Enzyme-Activated Biomimetic Vesicles Confining Mineralization for Bone Maturation.

Inspired by the crucial role of matrix vesicles (MVs), a series of biomimetic vesicles (BVs) fabricated by calcium glycerophosphate (CaGP) modified polyurethane were designed to mediate the mineralization through in situ enzyme activation for bone therapy. In this study, alkaline phosphatase (ALP) was harbored in the porous BVs by adsorption (Ad-BVs) or entrapment (En-BVs). High encapsulation of ALP on En-BVs was effectively self-activating by calcium ions of CaGP-modified PU that specifically hydrolyzed the organophosphorus (CaGP) to inorganic phosphate, thus promoting the formation of the highly oriented bone-like apatite in vitro. Enzyme-catalyzed kinetics confirms the regulation of apatite crystallization by the synergistic action of self-activated ALP and the confined microcompartments of BVs. This leads to a supersaturated microenvironment, with the En-BVs group exhibiting inorganic phosphate (Pi) levels 4.19 times higher and Ca2+ levels 3.67 times higher than those of simulated body fluid (SBF). Of note, the En-BVs group exhibited excellent osteo-inducing differentiation of BMSCs in vitro and the highest maturity with reduced bone loss in rat femoral defect in vivo. This innovative strategy of biomimetic vesicles is expected to provide valuable insights into the enzyme-activated field of bone therapy.

Effect of bone and analytical method on assessment of bone mineralization in response to dietary phosphorus, phytase, and vitamin D in finishing pigs.

A total of 882 pigs (PIC TR4 × [Fast LW × PIC L02]; initially 33.2 ±â€…0.31 kg) were used in a 112-d study to evaluate the effects of different bones and analytical methods on the assessment of bone mineralization response to changes in dietary P, phytase, and vitamin D in growing pigs. Pens of pigs (20 pigs per pen) were randomized to one of five dietary treatments with nine pens per treatment. Dietary treatments were designed to create differences in bone mineralization and included: 1) P at 80% of NRC (2012) standardized total tract digestible (STTD) P requirement, 2) NRC STTD P with no phytase, 3) NRC STTD P with phytase providing an assumed release of 0.14% STTD P from 2,000 FYT/kg, 4) high STTD P (128% of the NRC P) using monocalcium phosphate and phytase, and 5) diet 4 with additional vitamin D3 from 25(OH)D3. On day 112, one pig per pen was euthanized for bone, blood, and urine analysis. Additionally, 11 pigs identified as having poor body condition which indicated a history of low feed intake (unhealthy) were sampled. There were no differences between treatments for final body weight, average daily gain, average daily feed intake, gain to feed, or bone ash measurements (treatment × bone interaction) regardless of bone ash method. The response to treatment for bone density and bone mineral content was dependent upon the bone sampled (density interaction, P = 0.053; mineral interaction, P = 0.078). For 10th rib bone density, pigs fed high levels of P had increased (P < 0.05) bone density compared with pigs fed NRC levels with phytase, with pigs fed deficient P, NRC levels of P with no phytase, and high STTD P with extra 25(OH)D3 intermediate, with no differences for metacarpals, fibulas, or 2nd ribs. Pigs fed extra vitamin D from 25(OH)D3 had increased (P < 0.05) 10th rib bone mineral content compared with pigs fed deficient P and NRC levels of P with phytase, with pigs fed industry P and vitamin D, and NRC P with monocalcium intermediate. Healthy pigs had greater (P < 0.05) serum Ca, P, vitamin D concentrations, and defatted bone ash than those unhealthy, with no difference between the two health statuses for non-defatted bone ash. In summary, differences between bone ash procedures were more apparent than differences between diets. Differences in bone density and mineral content in response to dietary P and vitamin D were most apparent with 10th ribs.

