Procollagen Type Research Articles

Protaetia brevitarsis seulensis larvae ethanol extract inhibits RANKL-stimulated osteoclastogenesis and ameliorates bone loss in ovariectomized mice

Modulation of osteoclast formation could be a therapeutic target for inhibiting pathological bone destruction. The receptor activator of nuclear factor (NF)-κB ligand (RANKL) is known to be an essential factor in osteoclast differentiation and activation inducers. However, whether Protaetia brevitarsis seulensis (P. brevitarsis) larvae—a traditional animal-derived medicine used in many Asian countries—can inhibit RANKL-induced osteoclast formation and prevent ovariectomy (OVX)-induced bone loss has not been evaluated. Here, we aimed to investigate the anti-osteoporotic effects of P. brevitarsis larvae ethanol extract (PBE) in RANKL-stimulated RAW264.7 cells and OVX mice. In vitro, PBE (0.1, 0.5, 1, and 2 mg/mL) decreased RANKL‑induced tartrate-resistant acid phosphatase (TRAP) activity and expression of osteoclastogenesis-associated genes and proteins. Furthermore, PBE (0.1, 0.5, 1, and 2 mg/mL) significantly inhibited the phosphorylation of p38 and NF-κB. Female C3H/HeN mice were divided into five groups (n = 5 per group), namely, sham-operated, OVX, OVX+PBEL (100 mg/kg, oral gavage), OVX+PBEH (200 mg/kg, oral gavage), and OVX+estradiol (0.03 μg/day, subcutaneous injection). High doses of PBE significantly increased femoral bone mineral density (BMD) and bone volume/tissue volume (BV/TV), whereas femoral bone surface/bone volume (BS/BV) and osteoclastogenesis-associated protein expression decreased compared to those in the OVX group. Moreover, PBE (200 mg/kg) significantly increased estradiol and procollagen type I N-terminal propeptide and decreased N-terminal telopeptide of type I collagen and C-terminal telopeptide of type I collagen compared to those in the OVX group. Our results suggest that PBE can be an effective therapeutic candidate for preventing or treating postmenopausal osteoporosis.

Association of PFN1 Gene Polymorphisms with Bone Mineral Density, Bone Turnover Markers, and Osteoporotic Fractures in Chinese Population.

Recent studies have discovered an association between the PFN1 gene and Paget's disease. However, it is currently unknown whether the PFN1 gene is related to osteoporosis. This study was performed to investigate the association of Single-Nucleotide Polymorphisms (SNPs) in the PFN1 gene with Bone Mineral Density (BMD) as well as bone turnover markers and osteoporotic fractures in Chinese subjects. A total of 2836 unrelated Chinese subjects comprising 1247 healthy subjects and 1589 osteoporotic fractures patients (Fracture group) were enrolled in this study. Seven tagSNPs (rs117337116, rs238243, rs6559, rs238242, rs78224458, rs4790714, and rs13204) of the PFN1 gene were genotyped. The BMD of the lumbar spine 1-4 (L1-4), femoral neck, and total hip as well as bone turnover markers, such as β-C-Terminal telopeptide of type 1 collagen (β-CTX) and Procollagen type 1 N-terminal Propeptide (P1NP), were measured. The association between 7 tagSNPs and BMD and bone turnover markers was analyzed in 1247 healthy subjects only. After age matching, we selected 1589 osteoporotic fracture patients (Fracture group) and 756 nonfracture controls (Control group, selected from 1247 healthy subjects) for a case-control study, respectively. For the case-control study, we used logistic regression to investigate the relationship between 7 tagSNPs and osteoporotic fractures risk. In the All group, the PFN1 haplotype GAT was associated with the β-CTX (P = 0.007). In the Female group, the PFN1 haplotype GAT was associated with the β-CTX (P = 0.005). In the Male group, the rs13204, the rs78224458, and the PFN1 haplotype GAC were associated with the BMD of the L1-4 (all P = 0.012); the rs13204, the rs78224458, and the PFN1 haplotype GAC were associated with the BMD of the femoral neck (all P = 0.012); the rs13204 and rs78224458 were associated with the BMD of the total hip (both P = 0.015); and the PFN1 haplotype GAT was associated with the β-CTX (P = 0.013). In the subsequent case-control study, the rs13204 and rs78224458 in the male group were associated with the risk of L1-4 fracture (P = 0.016 and 0.010, respectively) and total hip fracture (P = 0.013 and 0.016, respectively). Our study reveals that PFN1 gene polymorphisms are associated with BMD in Chinese males and β-CTX in Chinese people and confirmed the relationship between PFN1 gene polymorphisms and Chinese male osteoporotic fractures in a case-control study.

