Verbascoside Inhibits/Repairs the Damage of LPS-Induced Inflammation by Regulating Apoptosis, Oxidative Stress, and Bone Remodeling.

Osteocytes play an important role as regulators of both osteoclasts and osteoblasts, and some proteins that are secreted from them play a role in bone remodeling and modeling. LPS affects bone structure because it is an inflammatory factor, despite verbascoside's potential for bone preservation and healing. Osteocytes may also be involved in the control of the bone's response to immunological changes in inflammatory situations. MLO-Y4 cells were cultured in either supplemented -MEM alone with a low serum to inhibit cell growth or media with LPS (10 ng/mL) and/or verbascoside (50 g/mL) to show the LPS effect. In our research, LPS treatment increased RANKL levels while decreasing OPG and RUNX2 expression. Treatment with verbascoside reduced RANKL expression. In our work, verbascoside increased the expression of OPG and RUNX2. In MLO-Y4 cells exposed to verbascoside, SOD, CAT, and GSH activities as well as the expression levels of bone mineralization proteins like PHEX, RUNX2, and OPG were all elevated.

Resveratrol prevents ovariectomy-induced bone quality deterioration by improving the microarchitectural and biophysicochemical properties of bone.

INTRODUCTION: Osteoporosis is a major health problem that is very common worldwide and is characterized by both low bone density and deterioration in bone quality. New treatment options without side effects have become an active area of research in recent years. This study was designed to investigate the preventive effects of resveratrol on bone quality deterioration caused by ovariectomy. MATERIALS AND METHODS: Sixty rats were randomly divided into five groups (12 animals per group): Control, Sham-operated (SHAM), ovariectomized (OVX), OVX + Resveratrol-40 mg/kg/day (OVX + Res40), OVX + Resveratrol-80 mg/kg/day (OVX + Res80). Resveratrol was administered by oral gavage (40 and 80 mg/kg/day) for ten weeks. Micro-CT measurements, biomechanical testing, Raman spectroscopy analysis, and RT-PCR analysis were performed. ALP, OCN, TAS, and TOS levels were also measured from blood serum. RESULTS: Bone strength, bone volume/total volume, trabecular volume, and trabecular thickness were higher in the OVX + RES-80 group than in the OVX group. Resveratrol increased osteogenic differentiation, as the expression of osteogenic markers ALP, Col1A1, Runx2, OPG, OCN increased in both OVX + RES-80 and OVX + RES-40 groups compared to the OVX group. 80 mg/kg/day resveratrol administration decreased the levels of ALP, OCN and TOS in ovariectomized rats. Raman spectroscopy findings showed a preventive effect of resveratrol administration against ovariectomy-induced deterioration in biophysiochemical properties of bone tissue. CONCLUSION: This study revealed that administration of different doses of 80 mg/kg/day and 40 mg/kg/day of resveratrol had protective effects on bone quality deterioration caused by ovariectomy.

Calcium­dependent activation of PHEX, MEPE and DMP1 in osteocytes.

Calcium (Ca2+) signaling is the first messenger signal exhibited by osteocytes. The present study aimed to better understand the link between Ca2+ concentration, and the levels of bone mineralization regulator proteins [phosphate­regulating neutral endopeptidase on chromosome X (PHEX), matrix extracellular phosphoglycoprotein (MEPE) and dentin matrix protein 1 (DMP1)] and the levels of oxidative stress in osteocytes. The viability of MLO­Y4 cells was determined using the live/dead assay following treatment with various Ca2+ concentrations (1.8, 6, 12, 18, 24 and 50 mM) for different durations (15 and 60 min, and 24 h). Superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and NADPH oxidase (NOX) enzymes were analyzed using a colorimetric method. Apoptosis was detected by caspase­3 analysis. Furthermore, the protein expression levels of PHEX, MEPE and DMP1 were analyzed using immunoblotting, and oxidative stress was examined using the total antioxidant and total oxidant status (TOS) assay. Notably, after 15 min, there were more live cells than dead cells; however, after 60 min, the number of dead cells was increased following treatment with 24 and 50 mM Ca2+. After 24 h, there were more dead cells than live cells following treatment with 50 mM Ca2+. After 24 h of Ca2+ treatment, the highest protein expression levels of PHEX, MEPE and DMP1 were measured in cells treated with 24 mM Ca2+. In addition, as Ca2+ concentration increased, the TOS and the oxidative stress index values were also increased. In conclusion, these results suggested that 24 mM Ca2+ may trigger bone mineralization proteins, such as PHEX, MEPE and DMP1, and could be considered an applicable dosage for the treatment of bone damage in the future.

