Effects of Different No-Ozone Cold Plasma Treatment Methods on Mouse Osteoblast Proliferation and Differentiation.

Background and Objectives: Enhanced osteoblast differentiation may be leveraged to prevent and treat bone-related diseases such as osteoporosis. No-ozone cold plasma (NCP) treatment is a promising and safe strategy to enhance osteoblast differentiation. Therefore, this study aimed to determine the effectiveness of direct and indirect NCP treatment methods on osteoblast differentiation. Mouse osteoblastic cells (MC3T3-E1) were treated with NCP using different methods, i.e., no NCP treatment (NT group; control), direct NCP treatment (DT group), direct NCP treatment followed by media replacement (MC group), and indirect treatment with NCP-treated media only (PAM group). Materials and Methods: The MC3T3-E1 cells were subsequently assessed for cell proliferation, alkaline phosphatase (ALP) activity, calcium deposition, and ALP and osteocalcin mRNA expression using real-time polymerase chain reaction. Results: Cell proliferation significantly increased in the NCP-treated groups (DT and PAM; MC and PAM) compared to the NT group after 24 h (p < 0.038) and 48 h (p < 0.000). ALP activity was increased in the DT and PAM groups at 1 week (p < 0.115) and in the DT, MC, and PAM groups at 2 weeks (p < 0.000) compared to the NT group. Calcium deposition was higher in the NCP-treated groups than in NT group at 2 and 3 weeks (p < 0.000). ALP mRNA expression peaked in the MC group at 2 weeks compared to the NP group (p < 0.014). Osteocalcin mRNA expression increased in the MC group at 2 weeks (p < 0.000) and was the highest in the PAM group at 3 weeks (p < 0.000). Thus, the effects of direct (DT and MC) and indirect (PAM) treatment varied, with MC direct treatment showing the most significant impact on osteoblast activity. Conclusions: The MC group exhibited enhanced osteoblast differentiation, indicating that direct NCP treatment followed by media replacement is the most effective method for promoting bone formation.

Osteocalcin and cross-linked C-terminal telopeptide of type I collagen in gingival crevicular fluid during piezocision accelerated orthodontic tooth movement: A randomized split-mouth study.

Background: Piezocision, a minimally invasive surgical procedure, has been used to accelerate tooth movement'' is appropriate as a background to the abstract section. Aim: The aim of this randomized split-mouth study was to evaluate gingival crevicular fluid (GCF) osteocalcin (OC) and type I collagen cross-linked C-terminal telopeptide (ICTP) levels during canine distalization with and without piezocision acceleration. Material and Methods: Fifteen systemically healthy subjects (M:F 7:8, 16.27 ± 1.14 years) requiring extraction of maxillary first premolars before retraction of canines were included in the study. Piezocisions were randomly carried out on one of the maxillary canines while bilateral canines served as controls. Canine distalization was conducted using closed-coil springs applying a force of 150 g/side by using miniscrews as anchorage. GCF sampling was performed from maxillary canine mesial and distal sites at baseline, 1, 7, 14, and 28 days. The GCF levels of OC and ICTP were detected by enzyme-linked immunosorbent assay (ELISA). The rate of tooth movement was evaluated at 2-week intervals. Results: The amounts of canine distalization from baseline to 14 and 28 days in the piezocision group were significantly higher than the control group (P < 0.05). The GCF OC level of the piezocision group on the tension side and the ICTP level of the same group on the compression side were higher than the respective sides of the control group on day 14 (P < 0.05). Conclusions: Piezocision was found to be an effective treatment procedure for accelerating canine distalization accompanied by increased levels of OC and ICTP.

Measurement of bioactive osteocalcin in humans using a novel immunoassay reveals association with glucose metabolism and ß-cell function.

