A quick and innovative pipeline for producing chondrocyte-homing peptide-modified extracellular vesicles by three-dimensional dynamic culture of hADSCs spheroids to modulate the fate of remaining ear chondrocytes in the M1 macrophage-infiltrated microenvironment.

BACKGROUND: Extracellular vesicles (EVs) derived from human adipose-derived mesenchymal stem cells (hADSCs) have shown great therapeutic potential in plastic and reconstructive surgery. However, the limited production and functional molecule loading of EVs hinder their clinical translation. Traditional two-dimensional culture of hADSCs results in stemness loss and cellular senescence, which is unfavorable for the production and functional molecule loading of EVs. Recent advances in regenerative medicine advocate for the use of three-dimensional culture of hADSCs to produce EVs, as it more accurately simulates their physiological state. Moreover, the successful application of EVs in tissue engineering relies on the targeted delivery of EVs to cells within biomaterial scaffolds. METHODS AND RESULTS: The hADSCs spheroids and hADSCs gelatin methacrylate (GelMA) microspheres are utilized to produce three-dimensional cultured EVs, corresponding to hADSCs spheroids-EVs and hADSCs microspheres-EVs respectively. hADSCs spheroids-EVs demonstrate excellent production and functional molecule loading compared with hADSCs microspheres-EVs. The upregulation of eight miRNAs (i.e. hsa-miR-486-5p, hsa-miR-423-5p, hsa-miR-92a-3p, hsa-miR-122-5p, hsa-miR-223-3p, hsa-miR-320a, hsa-miR-126-3p, and hsa-miR-25-3p) and the downregulation of hsa-miR-146b-5p within hADSCs spheroids-EVs show the potential of improving the fate of remaining ear chondrocytes and promoting cartilage formation probably through integrated regulatory mechanisms. Additionally, a quick and innovative pipeline is developed for isolating chondrocyte homing peptide-modified EVs (CHP-EVs) from three-dimensional dynamic cultures of hADSCs spheroids. CHP-EVs are produced by genetically fusing a CHP at the N-terminus of the exosomal surface protein LAMP2B. The CHP + LAMP2B-transfected hADSCs spheroids were cultured with wave motion to promote the secretion of CHP-EVs. A harvesting method is used to enable the time-dependent collection of CHP-EVs. The pipeline is easy to set up and quick to use for the isolation of CHP-EVs. Compared with nontagged EVs, CHP-EVs penetrate the biomaterial scaffolds and specifically deliver the therapeutic miRNAs to the remaining ear chondrocytes. Functionally, CHP-EVs show a major effect on promoting cell proliferation, reducing cell apoptosis and enhancing cartilage formation in remaining ear chondrocytes in the M1 macrophage-infiltrated microenvironment. CONCLUSIONS: In summary, an innovative pipeline is developed to obtain CHP-EVs from three-dimensional dynamic culture of hADSCs spheroids. This pipeline can be customized to increase EVs production and functional molecule loading, which meets the requirements for regulating remaining ear chondrocyte fate in the M1 macrophage-infiltrated microenvironment.

Co-culture of RhoA-overexpressed microtia chondrocytes and adipose-derived stem cells in the construction of tissue-engineered ear-shaped cartilage.

