Bone Marker Gene Expression Research Articles

The potent osteo-inductive capacity of bioinspired brown seaweed-derived carbohydrate nanofibrous three-dimensional scaffolds.

This study aimed to investigate the osteo-inductive capacity of a fucoidan polysaccharide network derived from brown algae on human adipose-derived stem cells (HA-MSCs) for bone regeneration. The physiochemical properties of the scaffold including surface morphology, surface chemistry, hydrophilicity, mechanical stiffness, and porosity were thoroughly characterized. Both in vitro and in vivo measurements implied a superior cell viability, proliferation, adhesion, and osteo-inductive performance of obtained scaffolds compared to using specific osteogenic induction medium with increased irregular growth of calcium crystallites, which mimic the structure of natural bones. That scaffold was highly biocompatible and suitable for cell cultures. Various examinations, such as quantification of mineralization, alkaline phosphatase, gene expression, and immunocytochemical staining of pre-osteocyte and bone markers confirmed that HAD-MSCs differentiate into osteoblasts, even without an osteogenic induction medium. This study provides evidence for the positive relationship and synergistic effects between the physical properties of the decellularized seaweed scaffold and the chemical composition of fucoidan in promoting the osteogenic differentiation of HA-MSCs. Altogether, the natural matrices derived from brown seaweed offers a sustainable, cost-effective, non-toxic bioinspired scaffold and holds promise for future clinical applications in orthopedics.

The Phenotype of Mesenchymal Stromal Cell and Articular Chondrocyte Cocultures on Highly Porous Bilayer Poly-L-Lactic Acid Scaffolds Produced by Thermally Induced Phase Separation and Supplemented with Hydroxyapatite.

Bilayer scaffolds could provide a suitable topology for osteochondral defect repair mimicking cartilage and subchondral bone architecture. Hence, they could facilitate the chondro- and osteogenic lineage commitment of multipotent mesenchymal stromal cells (MSCs) with hydroxyapatite, the major inorganic component of bone, stimulating osteogenesis. Highly porous poly-L-lactic acid (PLLA) scaffolds with two layers of different pore sizes (100 and 250 µm) and hydroxyapatite (HA) supplementation were established by thermally induced phase separation (TIPS) to study growth and osteogenesis of human (h) MSCs. The topology of the scaffold prepared via TIPS was characterized using scanning electron microscopy (SEM), a microCT scan, pycnometry and gravimetric analysis. HMSCs and porcine articular chondrocytes (pACs) were seeded on the PLLA scaffolds without/with 5% HA for 1 and 7 days, and the cell attachment, survival, morphology, proliferation and gene expression of cartilage- and bone-related markers as well as sulfated glycosaminoglycan (sGAG) synthesis were monitored. All scaffold variants were cytocompatible, and hMSCs survived for the whole culture period. Cross-sections revealed living cells that also colonized inner scaffold areas, producing an extracellular matrix (ECM) containing sGAGs. The gene expression of cartilage and bone markers could be detected. HA represents a cytocompatible supplement in PLLA composite scaffolds intended for osteochondral defects.

Titanium micro-nano textured surface with strontium incorporation improves osseointegration: an in vivo and in vitro study.

This study aimed to investigate the osseointegration of titanium (Ti) implants with micro-nano textured surfaces functionalized with strontium additions (Sr) in a pre-clinical rat tibia model. Ti commercially pure (cp-Ti) implants were installed bilaterally in the tibia of 64 Holtzman rats, divided into four experimental groups (n=16/group): (1) Machined surface - control (C); (2) Micro-nano textured surface treatment (MN); (3) Micro-nano textured surface with Sr2+ addition (MNSr); and (4) Micro-nano textured surface with a higher complementary addition of Sr2+ (MNSr+). In total, two experimental euthanasia periods were assessed at 15 and 45 days (n=8/period). The tibia was subjected to micro-computed tomography (μ-CT), histomorphometry with the EXAKT system, removal torque (TR) testing, and gene expression analysis by PCR-Array of 84 osteogenic markers. Gene expression and protein production of bone markers were performed in an in vitro model with MC3T3-E1 cells. The surface characteristics of the implants were evaluated by scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS), and laser scanning confocal microscopy. SEM, confocal, and EDS analyses demonstrated the formation of uniform micro-nano textured surfaces in the MN group and Sr addition in the MNSr and MNSr+ groups. TR test indicated greater osseointegration in the 45-day period for treated surfaces. Histological analysis highlighted the benefits of the treatments, especially in cortical bone, in which an increase in bone-implant contact was found in groups MN (15 days) and MNSr (45 days) compared to the control group. Gene expression analysis of osteogenic activity markers showed modulation of various osteogenesis-related genes. According to the in vitro model, RT-qPCR and ELISA demonstrated that the treatments favored gene expression and production of osteoblastic differentiation markers. Micro-nano textured surface and Sr addition can effectively improve and accelerate implant osseointegration and is, therefore, an attractive approach to modifying titanium implant surfaces with significant potential in clinical practice.

