Human Nasal Chondrocytes Research Articles

Nasal Chondrocyte-Derived Soluble Factors Affect Chondrogenesis of Cocultured Mesenchymal Stem Cells.

To investigate the effect of soluble factors released from human nasal chondrocytes (NCs) on cocultured human bone marrow mesenchymal stem cells (MSCs) and NC tissue-engineered constructs. Cartilage engineered from pure NCs on a three-dimensional (3D) porous collagen scaffold was cultured indirectly in a Transwell system with cartilage engineered from a direct coculture of human bone marrow-derived MSCs and NCs on a 3D porous collagen scaffold. The soluble factors were measured in the conditioned media from the different chambers of the Transwell system. Engineered cartilage from cocultures exposed to the pure NC construct exhibited reduced chondrogenic potential relative to control constructs, shown by reduced extracellular matrix deposition and increased expression of hypertrophic markers. Analysis of the soluble factors within the conditioned media showed an increase in inflammatory cytokines in the coculture chamber exposed to the pure NC construct. Principal component analysis revealed that the majority of the data variance could be explained by proinflammatory factors and hypertrophic chondrogenesis. In conclusion, our data suggest that inflammatory cytokines derived from NCs reduce the chondrogenic potential of coculture engineered cartilage through the induction of hypertrophic chondrogenesis. Impact statement The use of engineered cartilage from cocultured nasal chondrocytes (NCs) and mesenchymal stem cells for nasal cartilage reconstruction may be problematic. Our data suggest that the soluble factors from surrounding native NCs in the cartilage to be fixed can compromise the quality of the engineered cartilage if used in reconstructive surgery.

Intra-individual comparison of human nasal chondrocytes and debrided knee chondrocytes: Relevance for engineering autologous cartilage grafts.

Implantation of autologous chondrocytes for cartilage repair requires harvesting of undamaged cartilage, implying an additional joint arthroscopy surgery and further damage to the articular surface. As alternative possible cell sources, in this study we assessed the proliferation and chondrogenic capacity of debrided Knee Chondrocytes (dKC) and Nasal Chondrocytes (NC) collected from the same patients. Matched NC and dKC pairs from 13 patients enrolled in two clinical studies (NCT01605201 and NCT026739059) were expanded in monolayer and then chondro-differentiated in 3D collagenous scaffolds in medium with or without Transforming Growth Factor beta 1 (TGFβ1). Cell proliferation and amount of cartilage matrix production by these two cell types were assessed. dKC exhibited an inferior proliferation rate than NC, and a lower capacity to chondro-differentiate. Resulting dKC-grafts contained lower amounts of cartilage specific matrix components glycosaminoglycans and type II collagen. The cartilage forming capacity of dKC did not significantly correlate with specific clinical parameters and was only partially improved by medium supplemention with TGFβ1. dKC exhibit a reproducibly poor capacity to engineer cartilage grafts. Our in vitro data suggest that NC would be a better suitable cell source for the generation of autologous cartilage grafts.

Interleukin IL-1β stimulates revitalization of cartilage matrix <i>in vitro</i> with human nasal chondrocytes

Revitalization of decellularized or devitalized matrix scaffolds in tracheal tissue engineering typically involves seeding the autologous recipient cells or allogeneic cells under long-term cultivation. Objective: to study the capability of human nasal chondrocytes for colonization of devitalized scaffolds based on native human tracheal cartilage, with proinflammatory stimulation (cytokine) by adding Interleukin-1-beta (IL-1β) to the culture medium. Materials and methods. Scaffolds for tracheal tissue engineering were obtained from native human tracheal cartilage through devitalization and laser etching. The scaffold was revitalized by seeding the human nasal chondrocytes. Histological examination was performed after staining with hematoxylin and safranin-O, with further microscopy using a Nikon Eclipse L200 light microscope. X-ray microtomography was performed on a Phoenix nanotom m apparatus. Electron microscopy was performed on a Nova NanoSEM 230 setup. Results . There was statistically significant increase in the intensity of colonization (p = 0.0008) with nasal chondrocytes and stimulation of their migration activity (p < 0.0001) in the presence of IL-1β compared with the control groups. Conclusion. Addition of proinflammatory cytokine IL-1β (1 μg/ml) to the culture medium enhances volumetric seeding of devitalized cartilage scaffold with human nasal chondrocytes, allowing to create highly revitalized materials for tracheal tissue engineering.

