Quantitative T2 mapping monitoring the maturation of engineered elastic cartilage in a rabbit model.

BACKGROUND: Cartilage tissue engineering provides a promising approach to reconstruct craniofacial defects, and a noninvasive method is needed to assess its effectiveness. Although magnetic resonance imaging (MRI) has been used to evaluate articular cartilage in vivo, few studies focused on its feasibility in monitoring engineered elastic cartilage (EC). METHODS: Auricular cartilage, silk fibroin (SF) scaffold, and EC consisting of rabbit auricular chondrocytes and SF scaffold were transplanted subcutaneously into the rabbit back. In eight weeks after transplantation, grafts were imaged by MRI using PROSET, PDW VISTA SPAIR, 3D T2 VISTA, 2D MIXED T2 Multislice, and SAG TE multiecho sequences, followed by histological examination and biochemical analysis. Statistical analyses were performed to identify the association between T2 values and biochemical indicator values of EC. RESULTS: In vivo imaging shows that 2D MIXED T2 Multislice sequence (T2 mapping) clearly distinguished the native cartilage, engineered cartilage and fibrous tissue. T2 values showed high correlations with cartilage-specific biochemical parameters at different time points, especially the elastic cartilage specific protein elastin (ELN, r= -0.939, P < 0.001). CONCLUSION: Quantitative T2 mapping can effectively detect the in vivo maturity of engineered elastic cartilage after subcutaneously transplantation. This study would promote the clinical application of MRI T2 mapping in monitoring engineered elastic cartilage in the repair of craniofacial defects.

First evidence of a functional spiracle in stem chondrichthyan acanthodians, with the oldest known elastic cartilage.

Spiracles are a general character of gnathostomes (jawed fishes), being present in antiarch placoderms, commonly regarded as the most basal gnathostome group. The presence of spiracular tubes in acanthodians has been deduced from grooves on the neurocranium of the derived acanthodiform Acanthodes bronni from the Permian of Germany, but until now these tubes were presumed to lack an external opening, rendering them non-functional. Here we describe the external spiracular elements in specimens of the Middle Devonian acanthodiforms Cheiracanthus murchisoni, Cheiracanthus latus and Mesacanthus pusillus from northern Scotland, and the internal structure of these elements in C. murchisoni, demonstrating that the spiracle in acanthodiforms differed from all known extant and extinct fishes in having paired cartilage-pseudobranch structures. This arrangement represents a transitional state between the presumed basal gnathostome condition with an unconstricted first gill slit (as yet not identified in any fossil) and the derived condition with a spiracle and a single pseudobranch derived from the posterior hemibranch of the mandibular arch. We identify the main tissue forming the pseudobranch as elastic cartilage, a tissue previously unrecorded in fossils.

Development of a Method for Scaffold-Free Elastic Cartilage Creation.

Microtia is a congenital aplasia of the auricular cartilage. Conventionally, autologous costal cartilage grafts are collected and shaped for transplantation. However, in this method, excessive invasion occurs due to limitations in the costal cartilage collection. Due to deformation over time after transplantation of the shaped graft, problems with long-term morphological maintenance exist. Additionally, the lack of elasticity with costal cartilage grafts is worth mentioning, as costal cartilage is a type of hyaline cartilage. Medical plastic materials have been transplanted as alternatives to costal cartilage, but transplant rejection and deformation over time are inevitable. It is imperative to create tissues for transplantation using cells of biological origin. Hence, cartilage tissues were developed using a biodegradable scaffold material. However, such materials suffer from transplant rejection and biodegradation, causing the transplanted cartilage tissue to deform due to a lack of elasticity. To address this problem, we established a method for creating elastic cartilage tissue for transplantation with autologous cells without using scaffold materials. Chondrocyte progenitor cells were collected from perichondrial tissue of the ear cartilage. By using a multilayer culture and a three-dimensional rotating suspension culture vessel system, we succeeded in creating scaffold-free elastic cartilage from cartilage progenitor cells.

Effects of loading on chondrocyte hypoxia, HIF-1α and VEGF in the mandibular condylar cartilage of young rats.