Probiotics: Can it modulate fracture healing?

Fractures remain a huge burden and their management adversely affects individuals' function and productivity during the lengthy healing period. Gut microbiota exerts a systemic influence on diverse aspects of host physiology, including bone. The primary objective of this study was to evaluate if oral probiotic treatment before or after a fracture in a mouse model could increase cytokines and biomarkers essential for bone healing with subsequent improvement in the biomechanical properties of the healed callus. Femoral osteotomy and intramedullary pinning were performed on C57BL/6 mice. Group 1 received either control PBS or probiotic via oral gavage for 5 weeks before fracture (pre-fracture). Group 2 received equivalent treatments for 4 weeks only after fracture (post-fracture). Fracture calluses were harvested on day 3 and 7 for RT-qPCR to quantify osteogenic-related inflammatory cytokines and bone biomarkers. Fractured femurs were evaluated day 28 post-osteotomy via microstructural analysis (μCT) and biomechanical testing (torsion). Mice treated with probiotics pre-fracture (group 1) showed significantly increased gene expression on day 3 of cytokines TGF-β, IL-6 and IL-17F and a corresponding increase in gene expression on day 7 for Col1 and Runx2. Significant improvement was also seen in bone volume fraction, bone mineral density, tissue mineral density, maximum yield torque, stiffness and strain energy. Mice treated with probiotics post-fracture (group 2), demonstrated no changes in cytokine or bone marker gene expression with no significant changes on microstructural analysis. However, significant increases were seen in twist angle at failure and strain energy, with a corresponding reduction in torsional stiffness. Our results suggest that oral probiotic administration, before or after a fracture, may sufficiently alter the gut flora microenvironment leading to improved bone healing biomechanical properties. The use of probiotics may provide a cost-effective and low-risk adjunctive therapy to improve fracture healing.

The Combination of Ultraviolet-B and Vitamin K2 Exposure Effect on Fibroblast-like cell Number in Wistar Rats

Background: Vitamin D deficiency in children is one of the problems most often discussed and received attention from around the world today, especially during the Corona virus disease-19 (Covid-19) pandemic. Vitamin D3 increases the production of vitamin K2 protein and activates a protein involved in bone metabolism. Vitamin K2 increases bone formation by stimulating osteoblast differentiation, regulating mineralization of the extracellular matrix, regulating bone marker gene expression, and inhibiting osteoclastogenesis. Purpose: to analyzed the effect of sun exposure, Ultraviolet-B (UV-B) and vitamin K2 supplementation on fibroblast-like cell as bone formation marker in Wistar rats (Rattus novergicus). Methods: twenty-four samples divided into 4 groups namely control group, UV-B group, vitamin K2 group, combination of UV-B and vitamin K2 group. After 21 days, extraction of lower incisors was done to examine fibroblast-like cell number ​​after treatment. After being decalcified, specimens underwent histological evaluation using Haemotoxylin and Eosin staining to observe the fibroblast-like cell number. Result: Data analysis of fibroblast like cells number expression using one way analysis of variance (ANOVA) test showed a significant difference between sample groups (p<0.05). Conclusion: The combination of UV-B exposure and vitamin K2 administration group increased of fibroblast-like cells in Wistar rats (R. novergicus).

The Role of PEMFs on Bone Healing: An In Vitro Study.

