3D-Printed Magnesium Peroxide-Incorporated Scaffolds with Sustained Oxygen Release and Enhanced Photothermal Performance for Osteosarcoma Multimodal Treatments.

The hypoxic microenvironment in osteosarcoma inevitably compromises the antitumor effect and local bone defect repair, suggesting an urgent need for sustained oxygenation in the tumor. The currently reported oxygen-releasing materials have short oxygen-releasing cycles, harmful products, and limited antitumor effects simply by improving hypoxia. Therefore, the PCL/nHA/MgO2/PDA-integrated oxygen-releasing scaffold with a good photothermal therapy effect was innovatively constructed in this work to achieve tumor cell killing and bone regeneration functions simultaneously. The material distributes MgO2 powder evenly on the scaffold material through 3D printing technology and achieves the effect of continuous oxygen release (more than 3 weeks) through its slow reaction with water. The in vitro and in vivo results also indicate that the scaffold has good biocompatibility and sustained-release oxygen properties, which can effectively induce the proliferation and osteogenic differentiation of bone mesenchymal stem cells, achieving excellent bone defect repair. At the same time, in vitro cell experiments and subcutaneous tumorigenesis experiments also confirmed that local oxygen supply can promote osteosarcoma cell apoptosis, inhibit proliferation, and reduce the expression of heat shock protein 60, thereby enhancing the photothermal therapy effect of polydopamine and efficiently eliminating osteosarcoma. Taken together, this integrated functional scaffold provides a unique and efficient approach for antitumor and tumor-based bone defect repair for osteosarcoma treatment.

T-cell infiltration in the central nervous system and their association with brain calcification in Slc20a2-deficient mice.

Primary familial brain calcification (PFBC) is a rare neurodegenerative and neuropsychiatric disorder characterized by bilateral symmetric intracranial calcification along the microvessels or inside neuronal cells in the basal ganglia, thalamus, and cerebellum. Slc20a2 homozygous (HO) knockout mice are the most commonly used model to simulate the brain calcification phenotype observed in human patients. However, the cellular and molecular mechanisms related to brain calcification, particularly at the early stage much prior to the emergence of brain calcification, remain largely unknown. In this study, we quantified the central nervous system (CNS)-infiltrating T-cells of different age groups of Slc20a2-HO and matched wild type mice and found CD45+CD3+ T-cells to be significantly increased in the brain parenchyma, even in the pre-calcification stage of 1-month-old -HO mice. The accumulation of the CD3+ T-cells appeared to be associated with the severity of brain calcification. Further immunophenotyping revealed that the two main subtypes that had increased in the brain were CD3+ CD4- CD8- and CD3+ CD4+ T-cells. The expression of endothelial cell (EC) adhesion molecules increased, while that of tight and adherents junction proteins decreased, providing the molecular precondition for T-cell recruitment to ECs and paracellular migration into the brain. The fusion of lymphocytes and EC membranes and transcellular migration of CD3-related gold particles were captured, suggesting enhancement of transcytosis in the brain ECs. Exogenous fluorescent tracers and endogenous IgG and albumin leakage also revealed an impairment of transcellular pathway in the ECs. FTY720 significantly alleviated brain calcification, probably by reducing T-cell infiltration, modulating neuroinflammation and ossification process, and enhancing the autophagy and phagocytosis of CNS-resident immune cells. This study clearly demonstrated CNS-infiltrating T-cells to be associated with the progression of brain calcification. Impairment of blood-brain barrier (BBB) permeability, which was closely related to T-cell invasion into the CNS, could be explained by the BBB alterations of an increase in the paracellular and transcellular pathways of brain ECs. FTY720 was found to be a potential drug to protect patients from PFBC-related lesions in the future.

High expression of N-type calcium channel indicates a favorable prognosis in gliomas.

