Effect of ascorbic acid on bone marrow-derived mesenchymal stem cell proliferation and differentiation

Mesenchymal stem cells (MSCs) derived from bone marrow are an important tool in tissue engineering and cell-based therapies because of their multipotent capacity. Majority of studies on MSCs have investigated the roles of growth factors, cytokines, and hormones. Antioxidants such as ascorbic acid can be used to expand MSCs while preserving their differentiation ability. Moreover, ascorbic acid can also stimulate MSC proliferation without reciprocal loss of phenotype and differentiation potency. In this study, we evaluated the effects of ascorbic acid on the proliferation, differentiation, extracellular matrix (ECM) secretion of MSCs. The MSCs were cultured in media containing various concentrations (0-500 microM) of L-ascorbate-2-phosphate (Asc-2-P) for 2 weeks, following which they were differentiated into adipocytes and osteoblasts. Ascorbic acid stimulated ECM secretion (collagen and glycosaminoglycan) and cell proliferation. Moreover, the phenotypes of the experimental groups as well as the differentiation potential of MSCs remained unchanged. The apparent absence of decreased cell density or morphologic change is consistent with the toxicity observed with 5-250 microM concentrations of Asc-2-P. The results demonstrate that MSC proliferation or differentiation depends on ascorbic acid concentration.     

Mag-seeding of rat bone marrow stromal cells into porous hydroxyapatite scaffolds for bone tissue engineering

Bone tissue engineering has been investigated as an alternative strategy for autograft transplantation. In the process of tissue engineering, cell seeding into three-dimensional (3-D) scaffolds is the first step for constructing 3-D tissues. We have proposed a methodology of cell seeding into 3-D porous scaffolds using magnetic force and magnetite nanoparticles, which we term Mag-seeding. In this study, we applied this Mag-seeding technique to bone tissue engineering using bone marrow stromal cells (BMSCs) and 3-D hydroxyapatite (HA) scaffolds. BMSCs were magnetically labeled with our original magnetite cationic liposomes (MCLs) having a positive surface charge to improve adsorption to cell surface. Magnetically labeled BMSCs were seeded onto a scaffold, and a 1-T magnet was placed under the scaffold. By using Mag-seeding, the cells were successfully seeded into the internal space of scaffolds with a high cell density. The cell seeding efficiency into HA scaffolds by Mag-seeding was approximately threefold larger than that by static-seeding (conventional method, without a magnet). After a 14-d cultivation period using the osteogenic induction medium by Mag-seeding, the level of two representative osteogenic markers (alkaline phosphatase and osteocalcin) were significantly higher than those by static-seeding. These results indicated that Mag-seeding of BMSCs into HA scaffolds is an effective approach to bone tissue engineering.     

Application of computational fluid dynamics in tissue engineering

The process of tissue regeneration consists of a set of complex phenomena such as hydrodynamics, nutrient transfer, cell growth, and matrix deposition. Traditional cell culture and bioreactor design procedure follow trial-and-error analyses to understand the effects of varying physical, chemical, and mechanical parameters that govern the process of tissue regeneration. This trend has been changing as computational fluid dynamics (CFD) analysis can now be used to understand the effects of flow, cell proliferation, and consumption kinetics on the dynamics involved with in vitro tissue regeneration. Furthermore, CFD analyses enable understanding the influence of nutrient transport on cell growth and the effect of cell proliferation as the tissue regenerates. This is especially advantageous in improving and optimizing the design of bioreactors and tissue culture. Influence of parameters such as velocity, oxygen tension, stress, and strain on tissue growth can be effectively studied throughout the bioreactor using CFD as it becomes impractical and cumbersome to install probes at several locations in the bioreactor. Hence, CFD offers several advantages for the advancement of tissue engineering.     