Rapid Cartilage Regeneration of Spheroids Composed of Human Nasal Septum-Derived Chondrocyte in Rat Osteochondral Defect Model.

Cell-based therapies have been studied for articular cartilage regeneration. Articular cartilage defects have little treatments because articular cartilage was limited regenerative capacity. Damaged articular cartilage is difficult to obtain a successful therapeutic effect. In additionally these articular cartilage defects often cause osteoarthritis. Chondrocyte implantation is a widely available therapy used for regeneration of articular cartilage because this tissue has poor repair capacity after injury. Human nasal septum-drived chondrocytes (hNCs) from the septum show greater proliferation ability and chondrogenic capacity than human articular chondrocytes (hACs), even across different donors with different ages. Moreover, the chondrogenic properties of hNCs can be maintained after extensive culture expansion. In this study, 2 dimensional (2D) monolayer cultured hNCs (hNCs-2D) and 3 dimensional (3D) spheroids cultured hNCs (hNCs-3D) were examined for chondrogenic capacity in vitro by PCR and immunofluorescence staining for chondrogenic marker, cell survival during cultured and for cartilage regeneration ability in vivo in a rat osteochondral defect model. hNCs-3D showed higher viability and more uniform morphology than 3D spheroids cultured hACs (hACs-3D) in culture. hNCs-3D also showed greater expression levels of the chondrocyte-specific marker Type II collagen (COL2A1) and sex-determining region Y (SRY)-box 9 (SOX9) than hNCs-2D. hNCs-3D also expressed chondrogenic markers in collagen. Specially, in the osteochondral defect model, implantation of hNCs-3D led to greater chondrogenic repair of focal cartilage defects in rats than implantation of hNCs-2D. These data suggest that hNCs-3D are valuable therapeutic agents for repair and regeneration of cartilage defects.

Evaluation of Polycaprolactone-Associated Human Nasal Chondrocytes as a Therapeutic Agent for Cartilage Repair

In this study, we manufactured a complex of human nasal septal cartilage (hNC) with polycaprolactone (PCL) for transplantation into cartilaginous skeletal defects and evaluated their characteristics. Nasal septum tissue was obtained from five patients aged ≥ 20years who were undergoing septoplasty. hNCs were isolated and subcultured for three passages in vitro. To formulate the cell-PCL complex, we used type I collagen as an adhesive between chondrocyte and PCL. Immunofluorescence staining, cell viability and growth in the hNC-PCL complex, and mycoplasma contamination were assessed. hNCs in PCL showed viability ≥ 70% and remained at these levels for 9h of incubation at 4°C. Immunostaining of the hNC-PCL complex also showed high expression levels of chondrocyte-specific protein, COL2A1, SOX9, and aggrecan during 24h of clinically applicable conditions. The hNC-PCL complex may be a valuable therapeutic agent for implantation into injured cartilage tissue, and can be used clinically to repair cartilaginous skeletal defects. From a clinical perspective, it is important to set the short duration of the implantation process to achieve effective functional implantation.

In Vivo Human Cartilage Formation in Three-Dimensional Bioprinted Constructs with a Novel Bacterial Nanocellulose Bioink.

