Collagen type I and hyaluronic acid based hybrid scaffolds for heart valve tissue engineering

Tissue engineers have achieved limited success so far in designing an ideal scaffold for aortic valve; scaffolds lack in mechanical compatibility, appropriate degradation rate, and microstructural similarity. This paper, therefore, has demonstrated a carbodiimide-based sequential crosslinking technique to prepare aortic valve extracellular matrix mimicking (ECM) hybrid scaffolds from collagen type I and hyaluronic acid (HA), the building blocks of heart valve ECM, with tailorable crosslinking densities. Swelling studies revealed that crosslinking densities of parent networks increased with increasing the concentration of the crosslinking agents whereas crosslinking densities of hybrid scaffolds averaged from those of parent collagen and HA networks. Hybrid scaffolds also offered a wide range of pore size (66-126 μm) which fulfilled the criteria for valvular tissue regeneration. Scanning electron microscopy and images of Alcian blue-Periodic acid Schiff stained samples suggested that our crosslinking technique yielded an ECM mimicking microstructure with interlaced bands of collagen and HA in the hybrid scaffolds. The mutually reinforcing networks of collagen and HA also resulted in increased bending moduli up to 1660 kPa which spanned the range of natural aortic valves. Cardio sphere-derived cells (CDCs) from rat hearts showed that crosslinking density affected the available cell attachment sites on the surface of the scaffold. Increased bending moduli of CDCs seeded scaffolds up to two folds (2-6 kPa) as compared to the non-seeded scaffolds (1 kPa) suggested that an increase in crosslinking density of the scaffolds could not only increase the in vitro bending modulus but also prevented its disintegration in the cell culture medium.     

Proliferation of canine bone marrow derived mesenchymal stem cells on different nanomaterial based thin film scaffolds

Stem cell niche research uses nanotechnologies to mimic the extra-cellular microenvironment to promote proliferation and differentiation. The aim of designing different scaffolds is to simulate the best structural and environmental pattern for extracellular matrix. This experiment was designed to study the proliferative behaviour of canine bone marrow deriver mesenchymal stem cells (MSCs) on different nanomaterial based thin film scaffolds of carbon nanotubes (CNT), chitosan and poly ε-caprolactone. Similar number of cells was seeded on the scaffolds and standard cell culture flask, taken as control. Cells were maintained on DMEM media and relative number of metabolically active cells was determined by MTT assay up to day six of culture. Cells proliferated on control and all the scaffolds as the days progressed. Although proliferation rate was slow but no decline of cell number was noticed on the scaffolds during the study period. Initially, the cell proliferation was lower on CNT but as time progressed no significant difference was observed compared to control. The result indicated that nanomaterial based scaffolds reduce the proliferation rate of canine MSCs. However, canine MSCs adapted and proliferated better on CNT substrate in vitro and may be used as a scaffold component in canine tissue engineering in future.     

Bioactive nanofibers for fibroblastic differentiation of mesenchymal precursor cells for ligament/tendon tissue engineering applications

Mesenchymal stem cells and precursor cells are ideal candidates for tendon and ligament tissue engineering; however, for the stem cell-based approach to succeed, these cells would be required to proliferate and differentiate into tendon/ligament fibroblasts on the tissue engineering scaffold. Among the various fiber-based scaffolds that have been used in tendon/ligament tissue engineering, hybrid fibrous scaffolds comprising both microfibers and nanofibers have been recently shown to be particularly promising. With the nanofibrous coating presenting a biomimetic surface, the scaffolds can also potentially mimic the natural extracellular matrix in function by acting as a depot for sustained release of growth factors. In this study, we demonstrate that basic fibroblast growth factor (bFGF) could be successfully incorporated, randomly dispersed within blend-electrospun nanofibers and released in a bioactive form over 1 week. The released bioactive bFGF activated tyrosine phosphorylation signaling within seeded BMSCs. The bFGF-releasing nanofibrous scaffolds facilitated BMSC proliferation, upregulated gene expression of tendon/ligament-specific ECM proteins, increased production and deposition of collagen and tenascin-C, reduced multipotency of the BMSCs and induced tendon/ligament-like fibroblastic differentiation, indicating their potential in tendon/ligament tissue engineering applications.     