Bone health, body composition and physical fitness dose–response effects of 16 weeks of recreational team handball for inactive middle-to-older-aged males – A randomised controlled trial

ABSTRACT In this study we aimed at analysing the effects of different weekly exercise volumes (1, 2 or 3 times 60-min) on bone health, body composition and physical fitness of inactive middle-to-older-aged males, after 16 weeks of recreational team handball (RTH). Fifty-four men (68 ± 4 years, stature 169 ± 6 cm; body mass 78.4 ± 10.7 kg; fat mass 27.1 ± 5.3%; BMI 27.4 ± 2.9 kg/m2; VO2peak 27.3 ± 4.8 mL/min/kg) were randomised into three intervention groups (TH1, n = 13; TH2, n = 15; or TH3, n = 12, performing 1, 2 and 3 weekly 60-min training sessions, respectively), and a control group (CG, n = 14). The training sessions consisted mainly of RTH matches played as small-sided and formal game formats (4v4, 5v5, 6v6 or 7v7) with adapted rules. Matches’ mean and peak heart rate (HR) ranged from 78–80% and 86–89%HRmax, respectively, and distance covered from 4676 to 5202 m. A time x group interaction was observed for procollagen type-1 amino-terminal propeptide (P1NP), osteocalcin (OC), carboxy-terminal type-1 collagen crosslinks (CTX), sclerostin, upper and lower body dynamic strength, right arm fat mass, left and right arm, right leg and android total mass (TM; p ≤ 0.047) with the greatest effects being shown for TH2 and TH3 groups. Post-intervention group differences were observed in CTX, left arm and right leg TM (TH3 > TH1), P1NP (TH2 > CG), OC, right arm TM (TH3 > CG), upper (CG < TH1, TH2 and TH3) and lower body dynamic strength (CG < TH1 and TH3) (p ≤ 0.047). RTH was effective in enhancing bone health, body composition and physical fitness in middle-to-older-aged males, especially for the intervention groups that performed 2–3 weekly training sessions. ClinicalTrials.gov ID: NCT05295511. Trial registration: ClinicalTrials.gov identifier: NCT05295511. Highlights After 16 weeks of recreational team handball small-sided and formal matches, inactive middle-to-older-aged males improved bone health, body composition and physical fitness, by performing 1, 2 or 3 60-min weekly sessions, however, greater improvements were shown in the groups that performed 2 or 3 weekly training sessions. Training intensity was similar across the intervention groups that performed recreational team handball for 1, 2 or 3 60-min weekly sessions, which means that training volume is most likely to be the reason for the different health effects shown. The very high fun levels reported by all intervention groups shows that recreational team handball is a social and fun exercise modality for middle-to-older-aged males, with potential to intrinsically motivate the participants and assure long-term adherence to exercise.

Metabolic Syndrome in Aging Men as a Factor Affecting the Relationship between Mg, Ca, and P in Serum and Bone.

The objective of this study was to evaluate the relationship between the prevalence of metabolic syndrome (MetS) and selected hormonal disorders and concentrations of magnesium (Mg), calcium (Ca), and phosphorus (P) in both blood serum and bone tissue. This study involved 152 men with and without MetS. In the blood of the patients we examined, we determined levels of: testosterone (TT), estradiol (E2), sex hormone-binding globulin (SHBG), dehydroepiandrosterone sulfate (DHEAS), insulin (I), osteocalcin (OC), and concentrations of markers of bone turnover. The concentration of Mg, Ca, and P was determined in the serum and bone tissue. In patients with MetS, the serum Ca concentration correlated with procollagen type I N-terminal propeptide (PINP) and parathyroid hormone (PTH). Among patients without MetS, the serum Ca concentration correlated with SHBG and OC, while Ca concentration in bone correlated with the lipid accumulation product (LAP) index and the body mass index (BMI). After analyzing the serum Mg concentration, positive correlations were observed with E2, PINP, and PTH in patients with MetS. In patients without MetS, the Mg concentration in bone positively correlated with the BMI and the LAP index. Our study findings suggest that increased Mg levels could have an impact on bone tissue metabolism. Elevated serum Mg levels may be associated with changes in sex hormone concentrations and alterations in bone turnover markers.

Osteogenic Effects of Aerobic Both Sides Utilized Ball Versus Aerobic Step Training in Premenopausal Women

Study purpose. Exercise training plays an important role in increasing bone mass. Nevertheless, the osteogenic effects of exercise training using various bench surfaces are unknown. Therefore, this study aimed to compare the osteogenic effects of exercise with a soft surface bench and an aerobic both sides utilized (BOSU) ball with those of aerobic step exercise training (STEP). Materials and methods. Fifty-two sedentary female participants aged 30–45 years were recruited and randomly divided into three groups. Seventeen participants were in the STEP, 17 in the BOSU, and 18 in the control group. The exercise programs of the STEP and BOSU groups were designed to have the same intensity and heart rate range during each stage of the program. During training, music was used to set the tempo for workouts. Results. After week 24, both training groups showed significant improvements in physical fitness, body composition, and body stability (p&lt;0.05). Increased levels of procollagen type I N-terminal propeptide (P1NP), an osteogenesis marker, were observed in both STEP and BOSU groups. Increased bone mineral density was only seen in the BOSU group (p&lt;0.05). Conclusions. Both STEP and BOSU programs effectively improved P1NP levels, muscle strength, and postural control, but only the aerobic BOSU ball training improved bone mineral density in premenopausal women.

