Mouse Bone Mesenchymal Stem Cells Research Articles

Unveiling the role of hypoxia-inducible factor 2alpha in osteoporosis: Implications for bone health.

Osteoporosis (OP) has become a major public health problem worldwide. Most OP treatments are based on the inhibition of bone resorption, and it is necessary to identify additional treatments aimed at enhancing osteogenesis. In the bone marrow (BM) niche, bone mesenchymal stem cells (BMSCs) are exposed to a hypoxic environment. Recently, a few studies have demonstrated that hypoxia-inducible factor 2alpha (HIF-2α) is involved in BMSC osteogenic differentiation, but the molecular mechanism involved has not been determined. To investigate the effect of HIF-2α on the osteogenic and adipogenic differentiation of BMSCs and the hematopoietic function of hematopoietic stem cells (HSCs) in the BM niche on the progression of OP. Mice with BMSC-specific HIF-2α knockout (Prx1-Cre;Hif-2αfl/fl mice) were used for in vivo experiments. Bone quantification was performed on mice of two genotypes with three interventions: Bilateral ovariectomy, semilethal irradiation, and dexamethasone treatment. Moreover, the hematopoietic function of HSCs in the BM niche was compared between the two mouse genotypes. In vitro, the HIF-2α agonist roxadustat and the HIF-2α inhibitor PT2399 were used to investigate the function of HIF-2α in BMSC osteogenic and adipogenic differentiation. Finally, we investigated the effect of HIF-2α on BMSCs via treatment with the mechanistic target of rapamycin (mTOR) agonist MHY1485 and the mTOR inhibitor rapamycin. The quantitative index determined by microcomputed tomography indicated that the femoral bone density of Prx1-Cre;Hif-2αfl/fl mice was lower than that of Hif-2αfl/fl mice under the three intervention conditions. In vitro, Hif-2αfl/fl mouse BMSCs were cultured and treated with the HIF-2α agonist roxadustat, and after 7 d of BMSC adipogenic differentiation, the oil red O staining intensity and mRNA expression levels of adipogenesis-related genes in BMSCs treated with roxadustat were decreased; in addition, after 14 d of osteogenic differentiation, BMSCs treated with roxadustat exhibited increased expression of osteogenesis-related genes. The opposite effects were shown for mouse BMSCs treated with the HIF-2α inhibitor PT2399. The mTOR inhibitor rapamycin was used to confirm that HIF-2α regulated BMSC osteogenic and adipogenic differentiation by inhibiting the mTOR pathway. Consequently, there was no significant difference in the hematopoietic function of HSCs between Prx1-Cre;Hif-2αfl/fl and Hif-2αfl/fl mice. Our study showed that inhibition of HIF-2α decreases bone mass by inhibiting the osteogenic differentiation and increasing the adipogenic differentiation of BMSCs through inhibition of mTOR signaling in the BM niche.

Mesenchymal stem cells (MSCs) from the mouse bone marrow show differential expression of interferon regulatory factors IRF-1 and IRF-2.

Interferon regulatory factors (IRF-1 and IRF-2) are transcription factors widely implicated in various cellular processes, including regulation of inflammatory responses to pathogens, cell proliferation, oncogenesis, differentiation, autophagy, and apoptosis. We have studied the expression of IRF-1, IRF-2 mRNAs by RT-PCR, cellular localization of the proteins by immunofluorescence, and expression of mRNAs of genes regulated by IRF-1, IRF-2 by RT-PCR in mouse bone marrow cells (BMCs) and mesenchymal stem cells (MSCs). Higher level of IRF-1 mRNA was observed in BMCs and MSCs compared to that of IRF-2. Similarly, differential expression of IRF-1 and IRF-2 proteins was observed in BMCs and MSCs. IRF-1 was predominantly localized in the cytoplasm, whereas IRF-2 was localized in the nuclei of BMCs. MSCs showed nucleo-cytoplasmic distribution of IRF-1 and nuclear localization of IRF-2. Constitutive expression of IRF-1 and IRF-2 target genes: monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), cyclooxygenase-2 (COX-2), matrix metalloproteinase-9 (MMP-9), and caspase-1was observed in both BMCs and MSCs. MSCs showed constitutive expression of the pluripotency-associated factors, Oct3/4 and Sox-2. Lipopolysaccharide (LPS)-treatment of MSCs induced prominent cellular localization of IRF-1 and IRF-2. Our results suggest that IRF-1 and IRF-2 exhibit differential expression of their mRNAs and subcellular localization of the proteins in BMCs and MSCs. These cells also show differential levels of constitutive expression of IRF-1 and IRF-2 target genes. This may regulate immune-responsive properties of BMCs and MSCs through IRF-1, IRF-2-dependent gene expression and protein-protein interaction. Regulating IRF-1 and IRF-2 may be helpful for immunomodulatory functions of MSCs for cell therapy and regenerative medicine.

