Extracellular matrix production in vitro in cartilage tissue engineering.

Cartilage tissue engineering is arising as a technique for the repair of cartilage lesions in clinical applications. However, fibrocartilage formation weakened the mechanical functions of the articular, which compromises the clinical outcomes. Due to the low proliferation ability, dedifferentiation property and low production of cartilage-specific extracellular matrix (ECM) of the chondrocytes, the cartilage synthesis in vitro has been one of the major limitations for obtaining high-quality engineered cartilage constructs. This review discusses cells, biomaterial scaffolds and stimulating factors that can facilitate the cartilage-specific ECM production and accumulation in the in vitro culture system. Special emphasis has been put on the factors that affect the production of ECM macromolecules such as collagen type II and proteoglycans in the review, aiming at providing new strategies to improve the quality of tissue-engineered cartilage.

Interleukin-1beta gene in orange-spotted grouper, Epinephelus coioides: molecular cloning, expression, biological activities and signal transduction.

IL-1beta, a key mediator of inflammation, orchestrates a variety of immune responses by initiating gene expression. Herein, we have cloned and sequenced the IL-1beta in orange-spotted grouper (Epinephelus coioides), produced soluble mature recombinant IL-1beta in Escherichia coli, and characterized its biological properties and downstream signal transduction. The grouper IL-1beta cDNA was 1364bp in length, containing an open reading frame of 765bp. The predicted protein of 254 amino acids revealed the presence of the IL-1 family signature motif and the absence of a conventional ICE cut site. Phylogenetically, the grouper IL-1beta clustered closely with those of teleost belonging to Perciformes and apart from those of mammals. The grouper IL-1beta was constitutively expressed in almost all tissues examined, and was augmented in PBL after the addition of LPS or Poly I:C in vitro. The prokaryotically produced rIL-1beta significantly stimulated the proliferation of grouper head kidney cells, and activated gene expression of IL-1beta and COX-2. Moreover, the rIL-1beta-induced IL-1beta and COX-2 expression were reduced by p38 MAPK inhibitor (SB203580) and JNK inhibitor (SP600125), respectively. Taken together, the present study indicated that grouper IL-1beta may have an important role in grouper immune system and activate similar downstream cascades as its mammalian counterparts.

TGFß1 induces hypertrophic change and expression of angiogenic factors in human chondrocytes.

The transforming growth factor ß1 (TGFß1) plays an important role in cartilage development. However, whether TGFß1 stimulates chondrocyte proliferation and cartilage regeneration in osteoarthritis (OA) remains elusive, especially in the context of different treatment and tissue environments. In the present study, we investigated the role of TGFß1 in human chondrocyte culture in vitro, focusing on the morphological change of chondrocytes and the expression of angiogenic factors upon TGFß1 stimulation. We found increased expression of biomarkers indicating chondrocyte hypertrophy and the chondrocytes aggregated to form networks when they were treated with TGFß1. DNA microarray analysis revealed significantly increased expression of genes related to blood vessel formation in TGFß1 treatment group compared to control group. Matrigel assay further demonstrated that chondrocytes had the potential to form network-like structure. These results suggested that TGFß1 induces the hypertrophic change of chondrocytes culture in vitro and induce expression of angiogenic biomarkers. Therefore, application of TGFß1 for chondrocyte culture in practice should be considered prudentially and targeting TGFß1 or relevant receptors to block the signaling pathway might be a strategy to prevent or alleviate progression of osteoarthritis.

Cellular compatibility of magnetic nanocomposites.

OBJECTIVE: To study the cellular compatibility of the magnetic nanocomposite (n-HA/PLLA/Fe2O3) and to discuss the biological behaviors of cells including surface adhesion, proliferation, and expression. The present work provides an experimental basis for medical application. METHOD: Rat osteoblasts (OB) were co-cultured with the magnetic nanocomposite. Cell proliferation, cell adhesion, and the expression of type I collagen (Col-I) and osteocalcin (OCN) gene were characterized by the cell counting kit-8 (CCK-8) method, scanning electronic microscopy (SEM), and reverse transcription polymerase chain reaction (RT-PCR), respectively. RESULTS: CCK-8 detection showed that there was no difference in cell proliferation on the magnetic nanocomposite between the experimental group and control group (P > 0.05). SEM indicated that a large amount of cells adhered to the surface and in the pores of the magnetic nanocomposite. As the co-culture time increased, the cells adhering to the magnetic nanocomposite showed an obvious increase. RT-PCR detection showed that as the co-culture time increased, the expression of the Col-I gene was enhanced (P 0.05). There was no obvious difference in the expression of the OCN gene (P > 0.05). CONCLUSION: The magnetic nanocomposite is suitable for cell adhesion, growth, and differentiation with a high cellular compatibility.

The study on biocompatibility of porous nHA/PLGA composite scaffolds for tissue engineering with rabbit chondrocytes in vitro.

OBJECTIVE: To examine the biocompatibility of a novel nanohydroxyapatite/poly[lactic-co-glycolic acid] (nHA/PLGA) composite and evaluate its feasibility as a scaffold for cartilage tissue engineering. METHODS: Chondrocytes of fetal rabbit were cultured with nHA/PLGA scaffold in vitro and the cell viability was assessed by MTT assay first. Cells adhering to nHA/PLGA scaffold were then observed by inverted microscope and scanning electron microscope (SEM). The cell cycle profile was analyzed by flow cytometry. RESULTS: The viability of the chondrocytes on the scaffold was not affected by nHA/PLGA comparing with the control group as it was shown by MTT assay. Cells on the surface and in the pores of the scaffold increased in a time-dependent manner. Results obtained from flow cytometry showed that there was no significant difference in cell cycle profiles between the coculture group and control (P > 0.05). CONCLUSION: The porous nHA/PLGA composite scaffold is a biocompatible and good kind of scaffold for cartilage tissue engineering.

Molecular cloning and functional analysis of polymeric immunoglobulin receptor gene in orange-spotted grouper (Epinephelus coioides).

As one of the most important mucosal effectors, polymeric immunoglobulin receptor (pIgR) mediates the transcytosis of polymeric immunoglobulins (pIgs) to protect the organisms. In this study, a full-length cDNA of pIgR was isolated from orange-spotted grouper (Epinephelus coioides), and the sequence analysis of deduced protein revealed the presence of only two Ig-like domains (ILDs), and the absence of the conserved Ig-binding site and complementary determining region (CDR). The grouper pIgR mRNA was detected in almost all the peripheral tissues examined, especially the mucosal tissues by RT-PCR. Additionally, recombinant grouper pIgR was stably expressed in the COS-7 cell line and identified as a 40-kDa transmembrane receptor. Furthermore, the association of recombinant pIgR and purified grouper pIgM was demonstrated. Taken together, the present study provided strong evidence that grouper pIgR was produced as a transmembrane protein, and probably involved in the pIgM transport.