Branched Channels in Porous ß-Tricalcium Phosphate Scaffold Promote Vascularization.

Rapid and efficient vascularization is still considerably challenging for a porous ß-tricalcium phosphate (ß-TCP) scaffold to achieve. To overcome this challenge, branched channels were created in the porous ß-TCP scaffold by using 3D printing and a template-casting method to facilitate the instant flow of blood supply. Human bone mesenchymal stem cells (hBMSCs) and human umbilical vein endothelial cells (HUVECs) were seeded in the channeled porous scaffolds and characterized through a double-stranded DNA (dsDNA) assay, alkaline phosphatase (ALP) assay, and cell migration. Channeled porous ß-TCP scaffolds were then implanted in the subcutaneous pockets of mice. Histological staining and immunohistochemical staining on vascularization and bone-related markers were carried out on the embedded paraffin sections. Results from in vitro experiments showed that branched channels significantly promoted HUVECs' infiltration, migration, proliferation, and angiogenesis, and also promoted the proliferation and osteogenesis differentiation of hBMSCs. In vivo implantation results showed that, in the early stage after implantation, cells significantly migrated into branched channeled scaffolds. More matured blood vessels formed in the branched channeled scaffolds compared to that in nonchanneled and straight channeled scaffolds. Beside promoting vascularization, the branched channels also stimulated the infiltration of bone-related cells into the scaffolds. These results suggested that the geometric design of branched channels in the porous ß-TCP scaffold promoted rapid vascularization and potentially stimulated bone cells recruitment.

Investigation of the optimal light parameters for photobiomodulation to induce osteogenic differentiation of the human bone marrow stem cell and human umbilical vein endothelial cell co-culture.

Bones have an important role in the human body with their complex nature. Mesenchymal stem cells and endothelial cells together support their unique and complex nature. Photobiomodulation (PBM) is a promising method that provides cell proliferation, osteogenic differentiation, and bone regeneration. However, there are still unknowns in the mechanism of osteogenic differentiation induced by PBM. The main aim of the study is to understand the molecular mechanism of PBM at 655 and 808 nm of wavelengths and identify the most effective energy densities of both wavelengths for osteogenic differentiation. The effect of PBM on osteogenic differentiation of Human Bone Marrow Stem Cell (hBMSC) and Human Umbilical Vein Endothelial Cell (HUVEC) co-culture was examined at 1, 3, and 5 J/cm2 energy densities of red and near-infrared light through different analysis such as cell viability, scratch assay, intracellular reactive oxygen species production, and ATP synthesis, nitric oxide release, temperature monitoring, and osteogenic differentiation analyses. Even though all PBM-treated groups exhibited better results compared to the control group, 5 J/cm2 energy density induced faster cell proliferation and migration at both wavelengths. The increases in ATP and NO levels as signaling molecules, and the increases in DNA, ALPase, and calcium contents as osteogenic markers were higher in the groups treated with 5 J/cm2 energy density at both wavelengths. Only a slight change was obtained in the level of intracellular ROS after any light applications. It can be concluded that NO release has a very important role together with ATP production in PBM therapy to trigger DNA synthesis, ALPase activity, and mineralization for osteogenic differentiation of the hBMSC and HUVEC co-culture at 655 and 808 nm of wavelengths.

Ubiquitination of ASCL1 mediates CD47 transcriptional activation of the AKT signaling pathway, and glycolysis promotes osteogenic differentiation of hBMSCs.

Bones are extremely dynamic organs that continually develop and remodel. This process involves changes in numerous gene expressions. hBMSC cells can promote osteogenic differentiation. The purpose of this study was to elucidate the mechanism by which ASCL1 promotes osteogenic differentiation in hBMSC cells while decreasing glycolysis. hBMSCs were induced to differentiate into osteoblasts. The ASCL1 expression level during hBMSC osteogenic differentiation was measured by RT‒qPCR, Western blotting, and immunofluorescence. The differentiation level of osteoblasts was observed after staining with ALP and alizarin red. ChIP-qPCR were used to determine the relationship between ASCL1 and CD47, and the expression of glycolysis-related proteins was detected. Overexpression of ASCL1 was used to determine its impact on osteogenic differentiation. si-USP8 was used to verify the ubiquitination of ASCL1-mediated CD47/AKT pathway's impact on hBMSC glycolysis and osteogenic differentiation. The results showed that the expression of ASCL1 was upregulated after the induction of osteogenic differentiation in hBMSCs. From a functional perspective, knocking down USP8 can promote the ubiquitination of ASCL1, while the osteogenic differentiation ability of hBMSCs was improved after the overexpression of ASCL1, indicating that ASCL1 can promote the osteogenic differentiation of hBMSCs. In addition, USP8 regulates the ubiquitination level of ASCL1 and mediates CD47 transcriptional regulation of the AKT pathway to increase the glycolysis level of hBMSCs and cell osteogenic differentiation. USP8 ubiquitination regulates the level of ASCL1. In addition, ubiquitination of ASCL1 mediates CD47 transcription to activate the AKT signaling pathway and increase hBMSC glycolysis to promote osteogenic differentiation.