Osteocalcin (OCN) is a bone-derived hormone involved in the regulation of glucose metabolism. In serum, OCN exists in carboxylated and uncarboxylated forms (ucOCN), and studies in rodents suggest that ucOCN is the bioactive form of this hormone. Whether this is also the case in humans is unclear, because a reliable assay to measure ucOCN is not available. Here, we established and validated a new immunoassay (ELISA) measuring human ucOCN and used it to determine the level of bioactive OCN in two cohorts of overweight or obese subjects, with or without type 2 diabetes (T2D). The ELISA could specifically detect ucOCN concentrations ranging from 0.037 to 1.8 ng/mL. In a first cohort of overweight or obese postmenopausal women without diabetes (n = 132), ucOCN correlated negatively with fasting glucose (r = -0.18, P = 0.042) and insulin resistance assessed by the homeostatic model assessment of insulin resistance (r = -0.18, P = 0.038) and positively with insulin sensitivity assessed by a hyperinsulinemic-euglycemic clamp (r = 0.18, P = 0.043) or insulin sensitivity index derived from an oral glucose tolerance test (r = 0.26, P = 0.003). In a second cohort of subjects with severe obesity (n = 16), ucOCN was found to be lower in subjects with T2D compared with those without T2D (2.76 ± 0.38 versus 4.52 ± 0.06 ng/mL, P = 0.009) and to negatively correlate with fasting glucose (r = -0.50, P = 0.046) and glycated hemoglobin (r = -0.57, P = 0.021). Moreover, the subjects with ucOCN levels below 3 ng/mL had a reduced insulin secretion rate during a hyperglycemic clamp (P = 0.03). In conclusion, ucOCN measured with this novel and specific assay is inversely associated with insulin resistance and ß-cell dysfunction in humans.

Serum Osteocalcin Level is Negatively Associated with Vascular Reactivity Index by Digital Thermal Monitoring in Kidney Transplant Recipients.

BACKGROUND AND OBJECTIVES: Osteocalcin is the most abundant noncollagenous protein in bone matrix, which is considered a marker of bone formation. Previous studies indicate that circulating osteocalcin can be expressed by osteoblasts and even by osteoblast-like cells in vessel walls, and it is often associated with arterial stiffness. Our study aims to examine the potential association between osteocalcin levels and endothelial function among kidney transplant (KT) recipients. MATERIALS AND METHODS: Fasting blood samples were obtained from 68 KT recipients. To measure the endothelial function and vascular reactivity index (VRI), a digital thermal monitoring test (VENDYS) was used. A commercial enzyme-linked immunosorbent assay kit was also utilized to measure serum total osteocalcin levels. In this study, a VRI of less than 1.0 indicated poor vascular reactivity; a VRI of 1.0-2.0 indicated intermediate vascular reactivity; and a VRI of 2.0 or higher indicated good vascular reactivity. RESULTS: Our findings show that 8 KT recipients (11.8%) had poor vascular reactivity (VRI < 1.0), 26 (38.2%) had intermediate vascular reactivity (1.0 ≤ VRI < 2.0), and 34 (50%) had good vascular reactivity. Increased serum osteocalcin levels (p < 0.001) were found to be associated with poor vascular reactivity. Advanced age (r = -0.361, p = 0.002), serum alkaline phosphate level (r = -0.254, p = 0.037), and log-transformed osteocalcin levels (r = - 0.432, p < 0.001) were identified to be negatively correlated with VRI in KT recipients. Multivariable forward stepwise linear regression analysis revealed that the serum level of osteocalcin (ß = -0.391, adjusted R2 change = 0.174; p < 0.001) and advanced age (ß = -0.308, adjusted R2 change = 0.084; p = 0.005) were significantly and independently associated with VRI in KT recipients. CONCLUSIONS: Higher serum osteocalcin level was associated with lower VRI and poorer endothelial dysfunction among KT recipients.

Trabecular bone score and bone remodelling markers identify perimenopausal women at high risk of bone loss.

