Fibronectin-coating enhances attachment and proliferation of mesenchymal stem cells on a polyurethane meniscal scaffold.

INTRODUCTION: Partial meniscectomy is one of the most common surgical strategy for a meniscal injury, but sometimes, patients complain of knee pain due to an overload in the ablated compartment. In these cases, implantation of tissue engineering scaffold could be indicated. Currently, two commercial scaffolds, based on collagen or polycaprolactone-polyurethane (PCL-PU), are available for meniscus scaffolding. In short term follow-up assessments, both showed clinical improvement and tissue formation. However, long-term studies carried out in PCL-PU showed that the new tissue decreased in volume and assumed an irregular shape. Moreover, in some cases, the scaffold was totally reabsorbed, without new tissue formation.Mesenchymal stem cells (MSCs) combined with scaffolds could represents a promising approach for treating meniscal defects because of their multipotency and self-renewal. In this work, we aimed to compare the behaviour of MSCs and chondrocytes on a PCL-PU scaffold in vitro. MSCs express integrins that binds to fibronectin (FN), so we also investigate the effect of a FN coating on the bioactivity of the scaffold. METHODS: We isolated rabbit bone marrow MSCs (rBM-MSCs) from two skeletally mature New Zealand white rabbits and stablished the optimum culture condition to expand them. Then, they were seeded over non-coated and FN-coated scaffolds and cultured in chondrogenic conditions. To evaluate cell functionality, we performed an MTS assay to compare cell proliferation between both conditions. Finally, a histologic study was performed to assess extracellular matrix (ECM) production in both samples, and to compare them with the ones obtained with rabbit chondrocytes (rCHs) seeded in a non-coated scaffold. RESULTS: A culture protocol based on low FBS concentration was set as the best for rBM-MSCs expansion. The MTS assay revealed that rBM-MSCs seeded on FN-coated scaffolds have more cells on proliferation (145%; 95% CI: 107%-182%) compared with rBM-MSCs seeded on non-coated scaffolds. Finally, the histologic study demonstrated that rCHs seeded on non-coated scaffolds displayed the highest production of ECM, followed by rBM-MSCs seeded on FN-coated scaffolds. Furthermore, both cell types produced a comparable ECM pattern. CONCLUSION: These results suggest that MSCs have low capacity attachment to PCL-PU scaffolds, but the presence of integrin alpha5beta1 (FN-receptor) in MSCs allows them to interact with the FN-coated scaffolds. These results could be applied in the design of scaffolds, and might have important clinical implications in orthopaedic surgery of meniscal injuries.

[Andrographolide-releasing collagen scaffold enhance the ability of chondrocytes to maintain their specific phenotype under inflammatory environment in vitro].

The aim of this article is to study how andrographolide-releasing collagen scaffolds influence rabbit articular chondrocytes in maintaining their specific phenotype under inflammatory environment. Physical blending combined with vacuum freeze-drying method was utilized to prepare the andrographolide-releasing collagen scaffold. The characteristics of scaffold including its surface morphology and porosity were detected with environmental scanning electron microscope (ESEM) and a density instrument. Then, the release of andrographolide from prepared scaffolds was measured by UV-visible spectroscopy. Rabbit chondrocytes were isolated and cultured in vitro and seeded on andrographolide-releasing collagen scaffolds. Following culture with normal medium for 3 d, seeded chondrocytes were cultured with medium containing interleukin-1 beta (IL-1ß) to stimulate inflammation in vitro for 7 d. The proliferation, morphology and gene transcription of tested chondrocytes were detected with Alamar Blue assay, fluorescein diacetate (FDA) staining and reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) test respectively. The results showed that the collagen scaffolds prepared by vacuum freeze-dry possess a high porosity close to 96%, and well-interconnected chambers around (120.7±17.8) µm. The andrographolide-releasing collagen scaffold continuously released andrographolide to the PBS solution within 15 d, and collagen scaffolds containing 2.22% andrographolide significantly inhibit the proliferation of chondrocytes. Compared with collagen scaffolds, 0.44% andrographolide-containing collagen scaffolds facilitate chondrocytes to keep specific normal morphologies following 7 d IL-1ß induction. The results obtained by RT-qPCR confirmed this effect by enhancing the transcription of tissue inhibitor of metalloproteinase-1 ( TIMP-1), collagen II ( COL II), aggrecan ( Aggrecan) and the ratio of COL II/ collagen I( COL I), meanwhile, reversing the promoted transcription of matrix metalloproteinase-1 ( MMP-1) and matrix metalloproteinase-13 ( MMP-13). In conclusion, our research reveals that andrographolide-releasing (0.44%) collagen scaffolds enhance the ability of chondrocytes to maintain their specific morphologies by up-regulating the transcription of genes like COL II, Aggrecan and TIMP-1, while down-regulating the transcription of genes like MMP-1 and MMP-13 which are bad for phenotypic maintenance under IL-1ß simulated inflammatory environment. These results implied the potential use of andrographolide-releasing collagen scaffold in osteoarthritic cartilage repair.