Lameness is defined as impaired movement or deviation from normal gait. The evaluation of bone mineralization can be an important component of a diagnostic investigation of lameness. Lameness in growing pigs can cause an increase in morbidity and mortality, which leads to economic losses and animal welfare concerns for producers. Calcium and P are the primary minerals in skeletal tissue and their deficiency is considered to be one of the causes of lameness. To evaluate bone mineralization, it is important to know the differences between methodologies used to determine bone ash and the expected differences between the bones analyzed. Furthermore, there has been limited data comparing bone mineralization and serum Ca and P concentrations between healthy pigs and those exhibiting clinical signs of illness (unhealthy). By removing the lipid in the bone (defatting) before the bone is ashed, variation across bones is decreased compared with not removing lipid before ashing (non-defatted). The reduction in variation across bones allows for more differences to be detected among dietary treatments and health statuses of pigs. The 10th rib is more sensitive to detect dietary differences using bone density than metacarpals, fibulas, and 2nd ribs. When comparing healthy vs. unhealthy pigs exhibiting clinical signs of illness, healthy pigs have increased defatted percentage bone ash and serum Ca, P, and vitamin D.

17ß-Estradiol (E2) Activates Matrix Mineralization through Genomic/Nongenomic Pathways in MC3T3-E1 Cells.

Estrogen plays an important role in osteoporosis prevention. We herein report the possible novel signaling pathway of 17ß-estradiol (E2) in the matrix mineralization of MC3T3-E1, an osteoblast-like cell line. In the culture media-containing stripped serum, in which small lipophilic molecules such as steroid hormones including E2 were depleted, matrix mineralization was significantly reduced. However, the E2 treatment induced this. The E2 effects were suppressed by ICI182,780, the estrogen receptor (ER)α, and the ERß antagonist, as well as their mRNA knockdown, whereas Raloxifene, an inhibitor of estrogen-induced transcription, and G15, a G-protein-coupled estrogen receptor (GPER) 1 inhibitor, had little or no effect. Furthermore, the E2-activated matrix mineralization was disrupted by PMA, a PKC activator, and SB202190, a p38 MAPK inhibitor, but not by wortmannin, a PI3K inhibitor. Matrix mineralization was also induced by the culture media from the E2-stimulated cell culture. This effect was hindered by PMA or heat treatment, but not by SB202190. These results indicate that E2 activates the p38 MAPK pathway via ERs independently from actions in the nucleus. Such activation may cause the secretion of certain signaling molecule(s), which inhibit the PKC pathway. Our study provides a novel pathway of E2 action that could be a therapeutic target to activate matrix mineralization under various diseases, including osteoporosis.

Mineralization, degradation and osteogenic property of polylactide multicomponent porous composites for bone repair: In vitro and in vivo study.

Gelatin and hydroxyapatite were assembled into polylactide porous matrix to prepare multicomponent porous composites for bone repair (PLA-gH). PLA-gH possessed a superior ability of mineralization. During simulated body fluids (SBF), the spherical Ca-P depositions on surface of PLA-gH became bulk as Ca/P decreased, while they locally turned into the rod with different variation in Ca/P during SBF containing bovine serum albumin (SBF-BSA), indicating that the mineralization of PLA-gH could be regulated by BSA. Meanwhile, PLA-gH possessed good degradation behaviour, especially in SBF-BSA, the degradation of PLA porous matrix was higher than that in SBF after 14-day immersion, whose crystallinity (Xc) decreased to a slightly lower level. Gelatin and hydroxyapatite endowed PLA-gH with good osteogenic property, characterized by obvious osteogenic differentiation and bone regeneration. In terms of predicting the cytocompatibility, osteogenic differentiation and new bone mineralization of PLA-gH by in vitro methods, applying SBF-BSA may be more reliable than SBF.

Multiscale effects of the calcimimetic drug, etelcalcetide on bone health of rats with secondary hyperparathyroidism induced by chronic kidney disease.