CONTEXT: Bone loss is accelerated in the late perimenopause and early menopause. The date of the final menstrual period cannot be stated until 1 year after it has ended, and at that time, most of the rapid bone loss phase will have elapsed. Therefore, early detection of bone loss is crucial. OBJECTIVES: To evaluate the utility of bone turnover markers (BTM) to identify the women who are more likely to lose more bone mass during the transition to menopause and quantify the loss of bone quality measured by trabecular bone score (TBS). DESIGN, PATIENTS AND SETTING: Sixty-four healthy premenopausal women, mean age between 44 and 57 years old, were enrolled and followed up for 5 years. Clinical features, lifestyle, bone densitometry, TBS and BTM (CTX, P1NP and osteocalcin) were measured at baseline and follow-up. RESULTS: All women had densitometrically normal bone at the time of enrolment. After 5 years, 48.4% had normal bone mineral density, 45.8% low bone mass and 6.3% osteoporosis. Women with osteopenia/osteoporosis at follow-up had higher CTX and P1NP at enrolment compared with women with densitometrically normal bone. The areas under the curve for the prediction of low bone mass or osteoporosis were 0.69 (P = 0.011) for P1NP, 0.69 for CTX (P = 0.013) and 0.77 (P 0.001) for OC. A significant correlation was found between P1NP increase after 5 years and the decrease in lumbar bone density (r = -0.383, P = 0.002). At baseline, 7 (10.9%) women had deteriorated microarchitecture (TBS < 1.3). Three of these women developed osteoporosis and four osteopenia at follow-up. CONCLUSIONS: Women with higher P1NP and CTX and lower TBS at baseline had lower BMD in the transition to menopause suggesting these novel tools could have potential use in identifying women at high risk of rapidly decreasing bone mass.

Silicate-substituted strontium apatite nano coating improves osteogenesis around artificial ligament.

BACKGROUND: Treatment of anterior cruciate ligament injuries commonly involves the use of polyethylene terephthalate (PET) artificial ligaments for reconstruction. However, the currently available methods require long fixation periods, thereby necessitating the development of alternative methods to accelerate the healing process between tendons and bones. Thus, we developed and evaluated a novel technique that utilizes silicate-substituted strontium (SrSiP). METHODS: PET films, nano-coated with SrSiP, were prepared. Bone marrow mesenchymal cells (BMSCs) from femurs of male rats were cultured and seeded at a density of 1.0 × 104/cm2 onto the SrSiP-coated and non-coated PET film, and subsequently placed in an osteogenic medium. The osteocalcin concentration secreted into the medium was compared in each case. Next, PET artificial ligament, nano-coated with SrSiP, were prepared. BMSCs were seeded at a density of 4.5 × 105/cm2 onto the SrSiP-coated, and non-coated artificial ligament, and then placed in osteogenic medium. The osteocalcin and calcium concentrations in the culture medium were measured on the 8th, 10th, 12th, and 14th day of culture. Furthermore, mRNA expression of osteocalcin, alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP2), and runt-related transcription factor 2 (Runx2) was evaluated by qPCR. We transplanted the SrSiP-coated and non-coated artificial ligament to the tibiae of mature New Zealand white rabbits. Two months later, we sacrificed them and histologically evaluated them. RESULTS: The secretory osteocalcin concentration in the medium on the film was significantly higher for the SrSiP group than for the non-coated group. Secretory osteocalcin concentration in the medium on the artificial ligament was also significantly higher in the SrSiP group than in the non-coated group on the 14th day. Calcium concentration on the artificial ligament was significantly lower in the SrSiP group than in the non-coated group on the 8th, 10th, 12th, and 14th day. In qPCR as well, OC, ALP, BMP2, and Runx2 mRNA expression were significantly higher in the SrSiP group than in the non-coated group. Newly formed bone was histologically found around the artificial ligament in the SrSiP group. CONCLUSIONS: Our findings demonstrate that artificial ligaments using SrSiP display high osteogenic potential and thus may be efficiently used in future clinical applications.

Homogeneous Noncompetitive Luminescent Immunodetection of Small Molecules by Ternary Protein Fragment Complementation.

The homogeneous immunological detection of small molecules at high sensitivity is still a daunting task. Here, we tried sensitive noncompetitive detection of small peptides based on the open-sandwich immunoassay principle, which was combined with a bioluminescent protein-fragment complementation assay (PCA) in vitro. Since the detection of antigen-induced approximation of the two antibody variable region fragments VH and VL by the standard Nanoluc-based PCA utilizing larger (LgBiT) and shorter (SmBiT) fragments was not successful, we decided to further split LgBiT into two, yielding smaller N-terminal derivative (LnBiT) and two C-terminal, 11 residue peptides (LcBiT and SmBiT) corresponding to consecutive beta strands, to which VH and VL were each fused and expressed in Escherichia coli cells. Through the optimization of reaction conditions and peptide sequence, the antigen osteocalcin peptide can be noncompetitively detected with a low background signal and limit of detection, yielding a high light emission of 88% compared to that of the wild-type enzyme. Since the luminescence of this open sandwich bioluminescent immunoassay (OS-BLIA) can be observed with the naked eye, it could become the foundation of many point-of-care detection systems.