P93 Dupilumab treatment in children with moderate-to-severe atopic dermatitis increases levels of bone mineralization biomarkers

Abstract Children with atopic dermatitis (AD) are at risk for lower bone mineral density (BMD) and lower alkaline phosphatase (ALP) levels than healthy children. Low BMD and ALP in children could contribute to a higher lifetime prevalence of fractures, osteopenia and osteoporosis. Dupilumab is a fully humanized monoclonal anti-interleukin (IL)-4R antibody approved for the treatment of AD in children. The objective of this analysis is to report the impact of dupilumab treatment on markers of bone mineralization in children aged 6–12 years with moderate-to-severe AD vs. reference intervals (RIs). Analysis was performed retrospectively on sera from participants in LIBERTY AD PEDS, a phase III placebo-controlled trial of 16 weeks (NCT03345914) and LIBERTY AD PED-OLE (NCT02612454), an open-label extension trial in which eligible patients received dupilumab until week 52. Biomarkers, including bone ALP (BALP), procollagen type 1 N-terminal propeptide (P1NP), C-terminal crosslinking telopeptide of β-1 collagen (β-CTX), osteocalcin (OC) and insulin-like growth factor 1 (IGF-1), were analysed at baseline, 8, 12 and 16 weeks; only BALP was measured at 52 weeks. Both dupilumab dosing regimens led to significant increases in geometric mean (SE) levels of BALP at 8, 12 and 16 weeks compared with placebo. For the 100/200-mg Q2W group, at week 8 the BALP level was 72.7 (1.03) μg L−1 vs. 62.0 (1.05) μg L−1 (P &amp;lt; 0.0001), at week 12 it was 74.7 (1.03) μg L−1 vs. 64.3 (1.05) μg L−1 (P &amp;lt; 0.001) and at week 16 it was 78.0 (1.03) μg L−1 vs. 65.0 (1.04) μg L−1 (P &amp;lt; 0.001). For the 300-mg Q4W group, at week 8 the BALP level was 76.7 (1.03) μg L−1 vs. 62.0 (1.05) μg L−1 (P &amp;lt; 0.001), at week 12 it was 73.3 (1.04) μg L−1 vs. 64.3 (1.05) μg L−1 (P = 0.002) and at week 16 it was 77.3 (1.03) μg L−1 vs. 65.0 (1.04) μg L−1 (P &amp;lt; 0.001). At 52 weeks, BALP levels were statistically significantly increased compared with baseline [placebo vs. placebo transitioned to dupilumab: 64.2 (1.04) μg L−1 vs. 82.9 (1.04) μg L−1 (P &amp;lt; 0.001); 100/200 mg Q2W: 62.0 (1.05) μg L−1 vs. 83.8 (1.03) μg L−1 (P &amp;lt; 0.001); 300 mg Q4W: 64.1 (1.04) μg L−1 vs. 78.7 (1.04) μg L−1 (P &amp;lt; 0.001)] and also increased within RIs. From baseline to week 16, OC, P1NP and β-CTX levels improved from below to within the RIs, and levels of IGF-1 increased from the lower limit of the RI. However, data points were limited due to insufficient sera volumes, preventing statistical analyses for these biomarkers. The overall results suggest an increase in bone mineralization biomarkers in paediatric patients with moderate-to-severe AD following dupilumab treatment. Funding sources:research sponsored by Sanofi and Regeneron Pharmaceuticals Inc.

Impact of vitamin D supplementation on markers of bone turnover: Systematic review and meta-analysis of randomised controlled trials.

The effects of vitamin D administration on bone turnover markers (BTMs) in adults are controversial. Thus, we carried out a meta-analysis of available randomised controlled trials (RCTs) to examine the impact of vitamin D supplementation on BTMs. To identify relevant RCTs, we searched the PubMed/MEDLINE, Web of Science, Scopus, Cochrane Library and Embase databases for manuscripts published up to July 2022. The present study was conducted in agreement with the PRISMA guidelines. Weighed mean difference (WMD) and 95% confidence intervals (CI) were used to calculate the magnitude of the effect of the intervention. A total of 42 RCTs were included in the meta-analysis. The age of the participants enrolled in the RCTs ranged from 19.4 to 84 years. The pooled results depicted a decrease in deoxypyridinoline (DPD) concentrations (WMD: -1.58 nmol/mmol, 95% CI: -2.55, -.61, p = .001) following vitamin D supplementation. In addition, subgroup analyses demonstrated that vitamin D administration notably reduced procollagen type I N-terminal propeptide (PINP) levels in individuals aged >50 years and led to a pronounced decrease in alkaline phosphatase (ALP) values when the intervention lasted >12 weeks. No significant effect was observed on other BTMs, for example, collagen type 1 cross-linked C-telopeptide (CTX) and osteocalcin (OC) levels. Vitamin D administration decreases DPD, PINP and ALP levels, indicating a reduced bone turnover following the intervention. Other BTMs, for example, CTX or OC values, were not affected by vitamin D prescription. Vitamin D supplementation may exert a positive effect on some important BTMs.