3D bioprinting of cell-laden nano-attapulgite/gelatin methacrylate composite hydrogel scaffolds for bone tissue repair

Bone tissue engineering (BTE) has proven to be a promising strategy for bone defect repair. Due to its excellent biological properties, gelatin methacrylate (GelMA) hydrogels have been used as bioinks for 3D bioprinting in some BTE studies to produce scaffolds for bone regeneration. However, applications for load-bearing defects are limited by poor mechanical properties and a lack of bioactivity. In this study, 3D printing technology was used to create nano-attapulgite (nano-ATP)/GelMA composite hydrogels loaded into mouse bone mesenchymal stem cells (BMSCs) and mouse umbilical vein endothelial cells (MUVECs). The bioprintability, physicochemical properties, and mechanical properties were all thoroughly evaluated. Our findings showed that nano-ATP groups outperform the control group in terms of printability, indicating that nano-ATP is beneficial for printability. Additionally, after incorporation with nano-ATP, the mechanical strength of the composite hydrogels was significantly improved, resulting in adequate mechanical properties for bone regeneration. The presence of nano-ATP in the scaffolds has also been studied for cell-material interactions. The findings show that cells within the scaffold not only have high viability but also a clear proclivity to promote osteogenic differentiation of BMSCs. Besides, the MUVECs-loaded composite hydrogels demonstrated increased angiogenic activity. A cranial defect model was also developed to evaluate the bone repair capability of scaffolds loaded with rat BMSCs. According to histological analysis, cell-laden nano-ATP composite hydrogels can effectively improve bone regeneration and promote angiogenesis. This study demonstrated the potential of nano-ATP for bone tissue engineering, which should also increase the clinical practicality of nano-ATP.

Biomimetic hydroxyapatite coating on the 3D-printed bioactive porous composite ceramic scaffolds promoted osteogenic differentiation via PI3K/AKT/mTOR signaling pathways and facilitated bone regeneration in vivo

• Bionic hierarchical scaffold fabricated by 3D-printing and surface mineralization. • The obtained biomimetic multi-structured scaffolds facilitate bone regeneration. • Regulation of cell fate in activated PI3K/AKT/mTOR signaling were confirmed. • Hydroxyapatite coating accelerates osseointegration and enhance bone-augmentation. • A two-step method enriches and guides the design of modified bone graft materials. The architecture and surface modifications have been regarded as effective methods to enhance the biological response of biomaterials in bone tissue engineering. The porous architecture of the implantation was essential conditions for bone regeneration. Meanwhile, the design of biomimetic hydroxyapatite (HAp) coating on porous scaffolds was demonstrated to strengthen the bioactivity and stimulate osteogenesis. However, bioactive bio-ceramics such as β-tricalcium phosphate (β-TCP) and calcium silicate (CS) with superior apatite-forming ability were reported to present better osteogenic activity than that of HAp. Hence in this study, 3D-printed interconnected porous bioactive ceramics β-TCP/CS scaffold was fabricated and the biomimetic HAp apatite coating were constructed in situ via hydrothermal reaction, and the effects of HAp apatite layer on the fate of mouse bone mesenchymal stem cells (mBMSCs) and the potential mechanisms were explored. The results indicated that HAp apatite coating enhanced cell proliferation, alkaline phosphatase (ALP) activity, and osteogenic gene expression. Furthermore, PI3K/AKT/mTOR signaling pathway is proved to have an important impact on cellular functions. The present results demonstrated that the key molecules of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT) and mammalian target of rapamycin (mTOR) were activated after the biomimetic hydroxyapatite coating were constructed on the 3D-printed ceramic scaffolds. Besides, the activated influence on the protein expression of Runx2 and BMP2 could be suppressed after the treatment of inhibitor HY-10358. In vivo studies showed that the constructed HAp coating promoted bone formation and strengthen the bone quality. These results suggest that biomimetic HAp coating constructed on the 3D-printed bioactive composite scaffolds could strengthen the bioactivity and the obtained biomimetic multi-structured scaffolds might be a potential alternative bone graft for bone regeneration.