Effects of PDGF-BB delivery from heparinized collagen sutures on the healing of lacerated chicken flexor tendon in vivo.

Flexor tendon lacerations are traditionally repaired by using non-absorbable monofilament sutures. Recent investigations have explored to improve the healing process by growth factor delivery from the sutures. However, it is difficult to conjugate growth factors to nylon or other synthetic sutures. This study explores the performance of a novel electrochemically aligned collagen suture in a flexor tendon repair model with and without platelet derived growth factor following complete tendon laceration in vivo. Collagen suture was fabricated via electrochemical alignment process. Heparin was covalently bound to electrochemically aligned collagen sutures (ELAS) to facilitate affinity bound delivery of platelet-derived growth factor-BB (PDGF-BB). Complete laceration of the flexor digitorum profundus in the third digit of the foot was performed in 36 skeletally mature White Leghorn chickens. The left foot was used as the positive control. Animals were randomly divided into three groups: control specimens treated with standard nylon suture (n=12), specimens repaired with heparinated ELAS suture without PDGF-BB (n=12) and specimens repaired with heparinated ELAS suture with affinity bound PDGF-BB (n=12). Specimens were harvested at either 4weeks or 12weeks following tendon repair. Differences between groups were evaluated by the degree of gross tendon excursion, failure load/stress, stiffness/modulus, absorbed energy at failure, elongation/strain at failure. Quantitative histological scoring was performed to assess cellularity and vascularity. Closed flexion angle measurements demonstrated no significant differences in tendon excursion between the study groups at 4 or 12weeks. Biomechanical testing showed that the group treated with PDGF-BB bound heparinated ELAS suture had significantly higher stiffness and failure load (p<0.05) at 12-weeks relative to both heparinated ELAS suture and nylon suture. Similarly, the group treated with PDGF-BB bound suture had significantly higher ultimate tensile strength and Young's modulus (p<0.05) at 12-weeks relative to both ELAS suture and nylon suture. Compared to nylon controls, heparinized ELAS with PDGF-BB improved biomechanics and vascularity during tendon healing by 12-weeks following primary repair. The ability of ELAS to deliver PDGF-BB to the lacerated area of tendon presents investigators with a functional bioinductive platform to improve repair outcomes following flexor tendon repair. STATEMENT OF SIGNIFICANCE: A high strength aligned collagen suture was fabricated via linear electrocompaction and heparinized for prolonged delivery of PDFG-BB. When it was used to suture a complete lacerated flexor tendon in a chicken model controlled release of the PDGF-BB improved the strength of treated tendon after 12 weeks compared to tendon sutured with commercial nylon suture. Furthermore, Collagen suture with affinity bound PDGF-BB enhanced the vascularization and remodeling of lacerated tendon when it compare to synthetic nylon suture. Overall, electrocompacted collagen sutures holds potential to improve repair outcome in flexor tendon surgeries by improving repair strength and stiffness, vascularity, and remodeling via sustained delivery of the PDGF-BB. The bioinductive collagen suture introduces a platform for sustained delivery of other growth factors for a wide-array of applications.

Effects of losartan treatment on the physicochemical properties of diabetic rat bone.

