Fabrication and preliminary evaluation of the osteogenic potential for micro-/nano-structured porous BCP ceramics

Nanoscale interface of biomaterials exhibits superiorities in promoting tissue repair and healing. Bioceramics are one of the most commonly used biomaterials for biological purposes such as tissue engineering. The fabrication of nanoscale ceramics with smart topography is essential for modulating cells in contact with such surface. In this research, a gas foaming method was employed to fabricate porous biphasic calcium phosphate (BCP) ceramics as substrates with a controllable component of hydroxyapatite/beta tricalcium phosphate (HA/β-TCP). Hydrothermal reaction was then introduced to modify the surface morphology of the substrates to obtain a nanoscale topography at different pH values and ionic microenvironments. Three kinds of ceramics (BCP; BCP with a nanoneedle-like surface, BNN; BCP covered with a hollow nanorod-like surface, BHN) were prepared with different surface morphologies and roughness, degradation properties and the phase composition during the hydrothermal process. All of them possessed an interconnected pore structure of macropores and micropores. Importantly, the nano-scale surface structure increased the total pore volumes as well as the micropore volumes, thus enhanced the specific surface area (SSA) and subsequent biological performance. Their biological behavior has also been assessed through a series of pivotal events, i.e. protein adsorption, cell attachment, proliferation, and osteogenic differentiation. Consequently, we found the nano-crystallization of the hollow nanorod-like structured and nanoneedle-like structured ceramic surface enhance the adsorption of both total serum proteins and BSA in vitro compared to the original BCP ceramics with micro-sized grains, as well as exhibit a more persistent release of BSA. Additionally, all the three ceramics have displayed excellent biocompatibilities for MSCs, and the morphology of ceramics played a decisive role in cells spreading and morphology, in which BNN showed the strongest effect to facilitate MSCs differentiation towards osteogenesis.     

Stability and Bioaccessibility of β-Carotene in Oil in Water Emulsions: Impact of Antioxidant Type and Emulsifier

The objective of this study is to explore the influence of proteins type and antioxidants on the physicochemical properties, in vitro digestion, and bioaccessibility of β-carotene laden emulsions. The combination of rutin and whey protein isolate provides optimal bioaccessibility while also stabilizing the emulsion system and effectively preventing β-carotene degradation. This is due to the amphiphilic rutin adsorbed into the interface of the emulsion stabilized by whey protein, resulting in partially replacing and further improving the physical and oxidative stability of the β-carotene emulsion. Meanwhile, the rutin emulsions remain stable and carotene content is still higher (22.38–32.07%) than others even after digestion. Hence, these emulsions show great potential to be applied for delivering β-carotene based on oil-in-water emulsions. This study provides valuable information to build up stable excipient emulsions for functional foods and nutraceuticals. Practical applications: β-carotene is being exploited for a range of biomedical applications. Concentrate on the blend of antioxidants and proteins displaying various properties in β-carotene O/W emulsions that can more likely convey β-carotene to the body and better keep up with natural movement.     

人脂肪干细胞复合胶原/壳聚糖支架在生物反应器中扩增的研究

研究表明脂肪组织是多潜能干细胞的又一新来源,脂肪组织来源的干细胞可被用于细胞治疗及组织工程。然而,传统的培养方法很难满足临床需求。为获得大量的脂肪干细胞以满足临床需求,现将脂肪干细胞接种到胶原/壳聚糖支架上,比较细胞在静态环境和在转瓶中动态扩增的情况。通过CCK-8检测细胞增殖情况;并分析葡萄糖和乳酸的代谢情况;14d后扫描电镜观察细胞在支架内的生长情况;流式细胞仪检测干细胞相关表面标记表达;RT-PCR检测干细胞相关转录因子的表达;并检测扩增后的干细胞的多向分化潜能。结果表明:在动态微环境中扩增14d后,与静态条件下相比,支架内的细胞具有更强的增殖活性和更好的多向分化潜能。所扩增的细胞能够保持原有干细胞的特性。结论:所设计的支架-转瓶培养系统是一个简便有效的扩增脂肪干细胞的方法。     

The roles of integrin β1 in phenotypic maintenance and dedifferentiation in chondroid cells differentiated from human adipose-derived stem cells

Objective

The aim of this study is to probe the intrinsic mechanism of chondroid cell dedifferentiation in order to provide a feasible solution for this in cell culture.