Noncompetitive homogeneous immunodetection of small molecules based on beta-glucuronidase complementation.

In this study, a novel noncompetitive homogeneous immunoassay for antigen detection was developed. We utilized ß-glucuronidase (GUS), a homotetrameric enzyme, the assembly of all of whose subunits is necessary to attain its activity. By using a mutant GUS (GUSm), wherein the dimerization of dimers, which is a rate-limiting step, can be effectively inhibited by a set of interface mutations, we attempted to create a biosensor for detecting various molecules. Usually, the affinity between the two variable region domains (VH and VL) of an antibody, especially for a small molecule, is relatively low. However, in the presence of an antigen, the affinity increases so that they bind tighter to each other. A pair of fusion proteins, comprising the VH and VL regions of the antibody as the detector tethered to a GUSm subunit as the reporter, was constructed to detect antigen 4-hydroxy-3-nitrophenylacetyl (NP) and bone Gla protein (BGP) through GUS activity measurement. Colorimetric and fluorescence assays could detect NP, 5-iodo-NP, and BGP within 1 h without separation steps and with a higher signal/background ratio than conventional ELISA. The instantaneous response after simple mixing of the components makes this system convenient and high-throughput. The system could be effective for the analyses of various small molecules in environmental and clinical settings.

Systemic bone marker expression induced by grey and white mineral trioxide aggregate in normal and diabetic conditions.

AIM: To investigate the relationship between diabetes mellitus and local/systemic effects of both grey and white mineral trioxide aggregate (MTA) Angelus on bone marker expression. METHODOLOGY: Wistar rats were divided into two groups: healthy and diabetic (Alloxan induced), which were further divided into three subgroups (control, GMTA Angelus and WMTA Angelus). Polyethylene tubes filled with MTA materials or empty tubes were implanted in dorsal connective tissue. On days 7 and 30, blood samples were collected for calcium, phosphorus and ALP measurement. The animals were euthanized; implanted tubes were removed and processed for immunohistochemical analysis of osteocalcin (OCN) and osteopontin (OPN). Kruskal-Wallis followed by Dunn's multiple comparison test was performed for nonparametric data, and anova followed by Tukey's test for parametric data. RESULTS: No difference in systemic serum calcium levels between both groups was observed. On day 7, serum phosphorus levels within the WMTA healthy group were higher than that of the diabetic group. On day 30, healthy rats exhibited lower phosphorus levels than diabetic ones. At both time points, the diabetic group was associated with more ALP activity than the healthy group. Immunohistochemical analyses of the healthy group revealed OCN- and OPN-positive cells in the presence of both MTA materials. However, under diabetic conditions, both OCN and OPN were absent. CONCLUSION: Both MTA materials were associated with an increase in serum calcium, phosphorus and ALP, suggesting a potential systemic effect, along with triggered differentiation of OCN- and OPN-positive cells. Moreover, in diabetic conditions, an inhibitory effect on MTA-induced differentiation of OCN- and OPN-positive cells was detected.

Membrane-Based Microwave-Mediated Electrochemical Immunoassay for the In Vitro, Highly Sensitive Detection of Osteoporosis-Related Biomarkers.

Highly sensitive and multiplexed in vitro detection of osteoporosis-related biochemical markers were carried out based on the membrane-based microwave-mediated electrochemical immunoassay (MMeEIA), where we can dramatically reduce the sample preparation time by shortening the incubation time of conjugation to obtain sensitive detection based on three dimensional conjugation of antibodies with target antigens in nylon membrane disk. C-terminal cross-linked telopeptide of type I collagen (CTx), Osteocalcin (OC), parathyroid hormone (PTH), and N-terminal propeptide of type I collagen (P1NP), which can be utilized to monitor the progress of osteoporosis, were quantified using their corresponding antibody immobilized in membranes. Coefficient of variations in this intra- and inter-assays were within 8.0% for all markers. When compared with data obtained from clinically used standard equipment (Roche modular E170), their coefficients of determination, R² values, are mostly more than 0.9. They show that the results obtained from MMeEIA are in good agreement with that from the conventional clinical instruments.

