ZAKß is activated by cellular compression and mediates contraction-induced MAP kinase signaling in skeletal muscle.

Mechanical inputs give rise to p38 and JNK activation, which mediate adaptive physiological responses in various tissues. In skeletal muscle, contraction-induced p38 and JNK signaling ensure adaptation to exercise, muscle repair, and hypertrophy. However, the mechanisms by which muscle fibers sense mechanical load to activate this signaling have remained elusive. Here, we show that the upstream MAP3K ZAKß is activated by cellular compression induced by osmotic shock and cyclic compression in vitro, and muscle contraction in vivo. This function relies on ZAKß's ability to recognize stress fibers in cells and Z-discs in muscle fibers when mechanically perturbed. Consequently, ZAK-deficient mice present with skeletal muscle defects characterized by fibers with centralized nuclei and progressive adaptation towards a slower myosin profile. Our results highlight how cells in general respond to mechanical compressive load and how mechanical forces generated during muscle contraction are translated into MAP kinase signaling.

The Lysine Specific Demethylase-1 Negatively Regulates the COL9A1 Gene in Human Articular Chondrocytes.

Osteoarthritis (OA) is a degenerative disease of the joints which is associated with an impaired production of the cartilage matrix by the chondrocytes. Here, we investigated the role of Lysine-Specific Demethylase-1 (LSD1), a chromatin remodeling enzyme whose role in articular chondrocytes was previously associated with a catabolic activity and which is potentially involved during OA. Following a loss of function strategy and RNA sequencing analysis, we detail the genes which are targeted by LSD1 in human articular chondrocytes and identify COL9A1, a gene encoding the α1 chain of the cartilage-specific type IX collagen, as negatively regulated by LSD1. We show that LSD1 interacts with the transcription factor SOX9 and is recruited to the promoter of COL9A1. Interestingly, we observe that OA cartilage displays stronger LSD1 immunostaining compared with normal, and we demonstrate that the depletion of LSD1 in OA chondrocytes prevents the decrease in COL9A1 following Il-1ß treatment. These results suggest LSD1 is a new regulator of the anabolic activity of articular chondrocytes potentially destabilizing the cartilage matrix, since it negatively regulates COL9A1, a gene encoding a crucial anchoring collagen molecule. This newly identified role played by LSD1 may thus participate in the alteration of the cartilage matrix during OA.

Improvement of the Chondrocyte-Specific Phenotype upon Equine Bone Marrow Mesenchymal Stem Cell Differentiation: Influence of Culture Time, Transforming Growth Factors and Type I Collagen siRNAs on the Differentiation Index.

Articular cartilage is a tissue characterized by its poor intrinsic capacity for self-repair. This tissue is frequently altered upon trauma or in osteoarthritis (OA), a degenerative disease that is currently incurable. Similar musculoskeletal disorders also affect horses and OA incurs considerable economic loss for the equine sector. In the view to develop new therapies for humans and horses, significant progress in tissue engineering has led to the emergence of new generations of cartilage therapy. Matrix-associated autologous chondrocyte implantation is an advanced 3D cell-based therapy that holds promise for cartilage repair. This study aims to improve the autologous chondrocyte implantation technique by using equine mesenchymal stem cells (MSCs) from bone marrow differentiated into chondrocytes that can be implanted in the chondral lesion. The optimized protocol relies on culture under hypoxia within type I/III collagen sponges. Here, we explored three parameters that influence MSC differentiation: culture times, growth factors and RNA interference strategies. Our results suggest first that an increase in culture time from 14 to 28 or 42 days lead to a sharp increase in the expression of chondrocyte markers, notably type II collagen (especially the IIB isoform), along with a concomitant decrease in HtrA1 expression. Nevertheless, the expression of type I collagen also increased with longer culture times. Second, regarding the growth factor cocktail, TGF-ß3 alone showed promising result but the previously tested association of BMP-2 and TGF-ß1 better limits the expression of type I collagen. Third, RNA interference targeting Col1a2 as well as Col1a1 mRNA led to a more significant knockdown, compared with a conventional strategy targeting Col1a1 alone. This chondrogenic differentiation strategy showed a strong increase in the Col2a1:Col1a1 mRNA ratio in the chondrocytes derived from equine bone marrow MSCs, this ratio being considered as an index of the functionality of cartilage. These data provide evidence of a more stable chondrocyte phenotype when combining Col1a1 and Col1a2 siRNAs associated to a longer culture time in the presence of BMP-2 and TGF-ß1, opening new opportunities for preclinical trials in the horse. In addition, because the horse is an excellent model for human articular cartilage disorders, the equine therapeutic approach developed here can also serve as a preclinical step for human medicine.