Inhibitors of the renin-angiotensin system used to treat several diseases have also been shown to be effective on bone tissue, suggesting that angiotensin-converting enzyme inhibitors and angiotensin receptor blockers may reduce fracture risk. The present study investigated the effects of losartan on the physicochemical and biomechanical properties of diabetic rat bone. Losartan (5 mg/kg/day) was administered via oral gavage for 12 weeks. Bone mineral density (BMD) was measured using dual-energy X-ray absorptiometry. Whole femurs were tested under tension to evaluate the biomechanical properties of bone. The physicochemical properties of bone were analyzed by Fourier transform infrared spectroscopy. Although losartan did not recover decreases in the BMD of diabetic bone, it recovered the physicochemical (mineral and collagen matrix) properties of diabetic rat bone. Furthermore, losartan also recovered ultimate tensile strength of diabetic rat femurs. Losartan, an angiotensin II type 1 receptor blocker, has a therapeutic effect on the physicochemical properties of diabetic bone resulting in improvement of bone strength at the material level. Therefore, specific inhibition of this pathway at the receptor level shows potential as a therapeutic target for diabetic patients suffering from bone diseases such as osteopenia.

Heparinized collagen sutures for sustained delivery of PDGF-BB: Delivery profile and effects on tendon-derived cells In-Vitro.

UNLABELLED: Suturing is the standard of repair for lacerated flexor tendons. Past studies focused on delivering growth factors to the repair site by incorporating growth factors to nylon sutures which are commonly used in the repair procedure. However, conjugation of growth factors to nylon or other synthetic sutures is not straightforward. Collagen holds promise as a suture material by way of providing chemical sites for conjugation of growth factors. On the other hand, collagen also needs to be reconstituted as a mechanically robust thread that can be sutured. In this study, we reconstituted collagen solutions as suturable collagen threads by using linear electrochemical compaction. Prolonged release of PDGF-BB (Platelet derived growth factor-BB) was achieved by covalent bonding of heparin to the collagen sutures. Tensile mechanical tests of collagen sutures before and after chemical modification indicated that the strength of sutures following chemical conjugation stages was not compromised. Strength of lacerated tendons sutured with epitendinous collagen sutures (11.2±0.7N) converged to that of the standard nylon suture (14.9±2.9N). Heparin conjugation of collagen sutures didn't affect viability and proliferation of tendon-derived cells and prolonged the PDGF-BB release up to 15days. Proliferation of cells seeded on PDGF-BB incorporated collagen sutures was about 50% greater than those seeded on plain collagen sutures. Collagen that is released to the media by the cells increased by 120% under the effects of PDGF-BB and collagen production by cells was detectable by histology as of day 21. Addition of PDGF-BB to collagen sutures resulted in a moderate decline in the expression of the tendon-associated markers scleraxis, collagen I, tenomodulin, and COMP; however, expression levels were still greater than the cells seeded on collagen gel. The data indicate that the effects of PDGF-BB on tendon-derived cells mainly occur through increased cell proliferation and that longer term studies are needed to confirm whether this proliferation is outweighs the moderate reduction in the expression of tendon-associated genes. STATEMENT OF SIGNIFICANCE: A mechanically robust pure collagen suture was fabricated via linear electrocompaction and conjugated with heparin for prolonged delivery of PDFG-BB. Sustained delivery of the PDGF-BB improved the proliferation of tendon derived cells substantially at the expense of a moderate downregulation of tenogenic markers. The collagen threads were functionally applicable as epitendinous sutures when applied to chicken flexor tendons in vitro. Overall, electrocompacted collagen sutures holds potential to improve repair outcome in flexor tendon surgeries by improving cellularity and collagen production through delivery of the PDGF-BB. The bioinductive suture concept can be applied to deliver other growth factors for a wide-array of applications.

Effect of sodium tungstate on visual evoked potentials in diabetic rats.