Bone responses to pulsed electromagnetic fields (PEMFs) have been extensively studied by using devices that expose bone cells to PEMFs to stimulate extracellular matrix (ECM) synthesis for bone and cartilage repair. The aim of this work was to highlight in which bone healing phase PEMFs exert their action. Specifically, we evaluated the effects of PEMFs both on human adipose mesenchymal stem cells (hASCs) and on primary human osteoblasts (hOBs) by testing gene and protein expression of early bone markers (on hASCs) and the synthesis of late bone-specific proteins (on hOBs) as markers of bone remodeling. Our results indicate that PEMFs seem to exert their action on bone formation, acting on osteogenic precursors (hASCs) and inducing the commitment towards the differentiation pathways, unlike mature and terminally differentiated cells (hOBs), which are known to resist homeostasis perturbation more and seem to be much less responsive than mesenchymal stem cells. Understanding the role of PEMFs on bone regenerative processes provides important details for their clinical application.

Photobiomodulation treatments drive osteogenic versus adipocytic fate of bone marrow mesenchymal stem cells reversing the effects of hyperglycemia in diabetes.

Diabetes mellitus (DM) is a chronic metabolic disease that affects bone metabolism, which can be related to a reduced osteogenic potential of bone marrow mesenchymal stem cells (BM-MSCs). MSCs from diabetic rats (dBM-MSC) have shown a tendency to differentiate towards adipocytes (AD) instead of osteoblasts (OB). Since photobiomodulation (PBM) therapy is a non-invasive treatment capable of recovering the osteogenic potential of dBM-MSCs, we aimed to evaluate whether PBM can modulate MSC's differentiation under hyperglycemic conditions. BM-MSCs of healthy and diabetic rats were isolated and differentiated into osteoblasts (OB and dOB) and adipocytes (AD and dAD). dOB and dAD were treated with PBM every 3days (660nm; 5J/cm2; 0.14J; 20 mW; 0.714W/cm2) for 17days. Cell morphology and viability were evaluated, and cell differentiation was confirmed by gene expression (RT-PCR) of bone (Runx2, Alp, and Opn) and adipocyte markers (Pparγ, C/Ebpα, and C/Ebpβ), production of extracellular mineralized matrix (Alizarin Red), and lipid accumulation (Oil Red). Despite no differences on cell morphology, the effect of DM on cells was confirmed by a decreased gene expression of bone markers and matrix production of dOB, and an increased expression of adipocyte and lipid accumulation of dAD, compared to heatlhy cells. On the other hand, PBM reversed the effects of dOB and dAD. The negative effect of DM on cells was confirmed, and PBM improved OB differentiation while decreasing AD differentiation, driving the fate of dBM-MSCs. These results may contribute to optimizing bone regeneration in diabetic patients.

Effects of Dicationic Imidazolium-Based Ionic Liquid Coatings on Oral Osseointegration of Titanium Implants: A Biocompatibility Study in Multiple Rat Demographics.

Dicationic imidazolium-based ionic liquids with amino acid anions, such as IonL-phenylalanine (IonL-Phe), have been proposed as a multifunctional coating for titanium (Ti) dental implants. However, there has been no evaluation of the biocompatibility of these Ti coatings in the oral environment. This study aims to evaluate the effects of IonL-Phe on early healing and osseointegration of Ti in multiple rat demographics. IonL-Phe-coated and uncoated Ti screws were implanted into four demographic groups of rats to represent biological variations that could affect healing: young males (YMs) and females (YFs), ovariectomized (OVXFs) females, and old males (OMs). Samples underwent histopathological and histomorphometric analysis to evaluate healing at 7 and 30 days around IonL-coated and uncoated Ti. The real-time quantitative polymerase chain reaction was also conducted at the 2- and 7-day YM groups to evaluate molecular dynamics of healing while the IonL-Phe was present on the surface. IonL-coated and uncoated implants demonstrated similar histological signs of healing, while coated samples’ differential gene expression of immunological and bone markers was compared with uncoated implants at 2 and 7 days in YMs. While YMs presented suitable osseointegration for both uncoated and IonL-Phe-coated groups, decreased success rate in other demographics resulted from lack of supporting bone in YFs and poor bone quality in OVXFs and OMs. Overall, it was found that IonL-coated samples had increased bone-to-implant contact across all demographic groups. IonL-Phe coating led to successful osseointegration across all animal demographics and presented the potential to prevent failures in scenarios known to be challenged by bacteria.