鸡胚胎干细胞的分离、培养和鉴定

SNL cells (permanent line of irradiated mouse fibroblast cells), primary mice embryonic fibroblasts (PMEF) cells and primary chicken embryonic fibroblasts (PCEF) cells were respectively used as the feeder cells for chicken embryonic stem cell culture. The isolated blastoderm cells front the stage X embryos of chicken were cultured in Dulercco‘‘ s Modified Eagle Medium (DMEM) supplemented with leukemia inhibitory factor (LIF, 1 000 IU/ml), basic fibroblast growth factor (bFGF 10 ng/ml) and stem cell factor (SCF, 5 ng/ml). The alkaline phosphatase (AKP) test, differentiation experiment in vitro and chimeric chicken production were carried out. The resuts showed that culture on feeder layer of PMEF yielded high quality CES cell colonies. The shape of typical CES clone showed as follows: nested aggregation (clone) with clear edge and round surface as well as close arrangement within the clone. Strong positive AKP reactive cellswere observed. On the other hand, the fourth passage CES cells could differentiate into various cells in the absence of feeder layer cells and LIF in vitro. The third and fourth passage cells were injected into the subgerminal cavity of recipient embryos at stage X. The manipulated embryos were incubated until hatching. Of 269 Hailan embryos injected with CES cells of Shouguang Chickens, 8.2 % (22/269) survived to hatching, 3 feather chimeras had been produced, which suggests that an effective culture systems were established and it could promote the growth of CES cells and maintain them in an undifferentiated state .     

Fabrication of agar-gelatin hybrid scaffolds using a novel entrapment method for in vitro tissue engineering applications

Scaffolds of agar and gelatin were developed using a novel entrapment method where agar and gelatin molecules mutually entrapped one another forming stable cell adhesive matrices. Glutaraldehyde was used as a crosslinking agent for gelatin. Three types of hybrid matrices were prepared using agar and gelatin in different proportions in the weight ratio of 1:1, 2:1, and 3:1. Surface characterization of dry scaffolds was carried out by scanning electron microscope. Swelling studies were carried out in phosphate buffer saline (PBS) at physiological pH 7.4. The integral stability of the scaffolds was evaluated by estimating the released disintegrated gelatin from them in PBS at pH 7.4. The attachment kinetics of the cells was evaluated by culturing mouse fibroblast cell line NIH 3T3 on films. The cytocompatibility of these matrices was determined by studying growth kinetics of NIH 3T3 cells on them and morphology of cells was observed through optical photographs taken at various days of culture. It was found that the matrices containing agar and gelatin in 2:1 weight ratio exhibited best growth kinetics. The results obtained from these studies have suggested that the above-described method is a cheap and easy way to fabricate agar-gelatin hybrid scaffolds to grow cells which can be used in various in vitro tissue engineering applications like screening of drugs.     

Assessing the potential use of chitosan scaffolds for the sustained localized delivery of vitamin D

Vitamin D is a commonly used bone modulator in regenerative medicine. Several modalities have been explored for the delivery of vitamin D including nanoparticles and scaffold. The present study aimed to assess the potential use of a bio-degradable chitosan scaffold for the delivery of vitamin D. The objectives included fabrication of a bio-degradable chitosan scaffold, integration of vitamin D into the scaffold, characterization of the vitamin D integrated scaffold. Characterization was carried out using, X-ray diffraction, Fourier transform infrared spectroscopy, and differential scanning calorimetry. The structure of the scaffold was assessed by scanning electron microscopy. The scaffold was placed in phosphate buffer saline and the release duration of vitamin D was observed using UV spectrophotometry. Dental pulp mesenchymal stem cells were added to the scaffold to study the scaffold associated toxicity and the functionality of the scaffold released vitamin D. The vitamin D release period from the scaffold was estimated to be for 80 hrs. MTT assay of the stem cells was comparable to that of the control group (stem cells cultured in media) inferring that the scaffold is not toxic towards the stem cells. The positive alizarin red S staining, a higher expression of alkaline phosphatase, osteocalcin, and RunX2 confirmed the functional capability (osteogenic differentiation of the stem cells) of the released vitamin D. Based on the data from the present study, it can be inferred that chitosan scaffold can be used for the sustained delivery of functional vitamin D for 3–5 days.     