Chronic kidney disease-induced secondary hyperparathyroidism (CKD-SHPT) heightens fracture risk through impaired mineral homeostasis and elevated levels of uremic toxins (UTs), which in turn enhance bone remodeling. Etelcalcetide (Etel), a calcium-sensing receptor (CaSR) agonist, suppresses parathyroid hormone (PTH) in hyperparathyroidism to reduce excessive bone resorption, leading to increased bone mass. However, Etel's effect on bone quality, chemical composition, and strength is not well understood. To address these gaps, we established a CKD-SHPT rat model and administered Etel at a human-equivalent dose concurrently with disease induction. The effects on bone and mineral homeostasis were compared with a CKD-SHPT (vehicle-treated group) and a control group (rats without SHPT). Compared with vehicle-treated CKD-SHPT rats, Etel treatment improved renal function, reduced circulating UT levels, improved mineral homeostasis parameters, decreased PTH levels, and prevented mineralization defects. The upregulation of mineralization-promoting genes by Etel in CKD-SHPT rats might explain its ability to prevent mineralization defects. Etel preserved both trabecular and cortical bones with attendant suppression of osteoclast function, besides increasing mineralization. Etel maintained the number of viable osteocytes to the control level, which could also contribute to its beneficial effects on bone. CKD-SHPT rats displayed increased carbonate substitution of matrix and mineral, decreased crystallinity, mineral-to-matrix ratio, and collagen maturity, and these changes were mitigated by Etel. Further, Etel treatment prevented CKD-SHPT-induced deterioration in bone strength and mechanical behavior. Based on these findings, we conclude that in CKD-SHPT rats, Etel has multiscale beneficial effects on bone that involve remodeling suppression, mineralization gene upregulation, and preservation of osteocytes.

Controlled delivery of procyanidin through magnesium oxide nanoparticles (MgO NPs) to improve the activity and mineralization of osteoblasts under oxidative stressin vitro.

Excessive reactive oxygen species (ROS) in the microenvironment of osteoporosis (OP) not only accelerate the bone absorption, but also affect the osteogenic and mineralized effect of osteoblasts. Procyanidins (PC) have been reported to have anti-oxidation effects, but low bioavailability. This study aimed to explore the effect of magnesium oxide nanoparticles (MgO-PC NPs)-loaded PC on the osteogenesis and mineralization of osteoblasts that stimulated by H2O2. PC was loaded onto MgO NPs and characterized by transmission electron microscopy, energy dispersive spectroscopy, dynamic light scattering, and Fourier transform infrared spectroscopy. After primary screening by cytotoxicity assay, MgO-PC NPs containing 20 µM of PC were chosen for further studies. In H2O2-stimulated osteoblasts, dichlorodihydrofluorescein diacetate probe, Cell Counting Kit-8, quantitative real-time polymerase chain reaction, alkaline phosphatase staining/activity and Alizarin red staining were used to detect the ROS production, cell viability and osteogenic and mineralized markers of osteoblasts. PC was loaded onto MgO NPs to successfully receive MgO-PC NPs with a diameter of about 144 nm and negative potential. PC can sustain release from MgO-PC NPs for at least 16 d. The controlled release of PC from MgO-PC NPs can effectively eliminate ROS and thereby promoted the cell activity. Most importantly, the osteogenesis and mineralization of osteoblasts under oxidative stress were also significantly reversed by MgO-PC NPS. Thus, these findings indicate that MgO-PC NPs may be developed as a potential therapeutic strategy for OP.

Exposure to BPA and BPS during pregnancy disrupts the bone mineralization in the offspring.

Exposure to plastic-derived estrogen-mimicking endocrine-disrupting bisphenols can have a long-lasting effect on bone health. However, gestational exposure to bisphenol A (BPA) and its analogue, bisphenol S (BPS), on offspring's bone mineralization is unclear. The effects of in-utero bisphenol exposure were examined on the offspring's bone parameters. BPA and BPS (0.0, 0.4 µg/kg bw) were administered to pregnant Wistar rats via oral gavage from gestational day 4-21. Maternal exposure to BPA and BPS increased bone mineral content and density in the offspring aged 30 and 90 days (P < 0.05). Plasma analysis revealed that alkaline phosphatase, and Gla-type osteocalcin were significantly elevated in the BPS-exposed offspring (P < 0.05). The expression of BMP1, BMP4, and their signaling mediators SMAD1 mRNAs were decreased in BPS-exposed osteoblast SaOS-2 cells (P < 0.05). The expression of extracellular matrix proteins such as ALPL, COL1A1, DMP1, and FN1 were downregulated (P < 0.05). Bisphenol co-incubation with noggin decreased TGF-ß1 expression, indicating its involvement in bone mineralization. Altered mineralization could be due to dysregulated expression of bone morphogenetic proteins and signalling mediators in the osteoblast cells. Thus, bisphenol exposure during gestation altered growth and bone mineralization in the offspring, possibly by modulating the expression of Smad-dependent BMP/TGF-ß1 signalling mediators.