Inhibition of the Wnt Pathway Reduces Activation of the YAP/TAZ Mechanotransduction Pathways and Osteogenic Differentiation in Nanoparticulate Mineralized Collagen Glycosaminoglycan Materials

PURPOSE: Developing surgically practical regenerative materials requires in-depth understanding of cell-material interaction mechanisms. We previously identified a novel nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) material—inspired by the extracellular matrix—with the ability to induce calvarial bone healing without added exogenous growth factors or progenitor cells, suggesting that this material may support the development of off-the-shelf implants for skull defects. We have demonstrated that modulating the material’s stiffness activates the mechanotransduction pathway via YAP and TAZ, thus improving osteogenic differentiation. Given the strong relationship between the mechanotransduction pathways and Wnt activation, this work seeks to determine the necessity of the Wnt pathways for the activity of MC-GAG in relationship to differences in stiffness. METHODS: MC-GAG scaffolds were prepared with lyophilization. Crosslinking was performed with 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide at a molar ratio of 5:2:1 EDC:NHS:COOH; COOH represents the amount of collagen in the scaffold. Noncrosslinked (NX-MC) and crosslinked (MC) GAG scaffolds were cultured with primary bone-marrow-derived human mesenchymal stem cells. Osteogenic differentiation was assessed with a combination of quantitative real-time reverse transcriptase polymerase chain reaction for osteogenic differentiation markers, western blot and confocal microscopy for activation and subcellular localization of intracellular mediators, and alizarin red staining and micro-computed tomography for mineralization. Wnt pathways were inhibited using small molecular inhibitors IWR1 (specifically targeting the canonical Wnt/b-catenin pathway) and IWP2 (targeting both canonical and non-canonical Wnt pathways). Analyses of variance and Tukey post hoc testing were performed with a P < 0.05 significance threshold. RESULTS: Inhibiting the Wnt pathways with IWR1 and IWP2 resulted in decreased mineralization of primary human mesenchymal stem cells on MC scaffolds, whereas NX-MC scaffolds were unaffected. Both inhibitors downregulated, but did not eliminate, gene expression of bone markers, including alkaline phosphatase, collagen 1, and bone sialoprotein II primarily affecting MC over NX-MC. However, a reciprocal increase in BMP4 expression was found in the presence of both inhibitors. On western blot analysis, both inhibitors reduced protein expression of active b-catenin, YAP, and TAZ on MC materials, whereas the less stiff NX-MC material demonstrated no differences in expression. Despite the reduction in bone markers and mineralization, a reciprocal increase in Runx2 and Smad1/5 phosphorylation was noted in the presence of IWR1 and IWP2 specifically on the MC scaffolds, but not on NX-MC. On confocal microscopy, untreated materials exhibited colocalization of YAP and active b-catenin in the cytosolic compartment on NX-MC and in both cytosolic and nuclear compartments on MC. While IWR1 and IWP2 did not affect YAP and active b-catenin staining on NX-MC scaffolds, they reduced nuclear localization of active b-catenin on MC scaffolds, with IWP2 demonstrating more of an effect than IWR1. CONCLUSIONS: Mechanistic understanding of regenerative materials is required for safe clinical translation. We have shown that stiffness of MC-GAG, a promising skull regenerative material, can improve osteogenic capabilities via YAP/TAZ-mediated mechanotransduction. In this work, we showed that the osteogenic properties, imparted by the stiffness of MC-GAG, function through the Wnt signaling pathways, such that inhibition downregulates YAP/TAZ expression, mineralization, and expression of bone markers.

Effect of Amelotin on Bone Growth in the Murine Calvarial Defect Model.