Microtia is a congenital auricle dysplasia with a high incidence and tissue engineering technology provides a promising strategy to reconstruct auricles. We previously described that the engineered cartilage constructed from microtia chondrocytes exhibited inferior levels of biochemical and biomechanical properties, which was proposed to be resulted of the decreased migration ability of microtia chondrocytes. In the current study, we found that Rho GTPase members were deficient in microtia chondrocytes. By overexpressing RhoA, Rac1, and CDC42, respectively, we further demonstrated that RhoA took great responsibility for the decreased migration ability of microtia chondrocytes. Moreover, we constructed PGA/PLA scaffold-based cartilages to verify the chondrogenic ability of RhoA overexpressed microtia chondrocytes, and the results showed that overexpressing RhoA was of limited help in improving the quality of microtia chondrocyte engineered cartilage. However, coculture of adipose-derived stem cells (ADSCs) significantly improved the biochemical and biomechanical properties of engineered cartilage. Especially, coculture of RhoA overexpressed microtia chondrocytes and ADSCs produced an excellent effect on the wet weight, cartilage-specific extracellular matrix, and biomechanical property of engineered cartilage. Furthermore, we presented that coculture of RhoA overexpressed microtia chondrocytes and ADSCs combined with human ear-shaped PGA/PLA scaffold and titanium alloy stent fabricated by CAD/CAM and 3D printing technology effectively constructed and maintained auricle structure in vivo. Collectively, our results provide evidence for the essential role of RhoA in microtia chondrocytes and a developed strategy for the construction of patient-specific tissue-engineered auricular cartilage.

Profiling of Long Non-Coding RNAs in Auricular Cartilage of Patients with Isolated Microtia.

Introduction: Microtia is the second most common maxillofacial birth defect worldwide. However, the involvement of long non-coding RNAs (lncRNAs) in isolated microtia is not well understood. This study aimed at identifying lncRNAs that regulate the expression of genes associated with isolated microtia. Methods: We used our microarray data to analyze the expression pattern of lncRNA in the auricular cartilage tissues from 10 patients diagnosed with isolated microtia, alongside 15 control subjects. Five lncRNAs were chosen for validation using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Results: We identified 4651 differentially expressed lncRNAs in the auricular cartilage from patients with isolated microtia. By Gene Ontology/Kyoto Encyclopedia of Genes and Genomes pathway (GO/KEGG) analysis, we identified 27 differentially expressed genes enriched in pathways associated with microtia. In addition, we predicted 9 differentially expressed genes as potential cis-acting targets of 12 differentially expressed lncRNAs. Our findings by qRT-PCR demonstrate significantly elevated expression levels of ZFAS1 and DAB1-AS1, whereas ADIRF-AS1, HOTAIRM1, and EPB41L4A-AS1 exhibited significantly reduced expression levels in the auricular cartilage tissues of patients with isolated microtia. Conclusions: Our study sheds light on the potential involvement of lncRNAs in microtia and provides a basis for further investigation into their functional roles and underlying mechanisms.

Congenital microtia patients: the genetically engineered exosomes released from porous gelatin methacryloyl hydrogel for downstream small RNA profiling, functional modulation of microtia chondrocytes and tissue-engineered ear cartilage regeneration.

BACKGROUND: Mesenchymal stem cells (MSCs) exosomes were previously shown to be effective in articular cartilage repairing. However, whether MSCs exosomes promote mature cartilage formation of microtia chondrocytes and the underlying mechanism of action remains unknown. Additionally, some hurdles, such as the low yield and unsatisfactory therapeutic effects of natural exosomes have emerged when considering the translation of exosomes-therapeutics to clinical practices or industrial production. Herein, we investigated the roles of human adipose-derived stem cells (ADSCs) exosomes in modulating microtia chondrocytes and the underlying mechanism of action. Special attention was also paid to the mass production and functional modification of ADSCs exosomes. RESULTS: We firstly used porous gelatin methacryloyl (Porous Gelma) hydrogel with pores size of 100 to 200 µm for 3D culture of passage 2, 4 and 6 ADSCs (P2, P4 and P6 ADSCs, respectively), and obtained their corresponding exosomes (Exo 2, Exo 4 and Exo 6, respectively). In vitro results showed Exo 2 outperformed both Exo 4 and Exo 6 in enhancing cell proliferation and attenuating apoptosis. However, both Exo 4 and Exo 6 promoted chondrogenesis more than Exo 2 did. Small RNA sequencing results indicated Exo 4 was similar to Exo 6 in small RNA profiles and consistently upregulated PI3K/AKT/mTOR signaling pathway. Notably, we found hsa-miR-23a-3p was highly expressed in Exo 4 and Exo 6 compared to Exo 2, and they modulated microtia chondrocytes by transferring hsa-miR-23a-3p to suppress PTEN expression, and consequently to activate PI3K/AKT/mTOR signaling pathway. Then, we designed genetically engineered exosomes by directly transfecting agomir-23a-3p into parent P4 ADSCs and isolated hsa-miR-23a-3p-rich exosomes for optimizing favorable effects on cell viability and new cartilage formation. Subsequently, we applied the engineered exosomes to in vitro and in vivo tissue-engineered cartilage culture and consistently found that the engineered exosomes could enhance cell proliferation, attenuate apoptosis and promote cartilage regeneration. CONCLUSIONS: Taken together, the porous Gelma hydrogel could be applied to exosomes mass production, and functional modification could be achieved by selecting P4 ADSCs as parent cells and genetically modifying ADSCs. Our engineered exosomes are a promising candidate for tissue-engineered ear cartilage regeneration.