Proliferation and intracellular glutathione in Jurkat T cells with concentrated whey protein products

The effect of two whey protein concentrates (WPCs) and three whey protein isolates (WPIs) on the growth and intracellular glutathione concentration of Jurkat T cells was determined. Standard RPMI 1640 media containing foetal calf serum with no WPC or WPI supplementation was used as the control, while supplementation with N-acetyl cysteine—a known glutathione promoter—was included as a positive control. Both WPCs lowered the cell count-adjusted glutathione concentration following a 24 h incubation period and one significantly (p<0.05) increased cell proliferation. Only one of the three WPIs significantly (p<0.05) inhibited cell proliferation although its composition with respect to β-lactoglobulin, glycomacropeptide, α-lactalbumin, IgG, proteose peptone and BSA content was almost identical to another WPI, as determined by HPLC. Based on co-migration with standards under two different modes of chromatography, lactoferrin was detected in the WPI showing the inhibitory effect at a level of 0.4 mg mL⁻¹, but not in any of the other concentrated whey protein products. None of the whey protein products tested increased cell-adjusted intracellular glutathione concentration.     

Preparation and characterization of dexamethasone-immobilized chitosan scaffold

Chitosan film (CSF), 2-dimensional scaffold, was modified with dexamethasone (DEX) in the present work via amino-alcohol forming reaction between amino group of chitosan and carbonyl group of DEX. Successful immobilization was identified by ATR-FTIR spectroscopy and, the immobilized amounts were determined by weighting methods (WM) and integrated area analysis method (AM) of HPLC. For experiments of cell culture of osteogenic differentiation, our results showed that DEX immobilization has more efficiency than the other group (such as both groups of CSF and CSF with free DEX, CSF/fDEX), which was demonstrated as indicated by cell image analysis and ALP activity assay. The results show, as AD-MSCs cultured on DEX-CSF, its shape of cell transformed to polygonal or cubical. The ALP activity assay indicates that of DEX-CSF increase about 3.5 folds than that of CSF's. The results suggest that immobilization of DEX can make chitosan scaffolds to induce differentiation of AD-MSCs toward osteoblastic lineage for bone tissue engineering application.     

硒酸化乳清分离蛋白抗前列腺癌细胞活性

采用干燥加热法制备硒酸化乳清分离蛋白(selenized whey protein isolate,Se-WPI),通过Se-WPI中有机硒质量分数、硒酸根稳定性、Se-WPI消化率测定,⁷⁷Se核磁共振(nuclear magnetic resonance,NMR)分析以及Se-WPI抑制前列腺癌细胞增殖实验等,研究其理化性质及抗前列腺癌细胞活性。结果表明,在pH 3.0、加热温度80℃、加热时间24 h条件下制备得到的Se-WPI中有机硒质量分数为2.09%。⁷⁷Se-NMR和硒酸根稳定性分析结果表明,Se-WPI中的硒以亚硒酸酯的形式存在。经硒酸化的WPI多种生物活性得到改善,消化性明显提高。Se-WPI与亚硒酸钠均具有抑制人前列腺癌LNCaP细胞、DU145细胞增殖的活性。细胞周期、细胞凋亡和Caspase-3活力检测结果从不同角度验证了Se-WPI具有诱导LNCaP细胞凋亡的作用。另外,Se-WPI还能抑制癌细胞与基底膜成分的黏附能力。实验结果可为进一步研究有机硒化合物的抑癌机制提供依据。     

外泌体功能化串晶结构纤维膜的制备及其成骨分化性能

为提高合成可吸收引导骨再生膜的骨诱导活性,通过静电纺丝和溶剂诱导结晶法制备了聚己内酯(PCL)/β-磷酸三钙(β-TCP)串晶纳米纤维膜,并通过聚多巴胺的黏附作用将外泌体负载于纤维膜上进行改性,对复合纤维膜的微观形貌、化学组成、理化性能和细胞成骨分化性能进行测试与分析。结果表明:PCL/β-TCP纳米纤维上成功诱导出串晶结构,且经串晶和聚多巴胺双重修饰的纤维膜具有最佳的表面浸润性和优异的蛋白吸附能力,最终获得的外泌体功能化的串晶纳米纤维膜在串晶结构、聚多巴胺和外泌体共同作用下可促进骨髓间充质干细胞碱性磷酸酶活性的增加,有望应用于体内加速骨愈合。     