Cartilage tissue engineering: molecular control of chondrocyte differentiation for proper cartilage matrix reconstruction.

BACKGROUND: Articular cartilage defects are a veritable therapeutic problem because therapeutic options are very scarce. Due to the poor self-regeneration capacity of cartilage, minor cartilage defects often lead to osteoarthritis. Several surgical strategies have been developed to repair damaged cartilage. Autologous chondrocyte implantation (ACI) gives encouraging results, but this cell-based therapy involves a step of chondrocyte expansion in a monolayer, which results in the loss in the differentiated phenotype. Thus, despite improvement in the quality of life for patients, reconstructed cartilage is in fact fibrocartilage. Successful ACI, according to the particular physiology of chondrocytes in vitro, requires active and phenotypically stabilized chondrocytes. SCOPE OF REVIEW: This review describes the unique physiology of cartilage, with the factors involved in its formation, stabilization and degradation. Then, we focus on some of the most recent advances in cell therapy and tissue engineering that open up interesting perspectives for maintaining or obtaining the chondrogenic character of cells in order to treat cartilage lesions. MAJOR CONCLUSIONS: Current research involves the use of chondrocytes or progenitor stem cells, associated with "smart" biomaterials and growth factors. Other influential factors, such as cell sources, oxygen pressure and mechanical strain are considered, as are recent developments in gene therapy to control the chondrocyte differentiation/dedifferentiation process. GENERAL SIGNIFICANCE: This review provides new information on the mechanisms regulating the state of differentiation of chondrocytes and the chondrogenesis of mesenchymal stem cells that will lead to the development of new restorative cell therapy approaches in humans. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

The diazirine-mediated photo-crosslinking of collagen improves biomaterial mechanical properties and cellular interactions.

In tissue engineering, crosslinking with carbodiimides such as EDC is omnipresent to improve the mechanical properties of biomaterials. However, in collagen biomaterials, EDC reacts with glutamate or aspartate residues, inactivating the binding sites for cellular receptors and rendering collagen inert to many cell types. In this work, we have developed a crosslinking method that ameliorates the rigidity, stability, and degradation rate of collagen biomaterials, whilst retaining key interactions between cells and the native collagen sequence. Our approach relies on the UV-triggered reaction of diazirine groups grafted on lysines, leaving critical amino acid residues intact. Notably, GxxGER recognition motifs for collagen-binding integrins, ablated by EDC crosslinking, were left unreacted, enabling cell attachment, spreading, and colonization on films and porous scaffolds. In addition, our procedure conserves the architecture of biomaterials, improves their resistance to collagenase and cellular contraction, and yields material stiffness akin to that obtained with EDC. Importantly, diazirine-crosslinked collagen can host mesenchymal stem cells, highlighting its strong potential as a substrate for tissue repair. We have therefore established a new crosslinking strategy to modulate the mechanical features of collagen porous scaffolds without altering its biological properties, thereby offering an advantageous alternative to carbodiimide treatment. STATEMENT OF SIGNIFICANCE: This article describes an approach to improve the mechanical properties of collagen porous scaffolds, without impacting collagen's natural interactions with cells. This is significant because collagen crosslinking is overwhelmingly performed using carbodiimides, which results in a critical loss of cellular affinity. By contrast, our method leaves key cellular binding sites in the collagen sequence intact, enabling cell-biomaterial interactions. It relies on the fast, UV-triggered reaction of diazirine with collagen, and does not produce toxic by-products. It also supports the culture of mesenchymal stem cells, a pivotal cell type in a wide range of tissue repair applications. Overall, our approach offers an attractive option for the crosslinking of collagen, a prominent material in the growing field of tissue engineering.

