Yi-Xuan Chen

Strontium hydroxyapatite/chitosan nanohybrid scaffolds with enhanced osteoinductivity for bone tissue engineering

For the clinical application of bone tissue engineering with the combination of biomaterials and mesenchymal stem cells (MSCs), bone scaffolds should possess excellent biocompatibility and osteoinductivity to accelerate the repair of bone defects. Herein, strontium hydroxyapatite [SrHAP, Ca10-xSrx(PO4)6(OH)2]/chitosan (CS) nanohybrid scaffolds were fabricated by a freeze-drying method. The SrHAP nanocrystals with the different x values of 0, 1, 5 and 10 are abbreviated to HAP, Sr1HAP, Sr5HAP and Sr10HAP, respectively. With increasing x values from 0 to 10, the crystal cell volumes and axial lengths of SrHAP become gradually large because of the greater ion radius of Sr2+ than Ca2+, while the crystal sizes of SrHAP decrease from 70.4nm to 46.7nm. The SrHAP/CS nanohybrid scaffolds exhibits three-dimensional (3D) interconnected macropores with pore sizes of 100-400μm, and the SrHAP nanocrystals are uniformly dispersed within the scaffolds. In vitro cell experiments reveal that all the HAP/CS, Sr1HAP/CS, Sr5HAP/CS and Sr10HAP/CS nanohybrid scaffolds possess excellent cytocompatibility with the favorable adhesion, spreading and proliferation of human bone marrow mesenchymal stem cells (hBMSCs). The Sr5HAP nanocrystals in the scaffolds do not affect the adhesion, spreading of hBMSCs, but they contribute remarkably to cell proliferation and osteogenic differentiation. As compared with the HAP/CS nanohybrid scaffold, the released Sr2+ ions from the SrHAP/CS nanohybrid scaffolds enhance alkaline phosphatase (ALP) activity, extracellular matrix (ECM) mineralization and osteogenic-related COL-1 and ALP expression levels. Especially, the Sr5HAP/CS nanohybrid scaffolds exhibit the best osteoinductivity among four groups because of the synergetic effect between Ca2+ and Sr2+ ions. Hence, the Sr5HAP/CS nanohybrid scaffolds with excellent cytocompatibility and osteogenic property have promising application for bone tissue engineering.

Comparative evaluation of leukocyte- and platelet-rich plasma and pure platelet-rich plasma for cartilage regeneration

Platelet-rich plasma (PRP) has gained growing popularity in the treatment of articular cartilage lesions in the last decade. However, the potential harmful effects of leukocytes in PRP on cartilage regeneration have seldom been studied in vitro, and not at all in vivo yet. The objective of the present study is to compare the effects of leukocyte- and platelet-rich plasma (L-PRP) and pure platelet-rich plasma (P-PRP) on cartilage repair and NF-κB pathway, in order to explore the mechanism underlying the function of leukocytes in PRP in cartilage regeneration. The constituent analysis showed that P-PRP had significantly lower concentrations of leukocytes and pro-inflammatory cytokines compared with L-PRP. In addition, cell proliferation and differentiation assays indicated P-PRP promoted growth and chondrogenesis of rabbit bone marrow mesenchymal stem cells (rBMSC) significantly compared with L-PRP. Despite similarity in macroscopic appearance, the implantation of P-PRP combining rBMSC in vivo yielded better cartilage repair results than the L-PRP group based on histological examination. Importantly, the therapeutic effects of PRP on cartilage regeneration could be enhanced by removing leukocytes to avoid the activation of the NF-κB pathway. Thus, PRP without concentrated leukocytes may be more suitable for the treatment of articular cartilage lesions.

Enhanced antibacterial activity and osteoinductivity of Ag-loaded strontium hydroxyapatite/chitosan porous scaffolds for bone tissue engineering