OBJECTIVES: To investigate hypoxia-inducible factor 1-alpha (HIF-1α) and vascular endothelial growth factor (VEGF) expression under altered loading, and to explore the relationship between loading and hypoxia in the mandibular condylar cartilage of young rats. SETTING AND SAMPLE POPULATION: Eighty Sprague-Dawley rats. MATERIAL AND METHODS: The reduced loading group was fed soft food, and their incisors were cut to avoid occlusal contact. The increased loading group was fed hard food and had forced jaw-opening. Ten rats from each group (n = 10) were sacrificed at 12, 24, 48, and 96 hours after initiation of the experiment. Pimonidazole hydrochloride (Hypoxyprobe-1, HP-1) was used as a hypoxia marker to confirm the hypoxic state. Hypoxic chondrocytes as indicated by HP-1, HIF-1α and VEGF protein expressions were recognized by immunohistochemical detection. HIF-1α and VEGF mRNA expressions were detected by semi-quantitative RT-PCR. RESULTS: Hypoxyprobe-1 was confined in the upper layers of cartilage, and was most strongly expressed in the weight-bearing area of TMJ at 12 and 96 hours. Staining of HIF-1α and VEGF was most strongly expressed in the chondrocytes of the fibrous and proliferative layer at all time points. Furthermore, expressions were also displayed in the hypertrophic and calcified layers at 48 and 96 hours. The expressions of HIF-1α and VEGF mRNA were higher in the increased loading group than in the reduced loading group at 48 and 96 hours (P < . 05). CONCLUSION: Mechanical loading seems to directly induce weight-bearing area hypoxia followed by new vessel formation, which indicates that these factors are related and important for the development of cartilage.

Encapsulation of human elastic cartilage-derived chondrocytes in nanostructured fibrin-agarose hydrogels.

The generation of elastic cartilage substitutes for clinical use is still a challenge. In this study, we investigated the possibility of encapsulating human elastic cartilage-derived chondrocytes (HECDC) in biodegradable nanostructured fibrin-agarose hydrogels (NFAH). Viable HECDC from passage 2 were encapsulated in NFAH and maintained in culture conditions. Constructs were harvested for histochemical and immunohistochemical analyses after 1, 2, 3, 4 and 5 weeks of development ex vivo. Histological results demonstrated that it is possible to encapsulate HECDC in NFAH, and that HECDC were able to proliferate and form cells clusters expressing S-100 and vimentin. Additionally, histochemical and immunohistochemical analyses of the extracellular matrix (ECM) showed that HECDC synthetized different ECM molecules (type I and II collagen, elastic fibers and proteoglycans) in the NFAH ex vivo. In conclusion, this study suggests that NFAH can be used to generate biodegradable and biologically active constructs for cartilage tissue engineering applications. However, further cell differentiation, biomechanical and in vivo studies are still needed.

Brief report: reconstruction of joint hyaline cartilage by autologous progenitor cells derived from ear elastic cartilage.

In healthy joints, hyaline cartilage covering the joint surfaces of bones provides cushioning due to its unique mechanical properties. However, because of its limited regenerative capacity, age- and sports-related injuries to this tissue may lead to degenerative arthropathies, prompting researchers to investigate a variety of cell sources. We recently succeeded in isolating human cartilage progenitor cells from ear elastic cartilage. Human cartilage progenitor cells have high chondrogenic and proliferative potential to form elastic cartilage with long-term tissue maintenance. However, it is unknown whether ear-derived cartilage progenitor cells can be used to reconstruct hyaline cartilage, which has different mechanical and histological properties from elastic cartilage. In our efforts to develop foundational technologies for joint hyaline cartilage repair and reconstruction, we conducted this study to obtain an answer to this question. We created an experimental canine model of knee joint cartilage damage, transplanted ear-derived autologous cartilage progenitor cells. The reconstructed cartilage was rich in proteoglycans and showed unique histological characteristics similar to joint hyaline cartilage. In addition, mechanical properties of the reconstructed tissues were higher than those of ear cartilage and equal to those of joint hyaline cartilage. This study suggested that joint hyaline cartilage was reconstructed from ear-derived cartilage progenitor cells. It also demonstrated that ear-derived cartilage progenitor cells, which can be harvested by a minimally invasive method, would be useful for reconstructing joint hyaline cartilage in patients with degenerative arthropathies.

Auricular Chondrocytes as a Cell Source for Scaffold-Free Elastic Cartilage Tissue Engineering.

Current tissue engineering (TE) methods utilize chondrocytes primarily from costal or articular sources. Despite the robust mechanical properties of neocartilages sourced from these cells, the lack of elasticity and invasiveness of cell collection from these sources negatively impact clinical translation. These limitations invited the exploration of naturally elastic auricular cartilage as an alternative cell source. This study aimed to determine if auricular chondrocytes (AuCs) can be used for TE scaffold-free neocartilage constructs and assess their biomechanical properties. Neocartilages were successfully generated from a small quantity of primary neonatal AuCs of three minipig donors (n = 3). Neocartilage constructs had instantaneous moduli of 200.5 kPa ± 43.34 and 471.9 ± 92.8 kPa at 10% and 20% strain, respectively. TE constructs' relaxation moduli (Er) were 36.99 ± 6.47 kPa Er and 110.3 ± 16.99 kPa at 10% and 20% strain, respectively. The Young's modulus was 2.0 MPa ± 0.63, and the ultimate tensile strength was 0.619 ± 0.177 MPa. AuC-derived neocartilages contained 0.144 ± 0.011 µg collagen, 0.185 µg ± 0.002 glycosaminoglycans per µg dry weight, and 1.7e-3 µg elastin per µg dry weight. In conclusion, this study shows that AuCs can be used as a reliable and easily accessible cell source for TE of biomimetic and mechanically robust elastic neocartilage implants.