Andrographolide-releasing collagen scaffold enhance the ability of chondrocytes to maintain their specific phenotype under inflammatory environment / 生物医学工程学杂志

The aim of this article is to study how andrographolide-releasing collagen scaffolds influence rabbit articular chondrocytes in maintaining their specific phenotype under inflammatory environment. Physical blending combined with vacuum freeze-drying method was utilized to prepare the andrographolide-releasing collagen scaffold. The characteristics of scaffold including its surface morphology and porosity were detected with environmental scanning electron microscope (ESEM) and a density instrument. Then, the release of andrographolide from prepared scaffolds was measured by UV-visible spectroscopy. Rabbit chondrocytes were isolated and cultured and seeded on andrographolide-releasing collagen scaffolds. Following culture with normal medium for 3 d, seeded chondrocytes were cultured with medium containing interleukin-1 beta (IL-1β) to stimulate inflammation for 7 d. The proliferation, morphology and gene transcription of tested chondrocytes were detected with Alamar Blue assay, fluorescein diacetate (FDA) staining and reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) test respectively. The results showed that the collagen scaffolds prepared by vacuum freeze-dry possess a high porosity close to 96%, and well-interconnected chambers around (120.7±17.8) μm. The andrographolide-releasing collagen scaffold continuously released andrographolide to the PBS solution within 15 d, and collagen scaffolds containing 2.22% andrographolide significantly inhibit the proliferation of chondrocytes. Compared with collagen scaffolds, 0.44% andrographolide-containing collagen scaffolds facilitate chondrocytes to keep specific normal morphologies following 7 d IL-1β induction. The results obtained by RT-qPCR confirmed this effect by enhancing the transcription of tissue inhibitor of metalloproteinase-1 ( ), collagen II ( ), aggrecan ( ) and the ratio of / collagen I( ), meanwhile, reversing the promoted transcription of matrix metalloproteinase-1 ( ) and matrix metalloproteinase-13 ( ). In conclusion, our research reveals that andrographolide-releasing (0.44%) collagen scaffolds enhance the ability of chondrocytes to maintain their specific morphologies by up-regulating the transcription of genes like , and , while down-regulating the transcription of genes like and which are bad for phenotypic maintenance under IL-1β simulated inflammatory environment. These results implied the potential use of andrographolide-releasing collagen scaffold in osteoarthritic cartilage repair.

Xanthan gum protects rabbit articular chondrocytes against sodium nitroprusside-induced apoptosis in vitro.

We have previously reported that intra-articular injection of xanthan gum (XG) could significantly ameliorate the degree of joint cartilage degradation and pain in experimental osteoarthritis (OA) model in vivo. In this present study, we evaluated the protective effect of XG against Sodium nitroprusside (SNP)-induced rabbit articular chondrocytes apoptosis in vitro. Rabbit articular chondrocytes were incubated with various concentrations of XG for 24h prior to 0.5mmol/L SNP co-treatment for 24h. The proliferation of chondrocytes was analyzed using MTT assay. The chondrocytes early apoptosis rates were evaluated using Annexin V-FITC/PI flow cytometry. The morphology of apoptosis chondrocytes were observed by scanning electron microscopy (SEM). The loss/disruption of mitochondrial membrane potential was detected using rhodamin 123 by confocal microscope. The concentration of prostaglandin E2 (PGE2) in cell culture supernatants was evaluated using ELISA assay. The results showed that XG could significantly reverse SNP-reduced cell proliferation and inhibited cell early apoptosis rate in a dose-dependent manner. XG alleviated loss/disruption of mitochondrial membrane potential and decreased the PGE2 level of chondrocytes cell culture supernatants in SNP-induced chondrocytes. These results of the present research strongly suggest that XG can protect rabbit articular chondrocytes against SNP-induced apoptosis in vitro.

Culture of Rabbit Chondrocytes Using Chitosan Bead / 대한정형외과연구학회지

PURPOSE: To confirm the adhesion and matrix formation of chondrocytes which were cultured on chitosan beads and to elucidate the difference between the porous chitosan beads and non-porous chitsan beads as scaffold for chondrocytes. MATERIALS AND METHODS: Chondrocytes isolated from rabbit articular cartilage were cultured in vitro on porous and non-porous chitosan bead for 2 weeks. Histochemical (H&E stain, Toluidin blue stain) and scanning electromicroscopic approaches were used to compare the differences between two groups. RESULTS: In both groups, adhesion and proliferation of chondrocytes were observed on scanning electron microscopy. which were more active in the porous chitosan bead group. On histochemical staining with toluidine blue, the porous chitosan bead group showed stronger metachromasia than that of the non-porous chitosan bead. CONCLUSION: It is concluded that both chitosan beads could work as an effective scaffold for culturing chondrocytes, and that porous chitosan bead may be a better scaffold than non-porous chitosan bead because of cavities in former bead.