Amelotin (AMTN) is a protein that is expressed during the maturation of dental enamel and has important role in enamel hydroxyapatite mineralization. However, it is not well understood whether AMTN has a strong mineral-promoting ability in bone. In this study, the effect of AMTN on bone healing was investigated using mice calvarial defect model in vivo, and the expression of bone marker genes and cell proliferation were investigated to clarify the role of AMTN in bone mineralization using mouse osteogenic cells (MC3T3-E1) in vitro. Collagen membranes, with or without recombinant human (rh) AMTN, were applied to calvarial defects created on the parietal bones of C57BL/6N mice. Microcomputed tomography and histological observation revealed that the defect largely filled with mineralized tissue by the rhAMTN-containing membrane in eight weeks. Moreover, CD31 positive cells were observed in the newly formed mineralized tissue and around the rhAMTN-containing membrane. In the presence of rhAMTN, the expression of the Spp1gene in MC3T3-E1 cells significantly increased within ten days in an osteoinductive medium. Moreover, rhAMTN significantly enhanced MC3T3-E1 cell proliferation. These findings indicate that AMTN positively influences bone repair by promoting hydroxyapatite mineralization.

In vitro evaluation of ferutinin on proliferation and osteogenesis differentiation in human unrestricted Somatic stem cells

Osteoporosis is a common disease in post-menopausal women. The increased risk of breast cancer and malignancy with hormone replacement, hampers its wide-usage. Phytoestrogens are known to have selective estrogen receptor modulator activity. The present study aims to determine how ferutinin affects unrestricted human Somatic Stem Cells (USSCs) osteogenic differentiation. The effect of ferutinin on USSCs proliferation was assessed by MTT assay while osteogenesis was evaluated using Alkaline Phosphatase Activity (ALP), calcium deposition and Alizarin Red Staining. Quantitative real-time PCR was applied to examine the expression of bone specific genes such as osteocalcin, Runx2, and BMP-2. Ferutinin (5–15 µg/mL) could positively impact on the proliferation of cells in a dose-dependent manner. Also, ALP enzyme activity and calcium deposition were enhanced in the presence of ferutinin. Based on real-time PCR results, ferutinin could increase the expression of bone marker genes. The pattern of ferutinin effect on gene expression is similar to standard synthetic estrogen, 17-β-estradiol. In the presence of the estrogen activity inhibitor (ICI), the effect of ferutinin on ALP and gene level was diminished. In conclusion, ferutinin may be considered as a potential candidate for the stem cell therapy in osteoporosis.

Surface Topography of Titanium Affects Their Osteogenic Potential through DNA Methylation.

It is widely accepted that sandblasted/large-grit/acid-etched (SLA) surfaces of titanium (Ti) have a higher osteogenic potential than machined ones. However, most studies focused on differential gene expression without elucidating the underlying mechanism for this difference. The aim of this study was to evaluate how the surface roughness of dental Ti implants affects their osteogenic potential. Mouse preosteoblast MC3T3-E1 cells were seeded on machined and SLA Ti discs. The cellular activities of the discs were analyzed using confocal laser scanning microscopy, proliferation assays, and real-time polymerase chain reaction (PCR). DNA methylation was evaluated using a methylation-specific PCR. The cell morphology was slightly different between the two types of surfaces. While cellular proliferation was slightly greater on the machined surfaces, the osteogenic response of the SLA surfaces was superior, and they showed increased alkaline phosphatase (Alp) activity and higher bone marker gene expression levels (Type I collagen, Alp, and osteocalcin). The degree of DNA methylation on the Alp gene was lower on the SLA surfaces than on the machined surfaces. DNA methyltransferase inhibitor stimulated the Alp gene expression on the machined surfaces, similar to the SLA surfaces. The superior osteogenic potential of the SLA surfaces can be attributed to a different epigenetic landscape, specifically, the DNA methylation of Alp genes. This finding offers novel insights into epigenetics to supplement genetics and raises the possibility of using epidrugs as potential therapeutic targets to enhance osteogenesis on implant surfaces.

Recovering the osteoblastic differentiation potential of mesenchymal stem cells derived from diabetic rats by photobiomodulation therapy.

