Ex vivo cartilage defect model for the evaluation of cartilage regeneration using mesenchymal stem cells

An ex vivo cartilage defect model for the evaluation of cartilage regeneration using mesenchymal stem cells (MSCs) was developed. Porcine chondrocytes and human MSCs were transplanted into cartilage defects created on the porcine osteochondral and chondral discs and cultivated for 3 weeks. Although the regeneration of cartilage-like tissues was observed after the transplantation of chondrocytes to defects on both of the osteochondral and chondral discs, the transplanted MSCs formed cartilage-like tissues only in the defect on the chondral disc, in which an in vivo cartilage-like structure was partly observed, and a degraded tissue was observed in the defect on the osteochondral disc. The effects of medium additives such as serum, transforming growth factor-β3 (TGF-β3), and fibroblast growth factor-2, and the transfection of TGF-β3 gene to MSCs could be clearly estimated using the cartilage defect model. In conclusion, a chondral disc with defects is useful for evaluating cartilage regeneration using MSCs.     

Compare the effects of chondrogenesis by culture of human mesenchymal stem cells with various type of the chondroitin sulfate C

Chondroitin sulfate C (CSC) is a kind of glycosaminoglycans (GAGs) with molecular weights of 10,000 to 50,000 Da and a high charge density. GAGs are major components in extracellular matrix (ECM), which play important role in the regulation of cell proliferation, migration, and differentiation. In this study, we studied the effects of chondroitin sulfate C (CSC) on the differentiation of human mesenchymal stem cells (MSCs) toward the chondrocyte lineage. The MSCs were either cultured on type II collagen (COL II) scaffolds with high molecular weight CSC addition in the medium (free CSC) or with free oligosaccharide CSC. Special attention was given to the effects of MSCs cultured on CSC cross-linked type II scaffolds (cross-linked CSC). According to the analysis of histology stain, gene expression, and ECM secretion, our results showed that MSCs cultured with free CSC, free oligosaccharides CSC, and on the cross-linked CSC scaffolds all would be induced into chondrocytes. Moreover, free oligosaccharide CSC present in the microenvironment could significantly up-regulate MSC chondrogenesis gene expression and stimulate cartilage ECM accumulation more than free CSC with high molecular weight after 3-week induction. Importantly, cross-linked CSC had the most excellent effects on the MSC chondrogenesis. Thus, we believed that cross-linked CSC in the scaffold would play the similar roles with free oligosaccharide CSC in the medium. Cross-linked CSC would be a potential candidate for cartilage repair in the cell therapy and tissue engineering.     

Shrinkage-free preparation of scaffold-free cartilage-like disk-shaped cell sheet using human bone marrow mesenchymal stem cells

Aiming for the clinical application of cartilage regeneration, a culture method for mesenchymal stem cells (MSCs) derived from human bone marrow to obtain scaffold-free cartilage-like disk-shaped sheet of uniform sizes without the shrinkage was investigated. A disk-shaped cell sheet having the same diameter as that of the membrane without the shrinkage was formed after the cultivation of MSCs (18.6 × 10(5)cells/well) for 3 weeks in a cell culture insert (CCI) containing a flat membrane whose porosity was 12%, while 6.2 and 31.0 × 10(5)MSCs/well, respectively, resulted in the shrinkage of the aggregate and the hole formation in the center part of the sheet. Cell aggregates shrunk also in a 96-well plate and CCIs having lower porosity. The disk-shaped cell sheet showed the comparable thickness (1.2mm) and sulfated glycosaminoglycan (sGAG) density to those of the pellet formed in a pellet culture. The gene expression levels of aggrecan and type II collagen in the disk-shaped cell sheet were not lower than those in the pellet. In conclusion, the usage of CCI having 12% porosity and 18.6 × 10(5)MSCs/well could avoid the shrinkage from the formation of the scaffold-free cartilage-like disk-shaped cell sheet.     

High inoculation cell density could accelerate the differentiation of human bone marrow mesenchymal stem cells to chondrocyte cells

The effects of the density of human mesenchymal stem cells (MSCs) on their differentiation to chondrocytes in a differentiation medium supplemented with dexamethasone, TGFbeta3, and IGF-1 were investigated for the regenerative therapy of cartilage. The increase in the initial density of MSCs from 0.05 x 10(4) to 0.9 x 10(4) cells/cm(2) accelerated the increase in the expression level of aggrecan mRNA during the differentiation culture for 7 d. The conditioned medium harvested at 7 d from the differentiation culture with an initial MSC density of 0.3 x 10(4) cells/cm(2) accelerated the initial increase in the expression level for 3 d in the differentiation culture with an initial MSC density of 0.3 x 10(4) cells/cm(2), whereas the conditioned medium harvested at 7 d in the differentiation culture with an initial MSC density of 0.05 x 10(4) cells/cm(2) did not. The differentiation culture after 14 d with an initial MSC concentration of 0.3 x 10(4) cells/cm(2) showed an expression level 1.7-fold that in the case of the culture with an initial MSC concentration of 0.05 x 10(4) cells/cm(2). Thus, a high MSC inoculum density might be appropriate for the rapid differentiation of MSCs to chondrocytes.     