Urinary pentosidine level is associated with the risk of fracture in community-dwelling older adults: a prospective observational study.

This prospective observational study aimed to determine the factors associated with fragility fractures in community-dwelling older adults. Overall, 254 older adults who were participants of the Good Aging and Intervention Against Nursing Care and Activity Decline study in 2016 were included in this study. Grip strength, muscle mass, gait speed, calcaneal bone density, and the levels of parathyroid hormone, osteocalcin, 25-hydroxyvitamin D, total procollagen type I N-terminal propeptide, insulin-like growth factor-1 (IGF-1), tartrate-resistant acid phosphatase-5b, and urinary pentosidine were measured at baseline. Participants were classified as fracture ( +) or fracture (-) based on the data collected during a 5-year follow-up period. Excluding those who were lost to follow-up during the observation period, 182 participants (64 men and 118 women, mean age: 74.2years, range: 47-99years) were included in the analysis. During the observation period, 23 patients experienced 24 new fractures. In univariate analysis, sex, height, weight, history of fracture in adulthood, baseline grip strength, muscle mass, bone density, and the levels of urinary pentosidine and IGF-1 at baseline were significantly different between patients who developed a fracture during follow-up and those who did not. In multivariate analysis, a history of fracture in adulthood and urinary pentosidine levels were independently and significantly associated with fracture occurrence. High urine pentosidine levels and a history of fracture in adulthood are independent risk factors for fracture occurrence in community-dwelling older adults.

A Three-Year Follow-Up of Bone Density Among Thai Adolescents With Perinatally Acquired HIV After Completion of Vitamin D and Calcium Supplementation

PurposeTo determine changes in bone mineral density (BMD) and bone metabolism-related biomarkers among Thai adolescents with perinatally acquired HIV infection (PHIVA) at 3 years following completion of vitamin D and calcium (VitD/Cal) supplementation. MethodsAn observational follow-up study was conducted among PHIVA who received 48-week VitD/Cal supplementation (either high-dose [3,200 IU/1,200 mg daily] or standard-dose [400 IU/1,200 mg daily]). Lumbar spine BMD (LSBMD) was assessed by dual-energy x-ray absorptiometry. Serum 25-hydroxyvitamin D, intact parathyroid hormone, and bone turnover markers were measured. Changes in LSBMD z-scores and other bone parameters at 3 years after stopping VitD/Cal supplementation compared with baseline or week 48 of supplementation were assessed among participants previously receiving high-dose and standard-dose VitD/Cal supplementation. ResultsOf 114 enrolled PHIVA, 46% and 54% had previously received high-dose and standard-dose VitD/Cal supplementation, respectively. The median age was 20 years; 53% were male. At 3 years after completion of VitD/Cal supplementation, we observed a significant decline in 25-hydroxyvitamin D and increase in intact parathyroid hormone but no significant rebounds of C-terminal telopeptides of collagen type I and procollagen type I amino-terminal propeptides and no significant changes in LSBMD z-scores among PHIVA in both treatment groups, compared with the measurements at week 48 of supplementation. Notably, LSBMD z-scores at 3 years after stopping VitD/Cal supplements were not significantly altered from baseline evaluations in both PHIVA groups. DiscussionThree years after completion of high-dose or standard-dose VitD/Cal supplementation, LSBMD z-scores of our Thai PHIVA were not significantly changed from baseline and week 48 of supplementation. VitD/Cal supplementation of PHIVA during periods of peak bone mass accrual may have sustained and long-term skeletal benefits.

Clinical value of RANKL, OPG, IL-6 and sclerostin as biomarkers for fibrous dysplasia/McCune-Albright syndrome.