Low miR-182-5p Expressing Extracellular Vesicles Derived From Human Bone Marrow Stromal Cells of Subjects With Steroid-Induced Osteonecrosis of the Femoral Head Aggravate Disease Progression.

Steroid-induced osteonecrosis of the femoral head (SONFH) is a refractory, progressive disease. However, the underlying mechanisms that aggravate femoral head necrosis remain unclear. Extracellular vesicles (EVs) act as molecular carriers in intercellular communication. We hypothesize that EVs derived from human (h) bone marrow stromal cells (BMSC) resident in SONFH lesion areas promote the pathogenesis of SONFH. In the present study, we determined the modulatory effects of SONFH-hBMSCs-derived EVs on the pathogenesis of SONFH in vitro and in vivo. We found that the expression of hsa-miR-182-5p was downregulated in SONFH-hBMSCs and EVs isolated from those hBMSCs. After tail vein injection, EVs isolated from hBMSCs transfected with hsa-miR-182-5p inhibitor aggravated femoral head necrosis in the SONFH mouse model. We conclude that miR-182-5p regulates bone turnover in the SONFH mouse model via targeting MYD88 and subsequent upregulation of RUNX2 expression. We further assume that EVs derived from hBMSCs resident in SONFH lesion areas aggravate femoral head necrosis by downregulating miR-182-5p secreted from hBMSC located outside these lesions. We suggest that miR-182-5p could provide a novel target for future therapeutic approaches to treat or prevent SONFH. © 2023 American Society for Bone and Mineral Research (ASBMR).

Engineering Cell Microenvironment Using Nanopattern-Derived Multicellular Spheroids and Photo-Crosslinked Gelatin/Hyaluronan Hydrogels.

Cell cultures of dispersed cells within hydrogels depict the interaction of the cell-extracellular matrix (ECM) in 3D, while the coculture of different cells within spheroids combines both the effects of cell-cell and cell-ECM interactions. In this study, the cell co-spheroids of human bone mesenchymal stem cells/human umbilical vein endothelial cells (HBMSC/HUVECs) are prepared with the assistance of a nanopattern, named colloidal self-assembled patterns (cSAPs), which is superior to low-adhesion surfaces. A phenol-modified gelatin/hyaluronan (Gel-Ph/HA-Ph) hydrogel is used to encapsulate the multicellular spheroids and the constructs are photo-crosslinked using blue light. The results show that Gel-Ph/HA-Ph hydrogels with a 5%-to-0.3% ratio have the best properties. Cells in HBMSC/HUVEC co-spheroids are more favorable for osteogenic differentiation (Runx2, ALP, Col1a1 and OPN) and vascular network formation (CD31+ cells) compared to HBMSC spheroids. In a subcutaneous nude mouse model, the HBMSC/HUVEC co-spheroids showed better performance than HBMSC spheroids in angiogenesis and the development of blood vessels. Overall, this study paves a new way for using nanopatterns, cell coculturing and hydrogel technology for the generation and application of multicellular spheroids.

Photobiomodulation therapy at red and near-infrared wavelengths for osteogenic differentiation in the scaffold-free microtissues.