Challenges in Nasal Cartilage Tissue Engineering to Restore the Shape and Function of the Nose.

The repair of nasal septal cartilage is a key challenge in cosmetic and functional surgery of the nose, as it determines its shape and its respiratory function. Supporting the dorsum of the nose is essential for both the prevention of nasal obstruction and the restoration of the nose structure. Most surgical procedures to repair or modify the nasal septum focus on restoring the external aspect of the nose by placing a graft under the skin, without considering respiratory concerns. Tissue engineering offers a more satisfactory approach, in which both the structural and biological roles of the nose are restored. To achieve this goal, nasal cartilage engineering research has led to the development of scaffolds capable of accommodating cartilaginous extracellular matrix-producing cells, possessing mechanical properties close to those of the nasal septum, and retaining their structure after implantation in vivo. The combination of a non-resorbable core structure with suitable mechanical properties and a biocompatible hydrogel loaded with autologous chondrocytes or mesenchymal stem cells is a promising strategy. However, the stability and immunotolerance of these implants are crucial parameters to be monitored over the long term after in vivo implantation, to definitively assess the success of nasal cartilage tissue engineering. Here, we review the tissue engineering methods to repair nasal cartilage, focusing on the type and mechanical characteristics of the biomaterials; cell and implantation strategy; and the outcome with regard to cartilage repair.

The epigenetic players and the chromatin marks involved in the articular cartilage during osteoarthritis.

Epigenetics defines the modifications of the genome that do not involve a change in the nucleotide sequence of DNA. These modifications constitute a mechanism of gene regulation poorly explored in the context of cartilage physiology. They are now intensively studied by the scientific community working on articular cartilage and its related pathology such as osteoarthritis. Indeed, epigenetic regulations can control the expression of crucial gene in the chondrocytes, the only resident cells of cartilage. Some epigenetic changes are considered as a possible cause of the abnormal gene expression and the subsequent alteration of the chondrocyte phenotype (hypertrophy, proliferation, senescence…) as observed in osteoarthritic cartilage. Osteoarthritis is a joint pathology, which results in impaired extracellular matrix homeostasis and leads ultimately to the progressive destruction of cartilage. To date, there is no pharmacological treatment and the exact causes have yet to be defined. Given that the epigenetic modifying enzymes can be controlled by pharmacological inhibitors, it is thus crucial to describe the epigenetic marks that enable the normal expression of extracellular matrix encoding genes, and those associated with the abnormal gene expression such as degradative enzyme or inflammatory cytokines encoding genes. In this review, only the DNA methylation and histone modifications will be detailed with regard to normal and osteoarthritic cartilage. Although frequently referred as epigenetic mechanisms, the regulatory mechanisms involving microRNAs will not be discussed. Altogether, this review will show how this nascent field influences our understanding of the pathogenesis of OA in terms of diagnosis and how controlling the epigenetic marks can help defining epigenetic therapies.

Presence of type IIB procollagen in mouse articular cartilage and growth plate is revealed by immuno-histochemical analysis with a novel specific antibody.

Type II collagen is the major fibrillar collagen in cartilage. It is synthesized in the form of precursors (procollagens) containing N- and C-terminal propeptides. The two main isoforms of type II procollagen protein are type IIA and type IIB procollagens, generated in a developmentally regulated manner by differential splicing of the primary gene transcript. Isoform IIA contains exon 2 and is produced mainly by chondroprogenitor cells while isoform IIB lacks exon 2 and is produced by differentiated chondrocytes. Thus, expression of IIA and IIB isoforms are reliable markers for identifying the differentiation status of chondrocytes but their biological function in the context of skeletal development is still not yet fully understood. Specific antibodies against IIA and IIB procollagen isoforms are already available. In this study, a synthetic peptide spanning the junction between exon 1 and exon 3 of the murine sequence was used as an immunogen to generate a novel rabbit polyclonal antibody directed against procollagen IIB. Characterization of this antibody by Western-blotting analysis of murine cartilage extracts and ELISA tests demonstrated its specificity to the type IIB isoform. Furthermore, by immunohistochemical studies, this antibody allowed the detection of procollagen IIB in embryonic cartilage as well as in articular cartilage and growth plate of young adult mice. Interestingly, this is the first antibody that has allowed the detection of procollagen IIB at both the intra- and extracellular level. This antibody therefore represents an interesting new tool for monitoring the spatial and temporal distribution of IIB isoforms in skeletal tissues of mouse models and for tracking the trafficking and processing of type IIB procollagen.