Irisin promotes fracture healing by improving osteogenesis and angiogenesis.

BackgroundOsteogenesis and angiogenesis are important for bone fracture healing. Irisin is a muscle-derived monokine that is associated with bone formation.MethodsTo demonstrate the effect of irisin on bone fracture healing, closed mid-diaphyseal femur fractures were produced in 8-week-old C57BL/6 mice. Irisin was administrated intraperitoneally every other day after surgery, fracture healing was assessed by using X-rays. Bone morphometry of the fracture callus were assessed by using micro-computed tomography. Femurs of mice from each group were assessed by the three-point bending testing. Effect of irisin on osteogenic differentiation in mesenchymal stem cells in vitro was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), alkaline phosphatase staining and alizarin red staining. Angiogenesis of human umbilical vein endothelial cells (HUVECs) were evaluated by qRT-PCR, migration tests, and tube formation assays.ResultsIncreased callus formation, mineralization and tougher fracture healing were observed in the irisin-treated group than in the control group, indicating the better fracture callus healing due to Irisin treatment. The vessel surface and vessel volume fraction of the callus also increased in the irisin-treated group. The expression of BMP2, CD31, and VEGF in callus were enhanced in the irisin-treated group. In mouse bone mesenchymal stem cells, irisin promoted ALP expression and mineralization, and increased the expression of osteogenic genes, including OSX, Runx2, OPG, ALP, OCN and BMP2. Irisin also promoted HUVEC migration and tube formation. Expression of angiogenic genes, including ANGPT1, ANGPT2, VEGFb, CD31, FGF2, and PDGFRB in HUVECs were increased by irisin.ConclusionAll the results indicate irisin can promote fracture healing through osteogenesis and angiogenesis. These findings help in the understanding of muscle–bone interactions during fracture healing.The Translational Potential of this ArticleIrisin was one of the most important monokine secreted by skeletal muscle. Studies have found that irisin have anabolic effect one bone remodeling through affecting osteocyte and osteoblast. Based on our study, irisin could promote bone fracture healing by increasing bone mass and vascularization, which provide a potential usage of irisin to promote fracture healing and improve clinical outcomes.

Enhanced osteogenesis and angiogenesis of biphasic calcium phosphate scaffold by synergistic effect of silk fibroin coating and zinc doping.

Biphasic calcium phosphate (BCP) scaffold has been widely applied to bone regeneration because of its good biocompatibility and bone conduction property. However, the low mechanical strength and the lack of angiogenic and osteogenic induction properties have restricted its application in bone tissue regeneration. In this study, we combined zinc (Zn2+) doping and silk fibroin (SF) coating with expectation to enhance compressive strength, osteogenesis and angiogenesis of BCP scaffolds. The phase composition, morphology, porosity, compressive strength, in vitro degradation and cell behaviors were investigated systematically. Results showed that the scaffold coated with SF exhibited almost 3 times of compressive strength without compromising its porosity compared with the uncoated scaffold. Zn2+ doping and SF coating synergistically enhanced the alkaline phosphatase activity and osteogenesis-related genes expression of mouse bone mesenchymal stem cells (mBMSCs). Furthermore, SF coating notably improved the proliferation, cell viability and in vitro angiogenesis of human umbilical vein endothelial cells (HUVECs). This work provides a novel way to modify BCP scaffolds simultaneously with enhancing mechanical strength and biological properties.