One of the novel strategies for bone tissue regeneration is photobiomodulation (PBM) which depends on the red and near-infrared light absorption by mitochondria and may trigger bone tissue regeneration via the production of intracellular ROS and ATP, NO release, etc. It is also important to identify the changes in those signal molecule levels in an in vivo mimicking platform such as 3-Dimensional (3D) Scaffold Free Microtissues (SFMs) that may serve more natural osteogenic differentiation responses to PBM. Herein, we aimed to increase the osteogenic differentiation capability of the co-culture of Human Bone Marrow Stem Cells (hBMSC) and Human Umbilical Vein Endothelial Cells (HUVECs) on 3D SFMs by triple light treatment at 655 and 808-nm of wavelengths with the energy densities of 1, 3, and 5 J/cm2. We performed the analysis of cell viability, diameter measurements of SFMs, intracellular ROS production, NO release, ATP activity, temperature measurements, DNA content, ALPase activity, calcium content, and relative gene expressions of ALP, Collagen, and Osteopontin by qRT-PCR. It was found that both wavelengths were effective in terms of the viability of SFMs. 1 and 5 J/cm2 energy densities of both wavelengths increased the SFM diameter with significant changes in intracellular ROS, ATP, and NO levels compared to the control group. We concluded that PBM therapy was successful to induce osteogenesis. 1 J/cm2 at 655 nm of wavelength and 5 J/cm2 at 808 nm of wavelength were the most effective energy densities for osteogenic differentiation on SFMs with triple light treatment.

Competitive Hybridization of a Microarray Identifies CMKLR1 as an Up-Regulated Gene in Human Bone Marrow-Derived Mesenchymal Stem Cells Compared to Human Embryonic Fibroblasts.

Mesenchymal stem cells (MSCs) have been widely applied to the regeneration of damaged tissue and the modulation of immune response. The purity of MSC preparation and the delivery of MSCs to a target region are critical factors for success in therapeutic application. In order to define the molecular identity of an MSC, the gene expression pattern of a human bone marrow-derived mesenchymal stem cell (hBMSC) was compared with that of a human embryonic fibroblast (hEF) by competitive hybridization of a microarray. A total of 270 and 173 genes were two-fold up- and down-regulated with FDR < 0.05 in the hBMSC compared to the hEF, respectively. The overexpressed genes in the hBMSC over the hEF, including transcription factors, were enriched for biological processes such as axial pattern formation, face morphogenesis and skeletal system development, which could be expected from the differentiation potential of MSCs. CD70 and CD339 were identified as additional CD markers that were up-regulated in the hBMSC over the hEF. The differential expression of CD70 and CD339 might be exploited to distinguish hEF and hBMSC. CMKLR1, a chemokine receptor, was up-regulated in the hBMSC compared to the hEF. RARRES2, a CMKLR1 ligand, stimulated specific migration of the hBMSC, but not of the hEF. RARRES2 manifested as ~two-fold less effective than SDF-1α in the directional migration of the hBMSC. The expression of CMKLR1 was decreased upon the osteoblastic differentiation of the hBMSC. However, the RARRES2-loaded 10% HA-silk scaffold did not recruit endogenous cells to the scaffold in vivo. The RARRES2−CMKLR1 axis could be employed in recruiting systemically delivered or endogenous MSCs to a specific target lesion.

Evaluation of a hyaluronic acid hydrogel (Restylane Lyft) as a scaffold for dental pulp regeneration in a regenerative endodontic organotype model.

Scaffolds are crucial elements for dental pulp regeneration. Most of the currently used scaffolds in regenerative endodontic procedures (REPs) are unsuitable for chairside clinical use. This study aimed to evaluate the effect of an injectable synthetic scaffold (Restylane Lyft) on human bone marrow mesenchymal stem cell (hBMSC) viability, proliferation, and osteo/dentinogenic differentiation in a regenerative endodontic organotype model (REM). hBMSC were loaded in an REM either alone (hBMSC group) or mixed with the Restylane Lyft scaffold (Restylane/hBMSC group) and cultured in basal culture medium (n = 9/group). hMSC on culture plates served as controls. Cell viability and proliferation were measured using AlamarBlue assay. The loaded REM was cultured in an osteogenic differentiation medium to measure alkaline phosphatase activity (ALP) and examine the expression of the osteo/dentinogenic markers using real-time reverse transcriptase polymerase chain reaction. Cell viability in all groups increased significantly over 5 days. The Restylane/hBMSC group showed significantly higher ALP activity and dentin sialophosphoprotein, osteocalcin, and bone sialoprotein genes expression than the hBMSC and the control groups. Restylane Lyft, a hyaluronic acid (HA) injectable, FDA-approved hydrogel, maintained cell viability and proliferation and promoted osteo/dentinogenic differentiation of hBMSC when cultured in an REM. Henceforth, it could be a promising chairside scaffold material for REPs.