Fibrous dysplasia/McCune-Albright syndrome (FD/MAS) is a rare genetic bone disease caused by a somatic mutation in the GNAS gene. Currently used bone turnover markers (BTMs) do not correlate with the clinical picture and are not useful to predict or monitor therapy success. This study assessed the correlation of RANKL, OPG, RANKL/OPG ratio, IL-6 and sclerostin with the classic BTMs alkaline phosphatase (ALP), procollagen type 1 propeptide (P1NP) and beta crosslaps (CTX), with pain, skeletal burden score (SBS) and response to bisphosphonate or denosumab treatment. Ninety-six serum samples of adult patients >18years of age with any subtype of FD/MAS were included from the biobank facility of the Leiden University Medical Center, Center for Bone Quality between 2015 and 2021. Standard laboratory assessments were assessed as part of usual care. The concentrations of potential biomarkers RANKL, OPG, sclerostin, IL-6 were analyzed. Data on FD/MAS subtype, age, pain, treatment history and treatment response were retrieved from the electronic patient files. Baseline characteristics were summarized by descriptive statistics. Correlations of the concentrations of the potential biomarkers with classic bone turnover markers, SBS and pain scores were cross-sectionally assessed by Spearman rank order correlation. Correction for multiple testing was performed by Benjamini and Hochberg False Discovery Rate. A sensitivity analyses was performed by excluding patients with SBS below 15 and patients using antiresorptive medication at the time of blood withdrawal or within the wash-out period. In patients treated with bisphosphonates or denosumab after blood withdrawal, pre-treatment concentrations were compared in patients with and without therapy response by Mann Whitney U test. The median age of the patients was 41.2 (Q1-Q3 25.9-52.2) years, 62.5% was female. Median SBS was 2.5 (Q1-Q3 0.5-7.8). RANKL level correlated weakly with ALP (Spearman rho 0.309, p=0.004, n=84), but not with P1NP or CTX. The RANKL/OPG ratio, OPG, IL-6 and sclerostin did not correlate with ALP, P1NP or CTX. None of the potential biomarkers correlated with SBS or pain. Results of the sensitivity analyses were comparable. Pre-treatment biomarker levels were similar in patients with and without improvement in pain scores following bisphosphonate therapy. Pre-treatment RANKL and sclerostin were comparable between patients with and without improvement in pain scores after denosumab therapy. Pre-treatment IL-6 level and the RANKL/OPG ratio seemed to be higher in patients with response to denosumab (IL-6: median 0.64 (Q1-Q3 0.53-0.74) pg/mL, n=6, RANKL/OPG: median 0.062 (Q1-Q3 0.016-0.331), n=5) compared to patients without response (IL-6: median 0.35 (0.20-0.54) pg/mL, n=5, RANKL/OPG: 0.027 (0.024-0.046), n=4). Pre-treatment IL-6 correlated with the improvement in maximum pain scores (rho 0.962, p<0.001, n=9) and average pain scores (rho 0.895, p=0.001, n=9) reported during denosumab therapy. Increased concentrations of RANKL, IL-6, sclerostin and of the RANKL/OPG ratio do not indicate severity of FD/MAS, as no correlation was observed of these potential biomarkers with the classic BTMs and SBS. Biomarker levels did not correlate with pain and had no value in predicting bisphosphonate treatment response. These biomarkers are not superior over the currently used methods of assessing ALP, P1NP and CTX or evaluating SBS to establish disease extent or activity and provide no reliable results. Yet, possibly pre-treatment IL-6 and the RANKL/OPG ratio may have some predictive value for clinical response to denosumab. Therefore, studies investigating disease activity and treatment response should include lesional imaging and patient-reported outcome measures.

Liver Fibrosis Markers Represent Central Venous Pressure in Post-pubertal Patients With Congenital Heart Disease.

Background Central venous pressure (CVP) is one of the most important hemodynamic parameters in patients with congenital heart disease (CHD). In adults, it is well-known that liver fibrosis markers reflect CVP, but this is notwell-understood in children. We investigated the liver fibrosis markers in pediatric CHD patients and their ability to predict CVP. Methods We studied 160 patients who underwent cardiac catheterization in our hospital between January 2017 and December 2020. The levels of the fibrotic markers, including type IV collagen 7s, procollagen type III peptide, and hyaluronic acid, were measured. Results Procollagen type III peptide was markedly elevated in infants younger than one year of age. From one to 15 years of age, it was slightly lower than in the infant group, with a peak at around 10 years of age. In the age group of 16 years and older, most of its values were generally high. Type IV collagen 7s and hyaluronic acid levels were high in infants, with no significant differences at later ages. Procollagen type III peptide and hyaluronic acid showed no significant correlation with CVP in any of the age groups, whereas type IV collagen 7s significantly correlated with CVP in the age group above one yearold. Conclusions We found that elevated liver fibrosis markers, particularly type IV collagen 7s, correlated with central venous pressure in CHD patients older than one year. Measurement of liver fibrosis markers may allow the early detection of changes in CVP and liver function in patients with CHD.

The short-term effects of CGRP monoclonal antibodies on bone turnover: A prospective cohort study

Calcitonin gene-related peptide monoclonal antibodies (CGRP mAb) are an effective treatment of migraine however may have possible off-target effects. Pre-clinical studies implicate CGRP in several aspects of bone turnover and homeostasis. The clinical effect of CGRP mAb on bone turnover is not known, however. Between June 2021 and July 2022, a multi-centre prospective cohort study was undertaken with eligible patients undergoing paired testing of the validated bone turnover markers procollagen type I N-terminal propeptide (P1NP) and serum C-terminal telopeptide of type I collagen (CTX) prior to and at least three months following administration of a CGRP mAb. A total of 45 patients with a mean age of 41.8 (SD 11.9) were included in the final analysis, all of whom received a ligand-targeting CGRP mAb. Administration of a CGRP mAb was associated with a statistically significant increase in P1NP from 44.5 microg/L to 51.5 microg/L (p = 0.004), but no significant change in CTX. In otherwise homeostatic conditions, short-term administration of a CGRP mAb is associated with increased P1NP, a bone formation marker but not with increased CTX, a bone resorption marker. Further study is required to validate these findings over longer time periods, in a larger cohort, and in pre-existing states of increased calcium stress and bone-turnover.