Porous Nano-Fiber Structure of Modified Electrospun Chitosan GBR Membranes Improve Osteoblast Calcium Phosphate Deposition in Osteoblast-Fibroblast Co-Cultures.

Desirable characteristics of electrospun chitosan membranes (ESCM) for guided bone regeneration are their nanofiber structure that mimics the extracellular fiber matrix and porosity for the exchange of signals between bone and soft tissue compartments. However, ESCM are susceptible to swelling and loss of nanofiber and porous structure in physiological environments. A novel post-electrospinning method using di-tert-butyl dicarbonate (tBOC) prevents swelling and loss of nanofibrous structure better than sodium carbonate treatments. This study aimed to evaluate the hypothesis that retention of nanofiber morphology and high porosity of tBOC-modified ESCM (tBOC-ESCM) would support more bone mineralization in osteoblast-fibroblast co-cultures compared to Na2CO3 treated membranes (Na2CO3-ESCM) and solution-cast chitosan solid films (CM-film). The results showed that only the tBOC-ESCM retained the nanofibrous structure and had approximately 14 times more pore volume than Na2CO3-ESCM and thousands of times more pore volume than CM-films, respectively. In co-cultures, the tBOC-ESCM resulted in a significantly greater calcium-phosphate deposition by osteoblasts than either the Na2CO3-ESCM or CM-film (p < 0.05). This work supports the study hypothesis that tBOC-ESCM with nanofiber structure and high porosity promotes the exchange of signals between osteoblasts and fibroblasts, leading to improved mineralization in vitro and thus potentially improved bone healing and regeneration in guided bone regeneration applications.

Estradiol-17ß and bisphenol A affect growth and mineralization in early life stages of seabass.

Natural and synthetic estrogens are contaminants present in aquatic ecosystems. They can have significant consequences on the estrogen-sensitive functions of organisms, including skeletal development and growth of vertebrate larvae. Synthetic polyphenols represent a group of environmental xenoestrogens capable of binding the receptors for the natural hormone estradiol-17ß (E2). To better understand how (xeno-)estrogens can affect the skeleton in fish species with high ecological and commercial interest, 16 days post-hatch larvae of the seabass were experimentally exposed for 7 days to E2 and Bisphenol A (BPA), both used at the regulatory concentration of surface water quality (E2: 0.4 ng.L-1, BPA: 1.6 µg.L-1) or at a concentration 100 times higher. Skeletal mineralization levels were evaluated using Alizarin red staining, and expression of several genes playing key roles in growth, skeletogenesis and estrogen signaling pathways was assessed by qPCR. Our results show that E2 exerts an overall negative effect on skeletal mineralization at the environmental concentration of 0.4 ng.L-1, correlated with an increase in the expression of genes associated only with osteoblast bone cells. Both BPA exposures inhibited mineralization with less severe effects and modified bone homeostasis by regulating the expression of gene encoding osteoblasts and osteoclasts markers. Our results demonstrate that environmental E2 exposure inhibits larval growth and has an additional inhibitory effect on skeleton mineralization while both BPA exposures have marginal inhibitory effect on skeletal mineralization. All exposures have significant effects on transcriptional levels of genes involved in the skeletal development of seabass larvae.

Laser-assisted surface alloying of titanium with silver to enhance antibacterial and bone-cell mineralization properties of orthopedic implants.