Biomaterial functionalization with triple-helical peptides for tissue engineering.

In the growing field of tissue engineering, providing cells in biomaterials with the adequate biological cues represents an increasingly important challenge. Yet, biomaterials with excellent mechanical properties are often biologically inert to many cell types. To address this issue, researchers resort to functionalization, i.e. the surface modification of a biomaterial with active molecules or substances. Functionalization notably aims to replicate the native cellular microenvironment provided by the extracellular matrix, and in particular by collagen, its major component. As our understanding of biological processes regulating cell behavior increases, functionalization with biomolecules binding cell surface receptors constitutes a promising strategy. Among these, triple-helical peptides (THPs) that reproduce the architectural and biological properties of collagen are especially attractive. Indeed, THPs containing binding sites from the native collagen sequence have successfully been used to guide cell response by establishing cell-biomaterial interactions. Notably, the GFOGER motif recognizing the collagen-binding integrins is extensively employed as a cell adhesive peptide. In biomaterials, THPs efficiently improved cell adhesion, differentiation and function on biomaterials designed for tissue repair (especially for bone, cartilage and heart), vascular graft fabrication, wound dressing, drug delivery or immunomodulation. This review describes the key characteristics of THPs, their effect on cells when combined to biomaterials and their strong potential as biomimetic tools for regenerative medicine. STATEMENT OF SIGNIFICANCE: This review article describes how triple-helical peptides constitute efficient tools to improve cell-biomaterial interactions in tissue engineering. Triple helical peptides are bioactive molecules that mimic the architectural and biological properties of collagen. They have been successfully used to specifically recognize cell-surface receptors and provide cells seeded on biomaterials with controlled biological cues. Functionalization with triple-helical peptides has enabled researchers to improve cell function for regenerative medicine applications, such as tissue repair. However, despite encouraging results, this approach remains limited and under-exploited, and most functionalization strategies reported in the literature rely on biomolecules that are unable to address collagen-binding receptors. This review will assist researchers in selecting the correct tools to functionalize biomaterials, in efforts to guide cellular response.

Identification of four alternatively spliced transcripts of the Ucma/GRP gene, encoding a new Gla-containing protein.

The Ucma protein (Upper zone of growth plate and cartilage matrix associated protein) has recently been described as a novel secretory protein mainly expressed in cartilage and also as a novel vitamin-K-dependent protein named GRP (Gla-rich protein). This protein has the highest Gla content of any protein known to date. In this article, we identify four alternatively spliced variants of Ucma/GRP gene transcripts in mouse chondrocytes. We show that the expression of all four isoforms is associated with the early stages of chondrogenesis. The Ucma/GRP gene encodes four proteins named Ucma/GRP-F1, -F2, -F3, and -F4, which differ by exon 2, exon 4, or both. Among them, only Ucma/GRP-F1 and -F3 were secreted into the culture medium of transfected chondrocytes, while Ucma/GRP-F2 and -F4 accumulated in the cells. Using HeLa cells or freshly isolated embryonic mouse chondrocytes transfected with enhanced green fluorescent protein fusions, microscopy analysis revealed that Ucma/GRP-F1 and -F3 were localized in the Golgi complex, whereas Ucma/GRP-F2 and -F4 formed aggregates. This aggregation was microtubule-dependent since disruption of microtubules with nocodazole reduced Ucma/GRP-F2 and -F4 aggregation in a reversible manner. Using biochemical fractionation and Western blot analysis, Ucma/GRP-F1 and -F3 isoforms were detected in the soluble fraction while Ucma/GRP-F2 and -F4 were found in an insoluble-enriched fraction. We conclude that the co-expression of soluble and insoluble isoforms also Gla-rich and Gla-deleted isoforms may be finely tuned. Imbalance in isoform expression may therefore be involved in skeletal pathology.