MicroRNA-483-5p inhibits osteogenic differentiation of human bone marrow mesenchymal stem cells by targeting the RPL31-mediated RAS/MEK/ERK signaling pathway.

miR-483-5p has been shown to play a key regulatory role in mediating a variety of biological activities. However, there are only a few studies on how miR-483-5p regulates osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), hence there is a need to explore the role and mechanism of miR-483-5p in regulating the osteogenic differentiation of hBMSCs. To that end, we used bioinformatics and cell experiments to confirm our hypothesis, a miRNA microarray dataset (GSE74209) and a mRNA dataset (GSE56816) were obtained from the GEO database, and the differentially expressed miRNAs (DE-miRNAs) and mRNAs (DE-mRNAs) were screened. In total, We found that the up-regulated candidate target genes were significantly enriched in the MAPK signaling pathway. Using the MCODE plug-in from Cytoscape, we identified RPL31 as the seed gene in the third major module. And we successfully established a possible miRNA-hub gene regulatory network. More importantly, we confirmed that the expression of miR-483-5p was down-regulated while the expression of RPL31 was up-regulated during osteogenic induction. Overexpression of miR-483-5p significantly inhibited osteogenic differentiation. In addition, western blot was used to measure protein levels of RPL31 and phosphorylated MEK/ERK family members. We demonstrated that miR-483-5p inhibits the RAS/MEK/ERK signaling pathway by targeting RPL31 and inhibiting its expression, thereby playing an inhibitory role in osteogenic differentiation. In light of our findings, RPL31 can be used as a novel therapeutic target for bone defects and osteoporosis, we reveal a newly discovered mechanism of osteogenic differentiation and provide a new strategy for treating osteoporosis and related diseases.

Regulation of cell attachment, spreading, and migration by hydrogel substrates with independently tunable mesh size.

Hydrogels are widely used as substrates to investigate interactions between cells and their microenvironment as they mimic many attributes of the extracellular matrix. The stiffness of hydrogels is an important property that is known to regulate cell behavior. Beside stiffness, cells also respond to structural cues such as mesh size. However, since the mesh size of hydrogel is intrinsically coupled to its stiffness, its role in regulating cell behavior has never been independently investigated. Here, we report a hydrogel system whose mesh size and stiffness can be independently controlled. Cell behavior, including spreading, migration, and formation of focal adhesions is significantly altered on hydrogels with different mesh sizes but with the same stiffness. At the transcriptional level, hydrogel mesh size affects cellular mechanotransduction by regulating nuclear translocation of yes-associated protein. These findings demonstrate that the mesh size of a hydrogel plays an important role in cell-substrate interactions. STATEMENT OF SIGNIFICANCE: Hydrogels are ideal platforms with which to investigate interactions between cells and their microenvironment as they mimic many physical properties of the extracellular matrix. However, the mesh size of hydrogels is intrinsically coupled to their stiffness, making it challenging to investigate the contribution of mesh size to cell behavior. In this work, we use hydrogel-on-glass substrates with defined thicknesses whose stiffness and mesh size can be independently tuned. We use these substrates to isolate the effects of mesh size on cell behavior, including attachment, spreading, migration, focal adhesion formation and YAP localization in the nucleus. Our results show that mesh size has significant, yet often overlooked, effects, on cell behavior, and contribute to a further understanding of cell-substrate interactions.

Identification of intracellular glycosaminoglycan-interacting proteins by affinity purification mass spectrometry.

Glycosaminoglycans (GAGs) are essential functional components of the extracellular matrix (ECM). Artificial GAGs like sulfated hyaluronan (sHA) exhibit pro-osteogenic properties and boost healing processes. Hence, they are of high interest for supporting bone regeneration and wound healing. Although sulfated GAGs (sGAGs) appear intracellularly, the knowledge about intracellular effects and putative interaction partners is scarce. Here we used an affinity-purification mass spectrometry-based (AP-MS) approach to identify novel and particularly intracellular sGAG-interacting proteins in human bone marrow stromal cells (hBMSC). Overall, 477 proteins were found interacting with at least one of four distinct sGAGs. Enrichment analysis for protein localization showed that mainly intracellular and cell-associated interacting proteins were identified. The interaction of sGAG with α2-macroglobulin receptor-associated protein (LRPAP1), exportin-1 (XPO1), and serine protease HTRA1 (HTRA1) was confirmed in reverse assays. Consecutive pathway and cluster analysis led to the identification of biological processes, namely processes involving binding and processing of nucleic acids, LRP1-dependent endocytosis, and exosome formation. Respecting the preferentially intracellular localization of sGAG in vesicle-like structures, also the interaction data indicate sGAG-specific modulation of vesicle-based transport processes. By identifying many sGAG-specific interacting proteins, our data provide a resource for upcoming studies aimed at molecular mechanisms and understanding of sGAG cellular effects.