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.     

Glucose-stimulated insulin secretion of various mesenchymal stem cells after insulin-producing cell differentiation

Mesenchymal stem cells (MSCs) are capable of crossing germinative layer borders and are obtainable in high numbers via in vitro cultures. Therefore, many researchers have searched for diverse sources of MSCs. Recently the generation of glucose-responsive insulin-producing cells (IPCs) from MSCs has shown immense potential for the treatment of type 1 diabetes mellitus (T1DM) due to a lack of pancreas donors. In this study, we compared the growth potency of four kinds of MSCs derived from bone marrow, Wharton's jelly, adipose tissue, and the periosteum. In addition, in vitro differentiation of these MSCs into IPCs was also investigated. After 2weeks of IPCs differentiation, we compared the expression of the insulin gene and protein using RT-qPCR and immunofluorescence staining. Only IPCs derived from periosteum-derived progenitor cells (PDPCs) showed a response to glucose concentration. Glucose stimulated insulin secretion was conclusive evidence of the potential functionality of IPCs. Therefore, PDPCs are a promising alternative stem cell source for IPCs differentiation.     

Mesenchymal stem cells differentiate into tenocytes by bone morphogenetic protein (BMP) 12 gene transfer

Rhesus bone marrow mesenchymal stem cells (MSCs) were transfected with the BMP12 gene by electroporation, and the phenotype of the transfected cells was identified by morphological observation and molecular biological assay. After transfection, cells became slender, and their processes became thinner and were interwoven into a network. There were more organelles in the transfected cells than in the parental MSCs. The transfected cells exhibited mRNA expressions of BMP12, collagen type I and scleraxis, but not collagen type III mRNA expression. Immunocytochemical analysis also showed the presence of collagen type I, but not collagen type III in the transfected cells. The transfected cells were positive for CD44 and negative for HLA-DR. Therefore, MSCs can be introduced to differentiate into tenocytes by BMP12 gene transfection, and bone marrow MSCs can serve as an alternative seed cell for application in tendon tissue engineering.     

聚乳酸/胶原蛋白取向纳米纤维支架的性能

为研究取向纳米纤维的性能及取向纳米纤维对细胞生长的引导作用,利用可降解的聚乳酸和胶原蛋白为原料,通过静电纺丝方法制备了随机和取向排列的聚乳酸（PLLA）、聚乳酸 ∕ 胶原蛋白（PLLA ∕Coll）纳米纤维支架材料。利用扫描电镜、红外光谱仪等研究了纳米纤维的形态结构、化学性能和力学性能等,并在其表面培养间充质干细胞（MSCs）,研究细胞在不同的支架表面的生长形态。结果表明：取向纳米纤维具有较细的纤维直径,各向异性的结构性能,平行于纤维排列方向的润湿性能和优于垂直于纤维排列方向的断裂强度。复合 PLLA ∕ Coll 支架表面含有细胞识别基团,能增强细胞的黏附和增殖,培养在取向 PLLA ∕ Coll 纳米纤维支架上的 MSCs 呈现取向的纺锥形态,与体内 MSCs 的形态更类似。     

Novel gene-modified-tissue engineering of cartilage using stable transforming growth factor-beta1-transfected mesenchymal stem cells grown on chitosan scaffolds

Rabbit bone marrow-derived mesenchymal stem cells (MSCs) were stably transfected with the TGF-beta1 gene in monolayer culture using Lipofectamine 2000. After transfection, the expression of cartilage-specific extracellular matrix was upregulated, whereas matrix metalloproteinases 1 and 3 (MMP 1 and 3) protein expressions and enzymatic activities were downregulated. Autologous MSCs modified with the TGF-beta1 gene were seeded into chitosan scaffolds to construct gene-modified cartilage, which was then implanted into the full-thickness articular cartilage defects of rabbits' knees. Twelve weeks after implantation, the defects were filled with regenerated hyaline-like cartilage tissue as confirmed by the positive immunohistochemical staining of collagen type II and intense toluidine blue staining of proteoglycan. Our findings suggest that the repair of cartilage defects can be enhanced by TGF-beta1 gene-modified-tissue engineering of cartilage on the basis of a strategy using MSCs, chitosan, and liposomal transfection.     

Chlorinated phenols in human milk

The paper deals with the contamination of human milk with chlorinated phenols. The average and median concentrations of the chlorophenols investigated ranged from 0.75 to 9.74 g.kg^–1 and from being not detectable to 5.62 g.kg^–1, respectively, for the different compounds. The highest average and median levels were found for pentachlorophenol and 2,4,5-trichlorophenol. The concentrations determined were below the permissible levels.     