Analysis of collagen expression during chondrogenic induction of human bone marrow mesenchymal stem cells.

Adult mesenchymal stem cells (MSCs) are currently being investigated as an alternative to chondrocytes for repairing cartilage defects. As several collagen types participate in the formation of cartilage-specific extracellular matrix, we have investigated their gene expression levels during MSC chondrogenic induction. Bone marrow MSCs were cultured in pellet in the presence of BMP-2 and TGF-ß3 for 24 days. After addition of FGF-2, at the fourth passage during MSC expansion, there was an enhancing effect on specific cartilage gene expression when compared to that without FGF-2 at day 12 in pellet culture. A switch in expression from the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of the collagen type II gene was observed at day 24. A short-term addition of FGF-2 followed by a treatment with BMP-2/TGF-ß3 appears sufficient to accelerate chondrogenesis with a particular effect on the main cartilage collagens.

Repair of full-thickness articular cartilage defects using IEIK13 self-assembling peptide hydrogel in a non-human primate model.

Articular cartilage is built by chondrocytes which become less active with age. This declining function of the chondrocytes, together with the avascular nature of the cartilage, impedes the spontaneous healing of chondral injuries. These lesions can progress to more serious degenerative articular conditions as in the case of osteoarthritis. As no efficient cure for cartilage lesions exist yet, cartilage tissue engineering has emerged as a promising method aiming at repairing joint defects and restoring articular function. In the present work, we investigated if a new self-assembling peptide (referred as IEIK13), combined with articular chondrocytes treated with a chondrogenic cocktail (BMP-2, insulin and T3, designated BIT) could be efficient to restore full-thickness cartilage defects induced in the femoral condyles of a non-human primate model, the cynomolgus monkey. First, in vitro molecular studies indicated that IEIK13 was efficient to support production of cartilage by monkey articular chondrocytes treated with BIT. In vivo, cartilage implant integration was monitored non-invasively by contrast-enhanced micro-computed tomography, and then by post-mortem histological analysis and immunohistochemical staining of the condyles collected 3 months post-implantation. Our results revealed that the full-thickness cartilage injuries treated with either IEIK13 implants loaded with or devoid of chondrocytes showed similar cartilage-characteristic regeneration. This pilot study demonstrates that IEIK13 can be used as a valuable scaffold to support the in vitro activity of articular chondrocytes and the repair of articular cartilage defects, when implanted alone or with chondrocytes.

Stable subclones of the chondrogenic murine cell line MC615 mimic distinct stages of chondrocyte differentiation.

Fourteen stable subclones derived from the murine chondrogenic cell line MC615 were established and characterised regarding their differentiation stages and responsivity to BMP2. Based on their gene expression profiles which revealed remarkable variances in Col2a1 and Col10a1 expression, subclones could be grouped into at least three distinct categories. Three representative subclones (4C3, 4C6 and 4H4) were further characterised with respect to gene expression pattern and differentiation capacity. These subclones resembled (i) weakly differentiated chondrogenic precursors, strongly responding to BMP2 stimulation (4C3), (ii) collagen II expressing chondrocytes which could be induced to undergo maturation (4C6) and (iii) mature chondrocytes expressing Col10a1 and other markers of hypertrophy (4H4). Interestingly, BMP2 administration caused Smad protein phosphorylation and stimulated Col10a1 expression in all clones, but induced Col2a1 expression only in precursor-like cells. Most remarkably, these clones maintained a stable gene expression profile at least until the 30th passage of subconfluent culture, but revealed reproducible changes in gene expression and differentiation pattern in long term high density cultures. Thus, the newly established MC615 subclones may serve as a potent new tool for investigations on the regulation of chondrocyte differentiation and function.