Polo Like Kinase 4 (PLK4) impairs human bone marrow mesenchymal stem cell (BMSC) viability and osteogenic differentiation.

Human bone marrow mesenchymal stem cell (hBMSC) viability and osteogenic differentiation play a critical role in bone disorders such as osteoporosis. In the present study, we identified the aberrant PLK4 upregulation in osteoporosis and downregulation in BMSCs during osteogenic differentiation. In isolated hBMSCs, PLK4 overexpression significantly inhibited, whereas PLK4 knockdown promoted cell viability and hBMSC osteogenic differentiation. For molecular mechanism, PLK4 overexpression decreased, whereas PLK4 knockdown increased WNT1 and ß-catenin protein levels and the phosphorylation of Smad1/5/8. The Wnt/ß-catenin signaling antagonist Dickkopf 1 (DKK1) or the BMP-Smads antagonist LDN193189 dramatically suppressed hBMSC osteoblast differentiation, and partially attenuated the promotive effects of PLK4 knockdown on hBMSC osteogenic differentiation. Altogether, PLK4 overexpression impairs hBMSC viability and osteogenic differentiation potential, possibly through the Wnt/ß-catenin signaling and BMP/Smads signaling.

SNHG16/miR-485-5p/BMP7 axis modulates osteogenic differentiation of human bone marrow-derived mesenchymal stem cells.

BACKGROUND: Osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) is crucial for bone formation and its dysfunction is reported to be linked to osteoporosis (OP). The present study aimed to probe the function of the long non-coding RNA small nucleolar RNA host gene 16 (SNHG16) with respect to modulating the osteogenic differentiation of hBMSCs. METHODS: SNHG16 expression in hBMSCs obtained from OP patients was measured by a quantitative real-time polymerase chain reaction (qRT-PCR). Gain-of-function and loss-of-function models of SNHG16 were established with hBMSCs. The expression of OP-related genes (ALP, OCN and OPN) in hBMSCs was determined by qRT-PCR and western blotting. StarBase, TargetScan7.2, miRDB and PicTar databases were used to predict the binding sites between SNHG16 and miR-485-5p, miR-485-5p and 3'-UTR of bone morphogenetic protein 7 (BMP7), respectively. A dual-luciferase reporter assay was used to determine the regulatory relationships between SNHG16 and miR-485-5p, miR-485-5p and 3'-UTR of BMP7, respectively. RESULTS: SNHG16 was remarkably down-regulated in hBMSCs obtained from patients with OP. Overexpression of SNHG16 promoted the osteogenic differentiation of hBMSCs, whereas knockdown of SNHG16 suppressed it. Mechanistically, miR-485-5p is a target of SNHG16, and miR-485-5p can reverse the function of SNHG16. BMP7 is also identified as a target of miR-485-5p and can be indirectly modulated by SNHG16 in hBMSCs. CONCLUSIONS: SNHG16 promotes the osteogenic differentiation of hBMSCs via regulating the miR-485-5p/BMP7 axis and comprises a prospective therapy target for OP.

Concentrated Growth Factors (CGF) Induce Osteogenic Differentiation in Human Bone Marrow Stem Cells.

Bone regeneration is a complex process regulated by several factors that control overlapping biological processes, coordinating interactions among distinct cell populations. There is a great interest in identifying new strategies for inducing osteogenesis in a safe and efficient manner. Concentrated Growth Factor (CGF) is an autologous blood derived product obtained by centrifugation of venous blood following the procedure set on the Silfradent device. In this study the effects of CGF on osteogenic differentiation of human Bone Marrow Stem Cells (hBMSC) in vitro have been investigated; hBMSC were cultured with CGF or osteogenic medium, for 21 days. The osteogenic differentiation was evaluated measuring alkaline phosphatase (ALP) enzyme activity, matrix mineralization by alizarin red staining and through mRNA and protein quantification of osteogenic differentiation markers by Real-time PCR and Western blotting, respectively. The treatment with CGF stimulated ALP activity and promoted matrix mineralization compared to control and seems to be more effective than osteogenic medium. Also, hBMSC lost mesenchymal markers and showed other osteogenic features. Our study showed for the first time that CGF alone is able to induce osteogenic differentiation in hBMSC. The application of CGF on hBMSC osteoinduction might offer new clinical and biotechnological strategies in the tissue regeneration field.