Fabrication of chondrocytes/chondrocyte-microtissues laden fibrin gel auricular scaffold for microtia reconstruction.

Fibrin gel-based scaffolds have promising potential for microtia reconstruction. Autologous chondrocytes and chondrocyte cell sheets are frequently used seed cell sources for cartilage tissue engineering. However, the aesthetic outcome of chondrocyte-based microtia reconstruction is still not satisfactory. In this study, we aimed to fabricate the chondrocytes/chondrocyte-microtissues laden fibrin gel auricular scaffold for microtia reconstruction. We designed a unique auricular mold that could fabricate a fibrin gel scaffold resembling human auricle anatomy. Primary chondrocytes were harvested from rabbit auricular cartilage, and chondrocyte cell sheets were developed. Chondrocyte-microtissues were prepared from the cell sheets. The mixture of chondrocytes/chondrocyte-microtissues was laden in fibrin gel during the auricular scaffold fabrication. The protrusions and recessed structure in the auricular scaffold surface were still clearly distinguishable. After a one-week in vitro culture, the 3 D structure and auricular anatomy of the scaffold were retained. And followed by eight-week subcutaneous implantation, cartilaginous tissue was regenerated in the artificial auricular structure as indicated by the results of H&E, Toluidine blue, Safranin O, and type II collagen (immunohistochemistry) staining. Protrusions and depressions of the auricular scaffold were slightly deformed, but the overall auricular anatomy was maintained after 8-week in vivo implantation. Extracellular matrix components content were similar in artificial auricular cartilage and rabbit native auricular cartilage. In conclusion, the mixture of chondrocytes/chondrocyte-microtissues laden fibrin gel auricular scaffold showed a promising potential for cartilaginous tissue regeneration, suggesting this as an effective approach for autologous chondrocyte-based microtia reconstruction.

Preclinical assessment of clinically streamlined, 3D-printed, biocompatible single- and two-stage tissue scaffolds for ear reconstruction.

Auricular reconstruction is a technically demanding procedure requiring significant surgical expertise, as the current gold standard involves hand carving of the costal cartilage into an auricular framework and re-implantation of the tissue. 3D-printing presents a powerful tool that can reduce technical demands associated with the procedure. Our group compared clinical, radiological, histological, and biomechanical outcomes in single- and two-stage 3D-printed auricular tissue scaffolds in an athymic rodent model. Briefly, an external anatomic envelope of a human auricle was created using DICOM computed tomography (CT) images and modified in design to create a two-stage, lock-in-key base and elevating platform. Single- and two-stage scaffolds were 3D-printed by laser sintering poly-L-caprolactone (PCL) then implanted subcutaneously in five athymic rats each. Rats were monitored for ulcer formation, site infection, and scaffold distortion weekly, and scaffolds were explanted at 8 weeks with analysis using microCT and histologic staining. Nonlinear finite element analysis was performed to determine areas of high strain in relation to ulcer formation. Scaffolds demonstrated precise anatomic appearance and maintenance of integrity of both anterior and posterior auricular surfaces and scaffold projection, with no statistically significant differences in complications noted between the single- and two-staged implantation. While minor superficial ulcers occurred most commonly at the lateral and superior helix coincident with finite element predictions of high skin strains, evidence of robust tissue ingrowth and angiogenesis was visible grossly and histologically. This promising preclinical small animal model supports future initiatives for making clinically viable options for an ear tissue scaffold.

