Expression Of Osteogenic Marker Genes Research Articles

A laminin-211-derived bioactive peptide promotes the osseointegration of a sandblasted, large-grit, acid-etched titanium implant.

Early implant loading is very important for reducing the duration of missing teeth in human patients. The laminin-derived peptide, DLTIDDSYWYRI motif (Ln2-P3), accelerates bone healing. Therefore, to investigate the hypothesis that Ln2-P3 increases the bone response to sandblasted, large-grit, acid-etched (SLA) titanium implants, the effect of the Ln2-P3 peptide on the osseointegration of SLA titanium implants was evaluated in vitro and in vivo. Human osteoblast-like cells were cultured on untreated, scrambled peptide (SP)-treated, and Ln2-P3-treated SLA titanium discs, and the cellular responses of these cells were evaluated. The Ln2-P3 treatment augmented osteoblast attachment and spreading, alkaline phosphatase activity, and the expression of osteogenic marker genes. Furthermore, the untreated and Ln2-P3-treated SLA titanium implants were inserted into the tibiae of rabbits for 9 and 11 days. Compared with the untreated implants, the Ln2-P3-treated implants showed a significantly higher bone-to-implant contact ratio at Day 9 after implantation and an increased bone area. The Ln2-P3 treatment of the SLA titanium implant surface augmented osteoblastic activity and accelerated peri-implant bone formation at the bone-implant interface. Overall, these results indicated that compared with the SLA titanium surface alone, the Ln2-P3 peptide-treated SLA titanium surface enhances initial osseointegration, thereby facilitating earlier implant loading.

Three-dimensional printed multiphasic scaffolds with stratified cell-laden gelatin methacrylate hydrogels for biomimetic tendon-to-bone interface engineering

BackgroundThe anatomical properties of the enthesis of the rotator cuff are hardly regained during the process of healing. The tendon-to-bone interface is normally replaced by fibrovascular tissue instead of interposition fibrocartilage, which impairs biomechanics in the shoulder and causes dysfunction. Tissue engineering offers a promising strategy to regenerate a biomimetic interface. Here, we report heterogeneous tendon-to-bone interface engineering based on a 3D-printed multiphasic scaffold. MethodsA multiphasic poly(ε-caprolactone) (PCL)–PCL/tricalcium phosphate (TCP)–PCL/TCP porous scaffold was manufactured using 3D printing technology. The three phases of the scaffold were designed to mimic the graded tissue regions in the tendon-to-bone interface—tendon, fibrocartilage, and bone. Fibroblasts, bone marrow–derived mesenchymal stem cells, and osteoblasts were separately encapsulated in gelatin methacrylate (GelMA) and loaded seriatim on the relevant phases of the scaffold, by which a cells/GelMA-multiphasic scaffold (C/G-MS) construct, replicating the native interface, was fabricated. Cell proliferation, viability, and chondrogenic differentiation were evaluated in vitro. The C/G-MS constructs were further examined to determine the potential of regenerating a continuous interface with gradual transition of teno-, fibrocartilage- and osteo-like tissues in vivo. ResultsIn vitro tests confirmed the good cytocompatibility of the constructs. After seven days in culture, cellular microfilament staining indicated that not only could cells well proliferate in GelMA hydrogels ​but also efficiently attach to and grow on scaffold fibres. Moreover, by immunolocalizing collagen type II, chondrogenesis was identified in the intermediate phase of the C/G-MS construct that had been treated with transforming growth factor β3 for 21 days. After subcutaneous implantation in mice, the C/G-MS construct exhibited a heterogeneous and graded structure up to eight weeks, with distinguished matrix distribution observed at one week. Overall, gene expression of tenogenic, chondrogenic, and osteogenic markers showed increasing patterns for eight weeks. Among them, expression of collagen type X gene was found drastically increasing during eight weeks, indicating progressive formation of calcifying cartilage within the constructs. ConclusionOur findings demonstrate that the stratified manner of fabrication based on the 3D-printed multiphasic scaffold is an effective strategy for tendon-to-bone interface engineering in terms of efficient cell seeding, chondrogenic potential, and distinct matrix deposition in varying phases. The translational potential of this articleWe fabricated a biomimetic tendon-to-bone interface by using a 3D-printed multiphasic scaffold and adopting a stratified cell-seeding manner with GelMA. The biomimetic interface might have applications in tendon-to-bone repair in the rotator cuff. It can not only be an alternative to a biological fixation device ​but also offer an ex vivo living graft to replace the damaged enthesis.

