Nasal Septal Chondrocytes Research Articles

Engineered autologous nasal cartilage for repair of nasal septal perforations - a case series.

This phase I clinical trial assessed the use of autologous nasal chondrocyte tissue-engineered cartilage (N-TEC) for functional repair of nasal septal perforations (NSP). The most widely used technique to treat NSP, namely interposition grafting with a polydioxanone (PDS) plate combined with a deep temporal fascia (DTF) graft, is still suboptimal towards patient satisfaction and revision rates. Patients (n=5, all female, age range: 23-54 years) had a 0.5-2.0cm diameter NSP. N-TEC was manufactured by expansion and 3D culture of autologous nasal septum chondrocytes into Chondro-Gide® collagen membranes. N-TEC was then shaped intraoperatively and enveloped in the harvested DTF before suturing it into the NSP. Safety (primary outcome) was assessed by the number of serious adverse reactions (SAR) until 12 months. Secondary outcomes included feasibility, assessed by surgical graft manipulation, and efficacy, assessed using subjective scoring (Nasal Obstruction Symptom Evaluation, NOSE, and Visual Analogue Scale, VAS, scores) and objective breathing function tests. Structural closure of NSP after 12 months was defined using endoscopy and computed tomography (CT) scans. NSP treatment by N-TEC implantation was safe and feasible, as no SAR and no challenge in graft manipulation was recorded for any of the patients. One year postoperative, subjective scoring improved in all patients, unless already optimal (average improvement of 23 and 28.6 points out of 100 respectively for NOSE and VAS scores). Objective respiratory function overall confirmed - with the exception of one case - the observations above (average improvement of 172 ml/s). NSP were closed and the mucosae completely healed in three patients. Autologous N-TEC is a valid treatment for NSP and warrants further clinical tests.

Nuclear Factor-κB Decoy Oligodeoxynucleotide Attenuates Cartilage Resorption In Vitro.

Cartilage harvest and transplantation is a common surgery using costal, auricular, and septal cartilage for craniofacial reconstruction. However, absorption and warping of the cartilage grafts can occur due to inflammatory factors associated with wound healing. Transcription factor nuclear factor-κB (NF-κB) is activated by the various stimulation such as interleukin-1 (IL-1), and plays a central role in the transactivation of this inflammatory cytokine gene. Inhibition of NF-κB may have anti-inflammatory effects. The aim of this study was to explore the potential of an NF-κB decoy oligodeoxynucleotide (Decoy) as a chondroprotective agent. Safe and efficacious concentrations of Decoy were assessed using rabbit nasal septal chondrocytes (rNSChs) and assays for cytotoxicity, proteoglycan (PG) synthesis, and PG turnover were carried out. The efficacious concentration of Decoy determined from the rNSChs was then applied to human nasal septal cartilage (hNSC) in vitro and analyzed for PG turnover, the levels of inflammatory markers, and catabolic enzymes in explant-conditioned culture medium. Over the range of Decoy conditions and concentrations, no inhibition of PG synthesis or cytotoxicity was observed. Decoy at 10 μM effectively inhibited PG degradation in the hNSC explant, prolonging PG half-life by 63% and decreasing matrix metalloprotease 3 (MMP-3) by 70.7% (p = 0.027). Decoy may be considered a novel chondroprotective therapeutic agent in cartilage transplantation due to its ability to inhibit cartilage degradation due to inflammation cytokines.

Effects of Chitosan on Chondrocytes Derived from Human Nasal Septal Cartilage.

The aim of this study was to evaluate the influence of chitosan on the growth of nasal septal chondrocytes (NSCs). The final goal was to establish a novel methodology to enhance nasal septal cartilage regeneration. Human NSCs were isolated and their morphology was examined using Alcian blue staining and observed by light microscopy. The isolated NSCs were grown with various concentrations of chitosan and the expression of COL2A1 was investigated. NSCs were successfully isolated from nasal septal cartilage. Co-culture with 0.2% of chitosan greatly enhanced proliferation of NSCs compared to control cells. However, 0.5% of chitosan was harmful to NSCs, resulting in cell detachment from the culture plate. Furthermore, the addition of 0.2% chitosan significantly improved the expression of COL2A1 in NSCs. To our knowledge, this is the first report to demonstrate that chitosan could effectively guide the attachment and growth of human NSCs. Chitosan appears to be a promising additive for NSC culture, which sets the stage for studying tissue regeneration in nasal septal cartilage deficiency, rhinoplasty, and craniofacial reconstruction.