For the diagnosis and prognosis of glioma, the development of prognostic biomarkers is critical. The N-type calcium channel, whose predominant subunit is encoded by calcium voltage-gated channel subunit alpha1 B (CACNA1B), is mostly found in the nervous system and is closely associated with neurosensory functions. However, the link between the expression of CACNA1B and glioma remains unknown. We used ONCOMINE to explore the differences in CACNA1B expression among different cancers. We then conducted survival analysis and COX analysis using TCGA_LGG and TCGA_GBM datasets, which were divided into CACNA1Bhigh and CACNA1Blow based on the median. We examined the differences in other favorable prognostic markers or clinical characteristics between CACNA1Bhigh and CACNA1Blow using t tests. Differentially expressed genes were identified, and KEGG pathway enrichment was performed. We compared the expression of methyltransferases and analyzed the differentially methylated regions. Immunohistochemistry results were retrieved from the Human Protein Atlas database for validation purposes. CACNA1B was expressed at lower levels in gliomas, and, for the first time, we found that high expression of CACNA1B in gliomas predicts a good prognosis. Other favorable prognostic markers, such as isocitrate dehydrogenase mutation, 1p/19q codeletion, and O6-methylguanine-DNA methyltransferase promoter methylation, were increased in tandem with high expression of CACNA1B. Differentially expressed genes were enriched in multiple pathways related to cancer progression and aberrant epigenetic alterations were significantly associated with CACNA1B. High expression of N-type calcium channels indicates a favorable prognosis for gliomas. This study provides a better understanding of the link between gliomas and N-type calcium channels and may offer guidance for the future treatment of gliomas.

Stem Cell-Seeded 3D-Printed Scaffolds Combined with Self-Assembling Peptides for Bone Defect Repair.

Bone defects caused by infection, tumor, trauma, and so on remain difficult to treat clinically. Bone tissue engineering (BTE) has great application prospect in promoting bone defect repair. Polycaprolactone (PCL) is a commonly used material for creating BTE scaffolds. In addition, self-assembling peptides (SAPs) can function as the extracellular matrix and promote osteogenesis and angiogenesis. In the work, a PCL scaffold was constructed by 3D printing, then integrated with bone marrow mesenchymal stem cells (BMSCs) and SAPs. The research aimed to assess the bone repair ability of PCL/BMSC/SAP implants. BMSC proliferation in PCL/SAP scaffolds was assessed via Cell Counting Kit-8. In vitro osteogenesis of BMSCs cultured in PCL/SAP scaffolds was assessed by alkaline phosphatase staining and activity assays. Enzyme-linked immunosorbent assays were also performed to detect the levels of osteogenic factors. The effects of BMSC-conditioned medium from 3D culture systems on the migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) were assessed by scratch, transwell, and tube formation assays. After 8 weeks of in vivo transplantation, radiography and histology were used to evaluate bone regeneration, and immunohistochemistry staining was utilized to detect neovascularization. In vitro results demonstrated that PCL/SAP scaffolds promoted BMSC proliferation and osteogenesis compared to PCL scaffolds, and the PCL/BMSC/SAP conditional medium (CM) enhanced HUVEC migration and angiogenesis compared to the PCL/BMSC CM. In vivo results showed that, compared to the blank control, PCL, and PCL/BMSC groups, the PCL/BMSC/SAP group had significantly increased bone and blood vessel formation. Thus, the combination of BMSC-seeded 3D-printed PCL and SAPs can be an effective approach for treating bone defects. Impact statement Both polycaprolactone (PCL) and self-assembling peptides (SAPs) have been broadly applied in bone defect repair. However, the poor osteoinductivity of PCL and weak mechanical strength of SAPs have limited their clinical application. Here, a 3D-printed PCL scaffold was fabricated for seeding bone marrow mesenchymal stem cells (BMSCs), then combined with SAPs to construct a composite PCL/BMSC/SAP implant for treating the calvarial defect. We showed that transplantation of PCL/BMSC/SAP composite implants clearly promoted bone regeneration and neovascularization. To our knowledge, this is the first study to treat bone defects by combination of BMSC-seeded 3D-printed PCL and SAPs.

3D Printed Integrated Bionic Oxygenated Scaffold for Bone Regeneration.

The repair of large bone defects remains a challenging problem in bone tissue engineering. Ischemia and hypoxia in the bone defect area make it difficult for seed cells to survive and differentiate, which fail to perform effective tissue regeneration. Current oxygen-producing materials frequently encounter problems such as a rapid degradation rate, insufficient mechanical properties, difficult molding, and cumbersome fabrication. Here, a novel three-dimensional (3D) printed integrated bionic oxygenated scaffold was fabricated with gelatin-CaO2 microspheres, polycaprolactone (PCL), and nanohydroxyapatite (nHA) using low-temperature molding 3D printing technology. The scaffold had outstanding mechanical properties with bionic hierarchical porous structures. In vitro reports showed that the scaffold exhibited excellent cytocompatibility and could release O2 sustainably for more than 2 weeks, which significantly enhanced the survival, growth, and osteogenic differentiation of bone marrow mesenchymal stem cells under hypoxia. In vivo experiments revealed that the scaffold facilitated efficient bone repair after it was transplanted into a rabbit calvarial defect model. This result may be due to the scaffolds reducing hypoxia-inducible factor-1α accumulation, improving the expression of osteogenic regulatory transcription factors, and accelerating osteogenesis. In summary, the integrated bionic PCL/nHA/CaO2 scaffold had excellent capabilities in sustainable O2 release and bone regeneration, which provided a promising clinical strategy for bone defect repair.