DLL4 restores damaged liver by enhancing hBMSC differentiation into cholangiocytes.

BACKGROUND & AIMS: Biliary injury is one of the main pathological mechanisms of fulminant hepatic failure (FHF). Delta-like ligand 4 (DLL4)-mediated Notch activation contributes to reversing biliary injury; however, the specific role of DLL4 in biliary restoration is still unclear. This study aimed to determine whether human bone marrow mesenchymal stem cells (hBMSCs) can differentiate into biliary epithelial cells (cholangiocytes) in vitro and in vivo and to clarify the role of DLL4 in restoring damaged liver by enhancing cholangiocyte differentiation. METHODS: hBMSCs were transplanted into immunodeficient mice (FRGS) with FHF induced by the hamster-anti-mouse CD95 antibody JO2. The appearance of human cholangiocytes was evaluated in the generated hBMSC-FRGS mice by q-PCR expression, flow cytometry and immunohistochemistry. The potency of DLL4 in inducing cholangiocyte differentiation from hBMSCs was assessed by observing the cell morphology and measuring the expression of cholangiocyte-specific genes and proteins. RESULTS: Human KRT19- and KRT7-double-positive cholangiocyte-like cells appeared in hBMSC-FRGS mice at 12 weeks after transplantation. After these cells were separated and collected by fluorescent-activated cell sorting (FACS), there were high levels of expression of eight typical human cholangiocyte-specific genes and proteins (e.g., KRT19 and KRT7). Furthermore, hBMSC-derived cholangiocytes induced by DLL4 had a better shape with higher nucleus/cytoplasm ratios and showed a specific increase in the expression of cholangiocyte-specific genes and proteins (e.g., KRT19, KRT7, SOX9 and CFTR). CONCLUSIONS: Cholangiocytes can be efficiently differentiated from hBMSCs in vivo and in vitro. DLL4 restores damaged liver by enhancing cholangiocyte differentiation from hBMSCs and has the potential to be used in future clinical therapeutic applications.

Roles of Silk Fibroin on Characteristics of Hyaluronic Acid/Silk Fibroin Hydrogels for Tissue Engineering of Nucleus Pulposus.

Silk fibroin (SF) and hyaluronic acid (HA) were crosslinked by horseradish peroxidase (HRP)/H2O2, and 1,4-Butanediol di-glycidyl ether (BDDE), respectively, to produce HA/SF-IPN (interpenetration network) (HS-IPN) hydrogels. HS-IPN hydrogels consisted of a SF strain with a high content of tyrosine (e.g., strain A) increased viscoelastic modules compared with those with low contents (e.g., strain B and C). Increasing the quantities of SF in HS-IPN hydrogels (e.g., HS7-IPN hydrogels with weight ratio of HA/SF, 5:7) increased viscoelastic modules of the hydrogels. In addition, the mean pores size of scaffolds of the model hydrogels were around 38.96 ± 5.05 µm which was between those of scaffolds H and S hydrogels. Since the viscoelastic modulus of the HS7-IPN hydrogel were similar to those of human nucleus pulposus (NP), it was chosen as the model hydrogel for examining the differentiation of human bone marrow-derived mesenchymal stem cell (hBMSC) to NP. The differentiation of hBMSC induced by transforming growth factor ß3 (TGF-ß3) in the model hydrogels to NP cells for 7 d significantly enhanced the expressions of glycosaminoglycan (GAG) and collagen type II, and gene expressions of aggrecan and collagen type II while decreased collagen type I compared with those in cultural wells. In summary, the model hydrogels consisted of SF of strain A, and high concentrations of SF showed the highest viscoelastic modulus than those of others produced in this study, and the model hydrogels promoted the differentiation of hBMSC to NP cells.

Electrodeposition of Sr-substituted hydroxyapatite on low modulus beta-type Ti-45Nb and effect on in vitro Sr release and cell response.