The Releasate of Avascular Cartilage Demonstrates Inherent Pro-Angiogenic Properties In Vitro and In Vivo.

ObjectiveCartilage is avascular and numerous studies have identified the presence of single anti- and pro-angiogenic factors in cartilage. To better understand the maintenance hyaline cartilage, we assessed the angiogenic potential of complete cartilage releasate with functional assays in vitro and in vivo.DesignWe evaluated the gene expression profile of angiogenesis-related factors in healthy adult human articular cartilage with a transcriptome-wide analysis generated by next-generation RNAseq. The effect on angiogenesis of the releasate of cartilage tissue was assessed with a chick chorioallantoic membrane (CAM) assay as well as human umbilical vein endothelial cell (HUVEC) migration and proliferation assays using conditioned media generated from tissue-engineered cartilage derived from human articular and nasal septum chondrocytes as well as explants from bovine articular cartilage and human nasal septum. Experiments were done with triplicate samples of cartilage from 3 different donors.ResultsRNAseq data of 3 healthy human articular cartilage donors revealed that the majority of known angiogenesis-related factors expressed in healthy adult articular cartilage are pro-angiogenic. The releasate from generated cartilage as well as from tissue explants, demonstrated at least a 3.1-fold increase in HUVEC proliferation and migration indicating a pro-angiogenic effect of cartilage. Finally, the CAM assay demonstrated that cartilage explants can indeed attract vessels; however, their ingrowth was not observed.ConclusionUsing multiple approaches, we show that cartilage releasate has an inherent pro-angiogenic capacity. It remains vessel free due to anti-invasive properties associated with the tissue itself.

Human nasal septal chondrocytes (NSCs) preconditioned on NSC-derived matrix improve their chondrogenic potential

BackgroundExtracellular matrix (ECM) has a profound effect on cell behaviors. In this study, we prepare a decellularized human nasal septal chondrocyte (NSC)-derived ECM (CHDM), as a natural (N-CHDM) or soluble form (S-CHDM), and investigate their impact on NSCs differentiation.MethodsN-CHDM, S-CHDM were obtained from NSC. To evaluate function of NSC cultured on each substrate, gene expression using chondrogenic marker, and chondrogenic protein expression were tested. Preconditioned NSCs-loaded scaffolds were transplanted in nude mice for 3 weeks and analyzed.ResultsWhen cultivated on each substrate, NSCs exhibited similar cell spread area but showed distinct morphology on N-CHDM with significantly lower cell circularity. They were highly proliferative on N-CHDM than S-CHDM and tissue culture plastic (TCP), and showed more improved cell differentiation, as assessed via chondrogenic marker (Col2, Sox9, and Aggrecan) expression and immunofluorescence of COL II. We also investigated the effect of NSCs preconditioning on three different 2D substrates while NSCs were isolated from those substrates, subsequently transferred to 3D mesh scaffold, then cultivated them in vitro or transplanted in vivo. The number of cells in the scaffolds was similar to each other at 5 days but cell differentiation was notably better with NSCs preconditioned on N-CHDM, as assessed via real-time q-PCR, Western blot, and immunofluorescence. Moreover, when those NSCs-loaded polymer scaffolds were transplanted subcutaneously in nude mice for 3 weeks and analyzed, the NSCs preconditioned on the N-CHDM showed significantly advanced cell retention in the scaffold, more cells with a chondrocyte lacunae structure, and larger production of cartilage ECM (COL II, glycosaminoglycan).ConclusionsTaken together, a natural form of decellularized ECM, N-CHDM would present an advanced chondrogenic potential over a reformulated ECM (S-CHDM) or TCP substrate, suggesting that N-CHDM may hold more diverse signaling cues, not just limited to ECM component.