Lens-care-solution-induced alterations in dynamic interfacial properties of human tear-lipid films

PurposeTo evaluate the influence of lens care solutions (LCS) on interfacial dynamics and rheological properties of human tear-lipid films.MethodsTear lipids were extracted from Schirmer strips collected from 6 healthy subjects. Sessile bubble tensiometry was used to study interfacial properties at 22 °C. Lipids were deposited on an air bubble immersed into electrolytes solution to form 90 ± 20 nm films. Lipid films were subjected to expansion-compression cycles for dynamic interfacial properties and to step-strain relaxations for assessments of rheological properties. LCS (BioTrue [BT], PureMoist [PM], Revitalens [RL], ClearCare [CC]) were injected into optical chamber and equilibrated for 2 h without or with lipid films. Dynamic interfacial properties of films were measured. Then electrolyte solution was pumped through chamber and properties of films were re-evaluated.ResultsEquilibrium surface tension (EST), elasticity modulus (E), and relaxation times (τ) of tear lipids were 22 ± 2.1 mN/m, 10.7–14.8 mN/m, and 80–150 s, respectively. EST for LCS was 45.3 ± 0.8 for CC, 40.3 ± 0.8 for BT, 33.4 ± 1.0 for PM, and 30.1 ± 0.8 mN/m for RL. E for LCS varied within 0.5–6.7 mN/m, and τ varied from 49 to 68 ± 5 s. For mixed lipids + LCS films, EST remained unchanged whereas E and τ were reduced for all LCS types. Exposure to PM and RL noticeably altered the shape of lipid-film iso-cycles. These changes persisted after LCS washout.ConclusionsSome components of LCS bind irreversibly to lipid films and make them less viscous and less elastic. These findings suggest the possibility of tear-film destabilization upon LCS exposure.     

企业人力资本价值的计量研究

企业竞争,归根结底是人才的竞争。现代企业经营重心已从传统的物质资本经营转移到人力资本经营上,人力资本已取代物质资本成为企业最重要的战略性资本。然而,受各种因素影响,目前对人力资本重要性的认识还处在定性阶段,这极大地限制了对人力资本的开发和利用。该文着重研究人力资本计量问题,探索可行方法,力图从数量角度认识人力资本的价值,以充分发挥人力资本的作用。     

Influence of fetal calf serum on differentiation of mesenchymal stem cells to chondrocytes during expansion

Mesenchymal stem cells (MSCs) should be expanded in vitro while maintaining their multilineage potential before differentiation to one mesenchymal lineage for application to regeneration therapy. The effect of fetal calf serum (FCS) on undesirable differentiation during subcultivations for the expansion was investigated. The expression level of the aggrecan gene, which is a marker of chondrogenic differentiation, gradually and markedly increased during the subcultivations of MSCs with the addition of 10% FCS and without additional cytokines. The percentage of cells positive for CD90 and CD166, which are markers of MSCs, decreased, and the percentage of large polygonal cells and the average cell adhesion area increased during the expansion. There was a marked difference in the increase in the aggrecan expression level between the two expansion cultures employing different FCS lots, although their proliferation rates were almost the same. The decrease in FCS concentration resulted in a higher percentage of CD90(+)CD166(+) cells, a lower percentage of large polygonal cells, and a lower level of aggrecan expression. Consequently, FCS components could stimulate MSC differentiation to chondrocytes and a lower concentration could decrease this differentiation.     

Effects of ochratoxin A on micronucleus frequenncy in human lymphocytes

Ochratoxin A (OTA( a nephrotoxic and carcinogenic mycotoxin, was investigated to examine its potency to induce micronuclei (MN) in cultured human lymphocytes. Lymphocyte cultures were treated for the last 48 h with OTA at concentrations of 25 μM , 10 μM , 1 μM , 100 nM , 10 nM , 1 nM , and 100 pM and absolute ethanol. At the highest concentration, OTA was found to induce MN in cytokinesis‐blocked lymphocytes (p < 05). The 25 μM OTA concentration also led to a clear decrease in the percentage of binucleated cells, probably due to cytotoxicity. OTA at the other concentrations tested did not induce MN frequency. These results indicate that a high concentration of OTA is genotoxic in cultured human lymphocytes.     

