The bioextrusion of mesenchymal stromal cells (MSCs) directly seeded in a bioink enables the production of three-dimensional (3D) constructs, promoting their chondrogenic differentiation. Our study aimed to evaluate the effect of different type I collagen concentrations in the bioink on MSCs' chondrogenic differentiation. We printed 3D constructs using an alginate, gelatin, and fibrinogen-based bioink cellularized with MSCs, with four different quantities of type I collagen addition (0.0, 0.5, 1.0, and 5.0 mg per bioink syringe). We assessed the influence of the bioprinting process, the bioink composition, and the growth factor (TGF-ꞵ1) on the MSCs' survival rate. We confirmed the biocompatibility of the process and the bioinks' cytocompatibility. We evaluated the chondrogenic effects of TGF-ꞵ1 and collagen addition on the MSCs' chondrogenic properties through macroscopic observation, shrinking ratio, reverse transcription polymerase chain reaction, glycosaminoglycan synthesis, histology, and type II collagen immunohistochemistry. The bioink containing 0.5 mg of collagen produces the richest hyaline-like extracellular matrix, presenting itself as a promising tool to recreate the superficial layer of hyaline cartilage. The bioink containing 5.0 mg of collagen enhances the synthesis of a calcified matrix, making it a good candidate for mimicking the calcified cartilaginous layer. Type I collagen thus allows the dose-dependent design of specific hyaline cartilage layers.

Urothelium is a highly specialized epithelium covering the entire urinary excretory system. Tissue engineering of this urinary tract may allow to consider its reconstruction to perform in vitro studies or in vivo replacement. Therefore, the question of specific reconstruction of the urothelium arises in order to guarantee the neotissue’s ability to act as a barrier against highly cytotoxic urine. This literature review describes the different cell types and strategies available for this reconstruction. The non-reconstruction of urothelium relies on the colonization of a biomaterial by the adjacent healthy tissue but allows only incomplete reconstruction and fibrosis. The use of autologous urothelial cells requires preliminary surgery and has not been successful enough in humans. Research has therefore focused on the use of stem cells. Adipose Derived Stem Cells (ADSCs) and Bone Marrow Derived Stem Cells (BMSCs) allow the reconstruction of the smooth muscle layer, but have little effect on urothelium reconstruction. Urine Derived Stem Cells (UDSCs) and Bladder mesenchymal Stem Cells (BSCs) are very promising because they allow the achievement of a differentiated urothelium. Induced Pluripotent Stem Cells (IPSCs) and Embryonic Stem Cells (ESCs) can be differentiated towards urothelial phenotype but their use is restricted by ethics.

Objective: The aim of this study is to investigate the changes of human dental pulp stem cell (hDPSC) viability, proliferation and osteogenic differentiation in high glucose condition. Design: After 21 days of culture in low (5.5 mM) and high (20 mM) glucose medium, hDPSC viability and proliferation were assessed with respectively the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Hoechst assays. To investigate the influence of glucose on osteogenic differentiation hDPSCs were cultured for 28 days in low or high glucose medium with osteoinductive cocktail. Mineralization was examined by alizarin red staining/quantification and the expression of osteogenic-related genes [Runt-related transcription factor 2 (RUNX2), Osteocalcin (OCN), Collagen 1A1 (COL1A1)] analyzed by RT-qPCR. Results: We observed no significant difference (p > 0.05) on hDPSC proliferation or cell viability between low or high glucose groups. We did not highlight a significant difference after alizarin red staining and quantification between hDPSCs cultured with high or low glucose concentration in the culture medium. In the same manner, high glucose concentration did not appear to modify osteogenic gene expression: there was no significant difference in osteogenic-related gene expression between high or low glucose groups. Conclusion: Proliferation, viability, and osteogenic differentiation of hDPSCs were not changed by high glucose environment.