Comparative Evaluation of Different Bone Markers in Peri-implant Crevicular Fluid of Immediate Loaded and Nonloaded Dental Implants.

AIM: The present study was conducted to determine different bone markers in immediate loaded and nonloaded dental implants. MATERIALS AND METHODS: It comprised of 60 patients (males-30, females-30) which were divided into two groups of 30 each. Group I received immediate loaded dental implants, and group II received non-loaded dental implants. Modified bleeding on probing index, peri-implant sulcus depth was assessed in both groups at 1 month, 2 months, 3 months and 4 months. The crevicular fluid was obtained to determine bone markers levels such as transforming growth factor-alpha (TGF-a), osteocalcin (OCN), osteopontin (OPN), parathyroid hormone (PTH) and osteoprotegerin (OPG). RESULTS: Both groups revealed non-significant difference in modified bleeding on probing index and peri-implant sulcus depth (p > 0.05). Bone markers found to be elevated more in group I as compared to group II. However, the difference was non- significant (p > 0.05). CONCLUSION: Transforming growth factor alpha (TGF-a), OCN, OPN, OPG and PTH and parathyroid hormone (PTH) levels were higher in immediate loaded dental implants as compared to nonloaded dental implants. CLINICAL SIGNIFICANCE: Immediate loaded dental implants showed an increase in expression of bone markers such as TNF-a, OCN, OPN, PTH and OPG which may be useful in deciding future of immediate loaded dental implants.

Low-level laser therapy (780 nm) combined with collagen sponge scaffold promotes repair of rat cranial critical-size defects and increases TGF-ß, FGF-2, OPG/RANK and osteocalcin expression.

The aim of this study was to evaluate the effect of collagen sponge scaffold (CSS) implantation associated with low-level laser therapy (LLLT) on repairing bone defects. A single 5-mm cranial defect was surgically created in forty Wistar rats, which then received one of the following four interventions (n = 10 per group): no treatment (G0); bone defect implanted with collagen sponge scaffold (CSS) alone (G1); defect treated with low-level laser therapy (LLLT) (wavelength 780 nm; total energy density 120 J/cm2 ; power 50 mW) alone (G2); and CSS associated with LLLT treatment (G3). After surgery, animals in each group were euthanized at 21 days and 30 days (n = 5 per euthanasia time group). Bone formation was monitored by X-ray imaging analysis. Biopsies were collected and processed for histological analysis and immunohistochemical evaluation of transforming growth factor-beta (TGF-ß), fibroblast growth factor-2 (FGF-2), osteoprotegerin (OPG) and receptor activator of nuclear factor ƙ (RANK). Osteocalcin (OCN) was detected by immunofluorescence analysis. Compared to the G0 group, defects in the 30-day G3 group exhibited increased bone formation, both by increase in radiopaque areas (P < 0.01) and by histomorphometric analysis (P < 0.001). The histopathological analysis showed a decreased number of inflammatory cells (P < 0.001). The combined CCS + LLLT (G3) treatment also resulted in the most intense immunostaining for OPG, RANK, FGF-2 and TGF-ß, and the most intense and diffuse OCN immunofluorescent labelling at 30 days postsurgery (G3 vs. G0 group, P < 0.05). Therefore, the use of CCS associated with LLLT could offer a synergistic advantage in improving the healing of bone fractures.

Anti-Osteoporotic Activity of Harpagoside by Upregulation of the BMP2 and Wnt Signaling Pathways in Osteoblasts and Suppression of Differentiation in Osteoclasts.