Cell processing engineering for ex-vivo expansion of hematopoietic cells

The cell processing engineering for ex vivo expansion of hematopoietic cells is reviewed. All hematopoietic cells of different lineages and/or at various stages of differentiation are derived from the same precursor, pluripotent hematopoietic stem cells. Bone marrow stromal cells promote and regulate the self-renewal, commitment, differentiation, and proliferation of stem cells and progenitors through their secreted extracellular matrices and cytokine environment in the hematopoietic microenvironment. Although stroma-mediated hematopoiesis has been studied in vitro using the Dexter culture system in tissue culture flasks, hematopoiesis in the Dexter culture system is almost limited to a granulocyte lineage and the system could not expand primitive cells. The addition of large amounts of cytokines to the culture of hematopoietic cells enabled their expansion, but is too expensive. Some clonal stromal cell lines have been established from the Dexter culture of murine bone marrow cells in order to simplify and stimulate the ex vivo expansion of hematopoietic cells. In order to solve the problem regarding the usage of exogeneic stromal cell lines, a novel membrane-separated coculture system, in which stromal cells adhere onto the lower surface of a porous membrane and hematopoietic cells are incubated on the upper surface of the membrane, was proposed. In order to mimic the contact between stromal and hematopoietic cells in vivo in the bone marrow, several types of three-dimensional (3-D) culture of hematopoietic cells were developed. The 3-D coculture of hematopoietic cells with spatial development of stromal cells in nonwoven fabrics enabled the expansion of progenitors without cytokine addition. Progenitors in cord blood mononucleated cells were also successfully expanded without the addition in the 3-D coculture with primary human bone marrow stromal cells in 3-D. Heparin addition to the 3-D coculture and coating the nonwoven fabrics with N-(O-beta-(6-O-sulfogalactopyranosyl)-6-oxyhexyl)-3,5-bis(dodecyloxy)-benzamide further increased the number of progenitors.     

组织工程用人工支架表面改性的研究进展

组织工程用人工支架有着完美的三维结构、良好的细胞相容性和特殊的药理作用,一直受到生命科学和工程领域专家的重视。人工支架的表面改性研究关系到细胞生长、繁殖及迁移等状态,是组织工程的基础及热点。与普通人工支架相比,改性支架除了赋予细胞更好的黏附、增殖、迁移性能外,还赋予支架药物洗脱性能,使之能促进细胞生长或抑制病变细胞繁殖,为组织工程的设计研究提供了更广阔的应用空间。文章概述组织工程用人工支架表面改性的分类、制备方法、结构和性能,展望人工支架表面改性研究的前景。     

In vitro osteogenic differentiation of HOS cells on two types of collagen gels

HOS cell is a model strain of human osteoblasts derived from human osteosarcoma. We cultured the HOS cells on both the conventional collagen gel (neutral gel), and the gamma-crosslinked collagen gel without collagen fibrils (acidic gel). The shape of HOS cells on the neutral gel was similar to that on the culture dish. However, HOS cells on acidic gel had an elongated shape and attached each other to form a mesh-like pattern. The cells attached to the surface of both gels but scarcely penetrated their depths. We measured the biochemical markers for osteogenic differentiation in the HOS cells cultured on both the neutral gel and the acidic gel. The expressions of alkaline phosphatase and osteocalcin were detected in the HOS cells on both types of collagen gel. Deposition of the calcium also occurred on both gels although it was higher in the neutral gel than the acidic one. These results indicate the importance of collagen for the differentiation of HOS cells, but it is not dependent on the molecular structure (fibril formation) of collagen.     

