Hypoxia as a Stimulus for the Maturation of Meniscal Cells: Highway to Novel Tissue Engineering Strategies?

The meniscus possesses low self-healing properties. A perfect regenerative technique for this tissue has not yet been developed. This work aims to evaluate the role of hypoxia in meniscal development in vitro. Menisci from neonatal pigs (day 0) were harvested and cultured under two different atmospheric conditions: hypoxia (1% O2) and normoxia (21% O2) for up to 14 days. Samples were analysed at 0, 7 and 14 days by histochemical (Safranin-O staining), immunofluorescence and RT-PCR (in both methods for SOX-9, HIF-1α, collagen I and II), and biochemical (DNA, GAGs, DNA/GAGs ratio) techniques to record any possible differences in the maturation of meniscal cells. Safranin-O staining showed increments in matrix deposition and round-shape “fibro-chondrocytic” cells in hypoxia-cultured menisci compared with controls under normal atmospheric conditions. The same maturation shifting was observed by immunofluorescence and RT-PCR analysis: SOX-9 and collagen II increased from day zero up to 14 days under a hypoxic environment. An increment of DNA/GAGs ratio typical of mature meniscal tissue (characterized by fewer cells and more GAGs) was observed by biochemical analysis. This study shows that hypoxia can be considered as a booster to achieve meniscal cell maturation, and opens new opportunities in the field of meniscus tissue engineering.

Download Full-text

Fabrication of 3D-Printed Interpenetrating Hydrogel Scaffolds for Promoting Chondrogenic Differentiation

Polymers ◽

10.3390/polym13132146 ◽

2021 ◽

Vol 13 (13) ◽

pp. 2146

Author(s):

Jian Guan ◽

Fu-zhen Yuan ◽

Zi-mu Mao ◽

Hai-lin Zhu ◽

Lin Lin ◽

...

Keyword(s):

Tissue Engineering ◽

Elastic Modulus ◽

Chondrogenic Differentiation ◽

Marker Genes ◽

Self Healing ◽

Polyethylene Glycol Diacrylate ◽

3D Microenvironment ◽

3D Printed

The limited self-healing ability of cartilage necessitates the application of alternative tissue engineering strategies for repairing the damaged tissue and restoring its normal function. Compared to conventional tissue engineering strategies, three-dimensional (3D) printing offers a greater potential for developing tissue-engineered scaffolds. Herein, we prepared a novel photocrosslinked printable cartilage ink comprising of polyethylene glycol diacrylate (PEGDA), gelatin methacryloyl (GelMA), and chondroitin sulfate methacrylate (CSMA). The PEGDA-GelMA-CSMA scaffolds possessed favorable compressive elastic modulus and degradation rate. In vitro experiments showed good adhesion, proliferation, and F-actin and chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) on the scaffolds. When the CSMA concentration was increased, the compressive elastic modulus, GAG production, and expression of F-actin and cartilage-specific genes (COL2, ACAN, SOX9, PRG4) were significantly improved while the osteogenic marker genes of COL1 and ALP were decreased. The findings of the study indicate that the 3D-printed PEGDA-GelMA-CSMA scaffolds possessed not only adequate mechanical strength but also maintained a suitable 3D microenvironment for differentiation, proliferation, and extracellular matrix production of BMSCs, which suggested this customizable 3D-printed PEGDA-GelMA-CSMA scaffold may have great potential for cartilage repair and regeneration in vivo.

Download Full-text

Calcium/Cobalt Alginate Beads as Functional Scaffolds for Cartilage Tissue Engineering

Stem Cells International ◽

10.1155/2016/2030478 ◽

2016 ◽

Vol 2016 ◽

pp. 1-12 ◽

Cited By ~ 22

Author(s):

Stefano Focaroli ◽

Gabriella Teti ◽

Viviana Salvatore ◽

Isabella Orienti ◽

Mirella Falconi

Keyword(s):