In Vitro Fabrication of Hybrid Bone/Cartilage Complex Using Mouse Induced Pluripotent Stem Cells.

Cell condensation and mechanical stimuli play roles in osteogenesis and chondrogenesis; thus, they are promising for facilitating self-organizing bone/cartilage tissue formation in vitro from induced pluripotent stem cells (iPSCs). Here, single mouse iPSCs were first seeded in micro-space culture plates to form 3-dimensional spheres. At day 12, iPSC spheres were subjected to shaking culture and maintained in osteogenic induction medium for 31 days (Os induction). In another condition, the osteogenic induction medium was replaced by chondrogenic induction medium at day 22 and maintained for a further 21 days (Os-Chon induction). Os induction produced robust mineralization and some cartilage-like tissue, which promoted expression of osteogenic and chondrogenic marker genes. In contrast, Os-Chon induction resulted in partial mineralization and a large area of cartilage tissue, with greatly increased expression of chondrogenic marker genes along with osterix and collagen 1a1. Os-Chon induction enhanced mesodermal lineage commitment with brachyury expression followed by high expression of lateral plate and paraxial mesoderm marker genes. These results suggest that combined use of micro-space culture and mechanical stimuli facilitates hybrid bone/cartilage tissue formation from iPSCs, and that the bone/cartilage tissue ratio in iPSC constructs could be manipulated through the induction protocol.

Novel titanium-apatite hybrid scaffolds with spongy bone-like micro architecture intended for spinal application: In vitro and in vivo study.

Titanium alloy scaffolds with novel interconnected and non-periodic porous bone-like micro architecture were 3D-printed and filled with hydroxyapatite bioactive matrix. These novel metallic-ceramic hybrid scaffolds were tested in vitro by direct-contact osteoblast cell cultures for cell adhesion, proliferation, morphology and gene expression of several key osteogenic markers. The scaffolds were also evaluated in vivo by implanting them on transverse and spinous processes of sheep's vertebras and subsequent histology study. The in vitro results showed that: (a) cell adhesion, proliferation and viability were not negatively affected with time by compositional factors (quantitative MTT-assay); (b) the osteoblastic cells were able to adhere and to attain normal morphology (fluorescence microscopy); (c) the studied samples had the ability to promote and sustain the osteogenic differentiation, matrix maturation and mineralization in vitro (real-time quantitative PCR and mineralized matrix production staining). Additionally, the in vivo results showed that the hybrid scaffolds had greater infiltration, with fully mineralized bone after 6months, than the titanium scaffolds without bioactive matrix. In conclusion, these novel hybrid scaffolds could be an alternative to the actual spinal fusion devices, due to their proved osteogenic performance (i.e. osteoinductive and osteoconductive behaviour), if further dimensional and biomechanical optimization is performed.

An Ex Vivo Bone Defect Model to Evaluate Bone Substitutes and Associated Bone Regeneration Processes.

The increased incidence of bone defects, especially in cases of comminuted fractures or bone tumor resections demands suitable bone grafts and substitutes. The aim of this study was to establish an ex vivo bone defect model to evaluate new bone substitutes and associated repair processes under controlled conditions. Femoral heads derived from patients undergoing total hip replacement were cut into cylinders (20 mm diameter, 7 mm height). A central bone defect (6 mm diameter, 5 mm depth) was inserted centrally. The bone slides were cultured for 28 days and viability was evaluated by lactate dehydrogenase and alkaline phosphatase assay, and Calcein-AM viability staining and DNA quantification. Data revealed the viability of the bone tissue over the tested time period of 28 days, and an increase in cell numbers implicating active cell proliferation processes in the sections. To analyze the bone regeneration potential of this model in combination with a bone replacement material, we injected a collagen-type 1 hydrogel into the central defect. Cellular ingrowth into the gel was evaluated by microscopy and DNA quantification at different time points demonstrating an increase of cells in the defect over time. Finally, gene expression of osteogenic markers indicated an osteoblastic phenotype of the cells in the defect. In summary, the ex vivo bone defect model remains viable and shows active bone repair processes over 28 days. Additional advantages include high reproducibility, manageable costs, and a native bone-implant interface supporting the evaluation of bone substitute materials and associated regeneration processes. Impact statement Testing of new implant materials and bone repair strategies up to date rely mainly on in vivo and in vitro investigation models providing different pros and cons. In this study we established a novel human ex vivo bone defect model with a proven vitality of at least 28 days. The model provides a native bone implant interface and is designed to monitor cell invasion into a critically sized defect filled with the potential implant material. Furthermore, associated repair processes can be documented on the cell and molecular level, including additional advantages such as high reproducibility and manageable costs.