Monitoring wound healing of elastic cartilage using multiphoton microscopy.

OBJECTIVE: To demonstrate the ability of multiphoton microscopy (MPM) for monitoring wound healing of elastic cartilage. METHOD: In a rabbit ear model, four cartilage specimen groups at 1-day, 1-, 4-, 20-week healing time points as well as a normal elastic cartilage were examined with MPM without using labeling agents. MPM images at wound margins were obtained from specimens at different healing stages, compared with the Hematoxylin and Eosin (H&E) stained images. Image analysis was performed to characterize the collagen morphology for quantifying the wound healing progression of elastic cartilage. RESULTS: MPM provided high-resolution images of elastic cartilage at varying depths. Comparisons of the images of specimens at different healing stages show obvious cell growth and matrix deposition. The results are consistent with the histological results. Moreover, quantitative analysis results show significant alteration in the collagen cavity size or collagen orientation index during wound healing of elastic cartilage, indicating the possibility to act as indicators for monitoring wound healing. CONCLUSION: Our results suggested that MPM has the ability to monitor the wound healing progression of elastic cartilage, based on the visualization of cell growth and proliferation and quantitative characterization of collagen morphology during wound healing.

Upregulated desmin/integrin ß1/MAPK axis promotes elastic cartilage regeneration with increased ECM mechanical strength.

Elastic cartilage tissue engineering is promising for providing available scaffolds for plastic reconstructive surgery. The insufficient mechanical strength of regenerative tissue and scarce resources of reparative cells are two obstacles for the preparation of tissue-engineered elastic cartilage scaffolds. Auricular chondrocytes are important reparative cells for elastic cartilage tissue engineering, but resources are scarce. Identifying auricular chondrocytes with enhanced capability of elastic cartilage formation is conducive to reducing the damage to donor sites by decreasing the demand on native tissue isolation. Based on the biochemical and biomechanical differences in native auricular cartilage, we found that auricular chondrocytes with upregulated desmin expressed more integrin ß1, forming a stronger interaction with the substrate. Meanwhile, activated MAPK pathway was found in auricular chondrocytes highly expressing desmin. When desmin was knocked down, the chondrogenesis and mechanical sensitivity of chondrocytes were both impaired, and the MAPK pathway was downregulated. Finally, auricular chondrocytes highly expressing desmin regenerated more elastic cartilage with increased ECM mechanical strength. Therefore, desmin/integrin ß1/MAPK signaling can not only serve as a selection standard but also a manipulation target of auricular chondrocytes to promote elastic cartilage regeneration.

Cadherin-11 promotes the mechanical strength of engineered elastic cartilage by enhancing extracellular matrix synthesis and microstructure.

Limitations of current treatments for auricular cartilage defects have prompted the field of auricular cartilage tissue engineering. To date, inducing the formation of cartilaginous constructs with biochemical and biomechanical properties of native tissue is the final aim. Through hematoxylin-eosin and immunohistochemistry staining, Cadherin-11(CDH11) was confirmed highly expressed in the auricular cartilage tissue and chondrocytes. In vitro, by knockdown and overexpression of CDH11 in chondrocytes, CDH11 was demonstrated to promote the expression of collagen type II (COL2A), elastin (ELN), aggrecan (ACAN), and cartilage oligomeric matrix protein (COMP). In addition, the CDH11 overexpressed chondrocytes promoted neo-cartilage formation and its biomechanical property by increasing the key transcription factor of chondrogenesis SOX9 expression and cartilage extracellular matrix (ECM) production. The young's modulus and yield stress of the neo-cartilage in CDH11 overexpression group were about 1.7 times (p = 0.0152) and 2 times (p = 0.0428) higher than those in control group, respectively. Then, the immunohistochemistry staining, qRT-PCR and western blot examination results showed that the expression of COL2A and ELN were significantly increased. Notably, the electron microscopy results showed that the collagen and elastic fibers of the neo-cartilage in CDH11-OV group arranged in bunches and were more uniform and compact compared to the control group. Furthermore, CDH11 promoted elastic fiber assembly by increasing lysyl oxidase (LOX), fibrillin-1 (FBN1) expression. Taken together, our results demonstrated that CDH11 improves the mechanical strength of tissue-engineered elastic cartilage by promoting ECM synthesis and elastic fiber assembly.