Tissue Engineering ◽

Bone Morphogenetic Proteins ◽

Transforming Growth Factor ◽

Cartilage Tissue Engineering ◽

Biomechanical Properties ◽

Alginate Beads ◽

Cartilage Tissue ◽

Self Healing ◽

Tissue Replacement

Articular cartilage is a highly organized tissue with complex biomechanical properties. However, injuries to the cartilage usually lead to numerous health concerns and often culminate in disabling symptoms, due to the poor intrinsic capacity of this tissue for self-healing. Although various approaches are proposed for the regeneration of cartilage, its repair still represents an enormous challenge for orthopedic surgeons. The field of tissue engineering currently offers some of the most promising strategies for cartilage restoration, in which assorted biomaterials and cell-based therapies are combined to develop new therapeutic regimens for tissue replacement. The current study describes thein vitrobehavior of human adipose-derived mesenchymal stem cells (hADSCs) encapsulated within calcium/cobalt (Ca/Co) alginate beads. These novel chondrogenesis-promoting scaffolds take advantage of the synergy between the alginate matrix and Co+2ions, without employing costly growth factors (e.g., transforming growth factor betas (TGF-βs) or bone morphogenetic proteins (BMPs)) to direct hADSC differentiation into cartilage-producing chondrocytes.

Download Full-text

Identification of a gene cluster for cell-surface genes of the SRS superfamily inNeospora caninumand characterization of the novelSRS9gene

Parasitology ◽

10.1017/s0031182011001351 ◽

2011 ◽

Vol 138 (14) ◽

pp. 1832-1842 ◽

Cited By ~ 9

Author(s):

V. RISCO-CASTILLO ◽

V. MARUGÁN-HERNÁNDEZ ◽

A. FERNÁNDEZ-GARCÍA ◽

A. AGUADO-MARTÍNEZ ◽

E. JIMÉNEZ-RUIZ ◽

...

Keyword(s):

Western Blot ◽

Gene Cluster ◽

Neospora Caninum ◽

Pcr Analysis ◽

Rt Pcr ◽

Specific Expression ◽

Initial Characterization ◽

Hydrophilic Region

SUMMARYHere we present the detection of a gene cluster forNeospora caninumsurface genes, similar to theToxoplasma gondiiSRS9 locus, and the cloning and characterization of the NcSRS9gene. PCR genome walking, using NcBSR4gene as a framework, allows the identification, upstream NcBSR4, of 2 sequences homologous to theSRS5and the Ubiquinol-cytochrome C reductase genes and, downstream NcBSR4, of an ORF of 1191 bp coding for a 396-amino acid polypeptide with 59% similarity to the TgSRS9 antigen. A putative 39-residue signal peptide was found at the NH2-terminus followed by a hydrophilic region, and a potential site for a glycosylphosphatidylinositol anchor at the COOH-terminus. A recombinant NcSRS9 protein was produced and was recognized on a Western blot by a low proportion of sera from a panel of naturally infected cows and calves. In addition, Western blot analysis using polyclonal anti-rNcSRS9 revealed stage-specific expression of NcSRS9 in bradyzoites but not in tachyzoites, and immunohistochemistry on brain from a congenitally infected calf showed NcSRS9 recognition in bradyzoites contained in tissue cysts. However, bradyzoite-specific expression of NcSRS9 could not be proven by immunofluorescence on bradyzoites obtainedin vitroand RT-PCR analysis showed no significant variations of NcSRS9transcripts duringin vitrotachyzoite-bradyzoite switch, probably due to incomplete maturity ofin vitrobradyzoites. Initial characterization of NcSRS9 in this study may lead to further studies for a better understanding ofN. caninumpersistence.

Download Full-text

Mechano-Active Cartilage Tissue Engineering

Advances in Science and Technology ◽

10.4028/www.scientific.net/ast.49.189 ◽

2006 ◽

Vol 49 ◽

pp. 189-196

Author(s):

Soo Hyun Kim ◽

Young Mee Jung ◽

Sang Heon Kim ◽

Young Ha Kim ◽

Jun Xie ◽

...