The properties of bone scaffolds, including biocompatibility, osteoinductivity and antibacterial activity, are of great importance for reconstruction of large bone defects and prevention of implant-associated infections. Herein, we develop an Ag-loaded strontium hydroxyapatite (SrHAP)/chitosan (CS) porous scaffold (Ag-SrHAP/CS) according to the following steps: (i) freeze-drying fabrication of a SrHAP/CS porous scaffold; and (ii) deposition of Ag nanoparticles on the above scaffold. In addition, HAP/CS and Ag-HAP/CS porous scaffolds are prepared under the same conditions without doping Sr element. All the HAP/CS, Ag-HAP/CS, SrHAP/CS and Ag-SrHAP/CS porous scaffolds provide a friendly environment for the adhesion, spreading and proliferation of human bone marrow mesenchymal stem cells (hBMSCs). The three-dimensional (3D) interconnected macropores with a pore size of 100-400 μm allow the spreading of hBMSCs throughout the whole scaffolds. Interestingly, the Sr ions and Ag ions released from the Ag-SrHAP/CS porous scaffolds significantly enhance their osteoinductivity and antibacterial activity, respectively. The Sr element in the SrHAP/CS and Ag-SrHAP/CS porous scaffolds increase the alkaline phosphatase (ALP) activity of hBMSCs, extracellular matrix (ECM) mineralization, and the expression levels of osteogenic-related genes BMP-2 and COL-I. Moreover, the Ag ions released from the Ag-HAP/CS and Ag-SrHAP/CS scaffolds can effectively inhibit the growth and attachment of Staphylococcus aureus (S. aureus, ATCC 25923). In conclusion, the Ag-SrHAP/CS porous scaffold possesses excellent biocompatibility, osteoinductivity and antibacterial activity, so it has great potential for application in bone tissue engineering to repair bone defects and avoid infections.

SC79-loaded ZSM-5/chitosan porous scaffolds with enhanced stem cell osteogenic differentiation and bone regeneration

For effectively treating bone defects, the design of novel therapeutic scaffolds is an important strategy for enhancing stem cell osteogenic differentiation and new bone formation. Herein, we, for the first time, fabricated SC79-loaded ZSM-5/chitosan (ZSM-5/CS/SC79) porous scaffolds via the freeze-drying synthesis of ZSM-5/CS porous scaffolds followed by loading SC79 drug molecules. The ZSM-5/CS scaffolds possessed a three-dimensional (3D) interconnected porous structure, and the nanostructured ZSM-5 ellipsoids were uniformly dispersed on the CS films. The ZSM-5/CS/SC79 scaffolds had appropriate drug loading-release properties due to the hierarchically porous structures of ZSM-5 zeolites and the hydrogen bonding between the CS and SC97. In vitro cell tests demonstrated that both the ZSM-5/CS and ZSM-5/CS/SC79 scaffolds could promote the adhesion, spreading and proliferation of human bone mesenchymal stem cells (hBMSCs). Interestingly, the SC97 released from the scaffolds not only promoted the proliferation of hBMSCs, but also enhanced the osteogenic differentiation. As compared with the ZSM-5/CS control group, the ZSM-5/CS/SC79 scaffolds promoted the ALP activity of hBMSCs, improved the mRNA relative expression levels of osteocalcin (OCN), bone morphogenetic protein-2 (BMP-2) and alkaline phosphatise (ALP), and increased the protein level of β-catenin. The enhanced proliferation and osteogenic differentiation of hBMSCs contributed to the upregulation of Akt kinase by an activated Wnt/β-catenin signaling pathway. Moreover, in vivo animal tests indicated that SC79 released from the ZSM-5/CS/SC79 scaffolds promoted the new bone regeneration without systemic side effects in cranial defects. Therefore, ZSM-5/CS/SC79 scaffolds as novel and promising therapeutic scaffolds have promising applications in defined local bone regeneration.

Self-assembly of pifithrin-α-loaded layered double hydroxide/chitosan nanohybrid composites as a drug delivery system for bone repair materials

Bone repair materials for the effective treatment of bone defects should simultaneously possess excellent biocompatibility and promote osteogenic differentiation. Herein, we prepared pifithrin-α-loaded layered double hydroxide/chitosan (PFTα-LDH-CS) nanohybrid composites for the first time according to the following steps: (i) the immersion of LDH nanoplates and PFTα in a CS solution; and (ii) the self-assembly synthesis of PFTα-LDH-CS nanohybrid composites after the pH value of the mixed solution was adjusted to 7.4. Interestingly, the LDH nanoplates with a thickness of ∼20 nm and width of ∼300 nm agglomerated together into flower-like shapes by self-assembly, and the CS was dispersed around the LDH nanoplates. The mesopores with the pore size of 3.95 nm among the LDH nanoplates served as channels for loading PFTα. Moreover, the CS around the LDH nanoplates increased the drug loading efficiency and drug sustained release property compared with the pure LDH nanoplates. The in vitro tests demonstrated that the human bone marrow-derived mesenchymal stem cells (hBMSCs) had good adhesion, spreading and proliferating on the LDH-CS and PFTα-LDH-CS, suggesting that both samples had the desired cytocompatibility. Note that the PFTα released from the PFTα-LDH-CS rapidly improved the cell proliferation, ALP activity, ECM mineralization and protein level of the Runt-related transcription factor 2 (RUNX2) and β-catenin. The enhanced osteogenic differentiation of hBMSCs on the PFTα-LDH-CS may be attributed to the PFTα released from the abovementioned nanohybrid composites, which resulted in the accumulation of β-catenin and activation of the β-catenin-mediated transcription activity in the cell nucleus. Therefore, the PFTα-LDH-CS nanohybrid composites with excellent cytocompatibility and enhanced osteoinductivity have great applications for novel bone repair materials.