Newly Designed Decellularized Scaffolds for Scaffold-based Gene Therapy from Elastic Cartilages via Supercritical Carbon Dioxide Fluid and Alkaline/ Protease Treatments.

BACKGROUND: Scaffold-based gene therapy provides a promising approach for tissue engineering, which was important and popular as it combined medical applications and engineering materials' knowledge. OBJECTIVE: The decellularization techniques were employed to remove the cellular components from porcine elastic cartilages, leaving a native decellularized Extracellular Matrix (dECM) composition and architecture integrity of largely insoluble collagen, elastin, and tightly bound glycosaminoglycans. For newly designed collagen scaffold samples, elastic cartilages were hydrolyzed by protease with different concentrations to gain state completely and clearly. METHODS: An extraction process of Supercritical Carbon Dioxide (ScCO2) was used to remove cellular components from porcine elastic cartilage. The dECM scaffolds with collagen must be characterized by Fourier transform infrared spectroscopy(FTIR), Thermo-Gravimetric Analysis (TGA), and Scanning Electron Microscope (SEM). RESULTS: The study provided a new treatment combined with supercritical carbon dioxide and alkaline/ protease to prepare dECM scaffolds with hole-scaffold microstructures and introduce into a potential application on osteochondral tissue engineering using scaffold-based gene therapy. The new process is simple and efficient. The pore-scaffold microstructures were observed in dECM scaffolds derived from porcine elastic cartilages. The Tdmax values of the resulting dECM scaffolds were observed at over 330oC. CONCLUSION: A series of new scaffolds were successfully obtained from porcine tissue by using ScCO2 and alkaline/enzyme treatments such as a mixing aqueous solution of NH4OH and papain. The dECM scaffolds with high thermal stability were obtained. The resulting scaffold with clean pore-scaffold microstructure could be a potential application for scaffold-based gene therapy.

Fgf20b is required for the ectomesenchymal fate establishment of cranial neural crest cells in zebrafish.

In cranial skeletal development, the establishment of the ectomesenchymal lineage within the cranial neural crest is of great significance. Fgfs are polypeptide growth factors with diverse functions in development and metabolism. Fgf20b knockdown zebrafish embryos showed dysplastic neurocranial and pharyngeal cartilages. Ectomesenchymal cells from cranial neural crest cells were significantly decreased in Fgf20b knockdown embryos, but cranial neural crest cells with a non-ectomesnchymal fate were increased. However, the proliferation and apoptosis of cranial neural crest cells were essentially unchanged. Fgfr1 knockdown embryos also showed dysplastic neurocranial and pharyngeal cartilages. The present findings indicate that Fgf20b is required for ectomesenchymal fate establishment via the activation of Fgfr1 in zebrafish.

A review of diversity in the evolution and development of cartilage: the search for the origin of the chondrocyte.

Mammalian cartilage is a complex and developmentally important tissue type. Outside the mammalian lineage, cartilage may persist as an adult tissue, which shows a much wider diversity of histological structure. Tissues similar to vertebrate cartilage are also found within multiple invertebrate lineages, including mollusks, arthropods, and polychaetes, however the relationship of these tissues to vertebrate cartilage is unknown. Detailed molecular analysis of these invertebrate tissues is necessary to assess the degree of homology, if any, of cartilage throughout the metazoans. The purpose of the following review is to introduce readers to this diversity of cartilage and to synthesize the known genetic interactions that give rise to vertebrate cartilage into the format of a gene regulatory network (GRN). This chondrogenesis GRN highlights a large number of transcription factors known to be expressed during chondrogenesis, whose role in this process has yet to be elucidated. Verification and expansion of this initial GRN will assist in the identification of the core set of the genetic interactions necessary for the specification of the vertebrate chondrocyte. This is the necessary first step in allowing detailed comparison of the molecular signature of vertebrate chondrocytes with that of invertebrates with the ultimate goal of understanding the evolutionary origin of this important skeletal cell type.

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

OBJECTIVE: 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. DESIGN: 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"). RESULTS: 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. CONCLUSION: 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.

A novel elastic and controlled-release poly(ether-ester-urethane)urea scaffold for cartilage regeneration.