Keyword(s):

Tissue Engineering ◽

Dynamic Compression ◽

Cartilage Tissue Engineering ◽

Compressive Deformation ◽

Cartilage Tissue ◽

Cartilaginous Tissue ◽

Pressing Method ◽

Safranin O

To engineer cartilaginous constructs with a mechano-active scaffold and dynamic compression was performed for effective cartilage tissue engineering. Mechano-active scaffolds were fabricated from very elastic poly(L-lactide-co-ε-carprolactone)(5:5). The scaffolds with 85 % porosity and 300~500 μm pore size were prepared by a gel-pressing method. The scaffolds were seeded with chondrocytes and the continuous compressive deformation of 5% strain was applied to cell-polymer constructs with 0.1Hz to evaluate for the effect of dynamic compression for regeneration of cartilage. Also, the chondrocytes-seeded constructs stimulated by the continuous compressive deformation of 5% strain with 0.1Hz for 10 days and 24 days respectively were implanted in nude mice subcutaneously to investigate their biocompatibility and cartilage formation. From biochemical analyses, chondrogenic differentiation was sustained and enhanced significantly and chondrial extracellular matrix was increased through mechanical stimulation. Histological analysis showed that implants stimulated mechanically formed mature and well-developed cartilaginous tissue, as evidenced by chondrocytes within lacunae. Masson’s trichrome and Safranin O staining indicated an abundant accumulation of collagens and GAGs. Also, ECM in constructs was strongly immuno-stained with anti-rabbit collagen type II antibody. Consequently, the periodic application of dynamic compression can improve the quality of cartilaginous tissue formed in vitro and in vivo.

Download Full-text

Expression and homologous regulation of GH secretagogue receptor mRNA in rat adrenal gland

Acta Endocrinologica ◽

10.1530/eje.0.1470677 ◽

2002 ◽

pp. 677-688 ◽

Cited By ~ 23

Author(s):

ML Barreiro ◽

L Pinilla ◽

E Aguilar ◽

M Tena-Sempere

Keyword(s):

Biological Effects ◽

Biologically Active ◽

Pcr Analysis ◽

Mrna Levels ◽

R Gene ◽

Rt Pcr ◽

Short Term ◽

Opposite Pattern ◽

Corticosterone Secretion

OBJECTIVE: GH secretagogues (GHSs) elicit a variety of biological effects in several endocrine and non-endocrine target tIssues, including activation of the hypothalamic-pituitary-adrenal axis. The latter is mainly carried out through a central hypothalamic action; yet the possibility of additional effects directly at the adrenal level cannot be ruled out. The aims of this study were to evaluate the expression and homologous regulation of the GHS-receptor (GHS-R) gene in rat adrenal and to assess the effects of synthetic (GH releasing peptide-6 - GHRP-6) and natural (ghrelin) ligands of GHS-R upon basal and ACTH-stimulated corticosterone secretion in vitro. DESIGN AND METHODS: Analysis of adrenal expression of target mRNAs (GHS-R, GHS-R1a, ghrelin, and several steroidogenic factors) was conducted by means of primer-specific, semi-quantitative RT-PCR. Evaluation of corticosterone secretion by incubated adrenal tIssue was carried out by specific RIA. RESULTS: RT-PCR analysis demonstrated expression of the GHS-R gene, but not of the gene encoding the cognate ligand ghrelin, in rat adrenal. Moreover, expression of the mRNA coding for the type 1a GHS-R (GHS-R1a), i.e. the biologically active receptor form, was demonstrated. The adrenal expression of the GHS-R message appeared under the regulation of homologous signals in vitro, as short-term incubation of adrenal samples in serum-free medium induced a significant increase in GHS-R mRNA levels that was inhibited by exposure to different doses of GHRP-6 (10(-9)-10(-5) mol/l) or ghrelin (10(-7) mol/l). Notably, an opposite pattern of homologous regulation of GHS-R gene expression was observed at the pituitary. Finally, short-term stimulation with increasing concentrations of GHRP-6 (10(-9)-10(-5) mol/l) or ghrelin (10(-7) mol/l) failed to alter basal and ACTH-stimulated corticosterone secretion in vitro, neither did it modify ACTH-stimulated mRNA expression levels of several upstream elements in the steroidogenic route: the steroidogenic acute regulatory (StAR) protein, and the enzymes P450 cholesterol side-chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). CONCLUSIONS: Our study provides novel evidence for the expression and homologous regulation of the GHS-R gene in rat adrenal. However, our results cast doubts on the possibility of direct adrenal actions of ligands of the GHS-R in the regulation of corticosterone secretion in the rat.