Fate of parental mitochondria in embryonic stem hybrid cells

In hybrid cells, not only are the nuclear genomes of parent cells fused, but their cytoplasm is as well. Mitochondrial DNA (mtDNA) is a convenient marker of cytoplasm that allows us to gain insight into the organization of hybrid-cell cytoplasm. We analyzed the parental mtDNA in hybrid cells resulting from the fusion of Mus musculus embryonic stem (ES) cells with splenocytes and fetal fibroblasts of DD/c mice or with splenocytes of M. caroli. Identification of parental mtDNA in hybrid cells was based on polymorphism among parental mtDNA for certain restriction endonucleases. We found that intra- and interspecific ES cell-splenocyte hybrid cells either entirely or partially lost mtDNA derived from a somatic partner, whereas ES cell-fibroblast hybrids retained mtDNA from both parents in similar ratios with a slight bias. The loss of somatic mitochondria by ES-splenocyte hybrids implies a nonrandom segregation of parental mitochondria, which was supported by a computer simulation of genetic drift. In contrast, ES cell-fibroblast hybrids show bilateral random segregation of the parental mitochondria judging from the analysis of mtDNA in single cells. Preferential segregation of somatic mitochondria does not depend on the differences in sequences of the parental mtDNA, but rather on the replicative state of parental cells.     

Rapid establishment of highly migratory cells from cancer cells for investigating cellular functions

Abstract

Cell migration is closely involved in cancer cell invasion into surrounding tissue and metastasis to the distant organs. It is crucial for understanding the molecular mechanisms that regulate cell migration in cancer cells. The aim of this study is to establish a rapid induction method of highly migratory cells from cancer cells. Osteosarcoma MG-63 and colon cancer DLD1 cells were seeded at 1?×?10⁵ cells in 6-well plates. After 10?min, unattached cells were washed off three times with PBS. The cells which remained attached on the bottom of plates were cultured in DMEM containing 10% FBS. When the cells reached approximately 80% confluence, cells were harvested using trypsin/EDTA. The harvested cells were seeded in other 6-well plates and incubated for 10?min. The unattached cells were washed off and attached cells were further cultured. By repeating this procedure 11–12 times for 2?months, highly migratory MG63-A12 and DLD-A11 cells were obtained from MG-63 and DLD1 cells, respectively. In cell motility assay, the cell motile activities of MG63-A12 and DLD-A11 cells was 10.3 and 13.7 times higher than those of the parental cells, respectively. This procedure is useful to generate highly migratory cells for investigating cellular functions during tumor progression in cancer cells.     

Platelet-rich plasma-derived scaffolds increase the benefit of delayed mesenchymal stromal cell therapy after severe traumatic brain injury

Background

Cell therapy using mesenchymal stromal cells (MSCs) offers new perspectives in the treatment of traumatic brain injury (TBI). The aim of the present study was to assess the impact of platelet-rich plasma scaffolds (PRPS) as support of MSCs in a delayed phase after severe TBI in rats.

A monochromosomal hybrid cell assay for evaluating the genotoxicity of environmental chemicals

The development and utilization of a monochromosomal hybrid cell assay for detecting aneuploidy and chromosomal aberrations are described. The monochromosomal hybrid cell lines were produced by a two-step process involving transfer of a marker bacterial gene to a human chromosome and then by integration of that human chromosome into a mouse complement of chromosomes through microcell fusion. For chemically induced aneuploidy, the segregation of a single human chromosome among mouse chromosomes is used as a cytogenetic marker. The genetic assay for aneuploidy is based on the ability of the cells to grow in a medium that selects for the loss of the human chromosome. The assay for clastogenicity is based on survival of the cells after treatment with the chemicals in medium that selects for retention of the human chromosome but loss of its segment containing diphtheria toxin locus. The assays greatly simplify the detection of chromosomal aberrations induced by environmental factors at low-dose levels.     