Autologous cell-based therapy for bone regeneration might be impaired by diabetes mellitus (DM) due to the negative effects on mesenchymal stem cells (MSCs) differentiation. Strategies to recover their osteogenic potential could optimize the results. We aimed to evaluate the effect of photobiomodulation (PBM) therapy on osteoblast differentiation of rats with induced DM. Bone marrow MSCs of healthy and diabetic rats were isolated and differentiated into osteoblasts (OB and dOB, respectively). dOB were treated with PBM therapy every 72 hour (660 nm; 0.14 J; 20 mW; 0.714 W/cm2 , and 5 J/cm2 ). Cell morphology, viability, gene and protein expression of osteoblastic markers, alkaline phosphatase (ALP) activity, and the mineralized matrix production of dOB-PBM were compared to dOB. PBM therapy improved viability of dOB, increased the gene and protein expression of bone markers, the ALP activity and the mineralized matrix production. PBM therapy represents an innovative therapeutic approach to optimize the treatment of bone defects in diabetic patients.

Hierarchical dual-porous hydroxyapatite doped dendritic mesoporous silica nanoparticles based scaffolds promote osteogenesis in vitro and in vivo

Biomaterial based scaffolds for treating large bone defects require excellent biocompatibility and osteoconductivity. Here we report on the fabrication of hydroxyapatite-dendritic mesoporous silica nanoparticles (HA-DMSN) based scaffolds with hierarchical micro-pores (5 µm) and nano-pores (6.4 nm), and their application for bone regeneration. The in vitro studies demonstrated good biocompatibility of dissolution extracts, as well as enhanced osteogenic potential indicated by dose-dependent upregulation of bone marker gene expression (osteocalcin gene (OCN), osteopontin gene (OPN), collagen type I alpha 1 gene (CoL1A1), runt-related transcription factor 2 gene (RUNX2), and integrin-binding sialoprotein gene (IBSP)), alkaline phosphatise (ALP) activity, and alizarin red staining. The in vivo studies showed that HA-DMSN scaffolds significantly increased bone formation in a rat cranial bone defect model after 4 weeks healing. Our study provides a simple method to fabricate promising inorganic scaffolds with hierarchical pores for bone tissue engineering.

G9a is involved in the regulation of cranial bone formation through activation of Runx2 function during development.

The methyltransferase G9a was originally isolated as a histone methyltransferase that catalyzes the methylation of histone 3 lysine 9 (H3K9) to a dimethylated state (H3K9me2). Recent studies have revealed that G9a has multiple functions in various cells, including osteoblasts. Here, we investigated G9a function during cranial bone formation. Crossing Sox9-cre with G9aflox/flox (fl/fl) mice generated conditional knockout mice lacking G9a expression in Sox9-positive neural crest-derived bone cells. Sox9-Cre/G9afl/fl mice showed severe hypo-mineralization of cranial vault bones, including defects in nasal, frontal, and parietal bones with opened fontanelles. Cell proliferation was inhibited in G9a-deleted calvarial bone tissues. Expression levels of bone marker genes, i.e., alkaline phosphatase and osteocalcin, were suppressed, whereas Runx2 expression was not significantly decreased in those tissues. In vitro experiments using G9a-deleted calvarial osteoblasts showed decreased cell proliferation after G9a deletion. In G9a-deleted osteoblasts, expression levels of fibroblast growth factor receptors and several cyclins were suppressed. Moreover, the expression of bone marker genes was decreased, whereas Runx2 expression was not altered by G9a deletion in vitro. G9a enhanced the transcriptional activity of Runx2, whereas siRNA targeting G9a inhibited the transcriptional activity of Runx2 in C3H10T1/2 mesenchymal cells. We confirmed the direct association of endogenous Runx2 with G9a. Chromatin immunoprecipitation experiments showed that G9a bound to Runx2-target regions in promoters in primary osteoblasts. Furthermore, Runx2 binding to the osteocalcin promoter was abrogated in G9-deleted osteoblasts. These results suggest that G9a regulates proliferation and differentiation of cranial bone cells through binding to and activating Runx2.