Bacterial nanocellulose (BNC) is a 3D network of nanofibrils exhibiting excellent biocompatibility. Here, we present the aqueous counter collision (ACC) method of BNC disassembly to create bioink with suitable properties for cartilage-specific 3D-bioprinting. BNC was disentangled by ACC, and fibril characteristics were analyzed. Bioink printing fidelity and shear-thinning properties were evaluated. Cell-laden bioprinted grid constructs (5 × 5 × 1 mm3) containing human nasal chondrocytes (10 M mL-1) were implanted in nude mice and explanted after 30 and 60 days. Both ACC and hydrolysis resulted in significantly reduced fiber lengths, with ACC resulting in longer fibrils and fewer negative charges relative to hydrolysis. Moreover, ACC-BNC bioink showed outstanding printability, postprinting mechanical stability, and structural integrity. In vivo, cell-laden structures were rapidly integrated, maintained structural integrity, and showed chondrocyte proliferation, with 32.8 ± 13.8 cells per mm2 observed after 30 days and 85.6 ± 30.0 cells per mm2 at day 60 (p = 0.002). Furthermore, a full-thickness skin graft was attached and integrated completely on top of the 3D-bioprinted construct. The novel ACC disentanglement technique makes BNC biomaterial highly suitable for 3D-bioprinting and clinical translation, suggesting cell-laden 3D-bioprinted ACC-BNC as a promising solution for cartilage repair.

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.

Histological Image Processing for the Assessment of Tissue Engineered Cartilage

Abstract In a recently developed perfusion bioreactor, human nasal septum chondrocytes were seeded on decellularized porcine collagen scaffolds and cultivated for 6 weeks. Afterwards, the samples were stained with alcian blue and multiple histological microscope images were taken to assess the migration of the human cells and their redifferentiation. In this paper, we propose an image processing algorithm to objectively quantify the cultivation results. This algorithm performs a white balance, detects cell nuclei and their migration depth, and detects lacunae and their orientation. To evaluate performance, sensitivity and precision are calculated using manually labeled data over 50 images. The detection of cell nuclei has a reasonable precision (73%), but the level of sensitivity (42%) should be further improved. The proposed method demonstrates acceptable level of precision (83%) and sensitivity (86%) for the detection of lacunae.

Chondrogenic differentiation of human chondrocytes cultured in the absence of ascorbic acid.

Bioreactor systems will likely play a key role in establishing regulatory compliant and cost-effective production systems for manufacturing engineered tissue grafts for clinical applications. However, the automation of bioreactor systems could become considerably more complex and costly due to the requirements for additional storage and liquid handling technologies if unstable supplements are added to the culture medium. Ascorbic acid (AA) is a bioactive supplement that is commonly presumed to be essential for the generation of engineered cartilage tissues. However, AA can be rapidly oxidized and degraded. In this work, we addressed whether human nasal chondrocytes can redifferentiate, undergo chondrogenesis, and generate a cartilaginous extracellular matrix when cultured in the absence of AA. We found that when chondrocytes were cultured in 3D micromass pellets either with or without AA, there were no significant differences in their chondrogenic capacity in terms of gene expression or the amount of glycosaminoglycans. Moreover, 3D pellets cultured without AA contained abundant collagen Types II and I extracellular matrix. Although the amounts of Collagens II and I were significantly lower (34% and 50% lower) than in pellets cultured with AA, collagen fibers had similar thicknesses and distributions for both groups, as shown by scanning electron microscopy imaging. Despite the reduced amounts of collagen, if engineered cartilage grafts can be generated with sufficient properties that meet defined quality criteria without the use of unstable supplements such as AA, bioreactor automation requirements can be greatly simplified, thereby facilitating the development of more compact, user-friendly, and cost-effective bioreactor-based manufacturing systems.

Chondrocytes and stem cells in 3D-bioprinted structures create human cartilage in vivo.

Cartilage repair and replacement is a major challenge in plastic reconstructive surgery. The development of a process capable of creating a patient-specific cartilage framework would be a major breakthrough. Here, we described methods for creating human cartilage in vivo and quantitatively assessing the proliferative capacity and cartilage-formation ability in mono- and co-cultures of human chondrocytes and human mesenchymal stem cells in a three-dimensional (3D)-bioprinted hydrogel scaffold. The 3D-bioprinted constructs (5 × 5 × 1.2 mm) were produced using nanofibrillated cellulose and alginate in combination with human chondrocytes and human mesenchymal stem cells using a 3D-extrusion bioprinter. Immediately following bioprinting, the constructs were implanted subcutaneously on the back of 48 nude mice and explanted after 30 and 60 days, respectively, for morphological and immunohistochemical examination. During explantation, the constructs were easy to handle, and the majority had retained their macroscopic grid appearance. Constructs consisting of human nasal chondrocytes showed good proliferation ability, with 17.2% of the surface areas covered with proliferating chondrocytes after 60 days. In constructs comprising a mixture of chondrocytes and stem cells, an additional proliferative effect was observed involving chondrocyte production of glycosaminoglycans and type 2 collagen. This clinically highly relevant study revealed 3D bioprinting as a promising technology for the creation of human cartilage.