Physicochemical and cytological properties of poorly crystalline calcium-deficient hydroxyapatite with different Ca/P ratios

Compared to highly crystalline hydroxyapatite (HA), poorly crystalline hydroxyapatite has been proved to have better bioactivity and degradability, owing to its similar crystallographic structure to natural bone. However, there are few systematic comparative studies on poorly crystalline HA with different Ca/P ratios. In this work, poorly crystalline HA with different Ca/P molar ratios (1.50, 1.55, 1.60 and 1.67) was prepared by chemical precipitation method. The effects of Ca/P ratio on its structure, composition, morphology, surface properties, protein adsorption behaviors, ion adsorption and release abilities, cytological properties were systematically investigated. Results showed that the prepared HA was poorly crystalline, nano-sized and with the gradient change of Ca/P ratios. Protein adsorption capacity and P release of calcium deficient HA (CDHA) were effectively improved by reducing the Ca/P ratio, but CDHA with lower Ca/P ratio would also adsorb more Ca ions in the culture medium, which jointly affected the cytological properties of CDHA. In vitro cell experiments indicated that when mouse bone mesenchymal stem cells were co-cultured with CDHA with a Ca/P ratio of 1.55, their proliferation, ALP activity and osteogenesis-related genes expression were the strongest. This study provides a theoretical support and potential for further improving the biological performance of poorly crystalline CDHA materials.

Fabrication of strontium carbonate-based composite bioceramics as potential bone regenerative biomaterials

Strontium carbonate (SrC) bioceramics are proposed as potential biomaterials to efficaciously repair the bone defects. However, the development of SrC bioceramics is restricted by their intrinsic low mechanical strength. In this study, SrC-based composite bioceramics (SrC-SrP) were fabricated by incorporating strontium-containing phosphate glass (SrP). The results indicated that aside from the main crystalline phase SrC, new compounds were generated in the SrC-SrP bioceramics. Incorporating 10 wt% SrP promoted densification, thus dramatically improving compressive strength of SrC-SrP bioceramics. The SrC-SrP bioceramics facilitated apatite precipitation on their surface, and sustainedly released strontium, phosphorus and sodium ions. Compared with the well-known β-tricalcium phosphate bioceramics, the SrC-SrP bioceramics with certain amounts of SrP enhanced proliferation, alkaline phosphatase activity and osteogenesis-related gene expressions of mouse bone mesenchymal stem cells. The SrC-SrP bioceramics with appropriate constituent can serve as novel bone regenerative biomaterials.

Exosomes derived from platelet-rich plasma administration in site mediate cartilage protection in subtalar osteoarthritis

Subtalar osteoarthritis (STOA) is often secondary to chronic ankle sprains, which seriously affects the quality of life of patients. Due to its etiology and pathogenesis was not studied equivocally yet, there is currently a lack of effective conservative treatments. Although they have been used for tissue repair, platelet-rich plasma-derived exosomes (PRP-Exo) have the disadvantage of low retention and short-lived therapeutic effects. This study aimed to determine whether incorporation of PRP-Exo in thermosensitive hydrogel (Gel) increased their retention in the joint and thereby playing a therapeutic role on STOA due to chronic mechanical instability established by transecting lateral ligaments (anterior talofibular ligament (ATFL)/calcaneal fibular ligament (CFL)). PRP-Exo incorporated Gel (Exo-Gel) system, composed of Poloxamer-407 and 188 mixture-based thermoresponsive hydrogel matrix in an optimal ratio, was determined by its release ability of Exo and rheology of Gel response to different temperature. The biological activity of Exo-Gel was evaluated in vitro, and the therapeutic effect of Exo-Gel on STOA was evaluated in vivo. Exo released from Exo-Gel continuously for 28 days could promote the proliferation and migration of mouse bone mesenchymal stem cells (mBMSCs) and chondrocytes, at the same time enhance the chondrogenic differentiation of mBMSCs, and inhibit inflammation-induced chondrocyte degeneration. In vivo experiments confirmed that Exo-Gel increased the local retention of Exo, inhibited the apoptosis and hypertrophy of chondrocytes, enhanced their proliferation, and potentially played the role in stem cell recruitment to delay the development of STOA. Thus, Delivery of PRP-Exo incorporated in thermosensitive Gel provides a novel approach of cell-free therapy and has therapeutic effect on STOA.Graphical

Facile fabrication of near‐infrared light‐responsive shape memory nanocomposite scaffolds with hierarchical porous structures