Design and evaluation of Konjac glucomannan-based bioactive interpenetrating network (IPN) scaffolds for engineering vascularized bone tissues

Synthetic bone grafts are being developed to overcome the limitations of conventional treatments for bone defects. In this study, we have fabricated bioactive binary and novel ternary interpenetrating polymer network (IPN) scaffolds using a combination of natural and synthetic polymers. The binary IPN scaffolds were prepared using Konjac glucomannan (KGM) and polyvinyl alcohol (PVA). In the novel ternary IPN scaffolds, polycaprolactone (PCL) was added to PVA and KGM. SEM images showed that these scaffolds were microporous with good interconnectivity. Compression testing confirmed that both the scaffolds are mechanically strong, with the ternary scaffolds having moduli comparable to the natural bone. In vitro cytocompatibility studies performed with NIH/3T3 fibroblasts cells and MG-63 osteosarcoma cells demonstrated the non-toxic and osseointegrating nature of the scaffolds. Confocal images confirmed that the cells migrated into the interconnected pores of the scaffolds. RT-PCR analysis showed that both binary and ternary scaffolds enhanced the expression of the major bone marker genes, viz., ALP, BMP-2, COLLAGEN-1, and OSTEOCALCIN. However, the expression of these osteogenic markers was significantly enhanced in the ternary scaffolds compared to the binary scaffolds. In vivo chick chorioallantoic membrane (CAM) assay shows that these scaffolds possess excellent pro-angiogenic properties. Hence, these desirable biological properties, coupled with the suitable physicochemical properties, make these IPN scaffolds ideal for treating bone defects.

Histone acetylation together with DNA demethylation empowers higher plasticity in adipocytes to differentiate into osteoblasts

Bone regeneration has been a challenge for both researchers and clinicians. In the field of tissue engineering, much effort has been made to identify cell sources including stem cells. The present study aimed to induce trans-differentiation from adipocytes to osteoblasts using epigenetic modifiers; 5-aza-dC and/or trichostatin-A (TSA). 3 T3-L1 preadipocytes were treated with TSA (100 nM) and then with Wnt3a (50 ng/ml). Microscopic observation showed trans-differentiated cell morphology. Methylation-specific PCR and immunoblotting were performed to analyze the DNA methylation and histone acetylation patterns. The gene expression was determined by real-time PCR. Based on these in vitro experiments, in vivo mouse experiments supplemented the possibility of trans-differentiation by epigenetic modification. TSA induced the acetylation of lysine9 on histone H3, and a sequential Wnt3a treatment stimulated the expression of bone marker genes in adipocytes, suppressing adipogenesis and stimulating osteogenesis. Furthermore, TSA induced DNA hypomethylation, and a combined treatment with TSA and 5-aza-dC showed a synergistic effect in epigenetic modifications. The number of adipocytes and DNA methylation patterns of old (15 months) and young (6 weeks) mice were significantly different, and TSA and sequential Wnt3a treatments increased bone formation in the old mice. Collectively, our results confirmed cell trans-differentiation via epigenetic modifications and osteogenic signaling from adipocytes to osteoblasts for the bone regeneration in vitro and in vivo, and indicated that histone acetylation could induce DNA hypomethylation, enhancing the chance of trans-differentiation.

The NATO project: nanoparticle-based countermeasures for microgravity-induced osteoporosis

Recent advances in nanotechnology applied to medicine and regenerative medicine have an enormous and unexploited potential for future space and terrestrial medical applications. The Nanoparticles and Osteoporosis (NATO) project aimed to develop innovative countermeasures for secondary osteoporosis affecting astronauts after prolonged periods in space microgravity. Calcium- and Strontium-containing hydroxyapatite nanoparticles (nCa-HAP and nSr-HAP, respectively) were previously developed and chemically characterized. This study constitutes the first investigation of the effect of the exogenous addition of nCa-HAP and nSr-HAP on bone remodeling in gravity (1 g), Random Positioning Machine (RPM) and onboard International Space Station (ISS) using human bone marrow mesenchymal stem cells (hBMMSCs). In 1 g conditions, nSr-HAP accelerated and improved the commitment of cells to differentiate towards osteoblasts, as shown by the augmented alkaline phosphatase (ALP) activity and the up-regulation of the expression of bone marker genes, supporting the increased extracellular bone matrix deposition and mineralization. The nSr-HAP treatment exerted a protective effect on the microgravity-induced reduction of ALP activity in RPM samples, and a promoting effect on the deposition of hydroxyapatite crystals in either ISS or 1 g samples. The results indicate the exogenous addition of nSr-HAP could be potentially used to deliver Sr to bone tissue and promote its regeneration, as component of bone substitute synthetic materials and additive for pharmaceutical preparation or food supplementary for systemic distribution.