In vitro and in vivo study of pluripotency in intraspecific hybrid cells obtained by fusion of murine embryonic stem cells with splenocytes

Hypoxanthine phosphoribosyltransferase–deficient (HPRT^-) mouse embryonic stem (ES) cells, HM-1 cells (genotype XY), were fused with adult female DD/c mouse spleen cells. As a result, a set of HAT-resistant clones was isolated. Four hybrid clones most similar in morphology and growth characteristics to the HM-1 cells were studied in detail with respect to their pluripotency. Of these, three clones contained 41–43 chromosomes, and one clone was nearly tetraploid. All the clones had the XXY set of sex chromosomes and expressed the HPRT of the somatic partner only. The hybrid clones shared features with the HM-1 cells, indicating that they retained their pluripotent properties: (1) embryonic ECMA-7 antigen, not TROMA-1 antigen, was present in most cells; (2) the hybrid cells showed high activity of endogenous alkaline phosphatase (AP); (3) all the hybrid clones were able to form complex embryoid bodies containing derivatives of all the embryonic germinal layers; (4) the hybrid cells contained synchronously replicating X chromosomes, indicating that they were in an active state; and (5) a set of chimeric animals was generated by injecting hybrid cells into BALB/c and C57BL/6J mouse blastocysts. Evidence for chimerism was provided by the spotted coat derived from 129/Ola mice and identification of 129/Ola glucose phosphate isomerase (GPI) in many organs. Thus the results obtained demonstrated that the hybrid cells retain their high pluripotency level despite the close contact of the “pluripotent” HM-1 genome with the “somatic” spleen cell genome during hybrid cell formation and the presence of the “somatic” X chromosome during many cell generations. The presence of HPRT of the somatic partner in many organs and tissues, including the testes in chimeric animals, shows that the “somatic” X chromosome segregates weakly, if at all, during development of the chimeras. There were no individuals with the 129/Ola genotype among the more than 50 offspring from chimeric mice. The lack of the 129/Ola genotype is explained by the imbalance of the sex chromosomes in the hybrid cells rendering the passage of hybrid cell descendants through meiosis in chimeras impossible. As a result, chimeras become unable to produce gametes of the hybrid cell genotype. Mol. Reprod. Dev. 50:128–138, 1998. © 1998 Wiley-Liss, Inc.     

Embryonic stem cell/fibroblast hybrid cells with near-tetraploid karyotype provide high yield of chimeras

Ten primary clones of hybrid cells were produced by the fusion of diploid embryonic stem (ES) cells, viz., line E14Tg2aSc4TP6.3 marked by green fluorescent protein (GFP), with diploid embryonic or adult fibroblasts derived from DD/c mice. All the hybrid clones had many characteristics similar to those of ES cells and were positive for GFP. Five hybrid clones having ploidy close to tetraploidy (over 80% of cells had 76–80 chromosomes) were chosen for the generation of chimeras via injection into C57BL blastocysts. These hybrid clones also contained microsatellites marking all ES cell and fibroblast chromosomes judging from microsatellite analysis. Twenty chimeric embryos at 11–13 days post-conception were obtained after injection of hybrid cells derived from two of three clones. Many embryos showed a high content of GFP-positive descendents of the tested hybrid cells. Twenty one adult chimeras were generated by the injection of hybrid cells derived from three clones. The contribution of GFP-labeled hybrid cells was significant and comparable with that of diploid E14Tg2aSc4TP6.3 cells. Cytogenetic and microsatellite analyses of cell cultures derived from chimeric embryos or adults indicated that the initial karyotype of the tested hybrid cells remained stable during the development of the chimeras, i.e., the hybrid cells were mainly responsible for the generation of the chimeras. Thus, ES cell/fibroblast hybrid cells with near-tetraploid karyotype are able to generate chimeras at a high rate, and many adult chimeras contain a high percentage of descendants of the hybrid cells. A. A. Kruglova and E. A. Kizilova contributed equally to this work. This study was financially supported by grants from the Russian Academy of Sciences, Siberian Branch 5.2 and 14.0.     