In the tissue engineering of cartilage, scaffolds with appropriate elasticity and controlled-release properties are essential. Herein, we synthesized a poly(ether-ester-urethane)urea scaffold with a pendant amino group (PEEUUN) through a de-protection process from PEEUU-Boc polymers and grafted kartogenin (KGN) onto the PEEUUN scaffolds (PEEUUN-KGN). Characterization, performance tests, scaffold biocompatibility analysis, and chondrogenesis evaluation both in vitro and in vivo were conducted. The results revealed that the PEEUUN-KGN scaffolds were degradable and three-dimensional (3D) with interconnected pores, and possessed good elasticity, as well as excellent cytocompatibility. Meanwhile, KGN on the PEEUUN-KGN scaffolds underwent stable sustained release for a long time and promoted human umbilical cord mesenchymal stem cells (HUCMSCs) to differentiate into chondrocytes in vitro, thus enhancing cartilage regeneration in vivo. In conclusion, the present PEEUUN-KGN scaffolds would have application potential for cartilage tissue engineering.

Optimising the decellularization of human elastic cartilage with trypsin for future use in ear reconstruction.

Decellularized scaffolds can induce chondrogenic differentiation of stem cells. This study compares different methods to optimise the decellularization of auricular cartilage. The process consisted of an initial 12 hour dry freeze thaw which froze the cartilage specimens in an empty tube at -20 °C. Samples were allowed to thaw at room temperature followed by submersion in phosphate buffer solution in which they were frozen at -20 °C for a 12 hour period. They were then allowed to thaw at room temperature as before. Protocol A subsequently involved subjecting specimens to both deoxyribonuclease and sodium deoxycholate. Protocol B and C were adaptations of this using 0.25% trypsin (7 cycles) and a 0.5 molar solution of ethylenediaminetetraacetic acid (3 hours for each cycle) respectively as additional steps. Trypsin accelerated the decellularization process with a reduction in DNA content from 55.4 ng/µL (native) to 17.3 ng/µL (P-value < 0.05) after 14 days. Protocol B showed a faster reduction in DNA content when compared with protocol A. In comparison to protocol C after 14 days, trypsin also showed greater decellularization with a mean difference of 11.7 ng/µL (P-value < 0.05). Histological analysis with H&E and DAPI confirmed depletion of cells at 14 days with trypsin.

Pediatric auricular chondrocytes gene expression analysis in monolayer culture and engineered elastic cartilage.

OBJECTIVES: This study was aimed at regenerating autologous elastic cartilage for future use in pediatric ear reconstruction surgery. Specific attentions were to characterize pediatric auricular chondrocyte growth in a combination culture medium and to assess the possibility of elastic cartilage regeneration using human fibrin. STUDY DESIGN: Laboratory experiment using human pediatric auricular chondrocytes. METHODS: Pediatric auricular chondrocytes growth kinetics and quantitative gene expression profile in three different types of media were compared in primary culture and subsequent three passages. Large-scale culture-expanded chondrocytes from the combination medium were then mixed with human fibrin for the formation of elastic cartilage via tissue engineering technique. RESULTS: The equal mixture of Ham's F12 and Dulbecco's Modified Eagle Medium (FD) promoted the best chondrocyte growth at every passage compared to the individual media. Chondrocytes differentiation index; ratio of type II to type I collagen gene expression level, aggrecan and elastin expression gradually decreased while passaging but they were then restored in engineered tissues after implantation. The engineered cartilage was glistening white in color and firm in consistency. Histological evaluation, immunohistochemistry analysis and quantitative gene expression assessment demonstrated that the engineered cartilage resemble the features of native elastic cartilage. CONCLUSION: Pediatric auricular chondrocytes proliferate better in the combination medium (FD) and the utilization of human fibrin as a biomaterial hold promises for the regeneration of an autologous elastic cartilage for future application in ear reconstructive surgery.

A novel multiphoton microscopy images segmentation method based on superpixel and watershed.

Multiphoton microscopy (MPM) imaging technique based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) shows fantastic performance for biological imaging. The automatic segmentation of cellular architectural properties for biomedical diagnosis based on MPM images is still a challenging issue. A novel multiphoton microscopy images segmentation method based on superpixels and watershed (MSW) is presented here to provide good segmentation results for MPM images. The proposed method uses SLIC superpixels instead of pixels to analyze MPM images for the first time. The superpixels segmentation based on a new distance metric combined with spatial, CIE Lab color space and phase congruency features, divides the images into patches which keep the details of the cell boundaries. Then the superpixels are used to reconstruct new images by defining an average value of superpixels as image pixels intensity level. Finally, the marker-controlled watershed is utilized to segment the cell boundaries from the reconstructed images. Experimental results show that cellular boundaries can be extracted from MPM images by MSW with higher accuracy and robustness.