Harpagoside (1) is an iridoid glycoside isolated from the radix of Harpagophytum procumbens var. sublobatum, commonly called Devil's claw. The anti-osteoporotic effect of 1 was investigated in both in vitro cell cultures and in vivo using an ovariectomized (OVX) mouse model. Compound 1 induced bone formation by stimulating osteoblast proliferation, alkaline phosphatase activity, and mineralization in osteoblastic MC3T3-E1 cells. Treatment with 1 increased the mRNA and protein expression of bone formation biomarkers through regulation of the BMP2 and Wnt signaling pathway in MC3T3-E1 cells. Compound 1 also suppressed the RANKL-induced osteoclastogenesis of cultured mouse bone marrow cells. Oral administration of 1 restored the OVX-induced destruction of trabecular bone. The bone mineral density of the femur was also increased significantly by 1. The elevated serum levels of osteocalcin, C-terminal telopeptide, and tartrate-resistant acid phosphatase in the OVX mice were decreased by treatment with 1. These findings suggest that compound 1 may protect against bone loss induced by OVX in mice by regulating stimulation of osteoblast differentiation and inhibition of osteoclast resorption. Therefore, harpagoside (1) is a potential candidate for management of postmenopausal osteoporosis.

A novel approach for osteocalcin detection by competitive ELISA using porous silicon as a substrate.

In this study, porous silicon (PSi) was utilized instead of prevalent polystyrene platforms, and its capability in biomolecule screening was examined. Here, two types of porous structure, macroporous silicon (Macro-PSi) and mesoporous silicon (Meso-PSi), were produced on silicon wafers by electrochemical etching using different electrolytes. Moreover, both kinds of fresh and oxidized PSi samples were investigated. Next, osteocalcin as a biomarker of the bone formation process was used as a model biomarker, and the colorimetric detection was performed by competitive enzyme-linked immunosorbent assay (ELISA). Both Macro-PSi and Meso-PSi substrates in the oxidized state, specifically the Meso-porous structure, were reported to have higher surface area to volume ratio, more capacitance of surface-antigen interaction, and more ability to capture antigen in comparison with the prevalent platforms. Moreover, the optical density signal of osteocalcin detected by the ELISA technique was notably higher than the common platforms. Based on the findings of this study, PSi can potentially be used in the ELISA to achieve better results and consequently more sensitivity. A further asset of incorporating such a nanometer structure in the ELISA technique is that the system response to analyte concentration could be maintained by consuming lower monoclonal antibody (or antigen) and consequently reduces the cost of the experiment.

Cavity-Enhanced Immunoassay Measurements in Microtiter Plates Using BBCEAS.

We report on the first detailed use of broadband cavity enhanced absorption spectroscopy (BBCEAS) as a detection system for immunoassay. A vertical R ≥ 0.99 optical cavity was integrated with a motorized XY stage, which functioned as a receptacle for 96-well microtiter plates. The custom-built cavity enhanced microplate reader was used to make measurements on a commercially available osteocalcin sandwich ELISA kit. A 30-fold increase in path length was obtained with a minimum detectable change in the absorption coefficient, αmin(t), of 5.3 × 10(-5) cm(-1) Hz(-1/2). This corresponded to a 39-fold increase in the sensitivity of measurement when directly compared to measurements in a conventional microplate reader. Separate measurements of a standard STREP-HRP colorimetric reaction in microtiter plates of differing optical quality produced an increase in sensitivity of up to 115-fold compared to a conventional microplate reader. The sensitivity of the developed setup compared favorably with previous liquid-phase cavity enhanced studies and approaches the sensitivity of typical fluorometric ELISAs. It could benefit any biochemical test which uses single pass absorption as a detection method, through either the label free detection of biologically important molecules at lower concentrations or the reduction in the amount of expensive biochemicals needed for a particular test, leading to cheaper tests.

Reference intervals for bone turnover markers in Spanish premenopausal women.

