Auricular Chondrocytes Research Articles

M1-like macrophage contributes to chondrogenesis in vitro

Cartilage tissues have poor self-repairing abilities. Regenerative medicine can be applied to recover cartilage tissue damage in the oral and maxillofacial regions. However, hitherto it has not been possible to predict the maturity of the tissue construction after transplantation or to prepare mature cartilage tissues before transplantation that can meet clinical needs. Macrophages play an important role in cartilage tissue regeneration, although the exact mechanisms remain unknown. In this study, we established and verified an in vitro experimental system for the direct co-culture of cell pellets prepared from mouse auricular chondrocytes and macrophages polarized into four phenotypes (M1-like, M1, M2-like, and M2). We demonstrate that cartilage pellets co-cultured with M1-like promoted collagen type 2 and aggrecan production and induced the most significant increase in chondrogenesis. Furthermore, M1-like shifted to M2 on day 7 of co-culture, suggesting that the cartilage pellet supplied factors that changed the polarization of M1-like. Our findings suggest that cartilage regenerative medicine will be most effective if the maturation of cartilage tissues is induced in vitro by co-culture with M1-like before transplantation.

Three-Dimensional-Printed External Scaffolds Mitigate Loss of Volume and Topography in Engineered Elastic Cartilage Constructs.

A major obstacle in the clinical translation of engineered auricular scaffolds is the significant contraction and loss of topography that occur during maturation of the soft collagen-chondrocyte matrix into elastic cartilage. We hypothesized that 3-dimensional-printed, biocompatible scaffolds would "protect" maturing hydrogel constructs from contraction and loss of topography. External disc-shaped and "ridged" scaffolds were designed and 3D-printed using polylactic acid (PLA). Acellular type I collagen constructs were cultured in vitro for up to 3 months. Collagen constructs seeded with bovine auricular chondrocytes (BAuCs) were prepared in 3 groups and implanted subcutaneously in vivo for 3 months: preformed discs with ("Scaffolded/S") or without ("Naked/N") an external scaffold and discs that were formed within an external scaffold via injection molding ("Injection Molded/SInj"). The presence of an external scaffold or use of injection molding methodology did not affect the acellular construct volume or base area loss. In vivo, the presence of an external scaffold significantly improved preservation of volume and base area at 3 months compared to the naked group (P < 0.05). Construct contraction was mitigated even further in the injection molded group, and topography of the ridged constructs was maintained with greater fidelity (P < 0.05). Histology verified the development of mature auricular cartilage in the constructs within external scaffolds after 3 months. Custom-designed, 3D-printed, biocompatible external scaffolds significantly mitigate BAuC-seeded construct contraction and maintain complex topography. Further refinement and scaling of this approach in conjunction with construct fabrication utilizing injection molding may aid in the development of full-scale auricular scaffolds.

In vivo efficacy of 3D-printed elastin-gelatin-hyaluronic acid scaffolds for regeneration of nasal septal cartilage defects.

Nasal septal cartilage perforations occur due to the different pathologies. Limited healing ability of cartilage results in remaining defects and further complications. This study sought to assess the efficacy of elastin-gelatin-hyaluronic acid (EGH) scaffolds for regeneration of nasal septal cartilage defects in rabbits. Defects (4 × 7 mm) were created in the nasal septal cartilage of 24 New Zealand rabbits. They were randomly divided into four groups: Group 1 was the control group with no further intervention, Group 2 received EGH scaffolds implanted in the defects, Group 3 received EGH scaffolds seeded with autologous auricular chondrocytes implanted in the defects, and Group 4 received EGH scaffolds seeded with homologous auricular chondrocytes implanted in the defects. After a 4-month healing period, computed tomography (CT) and magnetic resonance imaging (MRI) scans were obtained from the nasal septal cartilage, followed by histological evaluations of new tissue formation. Maximum regeneration occurred in Group 2, according to CT, and Group 3, according to both T1 and T2 images with 7.68 ± 1.36, 5.44 ± 2.41, and 8.72 ± 3.02 mm2 defect area respectively after healing. The difference in the defect size was statistically significant after healing between the experimental groups. Group 3 showed significantly greater regeneration according to CT scans and T1 and T2 images. The neocartilage formed over the underlying old cartilage with no distinct margin in histological evaluation. The EGH scaffolds have the capability of regeneration of nasal cartilage defects and are able to integrate with the existing cartilage; yet, they present the best results when pre-seeded with autologous chondrocytes.