Background MSCs isolated from bone marrow (BM-MSCs) have well-established chondrogenic potential, but MSCs derived from the synovial membrane (SM-MSCs) and synovial fluid (SF-MSCs) are thought to possess superior chondrogenicity. This study aimed to compare the in vitro immunophenotype and trilineage and chondrogenic potential of BM-MSCs to SM-MSCs and SF-MSCs. Methods MSCs were isolated from bone marrow (BM-MSCs), synovial membrane (SM-MSCs), and synovial fluid (SF-MSCs) extracted from the hips (BM) and knees (SM and SF) of advanced OA patients undergoing arthroplasty. Flow cytometric analysis was used at P2 to evaluate cell stemness. The trilinear differentiation test was performed at P2. At P3, MSC-seeded collagen sponges were cultured in chondrogenic medium for 28 days. Chondrogenic gene expression was quantified by qRT-PCR. Finally, the implants were stained to assess the deposition of proteoglycans and type II collagen. Results Despite variability, the immunophenotyping of BM-MSCs, SM-MSCs, and SF-MSCs was quite similar. All cell types were positive for the expression of stem cell markers and negative for exclusion markers. Additionally, chondrogenic differentiation and hypertrophy were more pronounced in BM-MSCs ( ACAN , SOX9 , COL2B , and COL10A ) than in SF-MSCs, with SM-MSCs having intermediate characteristics. Concerning matrix synthesis, the three cell types were equipotent in terms of GAG content, while BM-MSC ECM synthesis of type II collagen was superior. Conclusions Chondrogenic MSCs are easily collected from SM and SF in advanced human OA, but in vitro chondrogenesis that is superior to age-matched BM-MSCs should not be expected. However, due to intra-articular priming, SF-MSCs did not overexpress hypertrophic gene.

Despite the promising applications of PLGA based particles, studies examining the fate and consequences of these particles after intra-articular administration in the joint are scanty. This study was carried out to evaluate the neutrality of the unloaded delivery system on different articular cell types. To facilitate tracking, we have thus developed a fluorescent core of particles, combined to a hyaluronate shell for cell recognition.

Proteomics users enjoy the rapid development of LC-MS-based label-free relative quantification methods but in practice these remain restricted to mass spectrometers using electrospray ionization. Here, tools dedicated to ion chromatogram extraction, time alignment, signal normalization and statistical analysis were used to interpret label-free relative difference between primary human chondrocyte secretomes and dilutions thereof, analyzed successively by LC-MALDI. The analysis of secretomes diluted into culture medium demonstrated that abundant proteins could be relatively quantified within 1.5 20-fold changes with satisfactory statistics. In addition, comparison of multiple samples requires analyzing most samples in TOP mode only, saving considerable machine-time usage. The method allowed identification and quantification of most secreted proteins relevant to the chondrocyte phenotype and evidenced their up- or down regulations by TGF131 and patient-to-patient differential expression. Novel targets of TGFB1 were evidenced, such as pro-collagen C-proteinase enhancer protein 1, Metalloproteinase inhibitor 1, Fibulin-3, Tetranectin and Cartilage Intermediate Layer Protein 1, while others match previous findings. Several were verified by Western blot. This whole workflow is non-invasive, compatible with many cell culture protocols, technically straightforward and rapid, particularly regarding mass spectrometer time usage and could make label-free LC-MALDI analysis of low-complexity proteomes a major tool for routine cell culture characterization.Biological significanceThe present work presents the adaptation of label free relative protein quantification principles to LC-MALDI data to rapidly measure protein fold-changes between samples of relative complexity and its utility to characterize the secreted proteome of human primary chondrocytes. The method was employed to characterize the chondrocyte secretome regulation by TGF beta 1 and is proposed as a routine tool to assess the quality of biomaterials designed for cartilage repair and to quantitatively investigate the influence of environmental factors upon it. (C) 2014 The Authors. Published by Elsevier B.V.