Orthopedic device-related infection (ODRI) poses a significant threat to patients with titanium-based implants. The challenge lies in developing antibacterial surfaces that preserve the bulk mechanical properties of titanium implants while exhibiting characteristics similar to bone tissue. In response, we present a two-step approach: silver nanoparticle (AgNP) coating followed by selective laser-assisted surface alloying on commonly used titanium alumina vanadium (TiAl6V4) implant surfaces. This process imparts antibacterial properties without compromising the bulk mechanical characteristics of the titanium alloy. Systematic optimization of laser beam power (8-40 W) resulted in an optimized surface (32 W) with uniform TiAg alloy formation. This surface displayed a distinctive hierarchical mesoporous textured surface, featuring cauliflower-like nanostructures measuring between 5-10 nm uniformly covering spatial line periods of 25 µm while demonstrating homogenous elemental distribution of silver throughout the laser processed surface. The optimized laser processed surface exhibited prolonged superhydrophilicity (40 days) and antibacterial efficacy (12 days) against Staphylococcus aureus and Escherichia coli. Additionally, there was a significant twofold increase in bone mineralization compared to the pristine Ti6Al4V surface (p < 0.05). Rockwell hardness tests confirmed minimal (<1%) change in bulk mechanical properties compared to the pristine surface. This innovative laser-assisted approach, with its precisely tailored surface morphology, holds promise for providing enduring antibacterial and osteointegration properties, rendering it an optimal choice for modifying load-bearing implant devices without altering material bulk characteristics.

Organic Matrices of Calcium Carbonate Biominerals Improve Osteoblastic Mineralization.

Many organisms incorporate inorganic solids into their tissues to improve functional and mechanical properties. The resulting mineralized tissues are called biominerals. Several studies have shown that nacreous biominerals induce osteoblastic extracellular mineralization. Among them, Pinctada margaritifera is well known for the ability of its organic matrix to stimulate bone cells. In this context, we aimed to study the effects of shell extracts from three other Pinctada species (Pinctada radiata, Pinctada maxima, and Pinctada fucata) on osteoblastic extracellular matrix mineralization, by using an in vitro model of mouse osteoblastic precursor cells (MC3T3-E1). For a better understanding of the Pinctada-bone mineralization relationship, we evaluated the effects of 4 other nacreous mollusks that are phylogenetically distant and distinct from the Pinctada genus. In addition, we tested 12 non-nacreous mollusks and one extra-group. Biomineral shell powders were prepared, and their organic matrix was partially extracted using ethanol. Firstly, the effect of these powders and extracts was assessed on the viability of MC3T3-E1. Our results indicated that neither the powder nor the ethanol-soluble matrix (ESM) affected cell viability at low concentrations. Then, we evaluated osteoblastic mineralization using Alizarin Red staining and we found a prominent MC3T3-E1 mineralization mainly induced by nacreous biominerals, especially those belonging to the Pinctada genus. However, few non-nacreous biominerals were also able to stimulate the extracellular mineralization. Overall, our findings validate the remarkable ability of CaCO3 biomineral extracts to promote bone mineralization. Nevertheless, further in vitro and in vivo studies are needed to uncover the mechanisms of action of biominerals in bone.

Six months of voluntary alcohol consumption in male cynomolgus macaques reduces intracortical bone porosity without altering mineralization or mechanical properties.

Chronic heavy alcohol consumption is a risk factor for low trauma bone fracture. Using a non-human primate model of voluntary alcohol consumption, we investigated the effects of 6 months of ethanol intake on cortical bone in cynomolgus macaques (Macaca fascicularis). Young adult (6.4 ± 0.1 years old, mean ± SE) male cynomolgus macaques (n = 17) were subjected to a 4-month graded ethanol induction period, followed by voluntary self-administration of water or ethanol (4 % w/v) for 22 h/d, 7 d/wk. for 6 months. Control animals (n = 6) consumed an isocaloric maltose-dextrin solution. Tibial response was evaluated using densitometry, microcomputed tomography, histomorphometry, biomechanical testing, and Raman spectroscopy. Global bone response was evaluated using biochemical markers of bone turnover. Monkeys in the ethanol group consumed an average of 2.3 ± 0.2 g/kg/d ethanol resulting in a blood ethanol concentration of 90 ± 12 mg/dl in longitudinal samples taken 7 h after the daily session began. Ethanol consumption had no effect on tibia length, mass, density, mechanical properties, or mineralization (p > 0.642). However, compared to controls, ethanol intake resulted in a dose-dependent reduction in intracortical bone porosity (Spearman rank correlation = -0.770; p < 0.0001) and compared to baseline, a strong tendency (p = 0.058) for lower plasma CTX, a biochemical marker of global bone resorption. These findings are important because suppressed cortical bone remodeling can result in a decrease in bone quality. In conclusion, intracortical bone porosity was reduced to subnormal values 6 months following initiation of voluntary ethanol consumption but other measures of tibia architecture, mineralization, or mechanics were not altered.