Inhibition of CRY2 by STAT3/miRNA-7-5p Promotes Osteoblast Differentiation through Upregulation of CLOCK/BMAL1/P300 Expression

Accumulating evidence indicates that cryptochrome circadian regulatory (CRY) proteins have emerged as crucial regulators of osteogenic differentiation. However, the associated mechanisms are quite elusive. In this study, we show that knockdown of CRY2 downregulated the expression of runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN) to facilitate osteoblast differentiation. Further study identified that CRY2 was directly targeted by microRNA (miR)-7-5p, which was highly induced during osteoblast differentiation. The expression of Runx2, ALP, collagen type I alpha 1 (Col1a1), and OCN was upregulated by overexpression of miR-7-5p and induction of osteoblast differentiation. Moreover, signal transducer and activator of transcription 3 (STAT3) transcriptionally activated miR-7-5p to significantly enhance the expression of above osteogenic marker genes and mineral formation. However, overexpression of CRY2 abolished the osteogenic differentiation induced by miR-7-5p overexpression. Silencing of CRY2 unraveled the binding of CRY2 with the circadian locomotor output cycles kaput (CLOCK)/brain and muscle ARNT-like 1 (BMAL1) complex to release CLOCK/BMAL1, which facilitated the binding of CLOCK/BMAL1 to the promoter region of the P300 E-box to stimulate the transcription of P300. P300 subsequently promoted the acetylation of histone 3 and the formation of a transcriptional complex with Runx2 to enhance osteogenesis. Taken together, our study revealed that CRY2 is repressed by STAT3/miR-7-5p to promote osteogenic differentiation through CLOCK/BMAL1/P300 signaling. The involved molecules may be potentially targeted for treatment of osteoporosis.

Electrospun poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)/silk fibroin film is a promising scaffold for bone tissue engineering

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters produced by microorganisms, under unbalanced growth conditions, as a carbon storage compound. PHAs are composed of various monomers such as 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx). Silk fibroin (SF) derived from Bombyx mori cocoons, is a widely studied protein polymer commonly used for biomaterial applications. In this study, non-woven electrospun films comprising a copolymer of 3HB and 3HHx [P(3HB-co-3HHx)], SF and their blends were prepared by electrospinning technique. The growth and osteogenic differentiation of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) were studied using different types of fabricated electrospun films. The differentiation study revealed that electrospun P(3HB-co-3HHx)/SF film supports the differentiation of hUC-MSCs into the osteogenic lineage, confirmed by histological analysis using Alizarin Red staining, energy dispersive X-ray (EDX) and quantitative real-time PCR analysis (qPCR). Electrospun P(3HB-co-3HHx)/SF film up-regulated the expression of osteogenic marker genes, alkaline phosphatase (ALP) and osteocalcin (OCN), by 1.6-fold and 2.8-fold respectively, after 21 days of osteogenic induction. In conclusion, proliferation and osteogenic differentiation of hUC-MSCs were enhanced through the blending of P(3HB-co-3HHx) and SF. The results from this study suggest that electrospun P(3HB-co-3HHx)/SF film is a promising biomaterial for bone tissue engineering.

Apolipoprotein E Inhibits Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells by Inhibiting β-Catenin Expression

Bone marrow mesenchymal stem cells (BMSCs) can differentiate into adipocytes, osteoblasts. Apolipoprotein E (ApoE) is closely related to bone metabolism and its effect on bone marrow mesenchymal stem cells is unclear. Therefore, this study investigated ApoE's effect on BMSCs osteogenic differentiation. BMSCs were isolated from ApoE – and WT mouse and cultured to induce osteogenic induction followed by analysis of expression of osteogenic differentiation marker genes by Real-time PCR, calcium nodules formation by ARS staining, ALP activity and -catenin protein level by Western blot. The number of bone differentiation markers, ALP activity and calcium nodules formation as well as β-catenin protein level in ApoE– group were significantly elevated compared with WT (P < 0 05). After treatment with DKK-1, β-catenin expression was significantly reduced (P < 0 05) without difference between ApoE– + DKK-1 group and WT group (P > 0 05). WT+ DKK1 group showed significantly reduced osteogenic differentiation marker expression, ALP activity and calcium nodule number compared to WT (P < 0 05) without difference between ApoE– + DKK1 group and WT group (P > 0 05). ApoE inhibits BMSCs osteogenic differentiation by inhibiting β-catenin expression.