Colonization of collagen scaffolds by adipocytes derived from mesenchymal stem cells of the common marmoset monkey

In regenerative medicine, human cell replacement therapy offers great potential, especially by cell types differentiated from immunologically and ethically unproblematic mesenchymal stem cells (MSCs). In terms of an appropriate carrier material, collagen scaffolds with homogeneous pore size of 65 μm were optimal for cell seeding and cultivating. However, before clinical application and transplantation of MSC-derived cells in scaffolds, the safety and efficiency, but also possible interference in differentiation due to the material must be preclinically tested. The common marmoset monkey (Callithrix jacchus) is a preferable non-human primate animal model for this aim due to its genetic and physiological similarities to the human.Marmoset bone marrow-derived MSCs were successfully isolated, cultured and differentiated in suspension into adipogenic, osteogenic and chondrogenic lineages by defined factors. The differentiation capability could be determined by FACS. Specific marker genes for all three cell types could be detected by RT-PCR. Furthermore, MSCs seeded on collagen I scaffolds differentiated in adipogenic lineage showed after 28 days of differentiation high cell viability and homogenous distribution on the material which was validated by calcein AM and EthD staining. As proof of adipogenic cells, the intracellular lipid vesicles in the cells were stained with Oil Red O. The generation of fat vacuoles was visibly extensive distinguishable and furthermore determined on the molecular level by expression of specific marker genes. The results of the study proved both the differential potential of marmoset MSCs in adipogenic, osteogenic and chondrogenic lineages and the suitability of collagen scaffolds as carrier material undisturbing differentiation of primate mesenchymal stem cells.     

Synthesis, characterization and bioactivity studies of novel beta-chitin scaffolds for tissue-engineering applications

Chitin is a biopolymer and it is non-toxic, biodegradable and biocompatible. Chitin has many potential industrial applications because of its abundance, biodegradability, non-toxicity, chemical inertness. beta-Chitin scaffolds were prepared by using saturated calcium chloride alcoholic solution (CaCl(2).6H(2)O/EtOH) and then followed by dialysis with lyophilization. The prepared beta-chitin scaffolds were characterized by FT-IR, scanning electron microscopy (SEM) and thermogravimetric (TGA). The preliminary bioactivity studies of beta-chitin scaffolds were studied by using simulated body fluid (SBF) solution for 7, 14 and 21 days. We also immersed the beta-chitin scaffolds in saturated aqueous CaCl(2) and Na(2)HPO(4) solution over 12h. After 7, 14 and 21 days, the scaffolds were characterized by SEM and FT-IR studies. The SEM studies showed that there is a calcium phosphate layer in the surface as well as in the cross-section of beta-chitin scaffolds. It seems that the beta-chitin scaffolds are useful in the tissue-engineering field.     

Media for cultivation of animal cells: an overview

The increasing interest in products from animal cells has caused an extensive research effort towards development of media for cell cultivation.The basic components in the media used for cultivation of animal cells vary depending upon the characters of the cells and the cultivation method. Basic components consist of an energy source, nitrogen source, vitamins, fats and fatty soluble components, inorganic salts, nucleic acid precursors, antibiotics, oxygen, pH buffering systems, hormones, growth factors and serum. Extensive efforts are directed towards developing serum-free or chemically defined media. Among the serum substitutes is a long list of hormones and growth factors.     

Immobilization of horseradish peroxidase on chitosan/silica sol-gel hybrid membranes for the preparation of hydrogen peroxide biosensor

A simple and effective strategy for fabrication of hydrogen peroxide (H₂O₂) biosensor has been developed by entrapping horseradish peroxidase (HRP) in chitosan/silica sol–gel hybrid membranes (CSHMs) doped with potassium ferricyanide (K₃Fe(CN)₆) and gold nanoparticles (GNPs) on platinum electrode surface. The hybrid membranes are prepared by cross-linking chitosan (CS) with 3-aminopropyltriethoxysilane (APTES), while the presence of GNPs improved the conductivity of CSHMs, and the Fe(CN)₆^3−/4− was used as a mediator to transfer electrons between the electrode and HRP due to its excellent electrochemistry activity. UV–Vis absorption spectroscopy was employed to characterize the different components in the CSHMs and their interaction. The parameters influencing the performance of the resulting biosensor were optimized and the characteristic of the resulting biosensor was characterized by cyclic voltammetry and chronoamperometry. Linear calibration for hydrogen peroxide was obtained in the range of 3.5 × 10^− 6 to 1.4 × 10^− 3 M under the optimized conditions with the detection limit (S/N = 3) of 8.0 × 10^− 7 M. The apparent Michaelis–Menten constant of the enzyme electrode was 0.93 mM. The enzyme electrode retained about 78% of its response sensitivity after 30 days. The system was applied for the determination of the samples, and the results obtained were satisfactory.