3D Printed Platelet-Rich Plasma-Loaded Scaffold with Sustained Cytokine Release for Bone Defect Repair.

The combination of three-dimensional (3D) printed scaffold materials and various cytokines can achieve the purpose of tissue reconstruction more efficiently. In this study, we prepared platelet-rich plasma (PRP)/gelatin microspheres combined with 3D printed polycaprolactone/ß-tricalcium phosphate scaffolds to solve the key problem that PRP cannot be released under control and the release time is too short, and thus better promote bone repair. Consequently, the composite scaffold displayed a good mechanical property and sustained cytokine release for ∼3 weeks. Increased survival, proliferation, migration, and osteogenic and angiogenic differentiation of bone marrow mesenchymal stem cells were observed compared with the control groups. The in vivo study demonstrated that the composite scaffold with PRP/gelatin microspheres led to greater positive effects in promoting large bone defect repair. In conclusion, in this study, a new type of PRP long-term sustained-release composite scaffold material was constructed that effectively improved the survival, proliferation, and differentiation of cells in the transplanted area, thereby better promoting the repair of large bone defects. Impact statement Reconstruction of bone tissue and blood vessels at bone defects takes time. Platelet-rich plasma (PRP) has been widely used in bone defect repair because it contains a variety of cytokine that can promote local osteogenesis and angiogenesis. In this study, we constructed a new type of polycaprolactone/ß-tricalcium phosphate/PRP/gelatin scaffold to solve the predicament of short cytokine release time in PRP-related materials. We proved that this scaffold can not only achieve long-term PRP-related cytokine release (more than 3 weeks) but also promote osteogenesis and bone defect repair. We believe that this is a novel concept of developing the sustained PRP-related cytokine releasing bioscaffold for treating large bone defect.

Effect of synthetic oxygen-generating system on cell survival under hypoxic condition in vitro.

A significant challenge in the tissue engineering of injured sites is the lack of vascularization in the engineered sites due to insufficient oxygen supply. A scaffolding system is required to support seeded cells as vascularization develops. In this study, we examined the effects of hypoxic conditions and oxygen release on cell survival in a synthetic system. We developed a three-dimensional system using CaO2/poly(lactic-co-glycolic acid) microspheres suspended in a hydrogel. The system material was evaluated using stem cells under hypoxic conditions alongside controls to evaluate its oxygen-generating potential over a period of 21 days. The hydrogel acted as a flexible carrier supporting cell attachment and growth, protecting microspheres, and prolonging oxygen release. The system generated oxygen and supported cell growth, which are together expected to promote stem cell survival and growth in the weeks following implantation.

Cotransplantation of mesenchymal stem cells and endothelial progenitor cells for treating steroid-induced osteonecrosis of the femoral head.

Steroid-induced osteonecrosis of the femoral head (ONFH) is characterized by decreased osteogenesis, angiogenesis, and increased adipogenesis. While bone tissue engineering has been widely investigated to treat ONFH, its therapeutic effects remain unsatisfactory. Therefore, further studies are required to determine optimal osteogenesis, angiogenesis and adipogenesis in the necrotic area of the femoral head. In our study, we developed a carboxymethyl chitosan/alginate/bone marrow mesenchymal stem cell/endothelial progenitor cell (CMC/ALG/BMSC/EPC) composite implant, and evaluated its ability to repair steroid-induced ONFH. Our in vitro studies showed that BMSC and EPC coculture displayed enhanced osteogenic and angiogenic differentiation. When compared with single BMSC cultures, adipogenic differentiation in coculture systems was reduced. We also fabricated a three-dimensional (3D) CMC/ALG scaffold for loading cells, using a lyophilization approach, and confirmed its good cell compatibility characteristics, that is, high porosity, low cytotoxicity and favorable cell adhesion. 3D coculture of BMSCs and EPCs also promoted secretion of osteogenic and angiogenic factors. Then, we established an rabbit model of steroid-induced ONFH. The CMC/ALG/BMSC/EPC composite implant was transplanted into the bone tunnel of the rabbit femoral head after core decompression (CD) surgery. Twelve weeks later, radiographical and histological analyses revealed CMC/ALG/BMSC/EPC composite implants had facilitated the repair of steroid-induced ONFH, by promoting osteogenesis and angiogenesis, and reducing adipogenesis when compared with CD, CMC/ALG, CMC/ALG/BMSC and CMC/ALG/EPC groups. Thus, our data show that cotransplantation of BMSCs and EPCs in 3D scaffolds is beneficial in treating steroid-induced ONFH.