Development of a rapid, efficient, and reusable magnetic bead-based immunocapture system for recombinant human procollagen type II isolation from yeast fermentation broth

Recombinant collagen production, especially using yeasts as expression systems, could represent a promising alternative over traditional extractive methods from animal sources, offering controllable, scalable, and high-quality products. Monitoring the efficiency and efficacy of procollagen/collagen expression, especially in the initial fermentation phases, can be difficult and time consuming, as biological matrices necessitate purification and commonly used analytical methods are only partially informative. We propose a straightforward, efficient, and reusable immunocapture system able to specifically isolate human procollagen type II from fermentation broths and to release it in few experimental steps. A recovered sample allows for a detailed characterization providing information on structural identity and integrity, which can strongly support the monitoring of fermentation processes. The immunocapture system relies on the use of protein A–coated magnetic beads which have been functionalized and cross-linked with a human anti-procollagen II antibody (average immobilization yield of 97.7%) to create a stable and reusable support for the specific procollagen fishing. We set up the binding and release conditions ensuring specific and reproducible binding with a synthetic procollagen antigen. The absence of non-specific interaction with the support and binding specificity was demonstrated, and the latter was also confirmed by a peptide mapping epitope study in reversed-phase liquid chromatography high-resolution mass spectrometry (RP-LC-HRMS). The bio-activated support proved to be reusable and stable over 21 days from the initial use. Finally, the system was successfully tested on a raw yeast fermentation sample to provide a proof of concept of the applicability within recombinant collagen production.Graphical

Abaloparatide prevents immobilization-induced cortical but not trabecular bone loss after spinal cord injury.

Spinal cord injury (SCI) causes severe and resistant sublesional disuse bone loss. Abaloparatide, a modified parathyroid hormone related peptide, is an FDA approved drug for treatment of severe osteoporosis with potent anabolic activity. The effects of abaloparatide on SCI-induced bone loss remain undefined. Thus, female mice underwent sham or severe contusion thoracic SCI causing hindlimb paralysis. Mice then received subcutaneous injection of vehicle or 20 μg/kg/day abaloparatide for 35 days. Micro-computed tomography (micro-CT) analysis of the distal and midshaft femoral regions of the SCI-vehicle mice revealed reduced trabecular fractional bone volume (56%), thickness (75%), and cortical thickness (80%) compared to sham-vehicle controls. Treatment with abaloparatide did not prevent SCI-induced changes in trabecular or cortical bone. However, histomorphometry evaluation of the SCI-abaloparatide mice demonstrated that abaloparatide treatment increased osteoblast (241%) and osteoclast (247%) numbers and the mineral apposition rate (131%) compared to SCI-vehicle animals. In another independent experiment, treatment with 80 μg/kg/day abaloparatide significantly attenuated SCI-induced loss in cortical bone thickness (93%) when compared to SCI-vehicle mice (79%) but did not prevent SCI-induced trabecular bone loss or elevation in cortical porosity. Biochemical analysis of the bone marrow supernatants of the femurs showed that SCI-abaloparatide animals had 2.3-fold increase in procollagen type I N-terminal propeptide, a bone formation marker than SCI-vehicle animals. SCI groups had 70% higher levels of cross-linked C-telopeptide of type I collagen, a bone resorption marker, than sham-vehicle mice. These findings suggest that abaloparatide protects the cortical bone against the deleterious effects of SCI by promoting bone formation.

Association of bone turnover biomarkers with severe intracranial and extracranial artery stenosis in type 2 diabetes mellitus patients

Intracranial and extracranial artery stenosis is associated with cerebral infarction. Vascular calcification and atherosclerosis are the main causes of stenosis and major risk factors for cardiovascular and cerebrovascular events in patients with type 2 diabetes mellitus (T2DM). Bone turnover biomarkers (BTMs) are associated with vascular calcification, atherosclerosis, glucose, and lipid metabolism. To investigate the association of circulating BTM levels with severe intracranial and extracranial artery stenosis in patients with T2DM. For this cross-sectional study including 257 T2DM patients, levels of the BTMs serum osteocalcin (OC), C-terminal cross-linked telopeptide of type I collagen (CTX), and procollagen type I N-peptide were measured by electrical chemiluminescent immunoassay, and artery stenosis was assessed by color Doppler and transcranial Doppler. Patients were grouped according to the existence and location (intracranial vs. extracranial) of artery stenosis. Correlations between BTM levels, previous stroke, stenosis location, and glucose and lipid metabolism were analyzed. T2DM patients with severe artery stenosis had a higher frequency of previous stroke and levels of all three tested BTMs (all P < 0.05) than patients without. Some differences in OC and CTX levels were observed according to the location of artery stenosis. Significant associations were also observed between BTM levels and some glucose and lipid homeostasis parameters. On multivariate logistic regression analysis, all BTMs were significant predictors of artery stenosis in T2DM patients with and without adjustment for confounding factors (all P < 0.001), and receiver operating characteristic curve analysis demonstrated the ability of BTM levels to predict artery stenosis in T2DM patients. BTM levels were found to be independent risk factors for severe intracranial and extracranial artery stenosis and were differentially associated with glucose and lipid metabolism in patients with T2DM. Therefore, BTMs may be promising biomarkers and potential therapeutic targets for artery stenosis.