BACKGROUND: The aims of this study were to establish robust reference intervals and to investigate the factors influencing bone turnover markers (BTMs) in healthy premenopausal Spanish women. METHODS: A total of 184 women (35-45 years) from 13 centers in Catalonia were analyzed. Blood and second void urine samples were collected between 8 a.m. and 10 a.m. after an overnight fast. Serum procollagen type I amino-terminal propeptide (PINP) and serum cross-linked C-terminal telopeptide of type I collagen (CTX-I) were measured by two automated assays (Roche and IDS), bone alkaline phosphatase (bone ALP) by ELISA, osteocalcin (OC) by IRMA and urinary NTX-I by ELISA. PTH and 25-hydroxyvitamin D (25OHD) levels were measured. All participants completed a questionnaire on lifestyle factors. RESULTS: Reference intervals were: PINP: 22.7-63.1 and 21.8-65.5 µg/L, bone ALP: 6.0-13.6 µg/L, OC: 8.0-23.0 µg/L, CTX-I: 137-484 and 109-544 ng/L and NTX-I: 19.6-68.9 nM/mM. Oral contraceptive pills (OCPs) influenced PINP (p=0.007), and low body mass index (BMI) was associated with higher BTMs except for bone ALP. Women under 40 had higher median values of most BTMs. CTX-I was influenced by calcium intake (p=0.010) and PTH (p=0.007). 25OHD levels did not influence BTMs. Concordance between the two automated assays for PINP and particularly CTX-I was poor. CONCLUSIONS: Robust reference intervals for BTMs in a Southern European country are provided. The effects of OCPs and BMI on their levels are significant, whilst serum 25OHD levels did not influence BTMs. Age, calcium intake, BMI and PTH influenced CTX-I. The two automated assays for measuring PINP and CTX-I are not interchangeable.

The biochemical signatures of stress: A preliminary analysis of osteocalcin concentrations and macroscopic skeletal changes associated with stress in the 13th - 17th centuries black friars population.

OBJECTIVE: As a chemical precursor to the hard tissue changes well-studied in bioarchaeological research, osteocalcin provides a unique opportunity to assess stress via fluctuations in bone metabolism. The main objectives of this research were 1) to successfully extract osteocalcin from the Black Friars skeletal population; 2) to assess the diagenetic change between individual bone samples; and 3) to compare osteocalcin concentrations across sex, age, time period and macroscopic indicators of stress. METHODS: Twenty adult individuals were selected from the 13th-17th centuries Black Friars skeletal population with bone samples taken from the clavicle and femur. Total protein was assessed through a MicroBCA analysis with osteocalcin quantified using a Human Quantikine ELISA kit. Diagenetic change was assessed using Fourier transform infrared spectroscopy and the attenuated total reflectance method. RESULTS: Osteocalcin concentrations showed no significant differences between sex or age groups; however, between time period the post-medieval individuals showed a significant reduction of osteocalcin in both the clavicle and the femur. There were no significant differences in osteocalcin concentrations between those with and without past stress indicators and only one significant difference among the chronic indicators. The diagenetic results demonstrated a similar degree of crystallinity between all samples. CONCLUSIONS: While preliminary in nature, this study was successful in demonstrating the potential use of osteocalcin in future health-related research and how the study of osteocalcin may contribute to a better understanding of how and when stress begins to affect the skeletal tissues.

Clinical and biochemical comparison of guided tissue regeneration versus guided tissue regeneration plus low-level laser therapy in the treatment of class II furcation defects: A clinical study.

OBJECTIVE: The present study was aimed to compare the clinical and biochemical effectiveness of guided tissue regeneration (GTR) alone and combined with low-level laser therapy (LLLT) application in the treatment of furcation II periodontal defects, over a period of 6 months. MATERIAL AND METHODS: Thirty-three furcation defects were included in the study. Seventeen of these defects were treated with GTR plus LLLT, and sixteen of them were treated with GTR alone. Probing pocket depth (PPD), clinical attachment level (CAL), horizontal probing depth (HPD), and alkaline phosphatase (ALP) and osteocalcin (OC) levels in the gingival crevicular fluid (GCF) were recorded at baseline and at postoperative 3rd and 6th months. RESULTS: Healing was uneventful in all cases. At the 3rd and 6th months, both treatment modalities-GTR and GTR plus LLLT--showed improved PPD, CAL, and HPD values compared to their baseline values. ALP and OC levels in GCF increased after the treatment in both groups (p < 0.05). When compared the two groups, at the 6th month, PPD, CAL, HPD, and ALP values showed significantly more improvement in laser group than non-laser group (p < 0.05). CONCLUSIONS: The results of this study showed that both treatments led to significantly favorable clinical improvements in furcation periodontal defects. LLLT plus GTR may be a more effective treatment modality compared to GTR alone.