Protein profile of ear auricle cartilage and the important role of ITGB1/PTK2 in microtia.

BACKGROUND: Microtia is a congenital malformation of the external ear that involves anything from a small reduction in size to a complete absence. The external ear is composed of elastic cartilage which is also the important skeleton of the outer ear. However no previous study explored the difference between abnormal elastic cartilage and normal cartilage in the molecular level. METHODS: Microtia cartilage and normal cartilage tissue samples from patients subjected to autologous costal cartilage reconstruction were obtained in surgery. Total proteins were extracted and purified, and then proteomic analyzed via LC-MS/MS using DDA/DIA data collection methods. Proteins were also isolated with lysis beads and then analyzed via antibody chip. Differentially expressed proteins were identified in both experiments and further analyzed with functional enrichment analysis and KEGG pathway analysis. Valuable regulatory gene expression level was verified by RT-PCR. RESULTS: A total of 4178 protein types were identified in the DDA experiment. A total of 2154 proteins were quantified, 172 of which were significantly upregulated and 82 downregulated in the microtia group (P < 0.05). Antibody chip detection allowed identification of 584 protein phosphorylation sites with 102 upregulation sites and 9 downregulation sites (P < 0.05). Differentially altered proteins were annotated to 143 KEGG pathways, while differentiated phosphate site-associated genes were annotated into 21 KEGG pathways. Two intersecting pathways, the PI3K/AKT/mTOR pathway and the focal adhesion pathway, may paly important role on ear auricle cartilage development. One item is significant in both differential protein expression and phosphorylation. Integrin beta-1, that is downregulated in protein quantification of the microtia group. The mean ITGB1 mRNA level of the microtia patient group was significantly lower than in the healthy control group (P = 0.0007 < 0.05). And the gene expression of downstream gene PTK2 was also decreased. (P = 0.0288 < 0.05). CONCLUSION: The research locates the key protein Integrin Beta-1, and verified it at the mRNA level. The increasing level of ITGB1 and decreasing of PTK2 may play an important role in congenital ear deformity. This research will inspire more otolaryngologists and orthopedics doctors to pay attention to the etiology and mechanism of microtia.

Design and fabrication of a hybrid alginate hydrogel/poly(ε-caprolactone) mold for auricular cartilage reconstruction.

The aim of this study was to design and manufacture an easily assembled cartilage implant model for auricular reconstruction. First, the printing accuracy and mechanical properties of 3D-printed poly-ε-caprolactone (PCL) scaffolds with varying porosities were determined to assess overall material properties. Next, the applicability of alginate as cell carrier for the cartilage implant model was determined. Using the optimal outcomes of both experiments (in terms of (bio)mechanical properties, cell survival, neocartilage formation, and printing accuracy), a hybrid auricular implant model was developed. PCL scaffolds with 600 µm distances between strands exhibited the best mechanical properties and most optimal printing quality for further exploration. In alginate, chondrocytes displayed high cell survival (~83% after 21 days) and produced cartilage-like matrix in vitro. Alginate beads cultured in proliferation medium exhibited slightly higher compressive moduli (6 kPa) compared to beads cultured in chondrogenic medium (3.5 kPa, p > .05). The final auricular mold could be printed with 300 µm pores and high fidelity, and the injected chondrocytes survived the culture period of 21 days. The presented hybrid auricular mold appears to be an adequate model for cartilage tissue engineering and may provide a novel approach to auricular cartilage regeneration for facial reconstruction. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1711-1721, 2019.

Roles of macrophage migration inhibitory factor in cartilage tissue engineering.