Combinatorial Delivery of miRNA-Nanoparticle Conjugates in Human Adipose Stem Cells for Amplified Osteogenesis.

It is becoming more apparent in tissue engineering applications that fine temporal control of multiple therapeutics is desirable to modulate progenitor cell fate and function. Herein, the independent temporal control of the co-delivery of miR-148b and miR-21 mimic plasmonic nanoparticle conjugates to induce osteogenic differentiation of human adipose stem cells (hASCs), in a de novo fashion, is described. By applying a thermally labile retro-Diels-Alder caging and linkage chemistry, these miRNAs can be triggered to de-cage serially with discrete control of activation times. The method relies on illumination of the nanoparticles at their resonant wavelengths to generate sufficient local heating and trigger the untethering of the Diels-Alder cycloadduct. Characterization of the photothermal release using fluorophore-tagged miRNA mimics in vitro is carried out with fluorescence measurements, second harmonic generation, and confocal imaging. Osteogenesis of hASCs from the sequential co-delivery of miR-21 and miR-148b mimics is assessed using xylenol orange and alizarin red staining of deposited minerals, and quantitative polymerase chain reaction for gene expression of osteogenic markers. The results demonstrate that sequential miRNA mimic activation results in upregulation of osteogenic markers and mineralization relative to miR-148b alone, and co-activation of miR-148b and miR-21 at the same time.

Icariin promotes osteogenic differentiation by suppressing Notch signaling

Osteoporosis is a bone disease characterized by microarchitectural deterioration, low bone mass, and increased risk of fractures. Icariin (ICA), an active flavonoid glucoside isolated from Herba epimedii (HEF), is a potent stimulator of osteogenic differentiation and has potential applications for preventing bone loss in postmenopausal women. However, the molecular mechanism underlying the osteogenic effect of ICA has not yet been fully elucidated. In this study, we report that ICA treatment significantly elevated gene expression of osteogenic markers and increased alkaline phosphatase (ALP) activity in MC3T3-E1 and C3H10T1/2 cells. RNA sequencing revealed that the expression of several genes involved in the Notch pathway was decreased following ICA treatment. Real-time PCR further demonstrated that the mRNA levels of Notch ligands Jagged-1 (Jag1), lunatic fringe (Lfng), and Notch signaling downstream target gene Hey-1 were significantly decreased following ICA treatment. In addition, we found that constitutive activation of Notch signaling through overexpression of the intracellular domain of Notch (NICD) fully blocked ICA-induced osteoblast differentiation. Moreover, inhibiting Notch signaling with DAPT markedly enhanced osteogenic differentiation following ICA treatment. We found that the mRNA levels of Notch pathway molecules (Lfng, Notch1, Rbpjk and Nfatc1) were increased in ovariectomized (OVX) mice, and administration of ICA significantly decreased the expression of these genes. Our results suggest that ICA promotes osteogenic differentiation in vitro and alleviates osteoporosis in vivo through inhibition of the Notch signaling pathway.

Deficiency of Macf1 in osterix expressing cells decreases bone formation by Bmp2/Smad/Runx2 pathway.