Beta-type Ti-based alloys are promising new materials for bone implants owing to their excellent mechanical biofunctionality and biocompatibility. For treatment of fractures in case of systemic diseases like osteoporosis the generation of implant surfaces which actively support the problematic bone healing is a most important aspect. This work aimed at developing suitable approaches for electrodeposition of Sr-substituted hydroxyapatite (Srx-HAp) coatings onto Ti-45Nb. Potentiodynamic polarization measurements in electrolytes with 1.67 mmol/L Ca(NO3)2, which was substituted by 0, 10, 50 and 100% Sr(NO3)2, and 1 mmol/L NH4H2PO4 at 333 K revealed the basic reaction steps for OH- and PO43- formation needed for the chemical precipitation of Srx-HAp. Studies under potentiostatic control confirmed that partial or complete substitution of Ca2+- by Sr2+-ions in solution has a significant effect on the complex reaction process. High Sr2+-ion contents yield intermediate phases and a subsequent growth of more refined Srx-HAp coatings. Upon galvanostatic pulse-deposition higher reaction rates are controlled and in all electrolytes very fine needle-like crystalline coatings are obtained. With XRD the incorporation of Sr-species in the hexagonal HAp lattice is evidenced. Coatings formed in electrolytes with 10 and 50% Sr-nitrate were chemically analyzed with EDX mapping and GD-OES depth profiling. Only a fraction of the Sr-ions in solution is incorporated into the Srx-HAp coatings. Therein, the Sr-distribution is laterally homogeneous but non-homogeneous along the cross-section. Increasing Sr-content retards the coating thickness growth. Most promising coatings formed in the electrolyte with 10% Sr-nitrate were employed for Ca, P and Sr release analysis in Tris-Buffered Saline (150 mM NaCl, pH 7.6) at 310 K. At a sample surface: solution volume ratio of 1:200, after 24 h the amount of released Sr-ions was about 30-35% of that determined in the deposited Srx-HAp coating. In vitro studies with human bone marrow stromal cells (hBMSC) revealed that the released Sr-ions led to a significantly enhanced cell proliferation and osteogenic differentiation and that the Sr-HAp surface supported cell adhesion indicating its excellent cytocompatibility.

Overexpression of RPN2 promotes osteogenic differentiation of hBMSCs through the JAK/STAT3 pathway.

Osteoporosis is characterized by decreased bone mass and degenerating bone structure, which cause severe bone fragility and increase the risk for fractures. Human bone mesenchymal stem cells (hBMSCs) differentiate into osteoblasts through osteogenesis, and disturbances in the balance between bone generation and degeneration underlie the pathogenesis of senile osteoporosis. The highly conserved glycoprotein Ribophorin II (RPN2) is involved in multiple biological reactions, but the role of RPN2 in the osteogenic differentiation of hBMSCs and their molecular etiology is incompletely understood. Here, we show that RPN2 expression is up-regulated in hBMSCs during osteogenic differentiation. In vitro assays revealed that silencing of RPN2 inhibited hBMSC differentiation into osteoblasts. Moreover, RPN2 overexpression enhanced the expression of linked genes and resulted in high alkaline phosphatase activity. Our results suggest that RPN2 targets Janus kinase 1 (JAK1), and RPN2 overexpression was observed to induce JAK1 ubiquitination. Depletion of JAK1 facilitated osteogenic differentiation of RPN2-silenced hBMSCs. Moreover, western blot analysis revealed that RPN2 silencing suppressed the stimulation and nuclear translocation of the downstream signal transducer and activator of transcription 3 sensor; this could be reversed via RPN2 overexpression. This research sheds light on an innovative molecular mechanism that is associated with hBMSC differentiation into osteoblasts and may facilitate bone anabolism through RPN2.

hBMSC-Derived Extracellular Vesicles Attenuate IL-1ß-Induced Catabolic Effects on OA-Chondrocytes by Regulating Pro-inflammatory Signaling Pathways.