Culture systems for pluripotent stem cells

Pluripotent stem cells have the capacity to self renew and to differentiate to cells of the three somatic germ layers that comprise an organism. Embryonic stem cells are the most studied pluripotent stem cells. Pluripotent stem cells have also been derived from adult tissues. Both embryonic and adult stem cells represent valuable sources of cells for applications in cell therapy, drug screening and tissue engineering. While expanding stem cells in culture, it is critical to maintain their self-renewal and differentiation capacity. In generating particular cell types for specific applications, it is important to direct their differentiation to the desired lineage. Challenges in expansion of undifferentiated stem cells for clinical applications include the removal of feeder layers and non-defined components in the culture medium. Our limited basic knowledge on the requirements for maintaining pluripotency of adult pluripotent stem cells and the lack of appropriate markers associated with pluripotency hinders the progress toward their wide spread application. In vitro differentiation of stem cells usually produces a mixed population of different cell lineages with the desired cell type present only at a small proportion. Use of growth factors that promote differentiation, expansion or survival of specific cell types is key in controlling the differentiation towards specific cell lineages. A variety of bioreactors for cell cultivation exist and can be readily adapted for stem cell cultivation and differentiation. They provide a well-controlled environment for studying the process of stem cell propagation and differentiation. Their wide use will facilitate the development of processes for stem cell application.     

Solanum Muricatum Promotes Osteogenic Differentiation of Rat Bone Marrow Stromal Cells

Solanum muricatum (SM), also known as pepino, is known for its antioxidative and anti‐inflammatory effects. The aim of this study was to evaluate the effects of SM extract in promoting osteogenic differentiation and regulating the Wnt and bone morphogenetic protein (BMP) signaling pathways. Ingredients of pepino were extracted and identified. SM extracts were used to treat rat bone marrow stromal cells (BMSCs), followed by evaluating alkaline phosphatase activities and mineralization levels. The mRNA levels of osteogenic biomarkers, including OPN and Collagen I, were also evaluated with real‐time polymerase chain reaction. After treatment with SM extracts, the expressions of key proteins in the Wnt and BMP signaling pathways were assessed. DKK‐1 and noggin, which are Wnt and BMP inhibitors, respectively, were added with SM extracts to investigate the role of Wnt and BMP pathways in the ameliorating effects of SM extract in osteogenesis. Treatment of BMSCs with SM extract promoted osteogenesis. Meanwhile, upregulations in the Wnt and BMP pathways were also observed. However, inhibiting both pathways compromised the effects of SM extract in promoting osteogenic differentiation. SM extract promotes osteogenic differentiation in BMSCs via promoting the Wnt and BMP signaling pathways.     

Proliferation and chondrogenic differentiation of human adipose-derived mesenchymal stem cells in porous hyaluronic acid scaffold

Human adipose-derived mesenchymal stem cells (AD-MSCs) attracted much interest as a promising alternative to autologous chondrocytes and bone marrow-derived mesenchymal stem cells for cartilage regeneration. Developing a suitable culture technique to direct AD-MSCs into the chondrogenic lineage could be a crucial prerequisite for the cartilage defect repair application of AD-MSCs. Herein, we prepared the PEGDG-crosslinked porous three-dimensional (3D) hyaluronic acid (HA) scaffold and evaluated for its feasibility to induce proliferation and chondrogenic differentiation of the AD-MSCs. In addition, the effect of bone-morphogenetic protein-2 (BMP-2) and platelet-derived growth factor (PDGF) on chondrogenic differentiation was further investigated. Proliferation and chondrogenic differentiation were evaluated by cell morphology, DNA contents, s-GAG contents, and level of mRNA expression of relevant marker genes. When cultured with reference chondrogenic medium (RCM; serum-free DMEM-HG supplemented with 10 ng/mL of transforming growth factor-β1 (TGF-β1), 50 nM ascorbate, 100 nM dexamethasone, and 5 μg/mL of ITS), better proliferation and chondrogenic differentiation of AD-MSCs were obtained in the 3D HA scaffold culture as compared to the micromass culture, a standard 3D culture system. Moreover, the level of chondrogenic differentiation of AD-MSCs in the HA scaffold-RCM culture system was further increased by BMP-2, and decreased by PDGF. These results suggested that the HA scaffold with RCM was a promising chondrogenic culture system of AD-MSCs, and that BMP-2 could potentially serve as a chondrogenic supplement for AD-MSCs. However, PDGF was determined to be an inappropriate supplement based on its inhibition of the chondrogenic differentiation of AD-MSCs.     