Calcified Cartilage-Guided Identification of Osteogenic Molecules and Geometries.

Calcified cartilage digested by chondroclasts provides an excellent scaffold to initiate bone formation. We analyzed bioactive proteins and microarchitecture of calcified cartilage either separately or in combination and evaluated biomimetic osteogenic culture conditions of surface-coated micropatterning. To do so, we prepared a crude extract from porcine femoral growth plates, which enhanced in vitro mineralization when coated on flat-bottom culture dishes, and identified four candidate proteins by fractionation and mass spectrometry. Murine homologues of two candidates, desmoglein 4 (DSG4) and peroxiredoxin 6 (PRDX6), significantly promoted osteogenic activity based on in vitro mineralization and osteoblast differentiation. Moreover, we observed DSG4 and PRDX6 protein expression in mouse femur. In addition, we designed circular, triangular, and honeycomb micropatterns with 30 or 50 µm units, either isolated or connected, to mimic hypertrophic chondrocyte-sized compartments. Isolated, larger honeycomb patterns particularly enhanced osteogenesis in vitro. Mineralization on micropatterns was positively correlated with the reduction of osteoblast migration distance in live cell imaging. Finally, we evaluated possible combinatorial effects of coat proteins and micropatterns and observed an additive effect of DSG4 or PRDX6 coating with micropatterns. These data suggest that combining a bioactive surface coating with osteogenic micropatterns may recapitulate initiation of bone formation during endochondral ossification.

Piezocatalytically-induced controllable mineralization scaffold with bone-like microenvironment to achieve endogenous bone regeneration.

Orderly hierarchical structure with balanced mechanical, chemical, and electrical properties is the basis of the natural bone microenvironment. Inspired by nature, we developed a piezocatalytically-induced controlled mineralization strategy using piezoelectric polymer poly-L-lactic acid (PLLA) fibers with ordered micro-nano structures to prepare biomimetic tissue engineering scaffolds with a bone-like microenvironment (pcm-PLLA), in which PLLA-mediated piezoelectric catalysis promoted the in-situ polymerization of dopamine and subsequently regulated the controllable growth of hydroxyapatite crystals on the fiber surface. PLLA fibers, as analogs of mineralized collagen fibers, were arranged in an oriented manner, and ultimately formed a bone-like interconnected pore structure; in addition, they also provided bone-like piezoelectric properties. The uniformly sized HA nanocrystals formed by controlled mineralization provided a bone-like mechanical strength and chemical environment. The pcm-PLLA scaffold could rapidly recruit endogenous stem cells, and promote their osteogenic differentiation by activating cell membrane calcium channels and PI3K signaling pathways through ultrasound-responsive piezoelectric signals. In addition, the scaffold also provided a suitable microenvironment to promote macrophage M2 polarization and angiogenesis, thereby enhancing bone regeneration in skull defects of rats. The proposed piezocatalytically-induced controllable mineralization strategy provides a new idea for the development of tissue engineering scaffolds that can be implemented for multimodal physical stimulation therapy.

Novel Peptide Derived from Gadus morhua Stimulates Osteoblastic Differentiation and Mineralization through Wnt/ß-Catenin and BMP Signaling Pathways.

Marine biodiversity offers a wide array of active ingredient resources. Gadus morhua peptides (GMPs) showed excellent osteoprotective effects in ovariectomized mice. However, the potential osteogenesis mechanisms of key osteogenic peptides in GMP were seldom reported. In this study, a novel osteogenic peptide (GETNPADSKPGSIR, P-GM-2) was screened from GMP. P-GM-2 has a high stability coefficient and a strong interaction with epidermal growth factor receptor. Cell culture experiments showed that P-GM-2 stimulated the expression of osteogenic differentiation markers to promote osteoblast proliferation, differentiation, and mineralization. Additionally, P-GM-2 phosphorylates GSK-3ß, leading to the stabilization of ß-catenin and its translocation to the nucleus, thus initiating the activation of the Wnt/ß-catenin signaling pathway. Meanwhile, P-GM-2 could also regulate the osteogenic differentiation of preosteoblasts by triggering the BMP/Smad and mitogen-activated protein kinase signaling pathways. Further validation with specific inhibitors (ICG001 and Noggin) demonstrated that the osteogenic activity of P-GM-2 was revealed by the activation of the BMP and Wnt/ß-catenin pathways. In summary, these results provide theoretical and practical insights into P-GM-2 as an effective antiosteoporosis active ingredient.