To obtain stable outcomes in regenerative medicine, understanding and controlling immunological responses in transplanted tissues are of great importance. In our previous study, auricular chondrocytes in tissue-engineered cartilage transplanted in mice were shown to express immunological factors, including macrophage migration inhibitory factor (MIF). Since MIF exerts pleiotropic functions, in this study, we examined the roles of MIF in cartilage regenerative medicine. We made tissue-engineered cartilage consisting of auricular chondrocytes of C57BL/6J mouse, atellocollagen gel and a PLLA scaffold, and transplanted the construct subcutaneously in a syngeneic manner. Localization of MIF was prominent in cartilage areas of tissue-engineered cartilage at 2 weeks after transplantation, though it became less apparent by 8 weeks. Co-culture with RAW264 significantly increased the expression of MIF in chondrocytes, suggesting that the transplanted chondrocytes in tissue-engineered cartilage could enhance the expression of MIF by stimulation of surrounding macrophages. When MIF was added in the culture of chondrocytes, the expression of type II collagen was increased, indicating that MIF could promote the maturation of chondrocytes. Meanwhile, toluidine blue staining of constructs containing wild type (Mif+/+) chondrocytes showed increased metachromasia compared to MIF-knockout (Mif-/-) constructs at 2 weeks. However, this tendency was reversed by 8 weeks, suggesting that the initial increase in cartilage maturation in Mif+/+ constructs deteriorated by 8 weeks. Since the Mif+/+ constructs included more iNOS-positive inflammatory macrophages at 2 weeks, MIF might induce an M1 macrophage-polarized environment, which may eventually worsen the maturation of tissue-engineered cartilage in the long term.

Cell yield, chondrogenic potential, and regenerated cartilage type of chondrocytes derived from ear, nasoseptal, and costal cartilage.

Functional reconstruction of large cartilage defects in subcutaneous sites remains clinically challenging because of limited donor cartilage. Tissue engineering is a promising and widely accepted strategy for cartilage regeneration. To date, however, this strategy has not achieved a significant breakthrough in clinical translation owing to a lack of detailed preclinical data on cell yield and functionality of clinically applicable chondrocytes. To address this issue, the current study investigated the initial cell yield, proliferative potential, chondrogenic capacity, and regenerated cartilage type of human chondrocytes derived from auricular, nasoseptal, and costal cartilage using a scaffold-free cartilage regeneration model (cartilage sheet). Chondrocytes from all sources exhibited high sensitivity to basic fibroblast growth factor within 8 passages. Nasoseptal chondrocytes presented the strongest proliferation rate, whereas auricular chondrocytes obtained the highest total cell amount using comparable cartilage sample weights. Importantly, all chondrocytes at fifth passage showed strong chondrogenic capacity both in vitro and in the subcutaneous environment of nude mice. Although some significant differences in histological structure, cartilage matrix content and cartilage type specific proteins were observed between the in vitro engineered cartilage and original tissue; the in vivo regenerated cartilage showed mature cartilage features with high similarity to their original native tissue, except for minor matrix changes influenced by the in vivo environment. The current study provides detailed preclinical data for choice of chondrocyte source and thus promotes the clinical translation of cartilage regeneration approach.

Comparisons of Auricular Cartilage Tissues from Different Species.

OBJECTIVES: Tissue engineering of auricular cartilage has great potential in providing readily available materials for reconstructive surgeries. As the field of tissue engineering moves forward to developing human tissues, there needs to be an interspecies comparison of the native auricular cartilage in order to determine a suitable animal model to assess the performance of engineered auricular cartilage in vivo. METHODS: Here, we performed interspecies comparisons of auricular cartilage by comparing tissue microstructure, protein localization, biochemical composition, and mechanical properties of auricular cartilage tissues from rat, rabbit, pig, cow, and human. RESULTS: Human, pig, and cow auricular cartilage have smaller lacunae compared to rat and rabbit cartilage ( P < .05). Despite differences in tissue microstructure, human auricular cartilage has similar biochemical composition to both rat and rabbit. Auricular cartilage from pig and cow, alternatively, display significantly higher glycosaminoglycan and collagen contents compared to human, rat, and rabbit ( P < .05). The mechanical properties of human auricular cartilage were comparable to that of all 4 animal species. CONCLUSIONS: This is the first study that compares the microstructural, biochemical, and mechanical properties of auricular cartilage from different species. This study showed that different experimental animal models of human auricular cartilage may be suitable in different cases.