Methods

Morphological and biomechanical properties of cells undergoing chondrogenic differentiation from human adipose-derived stem cells (ADSCs) were measured at the nanometer scale using atomic force microscopy and laser confocal scanning microscopy. Gene expression was determined by real-time quantitative polymerase chain reaction.

Results

The expression of COL II, SOX9, and Aggrecan mRNA began to increase gradually at the beginning of differentiation and reach a peak similar to that of normal chondrocytes on the 12th day, then dropped to the level of the 6th day at 18th day. Cell topography and mechanics trended resembled those of the genes’ expression. Integrin β1 was expressed in ADSCs and rapidly upregulated during differentiation but downregulated after reaching maturity.

Conclusions

The amount and distribution of integrin β1 may play a critical role in mediating both chondroid cell maturity and dedifferentiation. Integrin β1 is a possible new marker and target for phenotypic maintenance in chondroid cells.     

Complexation of poly(dimethylsiloxane)/poly(methyl methacrylate‐co‐butyl acrylate‐co‐methacrylic acid) latex with poly(dimethylsiloxane)/poly(vinyl acetate‐co‐butyl acrylate) latex

Two latices—the poly(dimethylsiloxane) (PDMS)/poly(methyl methacrylate‐co‐butyl acrylate‐co‐methacrylic acid) system (PA latex) and the PDMS/poly(vinyl acetate‐co‐butyl acrylate) system (PB latex)—were prepared by seeded emulsion polymerization, and PA/PB complex latices were obtained through the interparticle complexation of the PA latex with the PB latex. In addition, for the further study of the interparticle complexation of the PA latex with the PB latex, copolymer latices [PDMS/methyl methacrylate‐co‐butyl acrylate‐co‐vinyl acetate‐co‐methacrylic acid) (PC)] were prepared according to the monomer recipe of the complex latices and the polymerization process of the component latices. The properties of the obtained polymer latices and complex latices were investigated with surface‐tension, contact‐angle, and viscosity measurements. The mechanical properties of the coatings obtained from the latices were investigated with tensile‐strength measurements. The results showed that, in comparison with the two component latices (PA latex and PB latex) and the corresponding copolymer latices (PC latices), the PA/PB complex latices had lower surface tension, lower viscosities, and better wettability to different substrates. The tensile strengths of the coatings obtained from the complex latices were higher than the tensile strengths of the coatings from the two component latices and copolymer latices. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 2522–2527, 2004     

Inflammatory Regulation by TNF-α-Activated Adipose-Derived Stem Cells in the Human Bladder Cancer Microenvironment

Mesenchymal stem cells (MSCs), such as adipose-derived stem cells (ADSCs), have the most impressive ability to reduce inflammation through paracrine growth factors and cytokines that participate in inflammation. Tumor necrosis factor (TNF)-α bioactivity is a prerequisite in several inflammatory and autoimmune disease models. This study investigated the effects of TNF-α stimulate on ADSCs in the tumor microenvironment. The RNAseq analysis and cytokines assay demonstrated that TNF-α stimulated ADSCs proliferation and pro-inflammatory genes that correlated to leukocytes differentiation were upregulated. We found that upregulation of TLR2 or PTGS2 toward to IRF7 gene-associated with immunomodulatory and antitumor pathway under TNF-α treatment. In TNF-α-treated ADSCs cultured with the bladder cancer (BC) cell medium, the results showed that apoptosis ratio and OCT-4 and TLR2 genes which maintained the self-renewal ability of stem cells were decreased. Furthermore, the cell survival regulation genes including TRAF1, NF-kB, and IRF7 were upregulated in TNF-α-treated ADSCs. Additionally, these genes have not been upregulated in BC cell medium. A parallel study showed that tumor progressing genes were downregulated in TNF-α-treated ADSCs. Hence, the study suggests that TNF-α enhances the immunomodulatory potential of ADSCs during tumorigenesis and provides insight into highly efficacious MSC-based therapeutic options for BC.     