Unveiling the role of hydroxyapatite and hydroxyapatite/silver composite in osteoblast-like cell mineralization: An exploration through their viscoelastic properties.

Mechanical properties are becoming fundamental for advancing the comprehension of cellular processes. This study addresses the relationship between viscoelastic properties and the cellular mineralization process. Osteoblast-like cells treated with an osteogenic medium were employed for this purpose. Additionally, the study explores the impact of hydroxyapatite (HA) and hydroxyapatite/silver (HA/Ag) composite on this process. AFM relaxation experiments were conducted to extract viscoelastic parameters using the Fractional Zener (FZ) and Fractional Kelvin (FK) models. Our findings revealed that the main phases of mineralization are associated with alterations in the viscoelastic properties of osteoblast-like cells. Furthermore, HA and HA/Ag treatments significantly influenced changes in the viscoelastic properties of these cells. In particular, the HA/Ag treatment demonstrated a marked enhancement in cell fluidity, suggesting a possible role of silver in accelerating the mineralization process. Moreover, the study underscores the independence observed between fluidity and stiffness, indicating that modifications in one parameter may not necessarily correspond to changes in the other. These findings shed light on the factors involved in the cellular mineralization process and emphasize the importance of using viscoelastic properties to discern the impact of treatments on cells.

A synergistic strategy based on active hydroxymethyl amine compounds and fucoidan for bioprosthetic heart valves with enhancing anti-coagulation and anti-calcification properties.

With an aging population, the patients with valvular heart disease (VHD) are growing worldwide, and valve replacement is a primary choice for these patients with severe valvular disease. Among them, bioprosthetic heart valves (BHVs), especially BHVs trough transcatheter aortic valve replacement, are widely accepted by patients on account of their good hemodynamics and biocompatibility. Commercial BHVs in clinic are prepared by glutaraldehyde cross-linked pericardial tissue with the risk of calcification and thrombotic complications. In the present study, a strategy combines improved hemocompatibility and anti-calcification properties for BHVs has been developed based on a novel non-glutaraldehyde BHV crosslinker hexakis(hydroxymethyl)melamine (HMM) and the anticoagulant fucoidan. Besides the similar mechanical properties and enhanced component stability compared to glutaraldehyde crosslinked PP (G-PP), the fucoidan modified HMM-crosslinked PPs (HMM-Fu-PPs) also exhibit significantly enhanced anticoagulation performance with a 72 % decrease in thrombus weight compared with G-PP in ex-vivo shunt assay, along with the superior biocompatibility, satisfactory anti-calcification properties confirmed by subcutaneous implantation. Owing to good comprehensive performance of these HMM-Fu-PPs, this simple and feasible strategy may offer a great potential for BHV fabrication in the future, and open a new avenue to explore more N-hydroxymethyl compound based crosslinker with excellent performance in the field of biomaterials.

Interaction between p21-activated kinase 4 and ß-catenin as a novel pathway for PTH-dependent osteoblast activation.