Purpose: One of the major problems in treatment of osteoarticular diseases is to reach cells inside the matrix to provide drug. Indeed, cartilage is an avascular tissue with a few cells feed by diffusion through a dense protein network (collagens, glycosaminoglycans). In this work we have designed polymeric nanoparticles (NPs) of poly (D, L-lactic/glycolic acid)(PLGA) synthesized by a double emulsion method, which are biocompatible, biodegradable and can encapsulate water-soluble agents. Our NPs are labelled with BSA coupled to a fluorescent dye (Cyanine-3) to follow them by epifluorescent microscopy. As articular cells expressed CD44, one receptor of hyaluronic acid (HA)(a main component of synovial fluid), the nanoparticles are recovered with HA in order to enhance targeting of cells. Here, we have studied the internalization kinetics of "empty" nanoparticles, and we have evaluated their neutrality on chondrocytes matrix synthesis, mesenchymal stem cell (MSC) differentiation and inflammatory response. Innocuity has been also evaluated in healthy animals, after direct intraarticular injection of labelled nanoparticles (inflammatory response, Extracellular matrix integrity). Methods: Articular cells (chondrocytes, synoviocytes) and MSC are isolated from human donors, and cultured as primary culture. First, cells are exposed with 100 μg/mL of NPs from 2 to 12 hours. At the end of the kinetic immunofluorescence pictures with DAPI (nuclear staining) are realized to assess of the internalization of NPs, expression of inflammatory markers (IL1ß, TNFα and Cox2) are monitored by RT-qPCR analysis, and confirmed by PGE2 and nitrites measurement in supernatant. In other hand we evaluate, with pellets culture system, the effect of NPs exposition on extracellular matrix synthesis by chondrocytes, with RT-qPCR analysis of specific markers (Col2, Aggrecan and COMP), and by histological study of pellets (Alcian blue staining of proteoglycans, Sirius Red staining of collagen). Finally by growing MSC into 3 different differentiation media, we investigate if NPs pre-treatment can interfere with differentiation ability of MSC onto chondrogenic, adipogenic or osteogenic pathway. RT-qPCR assays for differentiation markers according to culture conditions and specific staining of lipid vesicles or calcium deposits, allow us to confirm the differentiation of cells. Intraarticular injections were realized in healthy rat's Knees. Structure of joint (synovium, cartilage, subchondral bone) was assessed by histological studies, performed at 7 and 10 days after injection (single and repeated). Results: For the different cell types, NPs are found into cytoplasm after 6 hours of exposition. Internalization of these NPs leads to an increase of inflammatory markers between 4 and 8 hours, basal level of expression being reached after 12 hours. Even if there is a weak increase of PGE2 and nitrites synthesis, that stay significantly lower than with LPS stimulation, our positive control of inflammation. NPs exposure, prior or after IL-1ß stimulation, does not aggravate the inflammatory response of these cells. When chondrocytes are exposed to NPs for 24 hours and then cultured in pellets for 28 days, there is no difference in matrix synthesis for the expression of mRNA and matrix deposition, as confirmed with histological exams. RT-qPCR and staining assays have also shown that despite a pretreatment with NPs, MSCs can be conducted onto adipogenic, osteogenic or chondrogenic differentiation pathways. Histological analyses of extracellular matrix integrity and inflammatory status do not demonstrate any differences for cartilage and subchondral bone structures but reveal a weak hyperplasia of synovial membrane, increasing with NPs concentration and the number of injection. Conclusions: These NPs are rapidly internalized by human articular cells, with only moderated and transient pro-inflammatory effects. In addition there is no side effects on (1) ECM synthesis by chondrocytes or MSC and (2) differentiation process due to the presence of NPs. Labelled NPs with Cya3, once injected in joint of healthy rat, do not lead to an inflammatory reaction and/or modification of extracellular matrix integrity. NPs are mainly found in synovium and repeated injections do not generate severe adverse reaction. This drug delivery system can be used to deliver an active molecule into the knee joint, thanks to the absence of side effects.