Differences in calcification and osteogenic potential of herniated discs according to the severity of degeneration based on Pfirrmann grade: a cross-sectional study.

BACKGROUND: Herniated discs may exhibit calcification, and calcified discs may complicate surgical treatment. However, the osteogenic potential and expression of osteogenic markers in degenerative discs of different degenerative grades are still unclear. Our purposes are to study the differences in calcification rate and osteogenic potential of herniated discs according to different degenerative grades. METHODS: Fifty-eight lumbar intervertebral discs were removed from 41 patients. After grading according to the Pfirrmann scale, calcification was analyzed by micro computed tomography (µ-CT), and expression of osteogenic markers was analyzed by immunohistochemistry and real-time quantitative polymerase chain reaction (qPCR). Data from µ-CT scans were compared with the Kruskal-Wallis test. The Mann-Whitney test was applied to compare data between any two groups. Differences in osteogenic mRNA expression in different regions of the removed discs (posterior vs. anterior) were analyzed by paired t tests. Differences in the posterior portion of removed discs of different Pfirrmann grades were analyzed by one-way analysis of variance (ANOVA), and comparisons of data between discs of any two grades were completed with least significant difference (LSD) tests. RESULTS: Significant differences in calcification according to µ-CT scanning were observed between discs of different degenerative grades. Nearly half of the discs of Pfirrmann grade V showed the highest degree of calcification compared to Pfirrman grade II discs. Bone morphogenetic protein (BMP)-2, Osterix, and Osteocalcin were detected histologically in discs of Pfirrmann grades III-V. Alkaline phosphatase (ALP) expression was observed in discs showing evidence of calcification. The qPCR analysis showed that BMP-2, Osterix, and Osteocalcin were expressed in most degenerated discs. We also observed greater expression of these osteogenic markers in the posterior portion of removed discs than in the anterior portion. CONCLUSIONS: The osteogenic potential of degenerated intervertebral discs appears to increase with the severity of degeneration and to be greater in the tissue near the spinal canal than in tissue in the inner portion of the disc.

Pre-coating deproteinized bovine bone mineral (DBBM) with bone-conditioned medium (BCM) improves osteoblast migration, adhesion, and differentiation in vitro.

OBJECTIVES: Autogenous bone grafting has remained the gold standard for bone augmentation procedures with ability to release growth factors to the surrounding microenvironment. Recent investigations have characterized these specific growth factors released by autogenous bone chips with further isolation into a "bone-conditioned medium" (BCM). The aim of the present investigation was to utilize autologous growth factors from bone chips (BCM) in combination with deproteinized bovine bone mineral (DBBM) and investigate the ability for BCM to enhance osteoblast behavior. MATERIALS AND METHODS: Mouse ST2 cells were seeded on (1) DBBM particles alone or (2) DBBM + BCM. Thereafter, samples were compared for cell recruitment, adhesion, proliferation, and real-time PCR for osteoblast differentiation markers including Runx2, collagen 1 alpha 2 (COL1A2), alkaline phosphatase (ALP), and osteocalcin (OCN). Alizarin red staining was used to assess mineralization. RESULTS: Coating BCM on DBBM particles improved cell migration of ST2 cells and significantly enhanced a 2-fold increase in cell adhesion. While no significant increase in cell proliferation was observed, BCM significantly increased mRNA levels of COL1A2, ALP, and OCN at 3 days post seeding. Furthermore, a 3-fold increase in alizarin red staining was observed on DBBM particles pre-coated with BCM. CONCLUSION: Pre-coating DBBM with BCM enhanced the osteoconductive properties of DBBM by mediating osteoblast recruitment, attachment, and differentiation towards bone-forming osteoblasts. Future animal study is necessary to further characterize the added benefit of BCM as an autogenous growth factor source for combination therapies. CLINICAL RELEVANCE: The application of BCM in combination with biomaterials may serve as an autogenous growth factor source for bone regeneration.