of POSTN mRNA in KFb infected with plasmid reduced 43% compared with negative control cells (1.98±0.03 vs 1.12±0.03, F=688.291, P<0.001). The mRNA and protein levels of POSTN in KFb infected with the lentivirus reduced 46% (0.44±0.09 vs 0.81±0.07, F=90.06, P<0.001) and 45% (0.29±0.04 vs 0.53±0.09, F=33.43, P<0.001), respectively, compared with negative control cells. Compared with negative control cells, the decreased proliferation rates were 19%, 14%, 18% in 48 h, 72 h, 96 h, respectively in KFb infected with the lentivirus. CONCLUSION: The expression of POSTN in primary keloid fibroblasts reduced after infecting plasmid or lentiviral shRNA expression vector targeting human POSTN, which will be a basis to the further function study of POSTN.

Inhibition of periostin gene expression via RNA interference suppressed the proliferation, apoptosis and invasion in U2OS cells.

BACKGROUND: Periostin originally designated osteoblast-specific factor 2 (OSF-2) is frequently found to be highly expressed in various types of human cancer cell lines in vitro and human cancer tissues in vivo. We proposed that periostin was a key factor during the process of proliferation and invasion in cancer cells. We investigated the effect of periostin on the function of human osteosarcoma cell line (U2OS), such as proliferation, apoptosis, invasion and the associated signal pathway. METHODS: A human PGCsi/U6 promoter-driven DNA template was adopted to induce short hairpin RNA (shRNA)-triggered RNA interference (RNAi) to block periostin gene expression in the cell line U2OS. U2OS cells were divided into three groups: cells transfected with phosphate buffered saline as control group (the U2OS group), cells transfected with pGCsi as negative control group (the NC group) and cells transfected with periostin/pGCsi as experimental group (the pGCsi-periostin group). Then, transfection efficiency of cell was observed under fluorescent microscope. The expressions of periostin and the related genes in cells were detected by reverse transcription polymerase chain reaction and Western Blotting. Cell viability was determined using the methyl-thiazolyl tetrazolium bromide (MTT) quantitative colorimetric assay. The invasion and migration capability of cells were tested by transwell plates with or without extracellular matrix gel. Furthermore, the changes of cell cycle and apoptosis were analyzed by flow cytometry. RESULTS: The transfection efficiency of periostin/pGCsi to U2OS cells was about 70% - 80%. When compared with the NC group, the levels of mRNA and protein of periostin in the pGCsi-periostin group decreased by 82% (F = 564.71, P < 0.001) and 58% (F = 341.51, P < 0.001), respectively. Meantime, the earlier apoptosis value increased by 417% (F = 28.69, P < 0.001). The percentage of S phase pGCsi-periostin cells decreased by 21% (F = 47.00, P < 0.001), however, that of G0 - G1 phase cells increased by 12% (F = 14.50, P < 0.001). The capability of migration and invasion reduced by 41% (F = 17.79, P < 0.001) and 72% (F = 197.08, P < 0.001), respectively. The cell proliferation in the pGCsi-periostin group decreased by 59% and 72% at 48 and 120 hours after transfection, respectively. The mRNA expressions of transforming growth factor-ß and vascular endothelial growth factor decreased by 17% (F = 73.99, P < 0.001) and 47% (F = 30.25, P < 0.001), respectively. A tendency of lower focal adhesion kinase (FAK) was shown in pGCsi-periostin cells but without any statistically significant difference. Otherwise the expression of p-FAK in those cells had markedly decreased by 21% (F = 16.81, P < 0.001). CONCLUSIONS: RNAi against periostin can effectively down-regulate periostin gene expression. Periostin increases the hyperplasia and invasion of cancer cells. Periostin might be involved in and served as a tumor promoter gene in the pathogenesis of osteosarcoma.

[Expression of periostin and the effect of hydrocortisone on it in human fibroblasts of scar].

OBJECTIVE: To probe into the role of periostin in the formation of scars and investigate its reaction to hydrocortisone. METHODS: Primary fibroblasts were cultured in vitro, then RT-PCR and immunocytochemical technique were used to examine the expressions of mRNA and preotein of periostin respectively in 24 samples of keloid fibroblasts (KFb), hypertrophic scar fibroblasts (HFb) and normal skin fibroblasts (SFb). RESULTS: The mRNA levels of periostin in KFb (1.645+/-0.549) and HFb (1.084+/-0.396) were both higher than that in SFb (0.274+/-0.215, P<0.05). After hydrocortisone was added, the mRNA levels of periostin in KFb and HFb were both decreased by 32% and 47% respectively (P<0.05 ); The protein of periostin distributed mainly in the endochylema around the nucleus of fibroblasts. The protein levels of periostin in KFb (91.815+/-0.961) and HFb (70.166+/-2.250) were both higher than that in SFb (41.011 +/- 1.576, P<0.01). CONCLUSION: The expressions of periostin were increased in the fibroblasts of hyperplasic scars, which could be inhibited by hydrocortisone. periostin may affect the function of fibroblasts of hyperplasic scars. periostin may be a cicatrix specific gene.