Microtubule actin cross‐linking factor 1 (Macf1) is a spectraplakin family member known to regulate cytoskeletal dynamics, cell migration, neuronal growth and cell signal transduction. We previously demonstrated that knockdown of Macf1 inhibited the differentiation of MC3T3‐E1 cell line. However, whether Macf1 could regulate bone formation in vivo is unclear. To study the function and mechanism of Macf1 in bone formation and osteogenic differentiation, we established osteoblast‐specific Osterix (Osx) promoter‐driven Macf1 conditional knockout mice (Macf1f/fOsx‐Cre). The Macf1f/fOsx‐Cre mice displayed delayed ossification and decreased bone mass. Morphological and mechanical studies showed deteriorated trabecular microarchitecture and impaired biomechanical strength of femur in Macf1f/fOsx‐Cre mice. In addition, the differentiation of primary osteoblasts isolated from calvaria was inhibited in Macf1f/fOsx‐Cre mice. Deficiency of Macf1 in primary osteoblasts inhibited the expression of osteogenic marker genes (Col1, Runx2 and Alp) and the number of mineralized nodules. Furthermore, deficiency of Macf1 attenuated Bmp2/Smad/Runx2 signalling in primary osteoblasts of Macf1f/fOsx‐Cre mice. Together, these results indicated that Macf1 plays a significant role in bone formation and osteoblast differentiation by regulating Bmp2/Smad/Runx2 pathway, suggesting that Macf1 might be a therapeutic target for bone disease.

In situ miRNA delivery from a hydrogel promotes osteogenesis of encapsulated mesenchymal stromal cells

Hydrogels are attractive candidates for use in tissue-engineering and the encapsulation and subsequent differentiation of mesenchymal stem/stromal cells (MSCs) is a strategy that holds great promise for the repair and regeneration of bone and cartilage. However, MSCs are well-known for their sensitivity to mechanical cues, particularly substrate stiffness, and so the inherent softness of hydrogels is poorly matched to the mechanical cues that drive efficient osteogenesis. One approach to overcome this limitation is to harness mechanotransductive signalling pathways and override the signals cells receive from their environment. Previous reports demonstrate that mechanosensitive miRNAs, miR-100–5p and miR-143–3p can enhance MSC osteogenesis, using a complex multi-step procedure to transfect, encapsulate and differentiate the cells.In this study, we develop and characterise a facile system for in situ transfection of MSCs encapsulated within a light-crosslinkable gelatin-PEG hydrogel. Comparing the influence of different transfection agents and hydrogel compositions, we show that particle size, charge, and hydrogel mechanical properties all influence the diffusion of embedded transfection agent complexes. By incorporating both MSCs and transfection agents into the hydrogels we demonstrate successful in situ transfection of encapsulated MSCs. Comparing the efficacy of pre- and in situ transfection of miR-100–5p/miR-143–3p on the osteogenic capacity of hydrogel-encapsulated MSCs, our data demonstrates superior mineralisation and osteogenic gene expression following in situ transfections. Overall, we demonstrate a simple, one-pot system for in situ transfection of miRNAs to enhance MSC osteogenic potential and thus demonstrates significant promise to improve the efficiency of MSC differentiation in hydrogels for bone tissue-engineering applications. Statement of significanceMesenchymal stromal cells (MSCs) are sensitive to cues from their surrounding microenvironment. Osteogenesis is enhanced in MSCs grown on stiffer substrates, but this is limited when using hydrogels for bone tissue-engineering. Modulating pro-osteogenic genes with mechanosensitive microRNAs (miRNAs) represents a potential tool to overcome this challenge. Here we report a hydrogel platform to deliver miRNAs to encapsulated MSCs. We characterise effects of hydrogel composition and transfection agent type on their mobility and transfection efficiency, demonstrating successful in situ transfection of MSCs and showing that miRNAs can significantly enhance osteogenic mineral deposition and marker gene expression. This system was simpler and more effective than conventional 2D transfection prior to encapsulation and therefore holds promise to improve MSC differentiation in bone tissue-engineering.

Intermittent compressive force promotes osteogenic differentiation in human periodontal ligament cells by regulating the transforming growth factor-\u03b2 pathway

Mechanical force regulates periodontal ligament cell (PDL) behavior. However, different force types lead to distinct PDL responses. Here, we report that pretreatment with an intermittent compressive force (ICF), but not a continuous compressive force (CCF), promoted human PDL (hPDL) osteogenic differentiation as determined by osteogenic marker gene expression and mineral deposition in vitro. ICF-induced osterix (OSX) expression was inhibited by cycloheximide and monensin. Although CCF and ICF significantly increased extracellular adenosine triphosphate (ATP) levels, pretreatment with exogenous ATP did not affect hPDL osteogenic differentiation. Gene-expression profiling of hPDLs subjected to CCF or ICF revealed that extracellular matrix (ECM)-receptor interaction, focal adhesion, and transforming growth factor beta (TGF-β) signaling pathway genes were commonly upregulated, while calcium signaling pathway genes were downregulated in both CCF- and ICF-treated hPDLs. The TGFB1 mRNA level was significantly increased, while those of TGFB2 and TGFB3 were decreased by ICF treatment. In contrast, CCF did not modify TGFB1 expression. Inhibiting TGF-β receptor type I or adding a TGF-β1 neutralizing antibody attenuated the ICF-induced OSX expression. Exogenous TGF-β1 pretreatment promoted hPDL osteogenic marker gene expression and mineral deposition. Additionally, pretreatment with ICF in the presence of TGF-β receptor type I inhibitor attenuated the ICF-induced mineralization. In conclusion, this study reveals the effects of ICF on osteogenic differentiation in hPDLs and implicates TGF-β signaling as one of its regulatory mechanisms.