Addition of Adipose-Derived Stem Cells to Mesenchymal Stem Cell Sheets Improves Bone Formation at an Ectopic Site

To determine the effect of adipose-derived stem cells (ADSCs) added to bone marrow-derived mesenchymal stem cell (MSC) sheets on bone formation at an ectopic site. We isolated MSCs and ADSCs from the same rabbits. We then prepared MSC sheets for implantation with or without ADSCs subcutaneously in the backs of severe combined immunodeficiency (SCID) mice. We assessed bone formation at eight weeks after implantation by micro-computed tomography and histological analysis. In osteogenic medium, MSCs grew to form multilayer sheets containing many calcium nodules. MSC sheets without ADSCs formed bone-like tissue; although neo-bone and cartilage-like tissues were sparse and unevenly distributed by eight weeks after implantation. In comparison, MSC sheets with ADSCs promoted better bone regeneration as evidenced by the greater density of bone, increased mineral deposition, obvious formation of blood vessels, large number of interconnected ossified trabeculae and woven bone structures, and greater bone volume/total volume within the composite constructs. Our results indicate that although sheets of only MSCs have the potential to form tissue engineered bone at an ectopic site, the addition of ADSCs can significantly increase the osteogenic potential of MSC sheets. Thus, the combination of MSC sheets with ADSCs may be regarded as a promising therapeutic strategy to stimulate bone regeneration.     

Emerging Role of HDACs in Regeneration and Ageing in the Peripheral Nervous System: Repair Schwann Cells as Pivotal Targets

The peripheral nervous system (PNS) has a remarkable regenerative capacity in comparison to the central nervous system (CNS), a phenomenon that is impaired during ageing. The ability of PNS axons to regenerate after injury is due to Schwann cells (SC) being reprogrammed into a repair phenotype called Repair Schwann cells. These repair SCs are crucial for supporting axonal growth after injury, myelin degradation in a process known as myelinophagy, neurotropic factor secretion, and axonal growth guidance through the formation of Büngner bands. After regeneration, repair SCs can remyelinate newly regenerated axons and support nonmyelinated axons. Increasing evidence points to an epigenetic component in the regulation of repair SC gene expression changes, which is necessary for SC reprogramming and regeneration. One of these epigenetic regulations is histone acetylation by histone acetyl transferases (HATs) or histone deacetylation by histone deacetylases (HDACs). In this review, we have focused particularly on three HDAC classes (I, II, and IV) that are Zn²⁺-dependent deacetylases. These HDACs are important in repair SC biology and remyelination after PNS injury. Another key aspect explored in this review is HDAC genetic compensation in SCs and novel HDAC inhibitors that are being studied to improve nerve regeneration.     

Biodegradable Poly-ε-Caprolactone Scaffolds with ECFCs and iMSCs for Tissue-Engineered Heart Valves

Clinically used heart valve prostheses, despite their progress, are still associated with limitations. Biodegradable poly-ε-caprolactone (PCL) nanofiber scaffolds, as a matrix, were seeded with human endothelial colony-forming cells (ECFCs) and human induced-pluripotent stem cells-derived MSCs (iMSCs) for the generation of tissue-engineered heart valves. Cell adhesion, proliferation, and distribution, as well as the effects of coating PCL nanofibers, were analyzed by fluorescence microscopy and SEM. Mechanical properties of seeded PCL scaffolds were investigated under uniaxial loading. iPSCs were used to differentiate into iMSCs via mesoderm. The obtained iMSCs exhibited a comparable phenotype and surface marker expression to adult human MSCs and were capable of multilineage differentiation. EFCFs and MSCs showed good adhesion and distribution on PCL fibers, forming a closed cell cover. Coating of the fibers resulted in an increased cell number only at an early time point; from day 7 of colonization, there was no difference between cell numbers on coated and uncoated PCL fibers. The mechanical properties of PCL scaffolds under uniaxial loading were compared with native porcine pulmonary valve leaflets. The Young’s modulus and mean elongation at F_max of unseeded PCL scaffolds were comparable to those of native leaflets (p = ns.). Colonization of PCL scaffolds with human ECFCs or iMSCs did not alter these properties (p = ns.). However, the native heart valves exhibited a maximum tensile stress at a force of 1.2 ± 0.5 N, whereas it was lower in the unseeded PCL scaffolds (0.6 ± 0.0 N, p < 0.05). A closed cell layer on PCL tissues did not change the values of F_max (ECFCs: 0.6 ± 0.1 N; iMSCs: 0.7 ± 0.1 N). Here, a successful two-phase protocol, based on the timed use of differentiation factors for efficient differentiation of human iPSCs into iMSCs, was developed. Furthermore, we demonstrated the successful colonization of a biodegradable PCL nanofiber matrix with human ECFCs and iMSCs suitable for the generation of tissue-engineered heart valves. A closed cell cover was already evident after 14 days for ECFCs and 21 days for MSCs. The PCL tissue did not show major mechanical differences compared to native heart valves, which was not altered by short-term surface colonization with human cells in the absence of an extracellular matrix.     