Melatonin increases collagen content accumulation and Fibroblast Growth Factor-2 secretion in cultured human cardiac fibroblasts

BackgroundThe extracellular matrix serves as a scaffold for cardiomyocytes, allowing them to work in accord. In rats, collagen metabolism within a myocardial infarction scar is regulated by melatonin. The present study determines whether melatonin influences matrix metabolism within human cardiac fibroblast cultures and examines the underlying mechanism.MethodsThe experiments were performed on cultures of cardiac fibroblasts. The Woessner method, 1,9-dimethylmethylene blue assay, enzyme-linked immunosorbent assay and quantitative PCR were used in the study.ResultsMelatonin treatment lowered the total cell count within the culture, elevated necrotic and apoptotic cell count as well as augmented cardiac fibroblast proliferation, and increased total, intracellular, and extracellular collagen within the fibroblast culture; it also elevated type III procollagen α1 chain expression, without increasing procollagen type I mRNA production. The pineal hormone did not influence matrix metalloproteinase-2 (MMP-2) release or glycosaminoglycan accumulation by cardiac fibroblasts. Melatonin increased the release of Fibroblast Growth Factor-2 (FGF-2) by human cardiac fibroblasts, but cardiotrophin release was not influenced.ConclusionWithin human cardiac fibroblast culture, collagen metabolism is regulated by melatonin. The profibrotic effect of melatonin depends on the elevation of procollagen type III gene expression, and this could be modified by FGF-2. Two parallel processes, viz., cell elimination and proliferation, induced by melatonin, lead to excessive replacement of cardiac fibroblasts.

Occult Hepatitis B Virus Infection and Liver Fibrosis in Chinese Patients.

The impact of hepatitis B surface antigen (HBsAg)-negative/hepatitis B virus (HBV) DNA-positive occult HBV infection (OBI) on the severity of liver fibrosis remains unclear. A total of 1772 patients negative for HBsAg but positive for antibody to hepatitis B core antigen (HBcAg), stratified by the presence or absence of OBI, were selected for long-term carriage leading to elevation of ≥2 of 4 liver fibrosis indexes-hyaluronic acid (HA), laminin, type III procollagen peptide (PCIII), and type IV collagen (CIV)-at testing in a Chinese hospital. Patients were tested for serum viral load, HBV markers, and histopathological changes in liver biopsy specimens. OBI was identified in 148 patients with liver fibrosis (8.4%), who had significantly higher levels of HA, laminin, PCIII, and CIV than 1624 fibrotic patients without OBI (P < .05). In 36 patients with OBI who underwent liver biopsy, significant correlations were observed between OBI viral load and serum HA levels (P = .01), PCIII levels (P = .01), and pathological histological activity index (HAI) scores (P < .001), respectively; HAI scores and PCIII levels (P = .04); HBcAg immunohistochemical scores and HA levels (P < .001); and HBcAg immunohistochemical scores and PCIII levels (P = .03). Positive fluorescent in situ hybridization results were significantly more frequent in patients with OBIs (80.6% vs 37.5% in those without OBIs). Among patients with OBIs, HBcAg was detected in the liver tissue in 52.8% and HBsAg in 5.6%. OBI status appears to be associated with liver fibrosis severity.

The efficacy of alendronate for the treatment of thalassemia-associated osteoporosis: a randomized controlled trial.