Age-related histologic and biochemical changes in auricular and septal cartilage.

OBJECTIVES/HYPOTHESIS: To characterize the histologic and biochemical properties of auricular and septal cartilage and analyze age-related changes in middle-aged to older adults. STUDY DESIGN: Cross-sectional study of auricular and septal cartilage from 33 fresh cadavers. METHODS: Auricular and septal cartilage specimens were stained using Safranin O for glycosaminoglycans, Verhoeff's stain for elastin, and Masson's trichrome for collagen. Percentage of tissue stained, cell density and size were quantified. Relationships between donor characteristics and histologic properties were evaluated using mixed model analyses. RESULTS: The average donor age was 75 years (standard deviation = 11 years; range, 55-93 years). In auricular cartilage, each 1-year increase in age was associated with a 0.97% decrease in glycosaminoglycans (P < .001) and a 0.98% decrease in elastin (P < .001). In septal cartilage, glycosaminoglycans decreased 2.4% per year (P < .001). Age did not affect collagen content significantly in auricular (P = .417) or septal cartilage (P = .284). Cell density and cell size declined with age in auricular (both P < .001) and septal cartilage (P = .044, P = .032, respectively). Compared to septal cartilage in patients of all ages, auricular cartilage had more glycosaminoglycans, less collagen, higher cell density, and smaller cells. CONCLUSIONS: In auricular and septal cartilage, glycosaminoglycans, elastin, cell density, and cell size decrease significantly with age in patients over 55 years of age. Glycosaminoglycan content declines faster with age in septal cartilage than auricular cartilage. These age-related changes may affect biomechanical properties and tissue viability, and thereby have implications for graft choice in functional, aesthetic, and reconstructive nasal surgery. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:E399-E407, 2017.

Cartilage engineering in reconstructive surgery: auricular, nasal and tracheal engineering from a surgical perspective.

This review provides an update on cartilage tissue engineering with particular focus on the head and neck. It is aimed at scientists and clinicians who are interested in tissue engineering and its clinical applicability. Principal tissue engineering strategies are summarized in the first part of this review. In the second part, current clinical approaches to auricular, nasal and tracheal reconstruction are discussed from a surgical perspective. By this approach, the requirements for clinical applicability are outlined and new insight into relevant aims of research is given to accelerate the transfer from bench to bedside.

Biological roles of glial fibrillary acidic protein as a biomarker in cartilage regenerative medicine.

Glial fibrillary acidic protein (GFAP) is an intermediate filament that is expressed in specifically expressed auricular chondrocytes, which are good cell sources of cartilage regenerative medicine. Although our group uses GFAP as a biomarker of matrix production in the cultured auricular chondrocytes, the biological roles of GFAP in auricular chondrocytes has remained unknown. In this study, we demonstrated the biological functions of GFAP in the human and mouse derived auricles to clarify the significance and role with the chondrocytes of GFAP in order to provide useful information for reliable and safe regenerative medicine. We examined the cell responses to stretch stress for these chondrocytes and completed a nuclear morphological analysis. Based on these results, GFAP seems to support the resistance to severe mechanical stress in the tissue which physiologically suffers from a stretch overload, and plays pivotal roles in the conservation of cell structures and functions through the maintenance of nuclear morphology.

Development of a 3D cell printed structure as an alternative to autologs cartilage for auricular reconstruction.