Download Full-text

Development and Use of a Reverse Transcription-PCR Assay To Study Expression of Tri5 byFusarium Species In Vitro and In Planta

Applied and Environmental Microbiology ◽

10.1128/aem.65.9.3850-3854.1999 ◽

1999 ◽

Vol 65 (9) ◽

pp. 3850-3854 ◽

Cited By ~ 72

Author(s):

F. M. Doohan ◽

G. Weston ◽

H. N. Rezanoor ◽

D. W. Parry ◽

P. Nicholson

Keyword(s):

Reverse Transcription ◽

Infected Plant ◽

Fusarium Culmorum ◽

Pcr Analysis ◽

Growth Stages ◽

In Planta ◽

Rt Pcr ◽

Pcr Assay ◽

Glucuronidase Activity

ABSTRACT The Tri5 gene encodes trichodiene synthase, which catalyzes the first reaction in the trichothecene biosynthetic pathway. In vitro, a direct relationship was observed between Tri5expression and the increase in deoxynivalenol production over time. We developed a reverse transcription (RT)-PCR assay to quantifyTri5 gene expression in trichothecene-producing strains ofFusarium species. We observed an increase inTri5 expression following treatment of Fusarium culmorum with fungicides, and we also observed an inverse relationship between Tri5 expression and biomass, as measured by β-d-glucuronidase activity, during colonization of wheat (cv. Avalon) seedlings by F. culmorum. RT-PCR analysis also showed that for ears of wheat cv. Avalon inoculated with F. culmorum, there were different levels of Tri5 expression in grain and chaff at later growth stages. We used the Tri5-specific primers to develop a PCR assay to detect trichothecene-producing Fusariumspecies in infected plant material.

Download Full-text

Characterization of regional meniscal cell and chondrocyte phenotypes and chondrogenic differentiation with histological analysis in osteoarthritic donor-matched tissues

Scientific Reports ◽

10.1038/s41598-020-78757-6 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Jingsong Wang ◽

Sally Roberts ◽

Jan Herman Kuiper ◽

Weiguo Zhang ◽

John Garcia ◽

...

Keyword(s):

Lateral Meniscus ◽

Pathological Process ◽

Knee Joints ◽

Meniscal Cell ◽

Meniscus Tissue ◽

Objective Metrics ◽

Meniscus Degeneration ◽

Cell Phenotypes ◽

Meniscal Cells

AbstractMeniscus degeneration is closely related to the progression of knee osteoarthritis (OA). However, there is currently a lack of quantitative and objective metrics to assess OA meniscal cell phenotypes. In this study we investigated the phenotypic markers and chondrogenic potency of avascular and vascular meniscal cells and chondrocytes from medial OA knee joints (n = 10). Flow cytometry results showed that a significantly greater percentage of meniscal cells were positive for CD49b, CD49c and CD166 compared to donor-matched chondrocytes after 14 days in monolayer culture. The integrins, CD49b and CD29, were expressed at a significantly higher level on avascular meniscal cells derived from tissues with a more degenerated inner border than non-degenerate menisci, suggesting that the integrin family may play an important role in meniscus OA pathology. Collagen fibres arranged in a “tree-like” formation within the meniscus appeared to have less blood vessels associated with them in the vascular region of the most degenerate menisci, which may indicate that such structures are involved in the pathological process. We have demonstrated that meniscal cells derived from the lateral meniscus in medial OA patients have chondrogenic capacity in vitro and hence could represent a potential cell source to consider for meniscus tissue engineering.