Background: Human bone marrow-derived mesenchymal stromal cells (hBMSCs) provide a promising therapeutic approach in the cell-based therapy of osteoarthritis (OA). However, several disadvantages evolved recently, including immune responses of the host and regulatory hurdles, making it necessary to search for alternative treatment options. Extracellular vesicles (EVs) are released by multiple cell types and tissues into the extracellular microenvironment, acting as message carriers during intercellular communication. Here, we investigate putative protective effects of hBMSC-derived EVs as a cell-free approach, on IL-1ß-stimulated chondrocytes obtained from OA-patients. Methods: EVs were harvested from the cell culture supernatant of hBMSCs by a sequential ultracentrifugation process. Western blot, scanning electron microscopy (SEM), and nanoparticle tracking analysis (NTA) were performed to characterize the purified particles as EVs. Intracellular incorporation of EVs, derived from PHK26-labeled hBMSCs, was tested by adding the labeled EVs to human OA chondrocytes (OA-CH), followed by fluorescence microscopy. Chondrocytes were pre-stimulated with IL-1ß for 24 h, followed by EVs treatment for 24 h. Subsequently, proliferation, apoptosis, and migration (wound healing) were analyzed via BrdU assay, caspase 3/7 assay, and scratch assay, respectively. With qRT-PCR, the relative expression level of anabolic and catabolic genes was determined. Furthermore, immunofluorescence microscopy and western blot were performed to evaluate the protein expression and phosphorylation levels of Erk1/2, PI3K/Akt, p38, TAK1, and NF-κB as components of pro-inflammatory signaling pathways in OA-CH. Results: EVs from hBMSCs (hBMSC-EVs) promote proliferation and reduce apoptosis of OA-CH and IL-1ß-stimulated OA-CH. Moreover, hBMSC-EVs attenuate IL-1ß-induced reduction of chondrocyte migration. Furthermore, hBMSC-EVs increase gene expression of PRG4, BCL2, and ACAN (aggrecan) and decrease gene expression of MMP13, ALPL, and IL1ß in OA-CH. Notably, COL2A1, SOX9, BCL2, ACAN, and COMP gene expression levels were significantly increased in IL-1ß+ EV groups compared with those IL-1ß groups without EVs, whereas the gene expression levels of COLX, IL1B, MMP13, and ALPL were significantly decreased in IL-1ß+ EV groups compared to IL-1ß groups without EVs. In addition, the phosphorylation status of Erk1/2, PI3K/Akt, p38, TAK1, and NF-κB signaling molecules, induced by IL-1ß, is prevented by hBMSC- EVs. Conclusion: EVs derived from hBMSCs alleviated IL-1ß-induced catabolic effects on OA-CH via promoting proliferation and migration and reducing apoptosis, probably via downregulation of IL-1ß-activated pro-inflammatory Erk1/2, PI3K/Akt, p38, TAK1, and NF-κB signaling pathways. EVs released from BMSCs may be considered as promising cell-free intervention strategy in cartilage regenerative medicine, avoiding several adverse effects of cell-based regenerative approaches.

Remodeling of Three-Dimensional Collagen I Matrices by Human Bone Marrow Stromal Cells during Osteogenic Differentiation In Vitro.

Cell fate is triggered by the characteristics of the surrounding extracellular matrix (ECM) including its composition and topological and mechanical properties. Human bone marrow stromal cells (hBMSC) are known to reside in a niche environment where they are maintained in a quiescent, multipotent state, also controlled by the ECM characteristics. In this in vitro study, three-dimensional (3D) fibrillary collagen I (Col)-based matrices with defined topological and mechanical characteristics were used (pore size of 3-4 µm, fibril diameter of ∼0.7 µm, ∼90 Pa (non-cross-linked), and ∼160 Pa (cross-linked)), mimicking conditions of the environment in the bone marrow. The performance of non-cross-linked and cross-linked scaffolds during osteogenic differentiation of hBMSC in terms of matrix stiffness and proteolytic degradability was investigated. Cell adhesion, morphology, and invasion as well as matrix remodeling were investigated on cross-linked and non-cross-linked Col matrices over 22 days. About 25% of the cells invaded the matrices and showed a spread morphology independent of cross-linking. Cellular proteolytic matrix degradation in terms of a decreased matrix layer thickness was only found for non-cross-linked matrices at constant pore size and fibril diameter. Osteogenic differentiation of hBMSC was examined by alkaline phosphatase staining and enzyme activity (early marker) and calcium phosphate deposition (late marker) and was similarly supported in both scaffolds. Furthermore, both matrices were strongly stiffened by about 10-fold because of high mineralization under osteogenic conditions. In summary, these results emphasize that fibrillary 3D Col matrices are a suitable model to study primary hBMSC behavior in terms of ECM remodeling during osteogenesis at defined in vitro conditions.