Suppression of Hypertrophy During in vitro Chondrogenesis of Cocultures of Human Mesenchymal Stem Cells and Nasal Chondrocytes Correlates With Lack of in vivo Calcification and Vascular Invasion.

ObjectiveHuman nasal septal chondrocytes (NC) are a promising minimally invasive derivable chondrogenic cell source for cartilage repair. However, the quality of NC-derived cartilage is variable between donors. Coculture of NC with mesenchymal stem cells (MSCs) mitigates the variability but with undesirable markers of chondrocyte hypertrophy, such as type X collagen, and the formation of unstable calcifying cartilage at ectopic sites. In contrast, monoculture NC forms non-calcifying stable cartilage. Formation of a stable NC-MSC coculture cartilage is crucial for clinical application. The aim of this study was to explore the utility of parathyroid hormone-related peptide (PTHrP) hormone to suppress chondrocyte hypertrophy in NC-MSC cocultures and form stable non-calcifying cartilage at ectopic sites.MethodsHuman NC and bone marrow MSCs, and cocultures of NC and MSC (1:3 ratio) were aggregated in pellet form and subjected to in vitro chondrogenesis for 3 weeks in chondrogenic medium in the presence and absence of PTHrP. Following in vitro chondrogenesis, the resulting pellets were implanted in immunodeficient athymic nude mice for 3 weeks.ResultsCoculture of NC and MSC resulted in synergistic cartilage matrix production. PTHrP suppressed the expression of hypertrophy marker, type X collagen (COL10A1), in a dose-dependent fashion without affecting the synergism in cartilage matrix synthesis, and in vivo calcification was eradicated with PTHrP. In contrast, cocultured control (CC) pellets without PTHrP treatment expressed COL10A1, calcified, and became vascularized in vivo.

Biosafety evaluation of culture-expanded human chondrocytes with growth factor cocktail: a preclinical study

The scarcity of chondrocytes is a major challenge for cartilage tissue engineering. Monolayer expansion is necessary to amplify the limited number of chondrocytes needed for clinical application. Growth factors are often added to improve monolayer culture conditions, promoting proliferation, and enhancing chondrogenesis. Limited knowledge on the biosafety of the cell products manipulated with growth factors in culture has driven this study to evaluate the impact of growth factor cocktail supplements in chondrocyte culture medium on chondrocyte genetic stability and tumorigenicity. The growth factors were basic fibroblast growth factor (b-FGF), transforming growth factor β2 (TGF β2), insulin-like growth factor 1 (IGF-1), insulin-transferrin-selenium (ITS), and platelet-derived growth factor (PD-GF). Nasal septal chondrocytes cultured in growth factor cocktail exhibited a significantly high proliferative capacity. Comet assay revealed no significant DNA damage. Flow cytometry showed chondrocytes were mostly at G0-G1 phase, exhibiting normal cell cycle profile with no aneuploidy. We observed a decreased tumour suppressor genes’ expression (p53, p21, pRB) and no TP53 mutations or tumour formation after 6 months of implantation in nude mice. Our data suggest growth factor cocktail has a low risk of inducing genotoxic and tumorigenic effects on chondrocytes up to passage 6 with 16.6 population doublings. This preclinical tumorigenicity and genetic instability evaluation is crucial for further clinical works.

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.

Temporomandibular joint regeneration: proposal of a novel treatment for condylar resorption after orthognathic surgery using transplantation of autologous nasal septum chondrocytes, and the first human case report