Physiological features of periodontal regeneration and approaches for periodontal tissue engineering utilizing periodontal ligament cells

Experimental studies have shown that the potential of periodontal regeneration seems to be limited by the regenerative capacity of the cells involved. The regeneration of damaged periodontal tissues is mediated by various periodontal cells and is regulated by a vast array of extracellular matrix informational molecules that induce both selective and nonselective responses in different cell lineages and their precursors. In this paper, we first review periodontal ligament tissue and its different cell subpopulations including fibroblasts and paravascular stem cells, and their functions during the development and homeostasis of periodontal tissues. Because conventional periodontal regeneration methods remain insufficient to obtain a complete and reliable periodontal regeneration, the concept of periodontal tissue engineering has been based on the generation of the conditions necessary to improve the healing of periodontal tissues. Additionally, the potential of periodontal ligament cells for use in periodontal tissue engineering to overcome the limitations of conventional periodontal regenerative therapies is discussed, followed by an update of the recent progress and future directions of research utilizing periodontal ligament cells for predictable periodontal regeneration.     

In vitro-digested milk proteins: Evaluation of angiotensin-1-converting enzyme inhibitory and antioxidant activities,peptidomic profile,and mucin gene expression in HT29-MTX cells

Over the past decades, several studies investigated the health-promoting functions of milk peptides. However, to date many hurdles still exist regarding the widespread use of milk-derived bioactive peptides, as they may be degraded during gastrointestinal digestion. Thus, the aim of our study was to in vitro digest intact whey protein isolate (WPI) and casein proteins (CNP), mimicking in vivo digestion, to investigate their bioactive effects and to identify the potential peptides involved. Whey protein isolate and CNP were digested using a pepsin–pancreatin protocol and ultra-filtered (3-kDa cutoff membrane). A permeate (<3 kDa) and a retentate (>3 kDa) were obtained. Soy protein was included as a control (CTR). Angiotensin-1-converting enzyme inhibitory (ACE1-I) and antioxidant activity (AOX) were assessed and compared with those observed in undigested proteins and CTR. Furthermore, the permeate was characterized by nano-liquid chromatography electrospray ionization tandem mass spectrometry (LC-nano ESI MS/MS) using a shotgun peptidomic approach, and retentate was further digested with trypsin and analyzed by MS using a shotgun proteomic approach to identify potentially bioactive peptides. Further, the effects of WPI, CNP, and CTR retentate on cell metabolic activity and on mucus production (MUC5AC and MUC2 gene expression) were assessed in intestinal goblet HT29-MTX-E12 cells. Results showed that WPI permeate induced a significant ACE1-I inhibitory effect [49.2 ± 0.64% (SEM)] compared with undigested WPI, CNP permeate, and retentate or CTR permeate (10.40 ± 1.07%). A significant increase in AOX (1.58 ± 0.04 and 1.61 ± 0.02 µmol of trolox AOX equivalents per mg of protein, respectively) upon digestion was found in WPI. Potentially bioactive peptides associated with ACE1-I and antihypertensive effects were identified in WPI permeate and CNP retentate. At specific concentrations, WPI, CNP, and CTR retentate were able to stimulate metabolic activity in HT29-MTX-E12 cells. Expression of MUC5AC was increased by CNP retentate and unaltered by WPI retentate; MUC2 expression was significantly increased by 0.33 mg/g of CNP and reduced by 1.33 mg/g of CNP. Our results confirm that milk proteins may be rich sources of bioactive compounds, with the greatest beneficial potential of CNP at the intestinal goblet cell level.     