Laser surface modification of decellularized extracellular cartilage matrix for cartilage tissue engineering.

The implantation of autologous cartilage as the gold standard operative procedure for the reconstruction of cartilage defects in the head and neck region unfortunately implicates a variety of negative effects at the donor site. Tissue-engineered cartilage appears to be a promising alternative. However, due to the complex requirements, the optimal material is yet to be determined. As demonstrated previously, decellularized porcine cartilage (DECM) might be a good option to engineer vital cartilage. As the dense structure of DECM limits cellular infiltration, we investigated surface modifications of the scaffolds by carbon dioxide (CO2) and Er:YAG laser application to facilitate the migration of chondrocytes inside the scaffold. After laser treatment, the scaffolds were seeded with human nasal septal chondrocytes and analyzed with respect to cell migration and formation of new extracellular matrix proteins. Histology, immunohistochemistry, SEM, and TEM examination revealed an increase of the scaffolds' surface area with proliferation of cell numbers on the scaffolds for both laser types. The lack of cytotoxic effects was demonstrated by standard cytotoxicity testing. However, a thermal denaturation area seemed to hinder the migration of the chondrocytes inside the scaffolds, even more so after CO2 laser treatment. Therefore, the Er:YAG laser seemed to be better suitable. Further modifications of the laser adjustments or the use of alternative laser systems might be advantageous for surface enlargement and to facilitate migration of chondrocytes into the scaffold in one step.

Vascular Endothelial Growth Factor Sequestration Enhances In Vivo Cartilage Formation.

Autologous chondrocyte transplantation for cartilage repair still has unsatisfactory clinical outcomes because of inter-donor variability and poor cartilage quality formation. Re-differentiation of monolayer-expanded human chondrocytes is not easy in the absence of potent morphogens. The Vascular Endothelial Growth Factor (VEGF) plays a master role in angiogenesis and in negatively regulating cartilage growth by stimulating vascular invasion and ossification. Therefore, we hypothesized that its sole microenvironmental blockade by either VEGF sequestration by soluble VEGF receptor-2 (Flk-1) or by antiangiogenic hyperbranched peptides could improve chondrogenesis of expanded human nasal chondrocytes (NC) freshly seeded on collagen scaffolds. Chondrogenesis of several NC donors was assessed either in vitro or ectopically in nude mice. VEGF blockade appeared not to affect NC in vitro differentiation, whereas it efficiently inhibited blood vessel ingrowth in vivo. After 8 weeks, in vivo glycosaminoglycan deposition was approximately two-fold higher when antiangiogenic approaches were used, as compared to the control group. Our data indicates that the inhibition of VEGF signaling, independently of the specific implementation mode, has profound effects on in vivo NC chondrogenesis, even in the absence of chondroinductive signals during prior culture or at the implantation site.

THE EFFECTS OF PALMVITEE ON HUMAN NASAL SEPTAL CHONDROCYTES CULTURE EXPANSION AND CARTILAGE RECONSTRUCTION

Palmvitee may act as a beneficial supplement to cartilage tissue engineering which is currently still has certain limitation on chondrocyte expansion and dedifferentiation. Our objectives in thisstudy were to evaluate the effects of Palmvitee on the growth kinetic and phenotype gene expression of human nasal septal chondrocytesin monolayer expansion as well as cartilage reconstruction via tissue engineering technology. Human chondrocytes were cultured inmediums containing various Palmvitee concentrations. Among the different test groups, the medium containing Palmvitee at 3 μg/ml supported the highest chondrocyte growth rate. The gene expression in monolayer chondrocytes culture supplemented with 3 μg/ml Palmviteedemonstrated similar results as in control. Cultured chondrocytes from the medium added with 3 μg/ml Palmvitee were then mixed with Pluronic F127 for cartilage reconstruction in nude mice model. Palmvitee supplementation supported the engineered cartilage development as shown in the histological and gene expression analysis on engineered tissue after 8 weeks of implantation. Therefore, Palmvitee at 3 μg/ml is beneficial for the human nasal septal chondrocytes monolayer expansion and cartilage engineering.