[The expression of fibrillin 1 in pathologic scars and its significance].

OBJECTIVE: To probe into the mechanism of fibrillin 1 in pathologic scar, by examining the expressions of fibrillin 1 and TGF-beta1 as well as their correlations in the tissues of keloid, hypertrophic scar and normal skin. METHODS: The tissues of keloid, hypertrophic scar and normal skin were tested. RT-PCR was used to assess the mRNA expression levels of the aimed genes. The distribution of fibrillin 1 in scars and normal skin was examined by immunohistochemistry staining. RESULTS: The mRNA level of fibrillin 1 in keloid (0.802 +/- 0.116) was increased by 218.25% (P < 0.01) than that in normal skin (0.252 +/- 0.067). The expression of the gene in hypertrophic scar (0.628 +/- 0.144) was higher by 149.21% (while, P > 0.05) than that in normal skin. The expression of TGF-beta1 in keloid and hypertrophic scar were more than that in normal skin. The expression of fibrillin 1 was related to that of TGF-beta1 positively (r = 0.820, P < 0.01). Fibrillin 1 protein was stained positively in basic membranes, endothelial cells, fibroblasts and extracellular matrix of skin tissues. In dermal, the protein levels of fibrillin 1 in keloid (0.117 +/- 0.042) was decreased than those in normal skin (0.185 +/- 0.043) and hypertrophic scar (0.181 +/- 0.048), the inhibition rates were 36.76%, 35.36% respectively (both P < 0.01). CONCLUSIONS: The expression of fibrillin 1 in keloid was changed and related to the expression of TGF-beta1 positively, which appears that fibrillin 1 was a cicatrix specific gene. Fibrillin 1 might play an important role in the formation of keloid.

[The expression of periostin in hyperplasic scars and the relations to TGF-beta1 and its receptors].

OBJECTIVE: To probe into periostin's role in the pathological mechanism of hyperplasic scars, by examining the expression of periostin in hyperplasic scar tissues. To investigate the correlations between periostin and TGF-beta1, TGF-beta R I, TGF-beta R II. METHODS: RT-PCR was used to assess the mRNA expression levels of TGF-beta1, TGF-beta R I, TGF-beta R II in three kinds of tissues, which are keloid (K), hypertrophic scar (HS) and normal skin (SK). The protein expression of periostin was measured with Western blotting. RESULTS: The mRNA level of periostin in K was higher than that in SK. The mRNA expression of TGF-beta1 in K was higher than that in HS and SK. The mRNA level of TGF-beta R I in K was higher than that in HS and SK. The significances above all was at P < 0.01. The protein expression level of periostin in HS increased, compared with that in SK (P < 0.05). Periostin was related to TGF-beta1 positively (P <0.01). CONCLUSIONS: The periostin's expression is increased in keloids. Periostin is a cicatrix specific gene. Periostin appears to play an important role in the formation of keloids, which is related to TGF-beta1 closely.

[Expression of thymosin beta 4 mRNA expression in keloid tissues and fibroblasts cultured from keloid and its significance].

OBJECTIVE: To investigate the relations between thymosin beta 4 and pathologic scars by comparing the mRNA levels of thymosin beta 4 in keloid, hypertrophic scar and normal skin. METHODS: The primary fibroblasts from the patients of keloid (KFB), hypertrophic scar (HFB) and normal skin (NFB, n=7) were cultured in vitro with tissue culture system. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay was used to assess the thymosin beta 4 mRNA levels in the tissues and fibroblasts obtained from the patients of three groups. RESULTS: The mRNA levels of thymosin beta 4 in the keloid tissues were lower than those in the tissues of hypertrophic scar and normal skin(both, P<0.01). The thymosin beta 4 mRNA level in keloid group was lower by 66.98% than hypertrophic scar, and 62.48% than normal skin. In addition, the same significant change was found in the cultured KFB compared with HFB group(the mean value was lower by 27.13%), but no difference was found between KFB and NFB. CONCLUSION: Expression of thymosin beta 4 is closely related with keloid. The inadequate expression of thymosin beta 4 may be one of the key factors for keloid formation.