AIM: To evaluate the effect of sodium tungstate on visual evoked potentials (VEPs) in diabetic rats. METHODS: Wistar rats were randomly divided into three groups as normal control, diabetic control and diabetic rats treated with sodium tungstate. Diabetes was induced by single intraperitoneal injection of streptozotocin (50 mg/kg). Sodium tungstate [40 mg/(kg·d)] was administered for 12wk and then VEPs were recorded. Additionally, thiobarbituric acid reactive substance (TBARS) levels were measured in brain tissues. RESULTS: The latencies of P1, N1, P2, N2 and P3 waves were significantly prolonged in diabetic rats compared with control group. Diabetes mellitus caused an increase in the lipid peroxidation process that was accompanied by changes in VEPs. However, prolonged latencies of VEPs for all components returned to control levels in sodium tungstate-treated group. The treatment of sodium tungstate significantly decreased brain TBARS levels and depleted the prolonged latencies of VEP components compared with diabetic control group. CONCLUSION: Sodium tungstate shows protective effects on visual pathway in diabetic rats, and it can be worthy of further study for potential use.

Inhibition of mammalian target of rapamycin signaling pathway decreases retinoic acid stimulated gene 8 expression in adult mouse testis.

OBJECTIVE: To evaluate the expression of mammalian target of rapamycin (mTOR) pathway molecules in mouse spermatogenesis and as well as its role during proliferation and meiotic initiation of spermatogenic cells. DESIGN: Experimental animal study. SETTING: University. ANIMAL(S): C57Balb-C adult male mice. INTERVENTION(S): Expressions of mTOR signaling pathway proteins in adult testis were evaluated. Then the effect of inhibition of this pathway on proliferation and differentiation of spermatogonial stem cells was investigated using seminiferous tubule culture. MAIN OUTCOME MEASURE(S): Immunohistochemistry was performed to evaluate the expressions of mTOR signaling pathway proteins. To inhibit mTOR signaling pathway by rapamycin, seminiferous tubule culture was done. Viability assay and terminal deoxynucleotidyl transferase dUTP nick end labeling was performed to evaluate the culture conditions and to examine cell death, respectively. Western blot was used to determine the expressions of the PCNA, STRA8, and VASA proteins. RESULT(S): Our results showed that spermatogonial stem cells and preleptotene spermatocytes express total mTOR, p-mTOR, total p70S6K, p-p70S6K, and p-4EBP1. Expressions of p-p70S6K, p-4EBP1, PCNA, and STRA8 decreased significantly in the rapamycin-treated group, where no difference was observed in VASA expression. Cell viability and the number of apoptotic cells were similar for all groups. CONCLUSION(S): Our findings suggest that the mTOR signaling pathway may have role in the proliferation and stimulation of meiotic initiation of spermatogonial stem cells. To the best of our knowledge, this is the first ex vivo study that reports the function of the mTOR pathway in adult mouse spermatogenesis.

Effect of angiotensin II type 1 receptor blocker on osteoporotic rat femurs.

BACKGROUND: Osteoblasts and osteoclasts are known to express Ang II type I (AT1) receptor in cell cultures, suggesting the existence of local renin-angiotensin system (RAS) in bone. This study was designed to investigate the effects of losartan as AT1 receptor blocker on ovariectomized rats' femur. METHODS: Losartan (5 mg/kg/day) was administered via oral gavage for 8 weeks. Bone mineral density (BMD) was measured using dual energy X-ray absorptiometry, while tensile and three-point bending tests were performed for evaluation of biomechanical properties of bone. The trabecular porosity was analyzed by scanning electron microscopy. RESULTS: There was a significant decrease in BMD values of ovariectomized rats' femurs which were reversed by losartan treatment. According to tensile test results, ultimate tensile strength and strain values of losartan treated ovariectomized rats' femurs increased and decreased, respectively, when compared to that of ovariectomized animals. Losartan treatment also caused a significant recovery in flexural strength and modulus parameters regarding respective control values, which mean losartan treated ovariectomized rats' femur had more force tolerance until break than ovariectomized rats' femur. Quantitative microscopic analysis showed larger trabecular porosity in ovariectomized rats than control rat femurs and it was significantly decreased after losartan treatment. CONCLUSION: Blockage of AT1 receptor increased strength, mass and trabecular connections of ovariectomized rat femurs. Therefore, it is tempting to speculate that drugs, including AT1 receptor blockers, may be used for the treatment of osteoporosis or reduction of its detrimental effects in the future.