Effect of alginate concentration on chondrogenesis of co-cultured human adipose-derived stem cells and nasal chondrocytes: a biological study

BackgroundThe three-dimensional (3D) system is one of the important factors to engineer a biocompatible and functional scaffold for the applications of cell-based therapies for cartilage repair. The 3D alginate hydrogels system has previously been shown to potentially promote chondrogenesis. The chondrocytic differentiation of co-cultured adipose-derived stem cells (ADSCs) and nasal chondrocytes (NCs) within alginate constructs are hypothesized to be influenced by concentration of alginate hydrogel. In this study, we evaluated the effects of alginate concentration on chondrogenic differentiation of ADSCs and NCs co-cultured in a biological approach.MethodThe co-cultured cells of 2:1 ADSCs-to-NCs ratio were encapsulated in alginate constructs in one of three concentrations (1.0%, 1.2% and 1.5%) and cultured under serum free conditions for 7 days. Cell viability, cell proliferation, immunohistochemical, gycosaminogylycans (GAG) synthesis, and gene expression were examined.ResultsOverall, the 1.2% alginate concentration group was relatively effective in chondrocytic differentiation in comparable to other groups. The cell morphology, cell viability, and cell proliferation revealed initial chondrogenic differentiation by the formation of cell clusters as well as the high permeability for exchange of solutes. The formation of newly synthesis cartilage-specific extracellular matrix in 1.2% group was demonstrated by positive immunohistochemical staining of collagen type II. The co-cultured cells in 1.2% group highly expressed COL II, ACP and SOX-9, compared to 1.0% and 1.5% groups, denote the retention of cartilaginous-specific phenotype by suppressing the undifferentiation stem cell markers of SOX-2 and OCT-4. The study showed 1.2% group was less likely to differentiate towards osteogenesis by downregulating hyperthrophy chondrocytic gene of COL X and osseous marker genes of OSC and OSP.ConclusionThis study suggests that variations in the alginate concentration of co-cultured ADSCs and NCs influenced the chondrogenesis. The remarkable biological performance on chondrogenic differentiation in regulating the concentration of alginate 3D culture provides new insights into the cell cross-talk and demonstrates the effectiveness in regenerative therapies of cartilage defects in tissue engineering.

In Vivo Chondrogenesis in 3D Bioprinted Human Cell-laden Hydrogel Constructs.

Background:The three-dimensional (3D) bioprinting technology allows creation of 3D constructs in a layer-by-layer fashion utilizing biologically relevant materials such as biopolymers and cells. The aim of this study is to investigate the use of 3D bioprinting in a clinically relevant setting to evaluate the potential of this technique for in vivo chondrogenesis.Methods:Thirty-six nude mice (Balb-C, female) received a 5- × 5- × 1-mm piece of bioprinted cell-laden nanofibrillated cellulose/alginate construct in a subcutaneous pocket. Four groups of printed constructs were used: (1) human (male) nasal chondrocytes (hNCs), (2) human (female) bone marrow–derived mesenchymal stem cells (hBMSCs), (3) coculture of hNCs and hBMSCs in a 20/80 ratio, and (4) Cell-free scaffolds (blank). After 14, 30, and 60 days, the scaffolds were harvested for histological, immunohistochemical, and mechanical analysis.Results:The constructs had good mechanical properties and keep their structural integrity after 60 days of implantation. For both the hNC constructs and the cocultured constructs, a gradual increase of glycosaminoglycan production and hNC proliferation was observed. However, the cocultured group showed a more pronounced cell proliferation and enhanced deposition of human collagen II demonstrated by immunohistochemical analysis.Conclusions:In vivo chondrogenesis in a 3D bioprinted human cell-laden hydrogel construct has been demonstrated. The trophic role of the hBMSCs in stimulating hNC proliferation and matrix deposition in the coculture group suggests the potential of 3D bioprinting of human cartilage for future application in reconstructive surgery.