Phenotypic Identification of Dental Pulp Mesenchymal Stem/Stromal Cells Subpopulations with Multiparametric Flow Cytometry.

Dental pulp (DP) is a specialized, highly vascularized, and innervated connective tissue mainly composed of undifferentiated mesenchymal cells, fibroblasts, and highly differentiated dentin-forming odontoblasts. Undifferentiated mesenchymal cells include stem/stromal cell populations usually called dental pulp mesenchymal stem cells (DP-MSCs) which differ in their self-renewal properties, lineage commitment, and differentiation capabilities. Analysis of surface antigens has been largely used to precisely identify these DP-MSC populations. However, a major difficulty is that these antigens are actually not specific for MSCs. Most of the markers used are indeed shared by other cell populations such as progenitor cells, mature fibroblasts, and/or perivascular cells. Accordingly, the detection of only one of these markers in a cell population is clearly insufficient to determine its stemness. Recent data reported that multiparametric flow cytometry, by allowing for the detection of several molecules on the surface of one single cell, is a powerful tool to elucidate the phenotype of a cell population both in vivo and in vitro. So far, DP-MSC populations have been characterized mainly based on the isolated expression of molecules known to be expressed by stem cells, such as Stro-1 antigen, melanoma cell adhesion molecule MCAM/CD146, low-affinity nerve growth factor receptor p75NTR/CD271, and the mesenchymal stem cell antigen MSCA-1. Using multiparametric flow cytometry, we recently showed that human DP-MSCs are indeed phenotypically heterogeneous and form several populations.The present paper describes the multiparametric flow cytometry protocol we routinely use for characterizing DP-MSCs. The description includes the design of the antibody panel and explains the selection of the different parameters related to the data quality control.

Evaluation of the biocompatibility and stability of allogeneic tissue-engineered cartilage in humanized mice.

Articular cartilage (AC) has poor capacities of regeneration and lesions often lead to osteoarthritis. Current AC reconstruction implies autologous chondrocyte implantation which requires tissue sampling and grafting. An alternative approach would be to use scaffolds containing off-the-shelf allogeneic human articular chondrocytes (HACs). To investigate tolerance of allogeneic HACs by the human immune system, we developed a humanized mouse model implanted with allogeneic cartilage constructs generated in vitro. A prerequisite of the study was to identify a scaffold that would not provoke inflammatory reaction in host. Therefore, we first compared the response of hu-mice to two biomaterials used in regenerative medicine, collagen sponge and agarose hydrogel. Four weeks after implantation in hu-mice, acellular collagen sponges, but not acellular agarose hydrogels, showed positive staining for CD3 (T lymphocytes) and CD68 (macrophages), suggesting that collagen scaffold elicits weak inflammatory reaction. These data led us to deepen our evaluation of the biocompatibility of allogeneic tissue-engineered cartilage by using agarose as scaffold. Agarose hydrogels were combined with allogeneic HACs to reconstruct cartilage in vitro. Particular attention was paid to HLA-A2 compatibility between HACs to be grafted and immune human cells of hu-mice: HLA-A2+ or HLA-A2- HACs agarose hydrogels were cultured in the presence of a chondrogenic cocktail and implanted in HLA-A2+ hu-mice. After four weeks implantation and regardless of the HLA-A2 phenotype, chondrocytes were well-differentiated and produced cartilage matrix in agarose. In addition, no sign of T-cell or macrophage infiltration was seen in the cartilaginous constructs and no significant increase in subpopulations of T lymphocytes and monocytes was detected in peripheral blood and spleen. We show for the first time that humanized mouse represents a useful model to investigate human immune responsiveness to tissue-engineered cartilage and our data together indicate that allogeneic cartilage constructs can be suitable for cartilage engineering.

Combination of bioactive factors and IEIK13 self-assembling peptide hydrogel promotes cartilage matrix production by human nasal chondrocytes.