Surgical technique using autologs cartilage is considered as the best treatment for cartilage tissue reconstruction, although the burdens of donor site morbidity and surgical complications still remain. The purpose of this study is to apply three-dimensional (3D) cell printing to fabricate a tissue-engineered graft, and evaluate its effects on cartilage reconstruction. A multihead tissue/organ building system is used to print cell-printed scaffold (CPS), then assessed the effect of the CPS on cartilage regeneration in a rabbit ear. The cell viability and functionality of chondrocytes were significantly higher in CPS than in cell-seeded scaffold (CSS) and cell-seeded hybrid scaffold (CSHS) in vitro. CPS was then implanted into a rabbit ear that had an 8 mm-diameter cartilage defect; at 3 months after implantation the CPS had fostered complete cartilage regeneration whereas CSS and autologs cartilage (AC) fostered only incomplete healing. This result demonstrates that cell printing technology can provide an appropriate environment in which encapsulated chondrocytes can survive and differentiate into cartilage tissue in vivo. Moreover, the effects of CPS on cartilage regeneration were even better than those of AC. Therefore, we confirmed the feasibility of CPS as an alternative to AC for auricular reconstruction. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1016-1028, 2017.

Biomechanical Characterisation of the Human Auricular Cartilages; Implications for Tissue Engineering.

Currently, autologous cartilage provides the gold standard for auricular reconstruction. However, synthetic biomaterials offer a number of advantages for ear reconstruction including decreased donor site morbidity and earlier surgery. Critical to implant success is the material's mechanical properties as this affects biocompatibility and extrusion. The aim of this study was to determine the biomechanical properties of human auricular cartilage. Auricular cartilage from fifteen cadavers was indented with displacement of 1 mm/s and load of 300 g to obtain a Young's modulus in compression. Histological analysis of the auricle was conducted according to glycoprotein, collagen, and elastin content. The compression modulus was calculated for each part of the auricle with the tragus at 1.67 ± 0.61 MPa, antitragus 1.79 ± 0.56 MPa, concha 2.08 ± 0.70 MPa, antihelix 1.71 ± 0.63 MPa, and helix 1.41 ± 0.67 MPa. The concha showed to have a significantly greater Young's Elastic Modulus than the helix in compression (p < 0.05). The histological analysis demonstrated that the auricle has a homogenous structure in terms of chondrocyte morphology, extracellular matrix and elastin content. This study provides new information on the compressive mechanical properties and histological analysis of the human auricular cartilage, allowing surgeons to have a better understanding of suitable replacements. This study has provided a reference, by which cartilage replacements should be developed for auricular reconstruction.

Morphological Characteristics of the Cartilaginous Tissue of Human Auricle in Different Age Periods.

A complex morphological study of the auricle to determine the human age was performed by evaluating the metric sizes between fixed points in each auricle with axial guidelines. The auricular elastic cartilage in different age periods was characterized by thickening of the cartilaginous plate, different mature and immature cartilage zone ratio, variations in the volume density of the intercellular substance and elastic fibers, and change in the numerical density of individual chondrocytes and isogroups. Aggrecan content in the cartilage was shown to increase in different age periods. Age-related structural changes in the auricular cartilage expand the possibilities of forensic medical examination and hold much promise for the identification of personality.

FT-IR Microspectroscopy of Rat Ear Cartilage.