Effects of Wnt-10b on proliferation and differentiation of adult murine skin-derived CD34 and CD49f double-positive cells

Although mouse Wnt-10b has been shown to play various roles in a wide range of biological actions, the effects on epithelial stem/progenitor cells in the skin have not been reported. In the present study, we investigated the effects of Wnt-10b on proliferation and differentiation of murine skin-derived CD34 and CD49f double-positive (CD34⁺CD49f⁺) cells, a supposed fraction as enriched epithelial stem/progenitor cells. The cells were prepared from dorsal skin samples obtained from young adult mice as α6 integrin (CD49f) and CD34 double-positive cells by fluorescent activated cell sorting (FACS), and they were cultured with or without Wnt-10b to investigate its effects on proliferation and differentiation. Involvement of canonical Wnt signaling pathway was confirmed by TOPFLASH assay, and differentiation of the CD34⁺CD49f⁺ cells was assessed by RT-PCR analysis and immunocytochemical examinations. The skin-derived CD34⁺CD49f⁺ cells were immunopositive for Lhx2 and expressed mRNA of classical markers for bulge stem cells, including Lhx2, keratin15, Sox9, S100a6, and NFATc1. Their proliferation was suppressed by Wnt-10b, and the markers for differentiated epithelial cells became to be expressed in the culture with Wnt-10b. These results suggest that Wnt-10b promotes differentiation of epithelial stem/progenitor cells in the skin.     

Effects of dried longan seed (Euphoria longana Lam.) extract on VEGF secretion and expression in colon cancer cells and angiogenesis in human umbilical vein endothelial cells

Angiogenesis is a critical event in cancer metastasis, via delivery of needed oxygen and nutrients to tumor cells. Anti-angiogenesis is one strategy for controlling cancer progression. We herein report anti-angiogenesis activity of dried longan seeds using colon adenocarcinoma cells (SW480 cells) and human umbilical vein endothelial cells (HUVECs). Sephadex LH-20 column chromatography was used for separate three dried longan seed fractions. We firstly evaluated vascular endothelial cell growth factor (VEGF) secretion, expression and colony formation of SW480 cells, using enzyme-linked immunosorbent assay (ELISA), Western blot analysis and soft agar assays. Meanwhile cell proliferation, gelatinase activity and tube formation of HUVECs were determined via proliferation assay, gelatin zymography and in vitro tube formation assay, respectively. The results suggest that dried longan seed fractions could be potential angiogenic inhibitors not only interruption of VEGF secretion and expression in SW480 cells but also abrogation of cell proliferation, the activity of gelatinase and tube formation of HUVECs.     

A secretion of the mollusc Cryptomphalus aspersa promotes proliferation, migration and survival of keratinocytes and dermal fibroblasts in vitro

Regenerative properties of skin decrease with age, and thus, the search for substances that minimize cutaneous ageing has increased in the last few years. The secretion of the mollusc Cryptomphalus Aspersa (SCA) is a natural product that bears regenerative properties when applied topically. The purpose of this work is to study the in vitro effects of SCA on cell proliferation and migration, as well as on cell-cell (E-cadherin and β-catenin) and cell-substrate (vinculin and β1-integrin) adhesion proteins expression, using a human keratinocyte cell line (HaCaT cells) and primary dermal fibroblasts (HF). We tested the effects of SCA on cell proliferation using a colorimetric assay. In addition, SCA-induced changes on cell migration were studied by wound-healing assays. Besides, Western blot and immunofluorescence microscopy were carried out to test the expression of different cell adhesion proteins. We found that SCA promotes proliferation and migration of HaCaT cells in a time- and dose-dependent manner. Moreover, treatment with SCA increases the migratory behaviour and the expression of adhesion molecules in both HaCaT and HF. Finally, SCA also improves cell survival and promotes phosphorylation of FAK and nuclear localization of β-catenin. These results shed light on the molecular mechanisms underlying the regenerative properties of SCA, based on its promoting effect on skin cell migration, proliferation and survival. Moreover, these results support future clinical uses of SCA in the regeneration of wounded tissues.