Nasal reconstruction remains a challenge for every reconstructive surgeon. Alloplastic implants are proposed to repair nasal cartilaginous defects but they are often associated with high rates of extrusion and infection and poor biocompatibility. In this context, a porous polymeric scaffold filled with an autologous cartilage gel would be advantageous. In this study, we evaluated the capacity of IEIK13 self-assembling peptide (SAP) to serve as support to form such cartilage gel. Human nasal chondrocytes (HNC) were first amplified with FGF-2 and insulin, and then redifferentiated in IEIK13 with BMP-2, insulin, and T3 (BIT). Our results demonstrate that IEIK13 fosters HNC growth and survival. HNC phenotype was assessed by RT-PCR analysis and neo-synthesized extracellular matrix was characterized by western blotting and immunohistochemistry analysis. BIT-treated cells embedded in IEIK13 displayed round morphology and expressed cartilage-specific markers such as type II and type IX collagens and aggrecan. In addition, we did not detect significant production of type I and type X collagens and gene products of dedifferentiated and hypertrophic chondrocytes that are unwanted in hyaline cartilage. The whole of these results indicates that the SAP IEIK13 represents a suitable support for hydrogel-based tissue engineering of nasal cartilage. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 893-903, 2019.

Design and characterization of a chitosan-enriched fibrin hydrogel for human dental pulp regeneration.

OBJECTIVE: Regenerating a functional dental pulp in the pulpectomized root canal has been recently proposed as a novel therapeutic strategy in dentistry. To reach this goal, designing an appropriate scaffold able to prevent the growth of residual endodontic bacteria, while supporting dental pulp tissue neoformation, is needed. Our aim was to create an innovative cellularized fibrin hydrogel supplemented with chitosan to confer this hydrogel antibacterial property. METHODS: Several fibrin-chitosan formulations were first screened by rheological analyses, and the most appropriate for clinical use was then studied in terms of microstructure (by scanning electron microscopy), antimicrobial effect (analysis of Enterococcus fæcalis growth), dental pulp-mesenchymal stem/stromal cell (DP-MSC) viability and spreading after 7 days of culture (LiveDead® test), DP-MSC ultrastructure and extracellular matrix deposition (transmission electron microscopy), and DP-MSC proliferation and collagen production (RT-qPCR and immunohistochemistry). RESULTS: A formulation associating 10mg/mL fibrinogen and 0.5% (w/w), 40% degree of acetylation, medium molar mass chitosan was found to be relevant in order to forming a fibrin-chitosan hydrogel at cytocompatible pH (# 7.2). Comparative analysis of fibrin-alone and fibrin-chitosan hydrogels revealed a potent antibacterial effect of the chitosan in the fibrin network, and similar DP-MSC viability, fibroblast-like morphology, proliferation rate and type I/III collagen production capacity. SIGNIFICANCE: These results indicate that incorporating chitosan within a fibrin hydrogel would be beneficial to promote human DP tissue neoformation thanks to chitosan antibacterial effect and the absence of significant detrimental effect of chitosan on dental pulp cell morphology, viability, proliferation and collagenous matrix production.

Characterization of Different Sources of Human MSCs Expanded in Serum-Free Conditions with Quantification of Chondrogenic Induction in 3D.

Mesenchymal stem cells (MSCs) represent alternative candidates to chondrocytes for cartilage engineering. However, it remains difficult to identify the ideal source of MSCs for cartilage repair since conditions supporting chondrogenic induction are diverse among published works. In this study, we characterized and evaluated the chondrogenic potential of MSCs from bone marrow (BM), Wharton's jelly (WJ), dental pulp (DP), and adipose tissue (AT) isolated and cultivated under serum-free conditions. BM-, WJ-, DP-, and AT-MSCs did not differ in terms of viability, clonogenicity, and proliferation. By an extensive polychromatic flow cytometry analysis, we found notable differences in markers of the osteochondrogenic lineage between the 4 MSC sources. We then evaluated their chondrogenic potential in a micromass culture model, and only BM-MSCs showed chondrogenic conversion. This chondrogenic differentiation was specifically ascertained by the production of procollagen IIB, the only type II collagen isoform synthesized by well-differentiated chondrocytes. As a pilot study toward cartilage engineering, we encapsulated BM-MSCs in hydrogel and developed an original method to evaluate their chondrogenic conversion by flow cytometry analysis, after release of the cells from the hydrogel. This allowed the simultaneous quantification of procollagen IIB and α10, a subunit of a type II collagen receptor crucial for proper cartilage development. This work represents the first comparison of detailed immunophenotypic analysis and chondrogenic differentiation potential of human BM-, WJ-, DP-, and AT-MSCs performed under the same serum-free conditions, from their isolation to their induction. Our study, achieved in conditions compliant with clinical applications, highlights that BM-MSCs are good candidates for cartilage engineering.