Automated bioreactor system for cartilage tissue engineering of human primary nasal septal chondrocytes.

An automated bioreactor system for three-dimensional (3D) cultivation of facial cartilage replacement matrices (e.g. whole human auricles) with automatised medium exchange, gas flow and temperature control was developed. The measurement of O2 saturation and pH value in the medium was performed with a non-invasive optical method. The whole system can be observed via remote monitoring worldwide. First results demonstrated that the complete system remained sterile throughout a period of 42 days. Human chondrocytes migrated into the employed cartilage replacement matrix consisting of decellularised porcine nasoseptal cartilage (pNSC). Furthermore, an improved migration and new synthesis of aggrecan was detected. A first evaluation of the system was conducted by comparison of the results from laboratory analysis with computational fluid dynamics (CFD).

New bioreactor vessel for tissue engineering of human nasal septal chondrocytes

AbstractCultivation of human nasal septal chondrocytes in a self-established automated bioreactor system with a new designed reactor glass vessel and the results of a computational fluid dynamics model are presented. The first results show the effect of a homogeneous fluidic condition of the continuous medium flow and the resulting stresses on the scaffolds’ surface and their influence on the migration of the cells into the scaffold matrix under these conditions. For this purpose computational models, generated with the computational fluid dynamics software STAR-CCM+, and the results of alcian blue staining for newly synthesized sulphated glycosaminoglycans have been compared during cultivation in the new and a first version of the glass reactor vessel with inhomogeneous fluidic conditions, with the same automated bioreactor system and under similar cultivation conditions.

3D bioprinting of human chondrocyte-laden nanocellulose hydrogels for patient-specific auricular cartilage regeneration

Auricular cartilage tissue engineering (TE) aims to provide an effective treatment for patients with acquired or congenital auricular defects. Bioprinting has gained attention in several TE strategies for its ability to spatially control the placement of cells, biomaterials and biological molecules. Although considerable advances have been made to bioprint complex 3D tissue analogues, the development of hydrogel bioinks with good printability and bioactive properties must improve in order to advance the translation of 3D bioprinting into the clinic. In this study, the biological functionality of a bioink composed of nanofibrillated cellulose and alginate (NFC-A) is extensively evaluated for auricular cartilage TE. 3D bioprinted auricular constructs laden with human nasal chondrocytes (hNC) are cultured for up to 28 days and the redifferentiation capacity of hNCs in NFC-A is studied on gene expression as well as on protein levels. 3D bioprinting with NFC-A bioink facilitates the biofabrication of cell-laden, patient-specific auricular constructs with an open inner structure, high cell density and homogenous cell distribution. The cell-laden NFC-A constructs exhibit an excellent shape and size stability as well as an increase in cell viability and proliferation during in vitro culture. Furthermore, NFC-A bioink supports the redifferentiation of hNCs and neo-synthesis of cartilage-specific extracellular matrix components. This demonstrated that NFC-A bioink supports redifferentiation of hNCs while offering proper printability in a biologically relevant aqueous 3D environment, making it a promising tool for auricular cartilage TE and many other biomedical applications.

Characterization of a migrative subpopulation of adult human nasoseptal chondrocytes with progenitor cell features and their potential for in vivo cartilage regeneration strategies.