AbstractIn this work, the near‐infrared (NIR) light‐responsive shape memory scaffolds with hierarchical porous structures are designed and facilely formed by freeze drying of 3D printed viscous gel‐like pickering emulsions, which are stabilized by hydrophobically modified graphene oxide (g‐GO) and silica nanoparticles, and contain thermo‐responsive poly(d, l‐lactic acid‐co‐trimethylene carbonate) (PLMC) in the oil phase. The prepared scaffolds display an interconnected filament structure with hierarchical pores and high porosity. The incorporation of g‐GO nanoparticles into PLMC matrix prompts that the scaffold shape memory can be triggered by NIR light with fast shape recovery. Moreover, the in vitro mineralization experiment shows that the scaffolds have biological activity, and the drug release study demonstrates that the scaffolds can be used as drug carriers with efficient drug release capacity. Furthermore, cell culture assays based on mouse bone mesenchymal stem cells exhibit that the scaffolds own good cytocompatibility. Therefore, the facile preparation and remote activation of the shape memory nanocomposite scaffolds with hierarchical porous structure and multifunctionality represents a highly attractive candidate as minimally invasive implantation scaffolds for bone tissue engineering applications.

Beraprost ameliorates postmenopausal osteoporosis by regulating Nedd4-induced Runx2 ubiquitination

Bone health requires adequate bone mass, which is maintained by a critical balance between bone resorption and formation. In our study, we identified beraprost as a pivotal regulator of bone formation and resorption. The administration of beraprost promoted differentiation of mouse bone mesenchymal stem cells (M-BMSCs) through the PI3K–AKT pathway. In co-culture, osteoblasts stimulated with beraprost inhibited osteoclastogenesis in a rankl-dependent manner. Bone mass of p53 knockout mice remained stable, regardless of the administration of beraprost, indicating that p53 plays a vital role in the bone mass regulation by beraprost. Mechanistic in vitro studies showed that p53 binds to the promoter region of neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4) to promote its transcription. As a ubiquitinating enzyme, Nedd4 binds to runt-related transcription factor 2 (Runx2), which results in its ubiquitination and subsequent degradation. These data indicate that the p53–Nedd4–Runx2 axis is an effective regulator of bone formation and highlight the potential of beraprost as a therapeutic drug for postmenopausal osteoporosis.

CircRNA-016901 silencing attenuates irradiation-induced injury in bone mesenchymal stem cells via regulating the miR-1249-5p/HIPK2 axis.

Currently, bone marrow transplantation remains the basic treatment for various hematological tumors and irradiation is one of the most important pretreatment methods. However, irradiation pretreatment may result in damage to bone mesenchymal stem cells (BMSCs). The present study aimed to investigate the effect of circular RNA-016901 (circ-016901) on the injury of irradiation-induced BMSCs and the underlying mechanism. The expression levels of circ-016901, microRNA-1249-5p (miR-1249-5p) and homeodomain interacting protein kinase 2 (HIPK2) in irradiation-induced mouse BMSCs at various irradiation doses were detected via reverse transcription-quantitative PCR (RT-qPCR). The effect of circ-016901 on cell proliferation was examined using Cell Counting Kit-8 assays following silencing or overexpression of circ-016901. Cell apoptosis was detected by flow cytometry and caspase-3/7 activity. The expression of autophagy-related markers, including Beclin-1 and LC3-II/I, was detected at the mRNA and protein levels by RT-qPCR and western blotting, respectively. Irradiation treatment upregulated the expression of circ-016901 and HIPK2 and downregulated miR-1249-5p expression. The expression levels of LC3-II/I and Beclin-1 in BMSCs were downregulated in a dose-dependent manner. Silencing of circ-016901 promoted proliferation of irradiation-induced BMSCs and attenuated irradiation-induced apoptosis. Moreover, silencing of circ-016901 elevated the expressions of LC3-II/I and Beclin-1 in irradiation-induced BMSCs. Similar results were obtained with miR-1249-5p overexpression and HIPK2 silencing. These results demonstrated that circ-016901 silencing attenuated injury in irradiation-induced mouse BMSCs by regulating the miR-1249-5p/HIPK2 axis, providing a novel target for future research on the mechanism of radiation resistance in BMSCs.

Quercetin promotes osteogenic differentiation and antioxidant responses of mouse bone mesenchymal stem cells through activation of the AMPK/SIRT1 signaling pathway.