Ethanol treatment of nanoPGA/PCL composite scaffolds enhances human chondrocyte development in the cellular microenvironment of tissue-engineered auricle constructs.

A major obstacle for tissue engineering ear-shaped cartilage is poorly developed tissue comprising cell-scaffold constructs. To address this issue, bioresorbable scaffolds of poly-ε-caprolactone (PCL) and polyglycolic acid nanofibers (nanoPGA) were evaluated using an ethanol treatment step before auricular chondrocyte scaffold seeding, an approach considered to enhance scaffold hydrophilicity and cartilage regeneration. Auricular chondrocytes were isolated from canine ears and human surgical samples discarded during otoplasty, including microtia reconstruction. Canine chondrocytes were seeded onto PCL and nanoPGA sheets either with or without ethanol treatment to examine cellular adhesion in vitro. Human chondrocytes were seeded onto three-dimensional bioresorbable composite scaffolds (PCL with surface coverage of nanoPGA) either with or without ethanol treatment and then implanted into athymic mice for 10 and 20 weeks. On construct retrieval, scanning electron microscopy showed canine auricular chondrocytes seeded onto ethanol-treated scaffolds in vitro developed extended cell processes contacting scaffold surfaces, a result suggesting cell-scaffold adhesion and a favorable microenvironment compared to the same cells with limited processes over untreated scaffolds. Adhesion of canine chondrocytes was statistically significantly greater (p ≤ 0.05) for ethanol-treated compared to untreated scaffold sheets. After implantation for 10 weeks, constructs of human auricular chondrocytes seeded onto ethanol-treated scaffolds were covered with glossy cartilage while constructs consisting of the same cells seeded onto untreated scaffolds revealed sparse connective tissue and cartilage regeneration. Following 10 weeks of implantation, RT-qPCR analyses of chondrocytes grown on ethanol-treated scaffolds showed greater expression levels for several cartilage-related genes compared to cells developed on untreated scaffolds with statistically significantly increased SRY-box transcription factor 5 (SOX5) and decreased interleukin-1α (inflammation-related) expression levels (p ≤ 0.05). Ethanol treatment of scaffolds led to increased cartilage production for 20- compared to 10-week constructs. While hydrophilicity of scaffolds was not assessed directly in the present findings, a possible factor supporting the summary data is that hydrophilicity may be enhanced for ethanol-treated nanoPGA/PCL scaffolds, an effect leading to improvement of chondrocyte adhesion, the cellular microenvironment and cartilage regeneration in tissue-engineered auricle constructs.

Chondrogenic Characteristics of Auricular Chondrocytes Cocultured With Adipose-Derived Stem Cells are Superior to Stromal Vascular Fraction of Adipose Tissue.

Reconstruction of craniofacial cartilage defects is among the most challenging operations in facial plastic surgery. The co-culture system of partial replacement of chondrocytes by stem cells has been confirmed effective in the repair of cartilaginous defects. The aim of this study is to compare chondrogenic properties of expanded adipose-derived stem cells (ADSCs) and stromal vascular fraction (SVF), including ADSCs/SVF monoculture and coculture with rabbit auricular chondrocytes (ACs). Analysis of morphology, histology, real-time polymerase chain reaction and glycosaminoglycans (GAG) quantification were performed to characterize the chondrogenesis of pellets. The triple differentiation potential of ADSCs had been confirmed. Further, using flow cytometry, the authors demonstrated that ADSCs and SVF have different characteristics in cell surface markers, and ADSCs are more enriched in cells from the mesenchymal lineage than SVF. GAG production of ADSCs is significantly higher than that of SVF in pellet monoculture, and pellet coculture of ADSCs and ACs are better in depositing cartilage matrix than the mixture of SVF and ACs. Our study suggests that ADSCs may be more suitable seed cells for craniofacial cartilage defect or deformity repair.

Biological Evaluation of Acellular Cartilaginous and Dermal Matrixes as Tissue Engineering Scaffolds for Cartilage Regeneration.