Effects of Bacillus megaterium L222 on quality and bacterial diversity of Sichuan paocai.

Bacillus sp. was found in the Sichuan paocai, but their possible effects on Sichuan paocai fermentation are still elusive. In this study, the effect of Bacillus megaterium L222 isolated from high-quality homemade Sichuan paocai on the flavor characteristics and bacterial diversity was investigated. Overall, 7 organic acids, 16 free amino acids, and 48 volatile substances were detected in the B. megaterium L222-inoculated paocai (BMP) and spontaneously fermented paocai (SP) within 7 days. The metabolites produced in BMP were significantly different from that in SP, and 13 main flavor-related metabolites were the discriminant markers. The contents of free amino acids in BMP were much higher than that in SP. Compared to the SP group, the BMP group could better maintain the high level of alcohols, which improved the synthesis of esters, and controlled the increase of the content of sulfides. The representative bacteria in BMP were Weissella, Lactococcus, Bacillus, Leuconostoc, and the inoculation of B. megaterium L222 could significantly increase the amount of Weissella and inhibit the growth of opportunistic pathogen and other bacteria during the fermentation process of paocai. This study presents an important basis for the development of B. megaterium L222 as a starter for paocai fermentation.

3D printed polycaprolactone/beta-tricalcium phosphate/magnesium peroxide oxygen releasing scaffold enhances osteogenesis and implanted BMSCs survival in repairing the large bone defect.

Ischemia and hypoxia in the bone defect area remain an intractable problem when treating large bone defects. Thus, oxygen-releasing biomaterials have been widely researched in recent years. Magnesium peroxide (MgO2) can release oxygen (O2), and magnesium ions (Mg2+), simultaneously, which is seen to have significant potential in bone substitutes. In this study, we used 3D printing technology to fabricate a MgO2-contained composite scaffold, which was composed of polycaprolactone (PCL), beta-tricalcium phosphate (ß-TCP) and magnesium peroxide (MgO2). Physical properties and O2/Mg2+ releasing behavior of the scaffold were studied. Then, we evaluated the effects of the scaffold on cell survival, proliferation, migration, adhesion and osteogenic differentiation by the co-culture of bone marrow mesenchymal stem cells (BMSCs) and scaffold under normoxia and hypoxia in vitro. Finally, the osteogenic properties of the scaffold in vivo were evaluated via the rat femoral condylar bone defect model. The PCL/ß-TCP/MgO2 scaffold showed good mechanical properties and sustained O2 and Mg2+ release for about three weeks. Meanwhile, the scaffold showed appreciable promotion on the survival, proliferation, migration and osteogenic differentiation of BMSCs under hypoxia compared with control groups. The results of imaging studies and histological analysis showed that implantation of PCL/ß-TCP/MgO2 scaffold could promote seed cell survival and significantly increased new bone formation. In sum, the PCL/ß-TCP/MgO2 scaffold is promising with great potential for treating large bone defects.

CaO2/gelatin oxygen slow-releasing microspheres facilitate tissue engineering efficiency for the osteonecrosis of femoral head by enhancing the angiogenesis and survival of grafted bone marrow mesenchymal stem cells.