Decrepitude and apoptosis of bone mesenchymal stem cells (BMSCs) induced by reactive oxygen species (ROS) lead to inhibited osteogenic differentiation, causing decreased bone density and osteoporosis. Quercetin, a bioactive component of Solanum muricatum extracts, promotes the osteogenic differentiation of BMSCs and ameliorates the symptoms of osteoporosis in vivo. However, the detailed mechanism underlying this process remains unclear. The study aims to reveal the regulatory mechanism of quercetin in BMSCs. Mouse BMSCs (mBMSCs) were isolated from the bone marrow and characterized by flow cytometry. QRT-PCR and western blot assays were performed to evaluate the expression levels of related genes and proteins. Alkaline phosphatase (ALP) staining and Oil Red O staining of lipids were used to estimate the osteogenesis and adipogenesis levels of mBMSCs, respectively. Quercetin treatment (2 and 5 μM) induced significant upregulation of antioxidant enzymes, SOD1 and SOD2, in mBMSCs. Quercetin promoted osteogenic differentiation and inhibited adipogenic differentiation of mBMSCs. Quercetin treatment enhanced the phosphorylation of AMPK protein and upregulated the expression of SIRT1, thus activating the AMPK/SIRT1 signaling pathway in mBMSCs. Quercetin promoted osteogenic differentiation and antioxidant responses of mBMSCs by activating the AMPK/SIRT1 signaling pathway.

Shape Memory and Osteogenesis Capabilities of the Electrospun Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Modified Poly(l-Lactide) Fibrous Mats.

Poly(l-lactide) (PLLA) as one of the most well-known biodegradable polyesters has been studied extensively for bone tissue engineering. If being properly programmed, scaffolds from PLLA can also be endowed with the capability of shape memory. However, several noted issues, for example, mechanical brittleness, high glass transition temperature Tg, and relatively poor shape retention and recovery properties, necessitate modification of the PLLA to improve its application efficacy in physiological conditions. This study is proposed to modify PLLA by having the biodegradable poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) incorporated to form ultrafine composite fibers (i.e., PLLA-PHBV) through electrospinning. Different pairs of PLLA-PHBV at the varying mass ratios of 10:0, 9:1, 8:2, 7:3, 6:4, and 0:10 can be successfully electrospun into fibrous form with the fineness of 2-3 μm. Incorporation of PHBV enables to give rise to desired Tg decreases and also, interestingly, increases in the Young's modulus of the PLLA-PHBV blends, while gradually increasing the PHBV mass ratios up to 30%. The PLLA-PHBV (7:3) formulation is identified to present excellent shape memory properties with high shape fixing ratio (>98%) and shape recovery ratio (>96%) compared to the unmodified PLLA fiber counterpart. Moreover, the PLLA-PHBV (7:3) fibers also show enhanced osteogenesis-inducing ability in the mouse bone mesenchymal stem cells, even under nonosteoinductive conditions. Collectively, for the first time this study demonstrates the enhanced shape memory and osteogenesis capabilities of the electrospun PLLA-PHBV composite fibers, and the researched PLLA-PHBV (7:3) fiber system could be potentially applied as a multifunctional scaffolding material for applications in bone tissue repair and regeneration. Impact statement By first converting the poly(l-lactide) (PLLA)-poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) hybrids into fibrous form at varied mass ratios followed by a thorough characterization, we reasonably demonstrated that incorporation of an appropriate amount of PHBV (i.e., 30%) into the PLLA fibers could give rise to significant improvement on the shape memory capability of the PLLA, along with the desired decreases in the transition temperature (Tg). Moreover, the fibrous PLLA-PHBV (7:3) scaffold was also found to significantly promote the osteogenic commitment in bone mesenchymal stem cells with osteoinductive factors in a synergistic manner. Our biomimicking and shape memory enabled fibrous scaffold of PLLA-PHBV could be used to construct multifunctional three-dimensional scaffold with shape memory effect for bone regeneration.

Fasudil enhanced differentiation of BMSCs in vivo and vitro, involvement of P38 signaling pathway

Bone mesenchymal stem cells (BMSCs) are a well-known donor graft source due to their potential for self-renewal and differentiation into multi-lineage cell types, including osteoblasts that are critical for fracture healing. Fasudil (FAS), a Rho kinase inhibitor, has been proven to induce the differentiation of bone marrow stem cells (BMSCs) into neuron-like cells. However, its role in the osteogenesis of BMSCs remain uncertain. Herein, we for the first time studied the effects of FAS on osteogenic differentiation in a mouse fracture model and further explored the involved mechanisms in mouse BMSCs. The results showed that FAS stimulated bone formation in the fracture mouse model. Additionally, at 30 μM, FAS significantly promotes alkaline phosphatase activity, mineralization, and the expression of osteogenic markers COL-1, RUNX2 and OCN in murine BMSCs. Blocking of P38 by SB202190 significantly reversed the effects of FAS, in vitro, suggesting that P38, but not ERK or JNK activation is required for FAS-induced osteogenesis. Collectively, our results indicate that FAS may be a promising agent for promoting fracture healing.