Retinoic acid disrupts osteogenesis in pre-osteoblasts by down-regulating WNT signaling

The skull bones are formed by osteoblasts by intramembranous ossification. WNT signaling is a regulator of bone formation. Retinoic Acid (RA) act as a teratogen affecting craniofacial development. We evaluated the effects of RA on the differentiation and mineralization of MC-3T3 cells, and on the expression of WNT components. MC-3T3 were cultured with or without 0.5 μM RA in osteogenic medium and mineralization was assessed by alizarin red staining. The expression of osteogenic marker genes and WNT genes was evaluated at several time points up to 28 days. RA significantly inhibited MC-3T3 mineralization (p < 0.01), without affecting ALP activity or Alp gene expression. Both parameters gradually increased in time. During culture, RA stimulated Runx2 expression at 14 and 28 days compared to the respective controls (p < 0.05). Also, RA significantly reduced Sp7 expression at days 14 and 21 (p < 0.05). Simultaneously, RA significantly reduced the expression of the WNT genes cMyc, Lef1, Lrp5, Lrp6 and Wnt11 compared to the controls (p < 0.05). In contrast, RA increased the expression of the WNT inhibitors Dkk1 at day 21 and Dkk2 at days 14 and 21 (p < 0.01). Our data indicate that RA disrupts osteogenic differentiation and mineralization by inhibiting WNT signaling.

The poly (l-lactid-co-glycolide; PLGA) fiber component of brushite-forming calcium phosphate cement induces the osteogenic differentiation of human adipose tissue-derived stem cells

A brushite-forming calcium phosphate cement (CPC) was mechanically stabilized by addition of poly (l-lactid-co-glycolide; PLGA) fibers (≤10% w/w). It proved highly biocompatible and its fiber component enhanced bone formation in a sheep lumbar vertebroplasty model. However, possible effects on the osteogenic differentiation of resident mesenchymal stem cells (MSCs) remained unexplored. The present study used a novel approach, simultaneously analyzing the influence of a solid CPC scaffold and its relatively low PLGA proportion (a mimicry of natural bone) on osteogenic, chondrogenic, and adipogenic differentiation, as well as the pluripotency of human adipose tissue-derived mesenchymal stem cells (hASCs). hASCs were cultured on CPC discs with/without PLGA fibers (5% and 10%) in the absence of osteogenic medium for 3, 7, and 14 d. Gene expression of osteogenic markers (Runx2, osterix, alkaline phosphatase, collagen I, osteonectin, osteopontin, osteocalcin), chondrogenic markers (collagen II, Sox9, aggrecan), adipogenic markers (PPARG, Leptin, and FABP4), and pluripotency markers (Nanog, Tert, Rex) was analyzed by RT-PCR. The ability of hASCs to synthesize alkaline phosphatase was also evaluated. Cell number and viability were determined by fluorescein diacetate/propidium iodide staining. Compared to pure CPC, cultivation of hASCs on fiber-reinforced CPC transiently induced the gene expression of Runx2 and osterix (day 3), and long-lastingly augmented the expression of alkaline phosphatase (and its enzyme activity), collagen I, and osteonectin (until day 14). In contrast, augmented expression of all chondrogenic, adipogenic, and pluripotency markers was limited to day 3, followed by significant downregulation. Cultivation of hASCs on fiber-reinforced CPC reduced the cell number, but not the proportion of viable cells (viability > 95%). The PLGA component of fiber-reinforced, brushite-forming CPC supports long-lasting osteogenic differentiation of hASCs, whereas chondrogenesis, adipogenesis, and pluripotency are initially augmented, but subsequently suppressed. In view of parallel animal results, PLGA fibers may represent an interesting clinical target for future improvement of CPC- based bone regeneration.