Effects of a Catechol-Functionalized Hyaluronic Acid Patch Combined with Human Adipose-Derived Stem Cells in Diabetic Wound Healing

Introduction: Chronic inflammation and impaired neovascularization play critical roles in delayed wound healing in diabetic patients. To overcome the limitations of current diabetic wound (DBW) management interventions, we investigated the effects of a catechol-functionalized hyaluronic acid (HA-CA) patch combined with adipose-derived mesenchymal stem cells (ADSCs) in DBW mouse models. Methods: Diabetes in mice (C57BL/6, male) was induced by streptozotocin (50 mg/kg, >250 mg/dL). Mice were divided into four groups: control (DBW) group, ADSCs group, HA-CA group, and HA-CA + ADSCs group (n = 10 per group). Fluorescently labeled ADSCs (5 × 10⁵ cells/100 µL) were transplanted into healthy tissues at the wound boundary or deposited at the HA-CA patch at the wound site. The wound area was visually examined. Collagen content, granulation tissue thickness and vascularity, cell apoptosis, and re-epithelialization were assessed. Angiogenesis was evaluated by immunohistochemistry, quantitative real-time polymerase chain reaction, and Western blot. Results: DBW size was significantly smaller in the HA-CA + ADSCs group (8% ± 2%) compared with the control (16% ± 5%, p < 0.01) and ADSCs (24% ± 17%, p < 0.05) groups. In mice treated with HA-CA + ADSCs, the epidermis was regenerated, and skin thickness was restored. CD31 and von Willebrand factor-positive vessels were detected in mice treated with HA-CA + ADSCs. The mRNA and protein levels of VEGF, IGF-1, FGF-2, ANG-1, PIK, and AKT in the HA-CA + ADSCs group were the highest among all groups, although the Spred1 and ERK expression levels remained unchanged. Conclusions: The combination of HA-CA and ADSCs provided synergistic wound healing effects by maximizing paracrine signaling and angiogenesis via the PI3K/AKT pathway. Therefore, ADSC-loaded HA-CA might represent a novel strategy for the treatment of DBW.     

Morphology of poly(styrene‐block‐dimethylsiloxane) copolymer films

The morphologies of poly(styrene‐block‐di‐methylsiloxane) (PS‐b‐PDMS) copolymer thin films were analyzed via atomic force microscopy and transition electron microscopy (TEM). The asymmetric copolymer thin films spin‐cast from toluene onto mica presented meshlike structures, which were different from the spherical structures from TEM measurements. The annealing temperature affected the surface morphology of the PS‐b‐PDMS copolymer thin films; the polydimethylsiloxane (PDMS) phases at the surface were increased when the annealing temperature was higher than the PDMS glass‐transition temperature. The morphologies of the PS‐b‐PDMS copolymer thin films were different from solvent to solvent; for thin films spin‐cast from toluene, the polystyrene (PS) phase appeared as pits in the PDMS matrix, whereas the thin films spin‐cast from cyclohexane solutions exhibited an islandlike structure and small, separated PS phases as protrusions over the macroscopically flat surface. The microphase structure of the PS‐b‐PDMS copolymer thin films was also strongly influenced by the different substrates; for an asymmetric block copolymer thin film, the PDMS and PS phases on a silicon substrate presented a lamellar structure parallel to the surface at intervals. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 104: 1010–1018, 2007     