Sodium alginate/collagen composite multiscale porous scaffolds containing poly(ε-caprolactone) microspheres fabricated based on additive manufacturing technology.

Biocompatible porous scaffolds with adjustable pore structures, appropriate mechanical properties and drug loading properties are important components of bone tissue engineering. In this work, biocompatible sodium alginate (SA)/collagen (Col) multiscale porous scaffolds containing poly(ε-caprolactone) microspheres (Ms-PCL) have been facilely fabricated based on 3D extrusion printing of the pre-crosslinked composite hydrogels. The prepared composite hydrogels can be 3D extrusion printed into porous scaffolds with different designed shapes and adjustable pore structures. The hydroxyapatite (HAP) nanoparticles have been added into the SA/Col hydrogels to achieve stress dispersion and form double crosslinking networks. SA-Ca2+ crosslinking networks and Col–genipin (GP) crosslinking networks have been constructed to improve the mechanical properties of the scaffolds (about 2557 kPa of compressive stress at 70% strain), and reduce the swelling rate and degradation rate of SA/Col scaffolds. Moreover, the SA/Col hydrogels contain hydrophobic antibacterial drug enrofloxacin loaded Ms-PCL, and in vitro drug release research shows a sustained-release function of porous scaffolds, indicating the potential application of SA/Col porous scaffolds as drug carriers. In addition, the antibacterial experiments show that the composite scaffolds display a distinguished and long-term antibacterial activity against Escherichia coli and Staphylococcus aureus. Furthermore, mouse bone mesenchymal stem cells (mBMSCs) are seeded on the SA/Col composite scaffolds, and an in vitro biocompatibility experiment shows that the mBMSCs can adhere well on the composite scaffolds, which indicate that the fabricated composite scaffolds are biocompatible. In short, all of the above results suggest that the biocompatible SA/Col composite porous scaffolds have enormous application and potential in bone tissue engineering.

GIT1 regulates angiogenic factor secretion in bone marrow mesenchymal stem cells via NF-κB/Notch signalling to promote angiogenesis.

ObjectivesOsteogenesis is coupled with angiogenesis during bone remodelling. G‐protein‐coupled receptor (GPCR) kinase 2‐interacting protein‐1 (GIT1) is an important protein that participates in fracture healing by regulating angiogenesis. This study investigated whether GIT1 could affect bone mesenchymal stem cells (BMSCs) to secrete angiogenic factors to enhance fracture healing by promoting angiogenesis and its possible mechanism.Materials and methodsThe angiogenesis of mice post‐fracture was detected by micro‐CT and immunofluorescence. Subsequently, vascular endothelial growth factor (VEGF) level in mouse and human BMSCs (hBMSCs) under TNF‐α stimulation was detected. The hBMSCs were transfected with GIT1 shRNAs to further explore the relationship between GIT1 and VEGF and angiogenesis in vitro. Furthermore, based on previous research on GIT1, possible signal pathways were investigated.ResultsGIT1 knockout mice exhibited impaired angiogenesis and delayed fracture healing. And GIT1 deficiency remarkably reduced the expression of VEGF mRNA in BMSCs, which affected the proliferation and migration of human umbilical vein endothelial cells. GIT1 knockdown inhibited the activation of Notch and NF‐κB signals by decreasing nuclear transportation of NICD and P65/P50, respectively. Overexpression of the canonical NF‐κB subunits P65 and P50 markedly increased NICD‐dependent activation of recombination signal‐binding protein‐jκ reporter. Finally, GIT1 enhanced the affinity of NF‐κB essential modulator (NEMO) for K63‐linked ubiquitin chains via interaction with NEMO coiled‐coil 2 domains.ConclusionThese data revealed a positive role for GIT1 by modulating the Notch/NF‐κB signals which promoting paracrine of BMSCs to enhance angiogenesis and fracture healing.