Parathyroid hormone (PTH) serves dual roles in bone metabolism, exhibiting both anabolic and catabolic effects. The anabolic properties of PTH have been utilized in the treatment of osteoporosis with proven efficacy in preventing fractures. Despite these benefits, PTH can be administered therapeutically for up to 2 years, and its use in patients with underlying malignancies remains a subject of ongoing debate. These considerations underscore the need for a more comprehensive understanding of the underlying mechanisms. p21-activated kinase 4 (PAK4) is involved in bone resorption and cancer-associated osteolysis; however, its role in osteoblast function and PTH action remains unknown. Therefore, in this study, we aimed to clarify the role of PAK4 in osteoblast function and its effects on PTH-induced anabolic activity. PAK4 enhanced MC3T3-E1 osteoblast viability and proliferation and upregulated cyclin D1 expression. PAK4 also augmented osteoblast differentiation, as indicated by increased mineralization found by alkaline phosphatase and Alizarin Red staining. Treatment with PTH (1-34), an active PTH fragment, stimulated PAK4 expression and phosphorylation in a protein kinase A-dependent manner. In addition, bone morphogenetic protein-2 (which is known to promote bone formation) increased phosphorylated PAK4 (p-PAK4) and PAK4 levels. PAK4 regulated the expression of both phosphorylated and total ß-catenin, which are critical for osteoblast proliferation and differentiation. Moreover, p-PAK4 directly interacted with ß-catenin, and disruption of ß-catenin's binding to T-cell factor impaired PAK4- and PTH-induced osteoblast differentiation. Our findings elucidate the effect of PAK4 on enhancing bone formation in osteoblasts and its pivotal role in the anabolic activity of PTH mediated through its interaction with ß-catenin. These insights improve the understanding of the mechanisms underlying PTH activity and should inform the development of more effective and safer osteoporosis treatments.

Chemical etching of Ti-6Al-4V biomaterials fabricated by selective laser melting enhances mesenchymal stromal cell mineralization.

Porous titanium scaffolds fabricated by powder bed fusion additive manufacturing techniques have been widely adopted for orthopedic and bone tissue engineering applications. Despite the many advantages of this approach, topological defects inherited from the fabrication process are well understood to negatively affect mechanical properties and pose a high risk if dislodged after implantation. Consequently, there is a need for further post-process surface cleaning. Traditional techniques such as grinding or polishing are not suited to lattice structures, due to lack of a line of sight to internal features. Chemical etching is a promising alternative; however, it remains unclear if changes to surface properties associated with such protocols will influence how cells respond to the material surface. In this study, we explored the response of bone marrow derived mesenchymal stem/stromal cells (MSCs) to Ti-6Al-4V whose surface was exposed to different durations of chemical etching. Cell morphology was influenced by local topological features inherited from the SLM fabrication process. On the as-built surface, topological nonhomogeneities such as partially adhered powder drove a stretched anisotropic cellular morphology, with large areas of the cell suspended across the nonhomogeneous powder interface. As the etching process was continued, surface defects were gradually removed, and cell morphology appeared more isotropic and was suggestive of MSC differentiation along an osteoblastic-lineage. This was accompanied by more extensive mineralization, indicative of progression along an osteogenic pathway. These findings point to the benefit of post-process chemical etching of additively manufactured Ti-6Al-4V biomaterials targeting orthopedic applications.

Periostin Promotes the Proliferation, Differentiation and Mineralization of Osteoblasts from Ovariectomized Rats.

Perimenopausal period causes a significant amount of bone loss, which results in primary osteoporosis (OP). The Periostin (Postn) may play important roles in the pathogenesis of OP after ovariectomized (OVX) rats. To identify the roles of Postn in the bone marrow mesenchymal stem cell derived osteoblasts (BMSC-OB) in OVX rats, we investigated the expression of Wnt/ß-catenin signaling pathways in BMSC-OB and the effects of Postn on bone formation by development of BMSC-OB cultures. Twenty-four female Sprague-Dawley rats at 6 months were randomized into 3 groups: sham-operated (SHAM) group, OVX group and OVX+Postn group. The rats were killed after 3 months, and their bilateral femora and tibiae were collected for BMSC-OB culture, Micro-CT Analysis, Bone Histomorphometric Measurement, Transmission Electron Microscopy and Immunohistochemistry Staining. The dose/time-dependent effects of Postn on the proliferation, differentiation and mineralization of BMSC-OB and the expression of osteoblastic markers were measured in in vitro experiments. We found increased Postn increased bone mass, promoted bone formation of trabeculae, Wnt signaling and the osteogenic activity in osteoblasts in sublesional femur. Postn have the function to enhance cell proliferation, differentiation and mineralization at a proper concentration and incubation time. Interestingly, in BMSC-OB from OVX rats treated with the different dose of Postn, the osteoblastic markers expression and Wnt/ß-catenin signaling pathways were significantly promoted. The direct effect of Postn may lead to inhibit excessive bone resorption and increase bone formation through the Wnt/ß-catenin signaling pathways after OVX. Postn may play a very important role in the pathogenesis of OP after OVX.