Characterization of Isoflavones in Membrane-processed Soy Protein Concentrate

ABSTRACT: Aqueous soy flour solutions (5% w/w) were treated for 3 h at 45 to 50 °C with Crystalzyme®, without enzyme at 45 to 50 °C, or blanched at 80 °C. Solutions were ultra‐ and dia‐filtered to produce membrane soy concentrates (MSC). Total isoflavones of MSCs were 3.02 mg/g, 3.12 mg/g, or 3.42 mg/g, respectively, on a dry weight basis. Membrane processing contributed to approximately 18, 15, or 8% decreases, respectively, in total isoflavones of MSCs compared with soy flour (3.71mg/g). This represents at least an 82% recovery. There was no significant difference in total isoflavone content in any MSC, however the profile of isoflavones as glucosides or aglycones changed with treatment conditions.     

The refractive index of the human cornea: A review

The refractive index of the cornea and overlying tear film are key factors affecting refraction and overall optical properties of the eye. A figure of 1.376 is often quoted for the refractive index of the human cornea over the visible spectrum. In the 19th century estimates for the average refractive index of the human cornea ranged from 1.335 to 1.4391. Over the last two decades data obtained from either ex or in vivo corneas (under local anaesthesia with or without stromal resection) by contact Abbé refractometry show the refractive index of the cornea changes along its’ depth undulating from around 1.400 at the epithelium to 1.380 at Bowman’s layer, a low of 1.369 in the mid stroma and 1.373 at the endothelium. The mean refractive index of harvested tear samples is 1.337 rising to 1.482 for the overlying lipid layer. Contemporary measurements obtained in vivo by non-invasive methods reveal the average, or equivalent, refractive index of the tear film-cornea complex along the antero-posterior direction ranges from 1.423 to 1.436. Over the last 200 years calculations, with respect to the optics of the human eye, were based on values for the refractive index of the cornea obtained from invasive techniques. The refractive index of the cornea and overlying tear film appears to be higher than previously accepted and varies from case to case.     

Pharmacokinetic study of mangiferin in human plasma after oral administration

Mangiferin, an active component of traditional Chinese herbal medicine, although it is reported to have various pharmacological effects, the limited number of pharmacokinetic studies limit its wide application. To evaluate the pharmacokinetics of mangiferin in human, a sensitive high performance liquid chromatography-mass spectrometry (HPLC-MS) method for the determination of mangiferin in human plasma was developed. The proposed HPLC-MS method is selective, precise and accurate enough and enables the identification and quantification of mangiferin for the use in clinical studies. After single oral administration of 0.1, 0.3 and 0.9 g mangiferin, respectively, the method was successfully applied for the pharmacokinetics of mangiferin in 21 healthy male Chinese volunteers. The pharmacokinetic of mangiferin was fit to the non-compartmental model. The pharmacokinetics parameters were calculated. Mangiferin concentration in plasma reached 38.64 ± 6.75 ng/mL about 1 h after oral administration of 0.9 g mangiferin and the the apparent elimination half-life (t_1/2) was 7.85 ± 1.72 h. The absorption of mangiferin was increased with the administration of a large dose and it was concluded that the pharmacokinetics of mangiferin in human was nonlinear.     

鱼蛋白对皮肤水份、油份的调节作用研究

为了研究鱼蛋白对人体皮肤水份、油份的调节作用,进行人体试食“鱼蛋白”４５ｄ的试验研究,结果显示试食组的皮肤水份自身对照前后改变差别有显性,试食组与对照组相比差别也有显性,说明“鱼蛋白”具有保持皮肤水份的作用。经对试食组的４５ｄ食用观察２,主我不良反应,客观上的各项体检和化验检查均未发现对人体靠官损害,证明“鱼蛋白”对人体健康无害。     

Concentrations of selenium in human milk

 Selenium (Se) concentrations in 58 samples of mature human milk from Canarian women were determined by spectrofluorimetry. According to the literature the Se concentrations found fall within the normal limits. The concentration of Se in human milk was compared with that in powdered infant formula and presented significantly lower concentrations in the latter. Babies fed with human milk had an adequate intake of Se. However, babies fed with powdered infant formula consumed only 56% of the requirements recommended by The National Research Council. No changes in Se concentration were observed between lactation stages. Human milk produced in springtime was found to be richer in Se than that produced in the autumn, which could be due to changes in the nutritional habits of the mothers. The mother's age, weight, height and number of previous children were not found to influence the Se levels in the milk. Received: 19 January 1998     

Concentrations of selenium in human milk

 Selenium (Se) concentrations in 58 samples of mature human milk from Canarian women were determined by spectrofluorimetry. According to the literature the Se concentrations found fall within the normal limits. The concentration of Se in human milk was compared with that in powdered infant formula and presented significantly lower concentrations in the latter. Babies fed with human milk had an adequate intake of Se. However, babies fed with powdered infant formula consumed only 56% of the requirements recommended by The National Research Council. No changes in Se concentration were observed between lactation stages. Human milk produced in springtime was found to be richer in Se than that produced in the autumn, which could be due to changes in the nutritional habits of the mothers. The mother's age, weight, height and number of previous children were not found to influence the Se levels in the milk. Received: 19 January 1998