An acellular matrix (AM) as a kind of natural biomaterial is gaining increasing attention in tissue engineering applications. An acellular cartilaginous matrix (ACM) and acellular dermal matrix (ADM) are two kinds of the most widely used AMs in cartilage tissue engineering. However, there is still debate over which of these AMs achieves optimal cartilage regeneration, especially in immunocompetent large animals. In the current study, we fabricated porous ADM and ACM scaffolds by a freeze-drying method and confirmed that ADM had a larger pore size than ACM. By recolonization with goat auricular chondrocytes and in vitro culture, ADM scaffolds exhibited a higher cell adhesion rate, more homogeneous chondrocyte distribution, and neocartilage formation compared with ACM. Additionally, quantitative polymerase chain reaction (qPCR) indicated that expression of cartilage-related genes, including ACAN, COLIIA1, and SOX9, was significantly higher in the ADM group than the ACM group. Furthermore, after subcutaneous implantation in a goat, histological evaluation showed that ADM achieved more stable and matured cartilage compared with ACM, which was confirmed by quantitative data including the wet weight, volume, and contents of DNA, GAG, total collagen, and collagen II. Additionally, immunological assessment suggested that ADM evoked a low immune response compared with ACM as evidenced by qPCR and immunohistochemical analyses of CD3 and CD68, and TUNEL. Collectively, our results indicate that ADM is a more suitable AM for cartilage regeneration, which can be used for cartilage regeneration in immunocompetent large animals.

Fibrin Gel Suspended Autologous Chondrocytes as Cell-based Material for long-term Injection Laryngoplasty.

Injection laryngoplasty of materials for unilateral vocal-fold paralysis has shown various results regarding the long-term stability of the injected material. We evaluated a fibrin-gel based cell suspension with autologous chondrocytes in-vitro and in-vivo as long-term-stable vocal-fold augmentation material in an animal model. This study compises an in vitro cell-culture part as well as an in vivo animal study with New Zealand White Rabbits. In in-vitro experiments, auricular chondrocytes harvested from 24 New Zealand White Rabbits cadavers were cultivated in pellet cultures to evaluate cartilage formation for 4 weeks using long-term-stable fibrin gel as carrier. Injectability and injection volume for the laryngoplasty was determined in-vitro using harvested cadaveric larynxes. In-vivo 24 Rabbits were biopsied for elastic cartilage of the ear and autologous P1 cells were injected lateral of one vocal cord into the paraglottic space suspended in a long-term-stable fibrin gel. Histologic evaluation was performed after 2, 4, 12, and 24 weeks. During 12-week pellet culture, we found extracellular matrix formation and weight-stable cartilage of mature appearance. In-vivo, mature cartilage was found in two larynxes (n = 6) at 4 weeks, in four (n = 6) at 12 weeks, and in five (n = 6) at 24 weeks mostly located in the paraglottic space and sometimes with spurs into the vocalis muscle. Surrounding tissue was often infiltrated with inflammatory cells. Material tended to dislocate through the cricothyroid space into the extraglottic surrounding tissue. A cell-based approach with chondrocytes for permanent vocal-fold augmentation has not previously been reported. We have achieved the formation of structurally mature cartilage in the paraglottic space, but this is accompanied by difficulties with dislocated material, deformation of the augmentation, and inflammation. N/A Laryngoscope, 131:E1624-E1632, 2021.

A photo-crosslinkable cartilage-derived extracellular matrix bioink for auricular cartilage tissue engineering