Gene expression profiles and bioinformatics analysis of insulin‐like growth factor‐1 promotion of osteogenic differentiation

BackgroundInsulin‐like growth factor‐1 (IGF‐1) promotes osteoblast differentiation and mineralization. The objective of this study was to investigate the effects of IGF‐1 on proliferation, mineralization, alkaline phosphatase (ALP) synthesis, and gene expression of osteoblast differentiation in MC3T3‐E1 osteoblasts cells, and to explore gene expression profiling differential genes.MethodsMC3T3‐E1 osteoblasts cells were cultured in medium with or without IGF‐1. The ALP assay was employed to determine the osteoblast mineralization, and Alizarin red S to stain for calcium deposits, which were the indicators of mature osteocytes. The living cell number was assessed by the Cell Counting Kit‐8 method. RNA‐seq analysis was applied to identify genes that were differentially expressed in with or without IGF‐1 as well as genes that varied between these two groups. The expression of osteogenic marker genes was determined by quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot analysis.ResultThe cell number of osteoblasts exposed to IGF‐1 at 200 μg/L significantly increased compared with the control group. The ALP activity in IGF‐1‐treated cells was higher than that in the control group. IGF‐1 can increase ALP synthesis in osteoblasts in vitro. RNA‐seq analysis showed that 677 triggered differentially expressed genes by IGF, of which 383 genes were downregulated and 294 genes were upregulated. Gene ontology (GO) analysis showed that IGF‐1 caused a significant change in gene expression patterns.ConclusionsThis result suggested that IGF‐1 could probably promote the synthesis of organic matrix and mineralize action of bone. Osteogenic‐related genes (DMP1, PHEX, SOST, BMP2, RUNX2, OPN, and OCN) were significantly upregulated both in GO analysis and in pathway analysis to perform qRT‐PCR. Western blot analysis demonstrated that the Notch pathway was highly upregulated in MC3T3‐E1 cells.

Study of bone repair mediated by recombination BMP-2/ recombination CXC chemokine Ligand-13-loaded hollow hydroxyapatite microspheres/chitosan composite

AimsThe present study aimed to investigate the mechanism of bone repair mediated by recombination BMP-2 (rhBMP-2)/recombination CXC chemokine ligand-13 (rhCXCL13)-loaded hollow hydroxyapatite (HA) microspheres/chitosan (CS) composite. Materials and methodsFirstly, the biological activity of rhBMP-2 and rhCXCL13 released from the complex was investigated. Secondly, the effect of rhBMP-2 sustained release solution on ALP activity and rhCXCL13 sustained release solution on cell migration of rat bone marrow mesenchyme stem cells was tested. Thirdly, osteoblasts differentiation test, X-ray scoring and three-point bending test were performed. Finally, the mRNAs expression of osteogenic marker genes and the protein expression of Runx2 was tested by reverse transcription-polymerase chain reaction (RT-PCR) and western blotting (WB), respectively. Key findingsRhBMP-2 could significantly promote the proliferation and differentiation, and RhCXCL13 could promote the migration of rat bone marrow MSCs. Detection of ALP activity and calcium salt deposition showed that rhBMP-2 and rhCXCL13 could significantly improve the biological activity and promote cell differentiation ability. X-ray scoring of radius and flexural strength test showed that rhBMP-2 and rhCXCL13 could promote bone healing and improve the bending resistance of bone tissue. The in vitro molecular experiments including RT-PCR and WB further demonstrated the roles of rhBMP-2 and rhCXCL13 in bone formation and bone repair. SignificanceOur results indicated that the hollow HA microspheres/CS composite could be effective as a delivery vehicle for rhBMP-2 and rhCXCL13 in bone regeneration and bone repair. In this process, rhBMP-2 may promote bone regeneration by regulating bone marrow MSCs cells recruited by rhCXCL13.