BackgroundUpon orthognathic mandibular advancement surgery the adjacent soft tissues can displace the distal bone segment and increase the load on the temporomandibular joint causing loss of its integrity. Remodeling of the condyle and temporal fossa with destruction of condylar cartilage and subchondral bone leads to postsurgical condylar resorption, with arthralgia and functional limitations. Patients with severe lesions are refractory to conservative treatments, leading to more invasive therapies that range from simple arthrocentesis to open surgery and prosthesis. Although aggressive and with a high risk for the patient, surgical invasive treatments are not always efficient in managing the degenerative lesions.MethodsWe propose a regenerative medicine approach using in-vitro expanded autologous cells from nasal septum applied to the first proof-of-concept patient. After the required quality controls, the cells were injected into each joint by arthrocentesis. Results were monitored by functional assays and image analysis using computed tomography.ResultsThe cell injection fully reverted the condylar resorption, leading to functional and structural regeneration after 6 months. Computed tomography images showed new cortical bone formation filling the former cavity space, and a partial recovery of condylar and temporal bones. The superposition of the condyle models showed the regeneration of the bone defect, reconstructing the condyle original form.ConclusionsWe propose a new treatment of condylar resorption subsequent to orthognathic surgery, presently treated only by alloplastic total joint replacement. We propose an intra-articular injection of autologous in-vitro expanded cells from the nasal septum. The proof-of-concept treatment of a selected patient that had no alternative therapeutic proposal has given promising results, reaching full regeneration of both the condylar cartilage and bone at 6 months after the therapy, which was fully maintained after 1 year. This first case is being followed by inclusion of new patients with a similar pathological profile to complete an ongoing stage I/II study.Trial registrationThis clinical trial is approved by the National Commission of Ethics in Medical Research (CONEP), Brazil, CAAE 12484813.0.0000.5245, and retrospectively registered in the Brazilian National Clinical Trials Registry and in the USA Clinical Trials Registry under the Universal Trial Number (UTN) U1111–1194-6997.

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.

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.

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.

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.

Factors Affecting the Expansion of Chondrocytes Harvested from Rabbit Auricle and Nasal Septum

Received October 6, 2014 Revised November 25, 2014 Accepted November 26, 2014 Address for correspondence Jeong-Hoon Oh, MD, PhD Department of OtorhinolaryngologyHead and Neck Surgery, College of Medicine, The Catholic University of Korea, 180 Wangsan-ro, Dongdaemun-gu, Seoul 130-709, Korea Tel +82-2-958-2148 Fax +82-2-959-5375 E-mail ojhent@catholic.ac.kr Background and ObjectivesZZIn tissue engineering of the cartilage, cellular expansion is an essential step for creating useful amounts of tissue required. Many variables such as media composition, growth factors and cell seeding density have been utilized to stimulate the ability of cellular proliferation and to reduce the number of expansion in monolayer. The object of this study was to evaluate the effects of human serum (HS) and rabbit serum (RS) on cellular proliferation of auricular and nasal septal chondrocytes of the rabbit. Materials and MethodZZAuricular and nasal septal chondrocytes of the New Zealand White Rabbit were expanded in monolayer in medium supplemented with 2% RS, 5% RS, 10% RS, or 10% HS with or without adding of growth factors. The effect of the supplements on the cellular proliferation was compared. ResultsZZThe proliferation yield of chondrocytes grown in RS was comparable to that with HS. Both serums results in increased proliferation; however, the amount of cellular proliferation was not correlated with the concentration of the serum. The proliferation rate of nasal septal chondrocytes was higher than that of auricular chondrocytes. ConclusionZZBoth HS and RS resulted in the increased proliferation of rabbit auricular and nasal septal chondrocytes. The results show that the determination of appropriate types and concentrations of serum is required for cell expansion in constructing tissue-engineered cartilage. Korean J Otorhinolaryngol-Head Neck Surg 2015;58(2):95-100

In VitroCytotoxicity andIn VivoEffects of a Decellularized Xenogeneic Collagen Scaffold in Nasal Cartilage Repair