Novel Akt activator SC-79 is a potential treatment for alcohol-induced osteonecrosis of the femoral head

Alcohol is a leading risk factor for osteonecrosis of the femoral head (ONFH). We explored the molecular mechanisms underlying alcohol-induced ONFH and investigated the protective effect of the novel Akt activator SC-79 against this disease. We found that ethanol inhibited expression of the osteogenic genes RUNX2 and OCN, downregulated osteogenic differentiation, impaired the recruitment of Akt to the plasma membrane, and suppressed Akt phosphorylation at Ser473, thereby inhibiting the Akt/GSK3β/β-catenin signaling pathway in bone mesenchymal stem cells. To assess SC-79′s ability to counteract the inhibitory effect of ethanol on Akt-Ser73 phosphorylation, we performed micro-computerized tomography and immunofluorescent staining of osteopontin, osteocalcin and collagen type 1 in a rat model of alcohol-induced ONFH. We found that SC-79 injections inhibited alcohol-induced osteonecrosis. These results show that alcohol-induced ONFH is associated with suppression of p-Akt-Ser473 in the Akt/GSK3β/β-catenin signaling pathway in bone mesenchymal stem cells. We propose that SC-79 treatment to rescue Akt activation could be tested in the clinic as a potential therapeutic approach to preventing the development of alcohol-induced ONFH.

The Protective Effect of Cordycepin On Alcohol-Induced Osteonecrosis of the Femoral Head

Background: Alcohol abuse is known to be a leading risk factor for atraumatic osteonecrosis of the femoral head (ONFH), in which the suppression of osteogenesis plays a critical role. Cordycepin benefits bone metabolism; however, there has been no study to determine its effect on osteonecrosis. Methods: Human bone mesenchymal stem cells (hBMSCs) were identified by multi-lineage differentiation. Alkaline phosphatase (ALP) activity, RT-PCR, western blots, immunofluorescent assay and Alizarin red staining of BMSCs were evaluated. A rat model of alcohol-induced ONFH was established to investigate the protective role of cordycepin against ethanol. Hematoxylin & eosin (H&E) staining and micro-computerized tomography (micro-CT) were performed to observe ONFH. Apoptosis was assessed by TdT-mediated dUTP nick end labeling (TUNEL). Immunohistochemical staining was carried out to detect OCN and COL1. Results: Ethanol significantly suppressed ALP activity, decreased gene expression of OCN and BMP2, lowered levels of RUNX2 protein, and reduced immunofluorescence staining of OCN and COL1 and calcium formation of hBMSCs. However, these inhibitory effects were attenuated by cordycepin co-treatment at concentrations of 1 and 10 µg/mL Moreover, it was revealed that the osteo-protective effect of cordycepin was associated with modulation of the Wnt/β-catenin pathway. In vivo, by micro-CT, TUNEL and immunohistochemical staining of OCN and COL1, we found that cordycepin administration prevented alcohol-induced ONFH. Conclusion: Cordycepin treatment to enhance osteogenesis may be considered a potential therapeutic approach to prevent the development of alcohol-induced ONFH.

Decreased extracellular pH inhibits osteogenesis through proton-sensing GPR4-mediated suppression of yes-associated protein

The pH of extracellular fluids is a basic property of the tissue microenvironment and is normally maintained at 7.40 ± 0.05 in humans. Many pathological circumstances, such as ischemia, inflammation, and tumorigenesis, result in the reduction of extracellular pH in the affected tissues. In this study, we reported that the osteogenic differentiation of BMSCs was significantly inhibited by decreases in the extracellular pH. Moreover, we demonstrated that proton-sensing GPR4 signaling mediated the proton-induced inhibitory effects on the osteogenesis of BMSCs. Additionally, we found that YAP was the downstream effector of GPR4 signaling. Our findings revealed that the extracellular pH modulates the osteogenic responses of BMSCs by regulating the proton-sensing GPR4-YAP pathway.