The osteonecrosis of femoral head (ONFH), a common refractory disease, is still not fully understood today. Hypoxia caused by ischemia is not only an important pathogenic factor but also a critical challenge for the survival of seed cells in the tissue engineering therapy of ONFH. To explore an efficient strategy to treat ONFH by targeting hypoxia, newly designed CaO2/gelatin microspheres were composited with 3D printed polycaprolactone/nano-hydroxyapatite (PCL/nHA) porous scaffold, sodium alginate/gelatin hydrogel, and bone marrow mesenchymal stem cells (BMSCs) to develop a novel tissue engineering scaffold and then transplanted into the core depression area of the ONFH rabbit model. The current data demonstrated that CaO2/gelatin microspheres can constantly release oxygen for 19 days. In vitro assays with BMSCs illustrated that scaffolds have high biocompatibility and are favorable for cell proliferation in extreme hypoxia (1% O2). The in vivo study demonstrated that the transplanted scaffold with oxygen-generating microspheres significantly enhanced the osteogenic and angiogenic effects compared to the scaffold without microspheres. Further assessments revealed that microspheres in the scaffold can reduce the local cell apoptosis and enhance the survival of grafted cells in the host. Collectively, the present study developed a novel oxygen slow-releasing composite scaffold, which can facilitate tissue engineering efficiency for treating the osteonecrosis of the femoral head by enhancing the angiogenesis and survival of grafted stem cells.

The Regulatory Effect of VEGF-Ax on Rat Bone Marrow Mesenchymal Stem Cells' Angioblastic Differentiation and Its Proangiogenic Ability.

Vascular endothelial growth factor A (VEGFA), which plays a key role in angiogenesis, is composed of many isoforms. Distinct VEGFA isoforms are generated by alternative splicing of VEGFA mRNA and named as VEGFxxx, where xxx represents the number of amino acids present in the final protein sequence. These isoforms have opponent pro- and antiangiogenic effects. VEGF-Ax, an additional isoform containing a 22-amino-acid extension in the COOH terminus, arising from VEGFA mRNA, programmed translational readthrough. The function of VEGF-Ax is not clear, especially the conclusion that VEGF-Ax regulates angiogenesis is contradictory. Thus, we investigated the effect of VEGF-Ax on differentiation and angiogenesis of rat bone marrow mesenchymal stem cells (BMMSCs). The results showed that VEGF-Ax could promote the proliferation and migration of BMMSCs, stimulate the differentiation of BMMSCs into endothelial cell-like cells, and protect BMMSCs from endoplasmic reticulum stress-induced apoptosis.

[Separation and purification of compounds from piper kadsura using preparative reversed-phase liquid chromatography and preparative supercritical fluid chromatography].

A method based on preparative reversed-phase liquid chromatography (prep-RPLC) and preparative supercritical fluid chromatography (prep-SFC) was developed for the separation and purification of compounds from piper kadsura. A pretreatment method was first developed, including methanol extraction, water precipitation, petroleum ether extraction, etc. Chlorophyll and other strong polar impurities were removed from the piper kadsura samples, and the target components were enriched in petroleum ether extracts. The piper kadsura samples were separated into 18 fractions on a Unitary C18 column (250 mm×20 mm, 5 µm) with water and methanol as the mobile phases. Then, the SFC parameters, including the column, modifier, temperature, and backpressure were optimized. The optimized conditions for prep-SFC were as follows:XAmide column (250 mm×20 mm, 5 µm), methanol as the modifier, 30℃ column temperature, and 15.0 MPa backpressure. Because of the good orthogonality of RPLC and SFC, six highly pure compounds were isolated, including kadsurenone, wallichinine, denudatin B, pellitorine, 2E-decenoic acid N-isobutylamide, and futoxide.

[Application of off-line two dimensional reversed-phase liquid chromatography/supercritical fluid chromatography to the separation of the seeds of Trichosanthes kirilowii Maxim].

An off-line two dimensional reversed-phase liquid chromatography/supercritical fluid chromatography (2D RPLC/SFC) method was developed for the separation of the seeds of Trichosanthes kirilowii Maxim. (T. kirilowii.). In the experiment, RPLC was used in the first dimension to analyze the samples. And 12 fractions (F1-F12) were obtained according to the chromatographic peak collection mode, the fractions were re-analyzed in both RPLC and SFC modes. The results clearly showed that this 2D method had good orthogonality. The SFC was used as the second dimension. Ethanol-n-hexane (3:7, v/v) was used as a modifier in SFC. The modifier could provide appropriate elution power. When the sample load increased, it could also ensure the solubility of samples. The 2D RPLC/SFC method had obvious advantages for separating the compounds with similar properties, enriching and purifying the trace components. Finally, 150 peaks could be detected. The off-line 2D RPLC/SFC method will be further enlarged to a preparative level for preparation of compounds. The method can provide a practical reference for the purification of chemical constituents and material basis research of the seeds of T. kirilowii. in the future.