Three-dimensional (3D) bioprinting of patient-specific auricular cartilage constructs could aid in the reconstruction process of traumatically injured or congenitally deformed ear cartilage. To achieve this, a hydrogel-based bioink is required that recapitulates the complex cartilage microenvironment. Tissue-derived decellularized extracellular matrix (dECM)-based hydrogels have been used as bioinks for cell-based 3D bioprinting because they contain tissue-specific ECM components that play a vital role in cell adhesion, growth, and differentiation. In this study, porcine auricular cartilage tissues were isolated and decellularized, and the decellularized cartilage tissues were characterized by histology, biochemical assay, and proteomics. This cartilage-derived dECM (cdECM) was subsequently processed into a photo-crosslinkable hydrogel using methacrylation (cdECMMA) and mixed with chondrocytes to create a printable bioink. The rheological properties, printability, and in vitro biological properties of the cdECMMA bioink were examined. The results showed cdECM was obtained with complete removal of cellular components while preserving major ECM proteins. After methacrylation, the cdECMMA bioinks were printed in anatomical ear shape and exhibited adequate mechanical properties and structural integrity. Specifically, auricular chondrocytes in the printed cdECMMA hydrogel constructs maintained their viability and proliferation capacity and eventually produced cartilage ECM components, including collagen and glycosaminoglycans (GAGs). The potential of cell-based bioprinting using this cartilage-specific dECMMA bioink is demonstrated as an alternative option for auricular cartilage reconstruction.

Improving In Vitro Cartilage Generation by Co-Culturing Adipose-Derived Stem Cells and Chondrocytes on an Allograft Adipose Matrix Framework.

Microtia is an inherited condition that results in varying degrees of external ear deformities; the most extreme form is anotia. Effective surgical reconstruction techniques have been developed. However, these usually require multistage procedures and have other inherent disadvantages. Tissue engineering technologies offer new approaches in the field of external ear reconstruction. In this setting, chondrocytes are cultured in the laboratory with the aim of creating bioengineered cartilage matrices. However, cartilage engineering has many challenges, including difficulty in culturing sufficient chondrocytes. To overcome these hurdles, the authors propose a novel model of cartilage engineering that involves co-culturing chondrocytes and adipose-derived stem cells on an allograft adipose-derived extracellular matrix scaffold. Auricular chondrocytes from porcine ear were characterized. Adipose-derived stem cells were isolated and expanded from human lipoaspirate. Then, the auricular chondrocytes were cultured on the allograft adipose matrix either alone or with the adipose-derived stem cells at different ratios and examined histologically. Cartilage induction was most prominent when the cells were co-cultured on the allograft adipose matrix at a ratio of 1:9 (auricular chondrocyte-to-adipose-derived stem cell ratio). Furthermore, because of the xenogeneic nature of the experiment, the authors were able to determine that the adipose-derived stem cells contributed to chondrogenesis by means of a paracrine stimulation of the chondrocytes. In this situation, adipose-derived stem cells provide sufficient support to induce the formation of cartilage when the number of auricular chondrocytes available is limited. This novel model of cartilage engineering provides a setting for using the patient's own chondrocytes and adipose tissue to create a customized ear framework that could be further used for surgical reconstruction.

Regeneration of human-ear-shaped cartilage with acellular cartilage matrix-based biomimetic scaffolds

Tissue engineering technology provides a promising approach for external ear reconstruction. Although the first clinical breakthrough of tissue engineered auricular reconstruction has been achieved based on polymer scaffold, the discrepant clinical efficacy among different patients triggered by residual scaffold mediated aseptic inflammatory reaction seriously hinders its further clinical application. A proper natural scaffold with low inflammatory reaction is urgently required to address this problem. Among all the natural materials, acellular cartilage matrix (ACM) is considered to be the ideal cartilage-specific microenvironmental biomimetic scaffold. However, no breakthroughs have been achieved for the regeneration of human-ear-shaped cartilage based on ACM so far. The main challenge is how to prepare ACM into a three-dimensional (3D) porous scaffold with precise human-ear shape and proper mechanical strength. In this study, ACM powder, prepared by freezing pulverization and decellularization, was successfully prepared into 3D porous scaffolds using gelatin as an auxiliary crosslinker. By optimizing proportion and concentration of ACM and gelatin, the scaffolds presented proper pore structure and degradation rate as well as good mechanical strength and biocompatibility. Furthermore, ACM/Gelatin could be fabricated into porous scaffolds with precise human-ear shape and proper mechanical strength by integrating 3D printing, polycaprolactone (PCL) inner core designing, cast molding, and freeze-drying technologies. Finally, human-ear-shaped cartilage with good elasticity and cartilage-specific extracellular matrices was successfully regenerated using the ACM/Gelatin-PCL scaffolds and auricular chondrocytes (elastic cartilage). All of these results provide important support for the application and clinical translation of human-ear-shaped cartilage.