Molecular analysis of chondrocytes cultured in agarose in response to dynamic compression.

BACKGROUND: Articular cartilage is exposed to high mechanical loads under normal physiological conditions and articular chondrocytes regulate the composition of cartilaginous matrix, in response to mechanical signals. However, the intracellular pathways involved in mechanotransduction are still being defined. Using the well-characterized chondrocyte/agarose model system and dynamic compression, we report protocols for preparing and characterizing constructs of murine chondrocytes and agarose, and analyzing the effect of compression on steady-state level of mRNA by RT-PCR, gene transcription by gene reporter assay, and phosphorylation state of signalling molecules by Western-blotting. The mouse model is of particular interest because of the availability of a large choice of bio-molecular tools suitable to study it, as well as genetically modified mice. RESULTS: Chondrocytes cultured in agarose for one week were surrounded by a newly synthesized pericellular matrix, as revealed by immunohistochemistry prior to compression experiments. This observation indicates that this model system is suitable to study the role of matrix molecules and trans-membrane receptors in cellular responsiveness to mechanical stress. The chondrocyte/agarose constructs were then submitted to dynamic compression with FX-4000C Flexercell Compression Plus System (Flexcell). After clearing proteins off agarose, Western-blotting analysis showed transient activation of Mitogen-activated protein kinases (MAPK) in response to dynamic compression. After assessment by capillary electrophoresis of the quality of RNA extracted from agarose, steady-state levels of mRNA expression was measured by real time PCR. We observed an up-regulation of cFos and cJun mRNA levels as a response to compression, in accordance with the mechanosensitive character observed for these two genes in other studies using cartilage explants submitted to compression. To explore further the biological response of mouse chondrocytes to the dynamic compression at the transcriptional level, we also developed an approach for monitoring changes in gene transcription in agarose culture by using reporter promoter constructs. A decrease in promoter activity of the gene coding for type II procollagen, the most abundant protein in cartilage, was observed in response to dynamic loading. CONCLUSION: The protocols developed here offer the possibility to perform an integrated analysis of the molecular mechanisms of mechanotransduction in chondrocytes, at the gene and protein level.

VEGF and VEGF receptors are differentially expressed in chondrocytes.

During long bone development, cartilage replacement by bone is governed in part by angiogenesis. Although it has been demonstrated that vascular endothelial growth factor (VEGF-A) is crucial during endochondral ossification, little is known about the involvement of the other VEGF family members. Thus, we examined the expression and production of these members on primary chondrocytes and ATDC5 chondrogenic cells. VEGF-A, VEGF-B, VEGF-C and VEGF-D were shown to be expressed and synthesized demonstrating that numerous angiogenic factors can be produced by chondrocytes. In ATDC5 VEGF-A, VEGF-B and VEGF-C were over-expressed in the presence of chondrogenic and bone morphogenetic protein (BMP)-2 treatment suggesting that these factors play an important role during chondrogenesis. In addition, neuropilin-1, VEGF receptor-2 and VEGF receptor-3 gene expression were observed with an increase in VEGF-R2 expression under chondrogenic and BMP-2 treatment, suggesting that VEGF proteins could act in an autocrine/paracrine manner in addition to their angiogenic function. In conclusion, we demonstrated for the first time that chondrocytes secreted the four members of the VEGF family. We also showed that VEGF-B, VEGF-C and VEGF-D were secreted as processed proteins. The up-regulation of VEGF-B and VEGF-C at the mRNA and protein levels under chondrogenic stimulation strongly suggests a major role for these proteins in growth plate physiology.