Tissue engineering is considered a promising future option for nasal cartilage repair. However, until now, an optimal material has not been identified for this specific purpose. Therefore, the aim of this study was to analyze a recently developed decellularized collagen matrix, which has promising material properties for septal cartilage repair. A tetrazolium dye based cytotoxicity assay using rat nasal septum chondrocytes was performed to examine the cytotoxic effects of decellularized cartilage matrices. Unseeded scaffolds as well as scaffolds seeded with chondrocytes were implanted in nasal septum defects in Lewis rats to investigate the cellular and humoral inflammatory responses in the surrounding tissue as well as the effect on the formation of nasal septum perforations. Samples were analyzed histochemically and immunohistochemically after 1, 4, and 12 weeks. Although cells for the cytotoxicity assay were cultured under serum-free conditions for 24 h to increase sensitivity, no cytotoxic effects were detected. Histological and immunohistochemical evidence displayed that the implanted scaffolds induced minor macrophage and lymphocyte infiltration and were well integrated at the contact site to native cartilage and between the mucosal membranes. The biocompatibility index revealed only slightly irritating effects during the study period. Septal perforations were prevented efficiently. In summary, our results provide evidence that decellularized xenogeneic collagen scaffolds are suitable for cartilage tissue engineering. The scaffolds were integrated well into septal cartilage defects without causing a strong inflammatory reaction and prevented the development of nasal septum perforations. Therefore, we envision the possibility to use them in nasal cartilage repair in the future.

Effect of oxygen tension on tissue-engineered human nasal septal chondrocytes.

Tissue-engineered nasal septal cartilage may provide a source of autologous tissue for repair of craniofacial defects. Although advances have been made in manipulating the chondrocyte culture environment for production of neocartilage, consensus on the best oxygen tension for in vitro growth of tissue-engineered cartilage has not been reached. The objective of this study was to determine whether in vitro oxygen tension influences chondrocyte expansion and redifferentiation. Proliferation of chondrocytes from 12 patients expanded in monolayer under hypoxic (5% or 10%) or normoxic (21%) oxygen tension was compared over 14 days of culture. The highest performing oxygen level was used for further expansion of the monolayer cultures. At confluency, chondrocytes were redifferentiated by encapsulation in alginate beads and cultured for 14 days under hypoxic (5 or 10%) or normoxic (21%) oxygen tension. Biochemical and histological properties were evaluated. Chondrocyte proliferation in monolayer and redifferentiation in alginate beads were supported by all oxygen tensions tested. Chondrocytes in monolayer culture had increased proliferation at normoxic oxygen tension (p = 0.06), as well as greater accumulation of glycosaminoglycan (GAG) during chondrocyte redifferentiation (p < 0.05). Chondrocytes released from beads cultured under all three oxygen levels showed robust accumulation of GAG and type II collagen with a lower degree of type I collagen immunoreactivity. Finally, formation of chondrocyte clusters was associated with decreasing oxygen tension (p < 0.05). Expansion of human septal chondrocytes in monolayer culture was greatest at normoxic oxygen tension. Both normoxic and hypoxic culture of human septal chondrocytes embedded in alginate beads supported robust extracellular matrix deposition. However, GAG accumulation was significantly enhanced under normoxic culture conditions. Chondrocyte cluster formation was associated with hypoxic oxygen tension.

Marine Collagen Scaffolds for Nasal Cartilage Repair: Prevention of Nasal Septal Perforations in a New Orthotopic Rat Model Using Tissue Engineering Techniques

Autologous grafts are frequently needed for nasal septum reconstruction. Because they are only available in limited amounts, there is a need for new cartilage replacement strategies. Tissue engineering based on the use of autologous chondrocytes and resorbable matrices might be a suitable option. So far, an optimal material for nasal septum reconstruction has not been identified. The aim of our study was to provide the first evaluation of marine collagen for use in nasal cartilage repair. First, we studied the suitability of marine collagen as a cartilage replacement matrix in the context of in vitro three dimensional cultures by analyzing cell migration, cytotoxicity, and extracellular matrix formation using human and rat nasal septal chondrocytes. Second, we worked toward developing a suitable orthotopic animal model for nasal septum repair, while simultaneously evaluating the biocompatibility of marine collagen. Seeded and unseeded scaffolds were transplanted into nasal septum defects in an orthotopic rat model for 1, 4, and 12 weeks. Explanted scaffolds were histologically and immunohistochemically evaluated. Scaffolds did not induce any cytotoxic reactions in vitro. Chondrocytes were able to adhere to marine collagen and produce cartilaginous matrix proteins, such as collagen type II. Treating septal cartilage defects in vivo with seeded and unseeded scaffolds led to a significant reduction in the number of nasal septum perforations compared to no replacement. In summary, we demonstrated that marine collagen matrices provide excellent properties for cartilage tissue engineering. Marine collagen scaffolds are able to prevent septal perforations in an autologous, orthotopic rat model. This newly described experimental surgical procedure is a suitable way to evaluate new scaffold materials for their applicability in the context of nasal cartilage repair.