Crocin inhibits titanium particle-induced inflammation and promotes osteogenesis by regulating macrophage polarization

Wear particle-induced periprosthetic inflammatory osteolysis and resultant aseptic loosening are major causes of orthopedic implant failure, for which there are no effective treatments other than revision surgery. Crocin, a carotenoid compound derived from crocus flowers, has anti-inflammatory properties, but its immunomodulatory function and role in particle-induced osteolysis are not well characterized. Here we report the effect of crocin on titanium (Ti) particle-induced macrophage polarization and osteogenic differentiation. We found that crocin induced anti-inflammatory (M2) macrophage polarization and attenuated Ti particle-induced inflammation by promoting the expression of anti-inflammatory cytokines in vitro as well as in vivo in a mouse air-pouch model. Additionally, crocin pre-treated macrophages promoted osteogenic differentiation of co-cultured mouse bone mesenchymal stem cells (BMSCs). These effects were mediated via inhibition of p38 and c-Jun N-terminal kinase signaling. Our results indicate that crocin suppresses Ti particle-induced inflammation and enhances osteogenic differentiation of BMSCs by inducing M2 macrophage polarization, highlighting its therapeutic potential for preventing wear particle-induced osteolysis.

Zinc doping induced differences in the surface composition, surface morphology and osteogenesis performance of the calcium phosphate cement hydration products.

In order to investigate the influence of Zn on the hydration reaction of calcium phosphate cement (CPC), the incompletely hydrated CPC tablets were kept soaking in varying zinc-containing tris-(hydroxymethyl)-aminomethane/hydrochloric acid (Zn-Tris-HCl) buffers. It was found that Zn could retard the CPC hydration, the inhibitory effect was in direct proportional to the Zn content in the Zn-Tris-HCl buffer, and overhigh concentration of Zn (≧800 μM) caused the CPC hydration products having different phase composition and surface morphology. Cell culture experimental results revealed the CPC tablets which were soaked in the Zn-Tris-HCl buffer containing relative low Zn content (≦320 μM) favored the mouse bone mesenchymal stem cells (mBMSCs) spreading. When Zn-doped CPC tablets released 10.91 to 27.15 μM of zinc ions into the cell culture medium, it greatly contributed to the improvement of the proliferation ability and the alkaline phosphatase (ALP) activity of the mBMSCs. In the same case, the expression of osteogenesis related genes such as collagen I and runt-related transcription factor 2 was remarkably up-regulated as well. However, the release of high concentration of Zn (128.58 μM) would significantly reduce the ALP activity of the mBMSCs. Therefore, Zn not only facilitates osteogenesis but also affects the CPC hydration behavior, and the CPC with suitable Zn dosage concentration has great potentials to be used for clinical bone repairing.

New BMSC-Laden Gelatin Hydrogel Formed in Situ by Dual-Enzymatic Cross-Linking Accelerates Dermal Wound Healing.

In situ forming hydrogel shows enormous potential as a therapeutic implant or carrier in tissue repair and regeneration. It can perfectly seal or fill the defective tissue, consequently functioning as a cell/drug delivery vehicle. In this contribution, a new gelatin hydrogel with dual-enzymatic cross-linking of horseradish peroxidase (HRP) and galactose oxidase (GalOx) was developed, and the therapeutic effect of this hydrogel encapsulated with bone mesenchymal stem cells (BMSC) in dermal wound healing was investigated. This hydrogel possesses a quick gelation process within 5 min, a high water content, and a uniform three-dimensional (3D) porous network. The 3D cell culture study indicated that gelatin hydrogel matrix of HRP(5U):GalOx(1U) or HRP(2U):GalOx(1U) could provide a friendly 3D microenvironment for supporting the survival, proliferation, and spread of mouse bone mesenchymal stem cells (BMSC) with negligible cytotoxicity. Hematoxylin and eosin staining test suggested that this hydrogel has superior histocompatibility and minimized immune response in vivo. Furthermore, wound-healing studies on a C57 mouse model of excised wound demonstrated that BMSC-laden gelatin hydrogel could significantly accelerate the wound closure as compared to other groups. These data suggest that this dual-enzymatically cross-linked gelatin hydrogel loaded with BMSC has a great potential in wound healing and other tissue-regeneration applications.