BackgroundProgenitor cells display interesting features for tissue repair and reconstruction. In the last years, such cells have been identified in different cartilage types. In this study, we isolated a migrative subpopulation of adult human nasoseptal chondrocytes with progenitor cell features by outgrowth from human nasal septum cartilage. These putative progenitor cells were comparatively characterized with mesenchymal stem cells (MSC) and human nasal septum chondrocytes with respect to their cellular characteristics as well as surface marker profile using flow cytometric analyses. Differentiation capacity was evaluated on protein and gene expression levels.ResultsThe migrative subpopulation differentiated into osteogenic and chondrogenic lineages with distinct differences to chondrocytes and MSC. Cells of the migrative subpopulation showed an intermediate surface marker profile positioned between MSC and chondrocytes. Significant differences were found for CD9, CD29, CD44, CD90, CD105 and CD106. The cells possessed a high migratory ability in a Boyden chamber assay and responded to chemotactic stimulation. To evaluate their potential use in tissue engineering applications, a decellularized septal cartilage matrix was either seeded with cells from the migrative subpopulation or chondrocytes. Matrix production was demonstrated immunohistochemically and verified on gene expression level. Along with secretion of matrix metalloproteinases, cells of the migrative subpopulation migrated faster into the collagen matrix than chondrocytes, while synthesis of cartilage specific matrix was comparable.ConclusionsCells of the migrative subpopulation, due to their migratory characteristics, are a potential cell source for in vivo regeneration of nasal cartilage. The in vivo mobilization of nasal cartilage progenitor cells is envisioned to be the basis for in situ tissue engineering procedures, aiming at the use of unseeded biomaterials which are able to recruit local progenitor cells for cartilage regeneration.

Si-HPMC and Si-chitosan hydrogel for tissue engineering

Event Abstract Back to Event Si-HPMC and Si-chitosan hydrogel for tissue engineering Gildas Rethore1, Cécile Boyer1, Fabienne Jordana1, Olivier Gauth1, 2, Jérome Guicheux1 and Pierre Weiss1 1 Université de Nantes - INSERM - Centre Hospitalier Universitaire de Nantes, LIOAD - UMRS 791, France 2 ONIRIS - École Vétérinaire, LIOAD - UMRS 791, France Introduction: It was believed that biomaterial selection was performed based on its macroporosity, biocompatibility and degradability properties but recently its ability to withstand biomechanical stress has also been considered. Discher et al[1]. have indeed shown that material stiffness induces critical effects on cell behaviour and differentiation. Therefore, we have recently embarked on the development of mechanically-competent hydrogels for bone and cartilage tissue engineering. We prepared a cellulose based polymer capable of self cross-linkage (Si-HPMC) to avoid any toxicity issues resulting from using cross-linking chemicals or photo cross-linking. Therefore, LIOAD has been working on the development of hydrogels as ECM for bone and cartilage regeneration. The objectives of the present work are to modulate the physico-chemical and biological properties of our hydrogels by mixing two silated polysaccharides Si-HPMC and Si-Chitosan. Experimental Methods: The self cross-linkable polymer (Si-HPMC) was prepared according to an already published method developed in LIOAD[2]. Silanol groups were linked to amine moieties of the chitosan via thio-urea bond. Hydrogel characterizations: Hydrogels and reinforced hydrogels were characterized physico chemically to determine their elastic moduli using MARS rheometer (G’), Dynamic Mechanical Analysis (E’) and microscopy. Biological investigations have been done with human nasal chondrocytes by determining the cell viability in 2D using MTS, 3D by confocal microscopy after Live&Dead staining. In vivo investigations have been performed in subcutaneous sockets in nude mice and in focal defects in rabbit. Results and Discussion: The characterizations showed that adding Si-Chitosan within Si-HPMC hydrogels increases the elastic moduli without interfering with the biocompatibility. Indeed, all the constructs that we used in this study showed no toxicity both in 2D and 3D (as shown in figure 1). Culturing cells in 3D within our hydrogel containing Si-Chitosan also induces cell adhesion onto the matrice as I can be observed on confocal micrograph in figure 2. It is worth noted that after 5 weeks of implantation, the chondrocytes showed a good viability and still express a chondrocyte phenotype. Moreover, during the surgery, due to the presence of chitosan, our constructs showed good adhesion behaviour onto the cartilage focal defect. Fig 1: left : 2D hASCs viability in contact with Si-HPMC/Si-Chitosan hydrogels (Positive control : cell alone, control for cell death: actinomycin). Right : Confocal micrograph showing living cells (green) and dead cells (red) after 7 days of culture inside hydrogels. Fig 2 : Confocal micrographs showing cell adhesions after 7 days culture in Si-HPMC/Si-Chitosan hydrogel. Conclusion: The study demonstrates the feasibility of our approach to modulate our self-setting hydrogel while keeping them biocompatible. ANR (HYCAR); Région Pays de la Loire (BIOREGOS)