Download Full-text

Expression and Localization of Plasma Transglutaminase Factor XIIIA in Bone

Journal of Histochemistry & Cytochemistry ◽

10.1369/jhc.6a7091.2007 ◽

2007 ◽

Vol 55 (7) ◽

pp. 675-685 ◽

Cited By ~ 34

Author(s):

Yukiko Nakano ◽

Hadil F. Al-Jallad ◽

Aisha Mousa ◽

Mari T. Kaartinen

Keyword(s):

Transglutaminase 2 ◽

Pcr Primers ◽

Cell Types ◽

Proteolytic Processing ◽

Factor Xiiia ◽

Pcr Analysis ◽

Rt Pcr ◽

Osteoblast Cell Line

Transglutaminases (TGs) are protein crosslinking enzymes involved in cell adhesion and signaling and matrix stabilization and maturation, in many cell types and tissues. We previously described that in addition to transglutaminase 2 (TG2), cultured MC3T3-E1 osteoblasts also express the plasma TG Factor XIIIA (FXIIIA). Here we report on the expression and localization of FXIIIA in bone in vivo and provide confirmatory in vitro data. Immuno-histochemistry and in situ hybridization demonstrated that FXIIIA is expressed by osteoblasts and osteocytes in long bones formed by endochondral ossification (femur) and flat bones formed primarily by intramembranous ossification (calvaria and mandible). FXIIIA immuno-reactivity was localized to osteoblasts, osteocytes, and the osteoid. RT-PCR analysis revealed FXIIIA expression by both primary osteoblasts and by the MC3T3-E1 osteoblast cell line. Western blot analysis of bone and MC3T3-E1 culture extracts demonstrated that FXIIIA is produced mainly as a small, 37-kDa form. Sequential RT-PCR analysis using overlapping PCR primers spanning the full FXIIIA gene showed that the entire FXIIIA gene is expressed, thus indicating that the 37-kDa FXIIIA is not a splice variant but a product of posttranslational proteolytic processing. Forskolin inhibition of osteoblast differentiation revealed that FXIIIA processing is regulated by the protein kinase A pathway.

Download Full-text

The Gonococcal Fur-Regulated tbpA and tbpB Genes Are Expressed during Natural Mucosal Gonococcal Infection

Infection and Immunity ◽

10.1128/iai.73.7.4281-4287.2005 ◽

2005 ◽

Vol 73 (7) ◽

pp. 4281-4287 ◽

Cited By ~ 19

Author(s):

Sarika Agarwal ◽

Carol A. King ◽

Ellen K. Klein ◽

David E. Soper ◽

Peter A. Rice ◽

...

Keyword(s):

Gene Expression ◽

Expression Profiles ◽

Iron Concentration ◽

Pcr Analysis ◽

Positive Trend ◽

Gonococcal Infection ◽

Rt Pcr ◽

Male Subjects

ABSTRACT Iron is limiting in the human host, and bacterial pathogens respond to this environment by regulating gene expression through the ferric uptake regulator protein (Fur). In vitro studies have demonstrated that Neisseria gonorrhoeae controls the expression of several critical genes through an iron- and Fur-mediated mechanism. While most in vitro experiments are designed to determine the response of N. gonorrhoeae to an exogenous iron concentration of zero, these organisms are unlikely to be exposed to such severe limitations of iron in vivo. To determine if N. gonorrhoeae expresses iron- and Fur-regulated genes in vivo during uncomplicated gonococcal infection, we examined gene expression profiles of specimens obtained from male subjects with urethral infections. RNA was isolated from urethral swab specimens and used as a template to amplify, by reverse transcriptase PCR (RT-PCR), gonococcal genes known to be regulated by iron and Fur (tbpA, tbpB, and fur). The constitutively expressed gonococcal rmp gene was used as a positive control. RT-PCR analysis indicated that gonorrhea-positive specimens where rmp expression was seen were also 93% (51/55) fbpA positive, 87% (48/55) tbpA positive, and 86% (14 of 16 tested) tbpB positive. In addition, we detected a fur transcript in 79% (37 of 47 tested) of positive specimens. We also measured increases in levels of immunoglobulin G antibody against TbpA (91%) and TbpB (73%) antigens in sera from infected male subjects compared to those in uninfected controls. A positive trend between tbpA gene expression and TbpA antibody levels in sera indicated a relationship between levels of gene expression and immune response in male subjects infected with gonorrhea for the first time. These results indicate that gonococcal iron- and Fur-regulated tbpA and tbpB genes are expressed in gonococcal infection and that male subjects with mucosal gonococcal infections exhibit antibodies to these proteins.

Download Full-text