A moldable thermosensitive hydroxypropyl chitin hydrogel for 3D cartilage regeneration in vitro and in vivo

Because of poor self-repair capacity, the repair of cartilage defect is always a great challenge in clinical treatment. In vitro cartilage regeneration provides a potential strategy for functional reconstruction of cartilage defect. Hydrogel has been known as an ideal cartilage regeneration scaffold. However, to date, in vitro cartilage regeneration based on hydrogel has not achieved satisfactory results. The current study explored the feasibility of in vitro 3D cartilage regeneration based on a moldable thermosensitive hydroxypropyl chitin (HPCH) hydrogel and its in vivo fate. The thermosensitive HPCH hydrogel was prepared and characterized. Goat auricular chondrocytes were encapsulated into the HPCH hydrogel to form a chondrocyte-hydrogel construct. The constructs were injected subcutaneously into nude mice or molded into different shapes for in vitro chondrogenic culture followed by in vivo implantation. The results demonstrated that the HPCH hydrogel possessed satisfactory gelation properties (gelation time < 18 s at 37 °C), biocompatibility (cell amount almost doubled within one week), and the ability to be applied as an injectable hydrogel for cartilage regeneration. All the constructs of in vitro culture basically maintained their original shapes (in vitro to initial: 110.8%) and displayed typical cartilaginous features with abundant lacunae and cartilage specific matrix deposition. These in vitro samples became more mature with prolonged in vivo implantation and largely maintained the original shape (in vivo to in vitro: 103.5%). These results suggested that the moldable thermosensitive HPCH hydrogel can serve as a promising scaffold for cartilage regeneration with defined shapes in vitro and in vivo. Statement of significanceBecause of avascular and non-nervous characteristic of cartilage, in vitro regeneration plays an important role in reconstructing cartilage function. Hydrogel has been known as an ideal cartilage regeneration scaffold. However, to date, in vitro cartilage regeneration based on hydrogel has not achieved satisfactory results. The current study demonstrated that the chondrocyte-hydrogel construct generated by high density of chondrocytes encapsulated into a thermosensitive HPCH hydrogel could successfully regenerate in vitro typical cartilage-like tissue with defined shapes and further mature to form homogeneous cartilage with their original shapes after in vivo implantation. The current study indicated that the moldable thermosensitive HPCH hydrogel could serve as a promising scaffold for in vitro and in vivo cartilage regeneration with different shapes.

Glial Fibrillary Acidic Protein as Biomarker Indicates Purity and Property of Auricular Chondrocytes.

Instead of the silicone implants previously used for repair and reconstruction of the auricle and nose lost due to accidents and disease, a new treatment method using tissue-engineered cartilage has been attracting attention. The quality of cultured cells is important in this method because it affects treatment outcomes. However, a marker of chondrocytes, particularly auricular chondrocytes, has not yet been established. The objective of this study was to establish an optimal marker to evaluate the quality of cultured auricular chondrocytes as a cell source of regenerative cartilage tissue. Gene expression levels were comprehensively compared using the microarray method between human undifferentiated and dedifferentiated auricular chondrocytes to investigate a candidate quality control index with an expression level that is high in differentiated cells, but markedly decreases in dedifferentiated cells. We identified glial fibrillary acidic protein (GFAP) as a marker that decreased with serial passages in auricular chondrocytes. GFAP was not detected in articular chondrocytes, costal chondrocytes, or fibroblasts, which need to be distinguished from auricular chondrocytes in cell cultures. GFAP mRNA expression was observed in cultured auricular chondrocytes, and GFAP protein levels were also measured in the cell lysates and culture supernatants of these cells. However, GFAP levels detected from mRNA and protein in cell lysates were significantly decreased by increases in the incubation period. In contrast, the amount of protein in the cell supernatant was not affected by the incubation period. Furthermore, the protein level of GFAP in the supernatants of cultured cells correlated with the in vitro and in vivo production of the cartilage matrix by these cells. The productivity of the cartilage matrix in cultured auricular chondrocytes may be predicted by measuring GFAP protein levels in the culture supernatants of these cells. Thus, GFAP is regarded as a marker of the purity and properties of cultured auricular chondrocytes.