Experimental measurement and correlation of solubility of β-carotene in pure and ethanol-modified subcritical water

For the first time, the solubility of β-carotene in pure and ethanol-modified subcritical water (SW) using the static method was determined. The experimental runs were performed at a temperature ranging from 298.15 to 403.15 K and 0–10% (w/w) of ethanol as a modifier at a constant pressure of 5 MPa. Samples were analyzed by UV–vis spectrophotometer. The solubility of β-carotene was found to range from 1.084 × 10⁻⁸ to 227.1 × 10⁻⁸ mol fractions in the subcritical water in above mentioned conditions. The obtained β-carotene solubility data were correlated using the linear model and modified Apelblat model. The obtained results showed the modified Apelblat model was better for estimating the solubility of β-carotene in SW. The values of the root-mean-square deviation (RMSD) between experimental and correlated data were calculated and used as the index of validity and accuracy for the model. Also, thermodynamic properties of the solution such as the Gibbs free energy of solution, enthalpy, and entropy of solution were estimated.     

Substrate contribution on carotenoids production in Blakeslea trispora cultivations

The present report gives an insight into the specific changes in the three main carotenoids (lycopene, γ-carotene and β-carotene) occurring in Blakeslea trispora cultures, with regard to medium composition. Various carbon sources and refined natural vegetable oils as co-substrates were used. The different carbon sources greatly affect the final composition of carotenoids, with lactose medium to preferentially accumulate the all-trans-β-carotene. Furthermore, the use of lactose and starch as carbon sources, respectively, gives a first indication that B. trispora is able to metabolise various homo- and hetero-saccharides, thus leading to different carotenoid percentages. The presence of oils as co-substrates resulted in enhanced fungus growth and subsequent higher carotenoid production. Substrates containing linoleic acid or other oils rich in it led to less lycopene accumulation. The data showed that the biosynthesis of lycopene, γ-carotene and β-carotene starts in most cases simultaneously in the early growth phase even in trace amounts and thus may play a role also as antioxidants for the B. trispora cells.     

Preparation and properties of polydimethylsiloxane/polyacrylate composite latex initiated by 60Co γ‐ray irradiation

In this study, polydimethylsiloxane (PDMS)/polyacrylate composite polymer latex was synthesized via polymerization of the acrylate monomer in the presence of vinyl‐containing PDMS seeded latex. The polymerization was initiated by ⁶⁰Co γ‐ray irradiation. The morphology of the PDMS/polyacrylate composite polymer latex was a core–shell structure with PDMS as the core and polyacrylate as the shell. There was an interpenetration layer between the PDMS core and the polyacrylate shell. The composition of the vinyl‐containing PDMS and the PDMS/polyacrylate composite latex were investigated with NMR and Fourier transform infrared spectroscopy, respectively. The effect of irradiation dose on the seeded emulsion polymerization conversion is discussed. Finally, the mechanical properties of latex film, such as water‐absorption ratio, tensile strength, pendulum hardness, and heat‐decomposed temperature, were tested. The results showed that the mechanical properties of the PDMS/polyacrylate film were remarkably improved when compared to the polyacrylate film. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 88: 2732–2736, 2003     

Application of Osmotic Dehydration Processes to Produce Apple Slices Enriched with β-Carotene

The objective of this work was the impregnation of β-carotene in apple slices by osmotic dehydration (OD) at atmospheric pressure and by pulsed vacuum osmotic dehydration (PVOD). Osmotic solutions were sucrose solutions of 30, 40, and 50 °Brix containing β-carotene (0.01 g/mL). Maximum impregnation using OD treatments was 1.5, 3.5, and 4.1 mg β-carotene/g ds. When using PVOD, significantly shorter processing times were required and impregnation reached 4.7, 5.5, and 6 mg β-carotene/g ds. Values of a_w obtained by OD were 0.973, 0.967, and 0.960 while by PVOD were 0.960, 0.930, and 0.880. Results showed that PVOD was a good option to impregnate apple with β-carotene.     