With adequate blood transfusion and iron chelation, thalassemia patients have a longer life expectancy and experience long-term metabolic complications, including osteoporosis, fractures, and bone pain. Alendronate, an oral bisphosphonate, is currently used to treat various types of osteoporosis. However, the efficacy for the treatment of thalassemia-associated osteoporosis remains unclear. We conducted a randomized controlled trial to evaluate the efficacy of alendronate for the treatment of osteoporosis in thalassemia patients. Patients were included if they were males (18-50 years) or premenopausal females with low bone mineral density (BMD) (Z-score < -2.0 SD) or positive vertebral deformities from vertebral fracture analysis (VFA). Stratified randomization was performed according to sex and transfusion status. Patients were 1:1 allocated to receive once weekly alendronate 70 mg orally or placebo for a total duration of 12 months. BMD and VFA were re-evaluated at 12 months. Markers of bone resorption (C-terminal crosslinking telopeptide of type I collagen; CTX) and bone formation (Procollagen type I N-terminal propeptide; P1NP), and pain scores were measured at baseline, 6 months, and 12 months. The primary outcome was the change of BMD. The secondary endpoints were changes in bone turnover markers (BTM) and pain scores. A total of 51 patients received the study drug, 28 patients were assigned to receive alendronate and 23 patients to receive placebo. At 12 months, patients in the alendronate group had significant improvement of BMD at L1-L4 compared to their baseline (0.72 ± 0.11 vs 0.69 ± 0.11 g/cm2, p = 0.004), while there was no change in the placebo group (0.69 ± 0.09 vs 0.70 ± 0.06 g/cm2, p = 0.814). There was no significant change of BMD at femoral neck in both groups. Serum BTMs were significantly decreased among patients receiving alendronate at 6 and 12 months. The mean back pain score was significantly reduced compared to the baseline in both groups (p = 0.003). Side effects were rarely found and led to a discontinuation of the study drug in 1 patient (grade 3 fatigue). Alendronate 70 mg orally once weekly for 12 months effectively improves BMD at L-spine, reduces serum BTMs, and alleviates back pain in thalassemia patients with osteoporosis. The treatment was well tolerated and had a good safety profile.

Chronic intermittent hypobaric hypoxia ameliorates osteoporosis after spinal cord injury through balancing osteoblast and osteoclast activities in rats.

As a common complication of spinal cord injury (SCI), most SCI patients suffer from osteoporosis. In our previous study, chronic intermittent hypobaric hypoxia (CIHH) could promote bone fracture healing. We speculated that it may act a role in the progression of osteoporosis. The current study purposed to explore the role of CIHH in the osteoporosis triggered by SCI in rats. A SCI-induced SCI model was established by completed transection at T9-T10 spinal cord of Wistar rats. One week after SCI, the rats were conducted toCIHH treatment (PB = 404 mmHg, Po2 = 84 mmHg) 6 hours a day for continuously 7 weeks. The results of X-radiography and Micro-CT assessment demonstrated that compared with sham rats, the areal bone mineral density (BMD), bone volume to tissue volume, volumetric BMD, trabecular thickness, trabecular number, and trabecular connectivity were decreased. Trabecular bone pattern factor, trabecular separation, as well as structure model index were increased at the distal femur and proximal tibia of SCI rats, which were effectively reversed by CIHH treatment. Histomorphometry showed that CIHH treatment increased bone formation of SCI rats, as evidenced by the increased osteoid formation, the decreased number and surface of TRAP-positive osteoclasts. Furthermore, ELISA and real time PCR results showed that the osteoblastogenesis-related biomarkers, such as procollagen type 1 N-terminal propeptide, osteocalcin in serum, as well as ALP and OPG mRNAs in bone tissue were decreased, while the osteoclastogenesis-related biomarkers, including scleorostin in serum and RANKL and TRAP mRNAs in bone tissue were increased in SCI rats. Importantly, the deviations of aforementioned biomarkers were improved by CIHH treatment. Mechanically, the protective effects of CIHH might be at least partly mediated by hypoxia-inducible factor-1 alpha (HIF-1α) signaling pathway. The present study testified that CIHH treatment ameliorates osteoporosis after SCI by balancing osteoblast and osteoclast activities in rats.

Comparison of the Effects of Metformin and Thiazolidinediones on Bone Metabolism: A Systematic Review and Meta-Analysis.

Studies have shown that people with diabetes have a high risk of osteoporosis and fractures. The effect of diabetic medications on bone disease cannot be ignored. This meta-analysis aimed to compare the effects of two types of glucose-lowering drugs, metformin and thiazolidinediones (TZD), on bone mineral density and bone metabolism in patients with diabetes mellitus. This systematic review and meta-analysis were prospectively registered on PROSPERO, and the registration number is CRD42022320884. Embase, PubMed, and Cochrane Library databases were searched to identify clinical trials comparing the effects of metformin and thiazolidinediones on bone metabolism in patients with diabetes. The literature was screened by inclusion and exclusion criteria. Two assessors independently assessed the quality of the identified studies and extracted relevant data. Seven studies involving 1656 patients were finally included. Our results showed that the metformin group had a 2.77% (SMD = 2.77, 95%CI [2.11, 3.43]; p < 0.00001) higher bone mineral density (BMD) than the thiazolidinedione group until 52 weeks; however, between 52 and 76 weeks, the metformin group had a 0.83% (SMD = -0.83, 95%CI: [-3.56, -0.45]; p = 0.01) lower BMD. The C-terminal telopeptide of type I collagen (CTX) and procollagen type I N-terminal propeptide (PINP) were decreased by 18.46% (MD = -18.46, 95%CI: [-27.98, -8.94], p = 0.0001) and 9.94% (MD = -9.94, 95%CI: [-16.92, -2.96], p = 0.005) in the metformin group compared with the TZD group.