Double-Network Hydrogels Including Enzymatically Crosslinked Poly-(2-alkyl-2-oxazoline)s for 3D Bioprinting of Cartilage-Engineering Constructs.

Double-network (DN) hydrogels are fabricated from poly(2-ethyl-2-oxazoline) (PEOXA)-peptide conjugates, which can be enzymatically crosslinked in the presence of Sortase A (SA), and physical networks of alginate (Alg), yielding matrices with improved mechanical properties with respect to the corresponding PEOXA and Alg single networks and excellent cell viability of encapsulated human auricular chondrocytes (hACs). The addition of a low content of cellulose nanofibrils (CNFs) within DN hydrogel formulations provides the rheological properties needed for extrusion-based three-dimensional (3D) printing, generating constructs with a good shape fidelity. In the presence of hACs, PEOXA-Alg-CNF prehydrogel mixtures can be bioprinted, finally generating 3D-structured DN hydrogel supports showing a cell viability of more than 90%. Expanding the application of poly(2-alkyl-2-oxazoline)-based formulations in the design of tissue-engineering constructs, this study further demonstrates how SA-mediated enzymatic crosslinking represents a suitable and fully orthogonal method to generate biocompatible hydrogels with fast kinetics.

Proliferation medium in three-dimensional culture of auricular chondrocytes promotes effective cartilage regeneration invivo.

IntroductionCartilage regeneration have been attracted attentions because of the poor ability of cartilage tissues to regenerate. Three-dimensional (3D) culture of chondrocytes is considered to be advantageous for cartilage regeneration. Although it is plausible that maturation of the constructs before transplantation positively affects the chondrogenesis, matured constructs after cultures for longer periods do not necessarily result in effective cartilage regeneration. In this study, we compared different types of culture media including growth factors which are clinically available. We prepared differentiation medium containing insulin-like growth factor-1 (IGF-1), proliferation medium containing fibroblast growth factor-2 (FGF-2) and insulin, and combination of them, and compared their efficacies on chondrogenesis when used in 3D culture of engineered cartilage constructs.MethodsCartilage constructs were fabricated by auricular chondrocytes and atelocollagen, and they were 3D-cultured with four types of media: control medium, differentiation medium, proliferation medium, and combination medium. After 3 weeks of culture, the constructs were analyzed for cell number, gene and protein expressions and mechanical properties. The constructs were also transplanted into nude mice. After 8 weeks, the degree of cartilage regeneration was evaluated. Constructs manufactured with canine auricular chondrocytes were subjected to autologous transplantation into beagles and examined for cartilage regeneration.ResultsDuring 3D culture, remarkably high gene expression of type II collagen was detected in the construct cultured with the differentiation medium whereas cell apoptosis were suppressed in the proliferation medium. When transplanted into nude mice, the constructs 3D-cultured in the proliferation medium produced abundant cartilage matrices. In autologous implantation model, the construct cultured in the proliferation medium again showed better chondrogenesis than those in other media.ConclusionsThe present study indicates that 3D culture with the proliferation medium maintains the cell viability to potentiate the subsequent cartilage regeneration. Here, we propose that not only differentiation but also high cell viability accompanied by proliferation factors should be taken into account to improve cartilage regeneration.

Regenerative medicine of tissue engineering： auricular cartilage regeneration and functional reconstruction

Summary Deformity of the auricle can result from congenital defects, oncologic resection, or traumatic injury. Current clinical strategy is to utilize autologous costal cartilage for reconstructing auricular scaffolds with long-term stability. However, this method is limited by donor-site morbidity, a complex surgical sculpting process, and differing mechanical properties compared to elastic auricular cartilage. Alternatively, alloplastic implants can offer precise shape and avoid donor-site surgery, but suffer from poor biocompatibility and high rates of infection and extrusion. A tissue engineering approach to auricular reconstructions would overcome the limitations of both autologous and alloplastic transplants. In this review we focus on tissue engineering auricular cartilage reconstruction, including auricular seed cells culture and their differentiated control strategy, scaffolds choices and design technique. Many studies found that auricular chondrocytes were the most suitable seed cells to fabricate elastic cartilage; progenitor cells from auricular cartilage could differentiate into elastic chondrocytes, and other types of chondrocytes had its limitation because of the final ossification. Many types of growth factors had the differing potential ability for accelerating chondrocytes growth, differentiation, or maintaining chondrocytes phenotype. Recent advances in tissue engineering auricular cartilage, including several clinic applications, implied that tissue engineering auricules would become a hopefully potential soulution for the future of auricular reconstruction.