Physicochemical and biological evaluation of plasma‐induced graft polymerization of acrylamide onto polydimethylsiloxane

Polydimethylsiloxane (PDMS) rubbers exhibit good mechanical properties for biomedical and industrial applications, but their inherently high hydrophobicity limits biomedical applications of this material despite its favorable mechanical properties. In this work, surface modification of PDMS by radio‐frequency glow discharge and subsequently graft polymerization of acrylamide was studied. PAAm‐grafted, oxygen plasma‐treated, and control (untreated) PDMS rubbers were characterized using attenuated total reflectance Fourier transform infrared, scanning electron microscopy, dynamic mechanical thermal analyses, zeta potential, and contact angle techniques. Fibroblast (L929) cell attachment and growth onto these surfaces were examined by optical microscopy. The data from in vitro assays showed that cell attachment onto control surface was very negligible while significant cell attachment and growth was observed onto oxygen plasma‐treated and PAAm‐grafted PDMS surfaces. The method developed in this work offers a convenient way of surface modifications of biomaterials to improve attachment of cells onto substrates. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2008     

Application of polymeric nanofibers in medical designs,part II: Neural and cardiovascular tissues

Nanotechnology has wide applications in many fields, especially in the biological sciences and medicine. Nanomaterials are applied as potential materials for treatment and diagnosis. Nanofibers mimic the porous topography of natural extracellular matrix, are advantageous for tissue regeneration and also for sustained release of encapsulated drug or growth factor. The author summarizes the currently available applications of nanofibers in two soft tissues of nerve and cardiovascular tissues. Shown in the graphic are Schwann cells seeded on different nanofibers under scanning electron microscope.     

Solubility of β-carotene in poly-(ɛ-caprolactone) particles produced in colloidal state by Supercritical Fluid Extraction of Emulsions (SFEE)

β-Carotene is one of the most common pigments in nature. In food applications, β-carotene formulations are very attractive because they add value to the product due to their antioxidant and pro-vitamin activities. On the other hand, miscibility and solubility data of the active compound in the carrier material are relevant for the design and development of optimum formulations. This work presents a study of the solubility of β-carotene in the range of temperature between 10 °C and 50 °C in poly-(ɛ-caprolactones) with different molecular weights (between 4000 and 25,000 g mol⁻¹) produced in colloidal state by Supercritical Fluid Extraction of Emulsions. The determination of the solubility of β-carotene was carried out by two different methods: equilibration-impregnation and equilibration-de-supersaturation. Results obtained by impregnation showed that β-carotene content impregnated into the PCLs increased gradually when the temperature was increased, obtaining maximum β-carotene contents between 87 and 191 ppm depending on the molecular weight of the polycaprolactone. However, results obtained by de-supersaturation experiments showed β-carotene contents considerably higher than those achieved in impregnation experiments, obtaining a maximum β-carotene content of 8800 ppm when polycaprolactone with the highest molecular weight was used. This result can be due to the slow release of entrapped β-carotene crystals from PCL particles.     

自组装多肽水凝胶支架中MSCs的三维培养及成骨分化

使用一种新型人工设计自组装多肽(RADA16)水凝胶作为三维培养支架评价MSCs成骨分化情况。将人骨髓MSCs培养增殖后接种到水凝胶中,在成骨分化培养液中进一步培养1～3周。荧光染色法观察细胞形态和存活情况;组织学染色检测MSCs ALP活性;半定量RT-PCR分析成骨特异性基因的表达。绝大多数MSCs在水凝胶支架内能够存活,呈纺锤样形态。诱导培养后蛋白和基因表达水平均检测到ALP活性,在14天时达到峰值。骨晚期分化特异性基因BSP也有表达,且表达量随培养时间延长而增多。自组装多肽水凝胶为MSCs的黏附生长及向成骨细胞分化提供良好的三维微环境,有望成为极具吸引力的骨组织工程支架材料。