Cell Therapy: Effect of Locally Injected Mesenchymal Stromal Cells Derived from Bone Marrow or Adipose Tissue on Bone Regeneration of Rat Calvarial Defects

Treatment of large bone defects is a challenging clinical situation that may be benefited from cell therapies based on regenerative medicine. This study was conducted to evaluate the effect of local injection of bone marrow-derived mesenchymal stromal cells (BM-MSCs) or adipose tissue-derived MSCs (AT-MSCs) on the regeneration of rat calvarial defects. BM-MSCs and AT-MSCs were characterized based on their expression of specific surface markers; cell viability was evaluated after injection with a 21-G needle. Defects measuring 5 mm that were created in rat calvaria were injected with BM-MSCs, AT-MSCs, or vehicle-phosphate-buffered saline (Control) 2 weeks post-defect creation. Cells were tracked by bioluminescence, and 4 weeks post-injection, the newly formed bone was evaluated by µCT, histology, nanoindentation, and gene expression of bone markers. BM-MSCs and AT-MSCs exhibited the characteristics of MSCs and maintained their viability after passing through the 21-G needle. Injection of both BM-MSCs and AT-MSCs resulted in increased bone formation compared to that in Control and with similar mechanical properties as those of native bone. The expression of genes associated with bone formation was higher in the newly formed bone induced by BM-MSCs, whereas the expression of genes involved in bone resorption was higher in the AT-MSC group. Cell therapy based on local injection of BM-MSCs or AT-MSCs is effective in delivering cells that induced a significant improvement in bone healing. Despite differences observed in molecular cues between BM-MSCs and AT-MSCs, both cells had the ability to induce bone tissue formation at comparable amounts and properties. These results may drive new cell therapy approaches toward complete bone regeneration.

Soluble yerba mate (Ilex Paraguariensis) extract enhances in vitro osteoblastic differentiation of bone marrow-derived mesenchymal stromal cells

Ethnopharmacological relevanceYerba mate (Ilex paraguariensis) consumption has been associated with beneficial effects on bone health. Aim of the studyThe purpose of this study was to evaluate the mechanism by which soluble yerba mate (SYM) stimulates osteoblast differentiation of bone marrow-derived mesenchymal stromal cells (BM-MSCs). Materials and methodsBM-MSCs from male Wistar rats were induced towards osteoblastic differentiation with different concentrations of SYM (10, 20, and 50 μg/mL). Osteoblastic differentiation was evaluated by measuring proliferation rates, alkaline phosphatase activity, MMP-2 activity, mineralization, and gene expression of Runx2, Osterix, β-catenin (Catnb), collagen type I (Col1a1), osteopontin (Opn), osteocalcin (Ocn), bone sialoprotein (Bsp), bone morphogenetic protein-2 (Bmp2), osteoprotegerin (Opg), and Rankl. We also analyzed cytokine production and MAP kinase pathways. ResultsSYM (10 μg/mL) did not show a cytotoxic effect and induced a slight increase in ALP activity; however, a great increase in mineralization was observed. SYM was also able to reduce TNF-α and IL-10 production; increase the expression of transcription factors Runx2, Osterix, and Catnb; and increase matrix proteins Opn, Bsp, Ocn, and Bmp2. We also observed a decrease in intracellular signaling of ERK, JNK, and p38 MAPK, which seemed to be related to the SYM response. ConclusionsTogether, these results help to explain the promoting effect on osteoblast differentiation produced by a low SYM concentration. However, a higher SYM concentration presented deleterious effects, including cytotoxicity, decreased ALP activity, increased cytokine production, decreased bone marker gene expression, increased MAPK signaling, and significant mineralization reduction. In conclusion, our results suggest a concentration-specific direct stimulatory effect of SYM on osteoblastic differentiation in vitro.