Processed xenogenic cartilage as innovative biomatrix for cartilage tissue engineering: effects on chondrocyte differentiation and function

One key point in the development of new bioimplant matrices for the reconstruction and replacement of cartilage defects is to provide an adequate microenvironment to ensure chondrocyte migration and de novo synthesis of cartilage-specific extracellular matrix (ECM). A recently developed decellularization and sterilization process maintains the three-dimensional (3D) collagen structure of native septal cartilage while increasing matrix porosity, which is considered to be crucial for cartilage tissue engineering. Human primary nasal septal chondrocytes were amplified in monolayer culture and 3D-cultured on processed porcine nasal septal cartilage scaffolds. The influence of chondrogenic growth factors on neosynthesis of ECM proteins was examined at the protein and gene expression levels. Seeding experiments demonstrated that processed xenogenic cartilage matrices provide excellent environmental properties for human nasal septal chondrocytes with respect to cell adhesion, migration into the matrix and neosynthesis of cartilage-specific ECM proteins, such as collagen type II and aggrecan. Matrix biomechanical stability indicated that the constructs retrieve full stability and function during 3D culture for up to 42 days, proportional to collagen type II and GAG production. Thus, processed xenogenic cartilage offers a suitable environment for human nasal chondrocytes and has promising potential for cartilage tissue engineering in the head and neck region.

Insulin-like Growth Factor-I and Growth Differentiation Factor-5 Promote the Formation of Tissue-Engineered Human Nasal Septal Cartilage

Tissue engineering of human nasal septal chondrocytes offers the potential to create large quantities of autologous material for use in reconstructive surgery of the head and neck. Culture with recombinant human growth factors may improve the biochemical and biomechanical properties of engineered tissue. The objectives of this study were to (1) perform a high-throughput screen to assess multiple combinations of growth factors and (2) perform more detailed testing of candidates identified in part I. In part I, human nasal septal chondrocytes from three donors were expanded in monolayer with pooled human serum (HS). Cells were then embedded in alginate beads for 2 weeks of culture in medium supplemented with 2% or 10% HS and 1 of 90 different growth factor combinations. Combinations of insulin-like growth factor-I (IGF-1), bone morphogenetic protein (BMP)-2, BMP-7, BMP-13, growth differentiation factor-5 (GDF-5), transforming growth factor β (TGFβ)-2, insulin, and dexamethasone were evaluated. Glycosaminoglycan (GAG) accumulation was measured. A combination of IGF-1 and GDF-5 was selected for further testing based on the results of part I. Chondrocytes from four donors underwent expansion followed by three-dimensional alginate culture for 2 weeks in medium supplemented with 2% or 10% HS with or without IGF-1 and GDF-5. Chondrocytes and their associated matrix were then recovered and cultured for 4 weeks in 12 mm transwells in medium supplemented with 2% or 10% HS with or without IGF-1 and GDF-5 (the same medium used for alginate culture). Biochemical and biomechanical properties of the neocartilage were measured. In part I, GAG accumulation was highest for growth factor combinations including both IGF-1 and GDF-5. In part II, the addition of IGF-1 and GDF-5 to 2% HS resulted in a 12-fold increase in construct thickness compared with 2% HS alone (p < 0.0001). GAG and type II collagen accumulation was significantly higher with IGF-1 and GDF-5. Confined compression modulus was greatest with 2% HS, IGF-1, and GDF-5. Supplementation of medium with IGF-1 and GDF-5 during creation of neocartilage constructs results in increased accumulation of GAG and type II collagen and improved biomechanical properties compared with constructs created without the growth factors.