Cartilage regeneration using decellularized cartilage matrix: Long-term comparison of subcutaneous and intranasal placement in a rabbit model

Autologous cartilage as donor tissue for various surgical reconstructions such as nasal septum regeneration is limited and associated with donor site morbidity. Our goal was to evaluate a new resorbable chondroconductive biomaterial made of decellularized porcine nasal septum cartilage compared with autologous native auricular cartilage as the gold standard. In order to examine the material and determine its long-term outcome further, we used subcutaneous implantation and septal implantation in an orthotopic rabbit model. In addition to non-seeded decellularized xenogenic cartilage, chondrocyte-seeded decellularized xenogenic cartilage was implanted as a septal replacement. After a three- or six-month period, the formation of newly synthesized cartilage extracellular matrix was evaluated immunohistochemically, whereas septal integrity and biocompatibility were evaluated histologically. The formation of the implanted neoseptum and form stability was analyzed by using 7-Tesla Magnetic Resonance Imaging.Good biocompatibility with no excessive rejection was demonstrated in all groups. Long-term stable and reliable septal reconstruction could be achieved in the study groups with or without cell seeding with autologous auricular chondrocytes. Autologous cell seeding was advantageous only with regard to septal perforations. Thus, cell seeding provides a benefit regarding long-term stability. However, because of slightly better biocompatibility, less pronounced septum deviation and the markedly lower effort involved, the non-seeded scaffold is favoured for possible clinical application.

New Developments in Understanding the Histological Structure of Human Ear Cartilage

Histological, immunohistochemical and molecular examination of bioptic samples of 30 normal adult auricular cartilages and small samples from 6 ear cartilages from aborted foetuses was performed. The adult cartilage was the tissue with minimal proliferative activity, which we were able to confirm with antibodies against Ki67 in contrast to a high proliferative activity in the auricular cartilage of foetal tissues. It may therefore be presumed that the process of foetal tissue maturation is undoubtedly associated with a significant reduction in proliferative activity. The mature lamella of the adult auricular cartilage has a histological tri-lamellar structure. There are a great number of elastic fibres in the intercellular matrix of the central zone, which are conversely present in only small amounts in both peripheral layers. While the external layer of the concave surface of the cartilage contains a fewer number of oval elements, the external layer of the convex side is composed of numerous fusiform chondrocytes. Antibodies against various subtypes of S-100 protein showed that auricular chondrocyte activity is modified depending on the configuration of individual distinct zones (isoforms A1, A6, B2 and P were positive in all layers, isoforms A2 and A2 in peripheral zones). The most active cells metabolically are most likely chondrocytes in both external layers adjacent to the perichondrium. We have also demonstrated α-smooth muscle actin (SMA)-positive chondrocytes in both peripheral layers of the auricular cartilage adjacent to the perichondrium. In addition, we found definite differences in the distribution of actin-positive cells depending on the external shape of the pinna. The majority of these fusiform cells were localised primarily in the areas of great curvature of the pinna, especially the convex side, as mentioned above. On the basis of these unique structural features we assume that the ear cartilage may embody an example of the socalled intelligent biological material, which has its internal structure made in such a way as to more easily develop and yet still maintain all the shape characteristics of the human auricle. The knowledge of these specific structural characteristics is important especially for use of auricular cartilage in auricular reconstruction.

Implant-type tissue-engineered cartilage derived from human auricular chondrocyte may maintain cartilaginous property even under osteoinductive condition.