Decreased osteogenic activity and mineralization of alveolar bone cells from a patient with amelogenesis imperfecta and FAM83H 1261G>T mutation

FAM83H mutations lead to autosomal dominant hypocalcified amelogenesis imperfecta (ADHCAI). However, the biological role of FAM83H remains unclear. The present study aimed to characterize the alveolar bone cells isolated from a patient with ADHCAI having the mutation, c.1261G > T, p.E421*, in FAM83H. We showed that FAM83H mutant cells had proliferation ability and morphology similar to the controls. The F-actin staining revealed that FAM83H mutant cells were remained in the earlier stages of cell spreading compared to the controls at 30 min, but their spreading was advanced comparable to the controls at later stages. After osteogenic induction, a significant decrease in mRNA levels of RUNX2 and ALP was observed in FAM83H mutant cells at day 7 compared with day 3 while their expressions were increased in the controls. The OPN levels in FAM83H mutant cells were not significantly changed at day 7 compared to day 3 while the controls showed a significant increase. After 14 days, the mineral deposition of FAM83H mutant cells was slightly lower than that of the controls. In conclusion, we identify that FAM83H bone cells have lower expression of osteogenic marker genes and mineralization while they maintain their morphology, proliferation, and spreading. Consistent with previous studies in the ameloblasts and periodontal ligamental cells, these evidences propose that FAM83H influences osteogenic differentiation across different cell types in oral cavity.

Iron oxide nanoparticle-calcium phosphate cement enhanced the osteogenic activities of stem cells through WNT/β-catenin signaling.

Calcium phosphate cement (CPC), functionalized with iron oxide nanoparticles (IONP), is of great promise to promote osteoinduction and new bone formation. In this work, the IONP powder was added into the CPC powder to fabricate CPC + IONP scaffolds and the effects of the novel composite on bone matrix formation and osteogenesis of human dental pulp stem cells (hDPSCs) were explored. A series of CPC + IONP magnetic scaffolds with different IONP contents (1%, 3% and 6%) were fabricated using 5% chitosan solution as the cement liquid. Western blotting and RT-PCR were used to analyze the signaling pathway. The IONP incorporation substantially enhanced the performance of CPC + IONP, with increases in both mechanical strength and cellular activities. The IONP addition greatly promoted the osteogenesis of hDPSCs, elevating the ALP activity, the expression of osteogenic marker genes and bone matrix formation with 1.5-2-fold increases. The 3% IONP incorporation showed the most enhancement among all groups. Activation of the extracellular signal-related kinases WNT/β-catenin in DPSCs was observed, and this activation was attenuated by the WNT inhibitor DKK1. The results indicated that the osteogenic behavior of hDPSCs was likely driven by CPC + IONP via the WNT signaling pathway. In conclusion, incorporate IONP into CPC scaffold remarkably enhanced the spreading, osteogenic differentiation and bone mineral synthesis of stem cell. Therefore, this method had great potential for bone tissue engineering. The novel CPC + IONP composite scaffolds with stem cells are promising to provide an innovative strategy to enhance bone regenerative therapies.

Inhibition of acetylation of histones 3 and 4 attenuates aortic valve calcification

Aortic valve calcification develops in patients with chronic kidney disease who have calcium and phosphate metabolic disorders and poor prognoses. There is no effective treatment except valve replacement. However, metabolic disorders put patients at high risk for surgery. Increased acetylation of histones 3 and 4 is present in interstitial cells from human calcific aortic valves, but whether it is involved in aortic valve calcification has not been studied. In this study, we found that treating cultured porcine aortic valve interstitial cells with a high-calcium/high-phosphate medium induced calcium deposition, apoptosis, and expression of osteogenic marker genes, producing a phenotype resembling valve calcification in vivo. These phenotypic changes were attenuated by the histone acetyltransferase inhibitor C646. C646 treatment increased the levels of class I histone deacetylase members and decreased the acetylation of histones 3 and 4 induced by the high-calcium/high-phosphate treatment. Conversely, the histone deacetylase inhibitor suberoylanilide hydroxamic acid promoted valve interstitial cell calcification. In a mouse model of aortic valve calcification induced by adenine and vitamin D treatment, the levels of acetylated histones 3 and 4 were increased in the calcified aortic valves. Treatment of the models with C646 attenuated aortic valve calcification by restoring the levels of acetylated histones 3 and 4. These observations suggest that increased acetylation of histones 3 and 4 is part of the pathogenesis of aortic valve calcification associated with calcium and phosphate metabolic disorders. Targeting acetylated histones 3 and 4 may be a potential therapy for inoperable aortic valve calcification in chronic kidney disease patients.