Rat ear cartilage was studied using Fourier transform-infrared (FT-IR) microspectroscopy to expand the current knowledge which has been established for relatively more complex cartilage types. Comparison of the FT-IR spectra of the ear cartilage extracellular matrix (ECM) with published data on articular cartilage, collagen II and 4-chondroitin-sulfate standards, as well as of collagen type I-containing dermal collagen bundles (CBs) with collagen type II, was performed. Ear cartilage ECM glycosaminoglycans (GAGs) were revealed histochemically and as a reduction in ECM FT-IR spectral band heights (1140-820 cm-1) after testicular hyaluronidase digestion. Although ear cartilage is less complex than articular cartilage, it contains ECM components with a macromolecular orientation as revealed using polarization microscopy. Collagen type II and GAGs, which play a structural role in the stereo-arrangement of the ear cartilage, contribute to its FT-IR spectrum. Similar to articular cartilage, ear cartilage showed that proteoglycans add a contribution to the collagen amide I spectral region, a finding that does not recommend this region for collagen type II quantification purposes. In contrast to articular cartilage, the symmetric stretching vibration of -SO3- groups at 1064 cm-1 appeared under-represented in the FT-IR spectral profile of ear cartilage. Because the band corresponding to the asymmetric stretching vibration of -SO3- groups (1236-1225 cm-1) overlapped with that of amide III bands, it is not recommended for evaluation of the -SO3- contribution to the FT-IR spectrum of the ear cartilage ECM. Instead, a peak (or shoulder) at 1027-1016 cm-1 could be better considered for this intent. Amide I/amide II ratios as calculated here and data from the literature suggest that protein complexes of the ear cartilage ECM are arranged with a lower helical conformation compared to pure collagen II. The present results could motivate further studies on this tissue under pathological or experimental states involving ear cartilage.

The formation of human auricular cartilage from microtic tissue: An in vivo study.

OBJECTIVES: This study aimed to isolate, culture-expand and characterize the chondrocytes isolated from microtic cartilage and evaluate its potential as a cell source for ear cartilage reconstruction. Specific attention was to construct the auricular cartilage tissue by using fibrin as scaffold. STUDY DESIGN: Cell culture experiment with the use of microtic chondrocytes. DESIGN: Cell culture experiment with the use of microtic chondrocytes. METHODS: After ear reconstructive surgery at the Universiti Kebangsaan Malaysia Medical Center, chondrocytes were isolated from microtic cartilage. Chondrocytes isolated from the tissue were cultured expanded until passage 4 (P4). Upon confluency at P4, chondrocytes were harvested and tissue engineered constructs were made with human plasma polymerized to fibrin. Constructs formed later is implanted at the dorsal part of nude mice for 8 weeks, followed by post-implantation evaluation with histology staining (Hematoxylin and Eosin (H&E) and Safranin O), immunohistochemistry and RT-PCR for chondrogenic associated genes expression level. RESULTS: Under gross assessment, the construct after 8 weeks of implantation showed similar physical characteristics that of cartilage. Histological staining showed abundant lacunae cells embedded in extracellular matrix similar to that of native cartilage. Safranin O staining showed positive staining which indicates the presence of proteoglycan-rich matrix. Immunohistochemistry analysis showed the strong positive staining for collagen type II, the specific collagen type in the cartilage. Gene expression quantification showed no significant differences in the expression of chondrogenic gene used which is collagen type I, collagen type II, aggrecan core protein (ACP), elastin and sox9 genes when compared to construct formed from normal auricular tissue. CONCLUSION: Chondrocytes isolated from microtia cartilage has the potential to be used as an alternative cell source for external ear reconstruction in future clinical application.

Identification of shorter length lamin A protein in mouse ear cartilage tissue.

Lamin A is an intermediate filament protein which is cleaved by the enzyme, FACE 1 at VTRSY↓L. The cleavage is the final step in the production of the mature lamin A protein. The mature lamin A protein localizes in the inner membrane of the nucleus. The mutation in the lamin A gene causes many diseases, including accelerated aging. It is known that the protein is not expressed in neuronal cells of the brain. Many splicing variants of the lamin A gene have been reported. In this study, the amino acid sequence VTRSY (a penta-peptide repeat) was found in three different sites of the C-terminal end of the lamin A protein, the protein expressed in cells of ear cartilage tissues is shorter than the protein expressed in cells of the skin tissues. Using two lamin A antibodies, it was found that the amino acid sequence between penta-peptide 2 and 3 is missing in lamin A protein that was expressed in the cells of mouse ear cartilage tissue, besides the RT-PCR data confirmed that the corresponding coding sequence between the penta repeat 2 and 3 is intact. Cleavage may occur at the penta-peptide (VTRSY) at site 3 in the lamin A tail of mouse ear cartilage.