Introduction: There is a growing need for chondrocyte implantation for reconstructing cartilage defect. However, ossification of the implanted cartilage is a challenging problem. Implant-type tissue-engineered cartilage from human auricular chondrocytes is a three-dimensional implant type cartilage using PLLA as a scaffold for chondrocytes. Although there is a study which evaluated the ossification of this cartilage in subcutaneous area, there is no study which clarify the possibility of ossification in osteoinductive surroundings. The purpose of this study was to elucidate the possibility of the ossification of implant-type tissue-engineered cartilage using human auricular chondrocyte in an osteoinductive environment. Methods: Human chondrocytes were harvested from ear cartilage. After dispersion by enzyme digestion, they were put into either a poly-L-lactic acid (PLLA) or poly lactic-co-glycolic acid (PLGA) scaffold, with collagen gel. Implant-type tissue-engineered cartilage was interposed between pieces of human iliac bone harvested from the same donor and implanted subcutaneously in nude rats. Scaffold without chondrocytes was used as a control. After 1, 3, and 6 months, ossification and cartilage formation were evaluated by X-ray, hematoxylin-eosin (HE) stain and toluidine blue (TB) stain. Results: There was no ossification of implant-type cartilage using human chondrocytes, even under osteoinductive conditions. HE staining showed that perichondrium formed around the constructs and chondrocytes were observed 6months after the implantation. TB staining showed metachromasia in every sample, with the area of metachromasia increasing over time, suggesting maturation of the cartilage. Conclusions: In conclusion, adjacent iliac bone had no apparent effect on the maturation of cartilage in implant-type tissue-engineered cartilage. Cartilage retention and maturation even in the presence of iliac bone could have been due to a scarcity of mesenchymal stem cells in the bone and surrounding area.

Microtia patients: Auricular chondrocyte ECM is promoted by CGF through IGF-1 activation of the IGF-1R/PI3K/AKT pathway.

To explore the effectiveness of the insulin-like growth factor 1receptor (IGF-1R)/PI3K/AKT pathway in promoting the synthesis of the auricular chondrocyte extracellular matrix (ECM) using concentrated growth factor (CGF). Chondrocytes isolated from the remnant auricular cartilage of microtia patients were randomly divided into different experimental and control groups, then stimulated with a reagent.IGF-1 released by CGF was quantified by enzyme-linked immunosorbent assay. Glycosaminoglycan (GAG), proteoglycan, and type II collagen (COLAII) were examined by histological and immunohistological analysis. Expression levels of IGF-1R, pIGF-1R, PI3K, pPI3K, AKT, pAKT, COLAII, and Aggrecan were detected by western blot analysis technique andgene expression was tested by real-time polymerase chain reaction. CGF significantly stimulated the synthesis of COLAII and Aggrecan and increased the phosphorylation levels of IGF-1R, PI3K, and AKT. Small interfering RNA IGF-1 blocked ECM synthesis, COLAII and Aggrecan gene expression, and IGF-1R/PI3K/AKT activation. Inhibitor AG1024 and LY294002 significantly inhibited ECM synthesis and the phosphorylation of IGF-1R, PI3K, and AKT. CGF-released IGF-1 stimulates the synthesis of the auricular chondrocyte ECM via the IGF-1R/PI3K/AKT signaling pathway.

Tissue engineering the human auricle by auricular chondrocyte-mesenchymal stem cell co-implantation.

Children suffering from microtia have few options for auricular reconstruction. Tissue engineering approaches attempt to replicate the complex anatomy and structure of the ear with autologous cartilage but have been limited by access to clinically accessible cell sources. Here we present a full-scale, patient-based human ear generated by implantation of human auricular chondrocytes and human mesenchymal stem cells in a 1:1 ratio. Additional disc construct surrogates were generated with 1:0, 1:1, and 0:1 combinations of auricular chondrocytes and mesenchymal stem cells. After 3 months in vivo, monocellular auricular chondrocyte discs and 1:1 disc and ear constructs displayed bundled collagen fibers in a perichondrial layer, rich proteoglycan deposition, and elastin fiber network formation similar to native human auricular cartilage, with the protein composition and mechanical stiffness of native tissue. Full ear constructs with a 1:1 cell combination maintained gross ear structure and developed a cartilaginous appearance following implantation. These studies demonstrate the successful engineering of a patient-specific human auricle using exclusively human cell sources without extensive in vitro tissue culture prior to implantation, a critical step towards the clinical application of tissue engineering for auricular reconstruction.