Ag-loaded MgSrFe-layered double hydroxide/chitosan composite scaffold with enhanced osteogenic and antibacterial property for bone engineering tissue

Bone tissue engineering scaffolds for the reconstruction of large bone defects should simultaneously promote osteogenic differentiation and avoid postoperative infection. Herein, we develop, for the first time, Ag-loaded MgSrFe-layered double hydroxide/chitosan (Ag-MgSrFe/CS) composite scaffold. This scaffold exhibits three-dimensional interconnected macroporous structure with a pore size of 100-300 μm. The layered double hydroxide nanoplates in the Ag-MgSrFe/CS show lateral sizes of 200-400 nm and thicknesses of ∼50 nm, and the Ag nanoparticles with particle sizes of ∼20 nm are uniformly dispersed on the scaffold surfaces. Human bone marrow-derived mesenchymal stem cells (hBMSCs) present good adhesion, spreading, and proliferation on the Ag-MgSrFe/CS composite scaffold, suggesting that the Ag and Sr elements in the composite scaffold have no toxicity to hBMSCs. When compared with MgFe/CS composite scaffold, the Ag-MgSrFe/CS composite scaffold has better osteogenic property. The released Sr2+ ions from the composite scaffold enhance the alkaline phosphatase activity of hBMSCs, promote the extracellular matrix mineralization, and increase the expression levels of osteogenic-related RUNX2 and BMP-2. Moreover, the Ag-MgSrFe/CS composite scaffold possesses good antibacterial property because the Ag nanoparticles in the composite scaffold effectively prevent biofilm formation against S. aureus. Hence, the Ag-MgSrFe/CS composite scaffold with excellent osteoinductivity and antibacterial property has a great potential for bone tissue engineering.

The protective effect of PFTα on alcohol-induced osteonecrosis of the femoral head

Epidemiologic studies have shown alcohol plays a pivotal role in the development of osteonecrosis of the femoral head (ONFH). The aim of this study was to explore the underlying mechanism of alcohol-induced ONFH and the protective effect of pifithrin-α (PFTα). In vitro, we found ethanol treatment significantly activated p53, suppressed Wnt/β-catenin signaling and inhibited osteogenic-related proteins. Furthermore, by separating the cytoplasmic and nuclear proteins, we found ethanol inhibited osteogenesis by impairing the accumulation of β-catenin in both the cytoplasm and nucleus in human bone mesenchymal stem cells (hBMSCs), which resulted from activating glycogen synthase kinase-3β (GSK-3β). Therefore, PFTα, a p53 inhibitor, was introduced in this study to block the ethanol-triggered activation of p53 in hBMSCs and alcohol-induced ONFH in a rat model. In vivo, we established alcohol-induced ONFH in rats and investigated the protective effect of PFTα. Hematoxylin & eosin (H&E) staining combined with TdT-mediated dUTP nick end labeling (TUNEL), cleaved caspase-3 immunohistochemical staining, and micro-CT images revealed substantial ONFH in the alcohol-administered rats, whereas significantly less osteonecrosis developed in the rats injected with PFTα. Osteogenic-related proteins, including osteocalcin, osteopontin and collagen I, were significantly decreased in the alcohol-administered rats, whereas these results were reversed in the PFTα-injected rats. Fluorochrome labeling similarly showed that alcohol significantly reduced the osteogenic activity in the rat femoral head, which was blocked by the injection of PFTα. In conclusion, PFTα had an antagonistic effect against the effects of ethanol on hBMSCs and could be a clinical strategy to prevent the development of alcohol-induced ONFH.Epidemiologic studies have shown alcohol plays a pivotal role in the development of osteonecrosis of the femoral head (ONFH). The aim of this study was to explore the underlying mechanism of alcohol-induced ONFH and the protective effect of pifithrin-α (PFTα). In vitro, we found ethanol treatment significantly activated p53, suppressed Wnt/β-catenin signaling and inhibited osteogenic-related proteins. Furthermore, by separating the cytoplasmic and nuclear proteins, we found ethanol inhibited osteogenesis by impairing the accumulation of β-catenin in both the cytoplasm and nucleus in human bone mesenchymal stem cells (hBMSCs), which resulted from activating glycogen synthase kinase-3β (GSK-3β). Therefore, PFTα, a p53 inhibitor, was introduced in this study to block the ethanol-triggered activation of p53 in hBMSCs and alcohol-induced ONFH in a rat model. In vivo, we established alcohol-induced ONFH in rats and investigated the protective effect of PFTα. Hematoxylin & eosin (H&E) staining combined with TdT-mediated dUTP nick end labeling (TUNEL), cleaved caspase-3 immunohistochemical staining, and micro-CT images revealed substantial ONFH in the alcohol-administered rats, whereas significantly less osteonecrosis developed in the rats injected with PFTα. Osteogenic-related proteins, including osteocalcin, osteopontin and collagen I, were significantly decreased in the alcohol-administered rats, whereas these results were reversed in the PFTα-injected rats. Fluorochrome labeling similarly showed that alcohol significantly reduced the osteogenic activity in the rat femoral head, which was blocked by the injection of PFTα. In conclusion, PFTα had an antagonistic effect against the effects of ethanol on hBMSCs and could be a clinical strategy to prevent the development of alcohol-induced ONFH.