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.

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.

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.

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.

Chondrogenic differentiation of bone marrow­derived stem cells cultured in the supernatant of elastic cartilage cells.

Repair of cartilage defects remains a challenge for surgeons, owing to its poor self­repairing capacity. Cartilage tissue engineering, particularly marrow stem cell­based cartilage regeneration, provides a promising option for the regeneration of damaged cartilage. Although producing tissue­engineered cartilage from marrow stem cells appeared to be a feasible method, constructing certain sub­types of cartilage, including elastic cartilage, remains difficult. Therefore, the present study explored the feasibility of constructing elastic cartilage by culturing bone marrow­derived stem cells (BMSCs) in the supernatant of elastic cartilage cells to generate elastic cartilage. The elastic cartilage cells were obtained from the auricle cartilage of a newborn pig, and BMSCs were isolated from pig bone marrow aspirate. The supernatant of the chondrocytes was collected and then used to the culture BMSCs. At various time­points, the differentiation of BMSCs was evaluated by gross view, histological examination and quantitative polymerase chain reaction. BMSCs changed from spindle­shaped cells into polygonal cells with increasing culture time. The expression of collagen II and elastin was observed in the cells cultured in the supernatant of elastic chondrocytes, while no expression was observed in the control cells. Furthermore, the expression of collagen I and collagen X was downregulated in the cells cultured in the supernatant of elastic cartilage cells. The supernatant of elastic cartilage cells promoted the differentiation of BMSCs into elastic cartilage cells, which may be a promising method for constructing certain sub­types of tissue­engineered cartilage.

Prefabricated, ear-shaped cartilage tissue engineering by scaffold-free porcine chondrocyte membrane.

BACKGROUND: Ear defects caused by traumatic injury, tumor ablation, and congenital deficiency are still challenging problems for the plastic and reconstructive surgeon. The authors developed a scaffold-free, ear-shaped cartilage by tailoring a multilayered chondrocyte membrane on an ear-shaped titanium alloy model and investigated the possibility of long-term ear-shaped maintenance in nude mice. METHODS: High-density chondrocytes (approximately 30 × 10 cells) were seeded to produce chondrocyte membranes after cultivation under chondrogenic medium for 2 weeks. Then, three-layer chondrocyte membranes were tailored on the ear-shaped titanium mold and fixed by 6-0 nylon. The constructs were implanted onto the dorsal pockets of nude mice for 8 and 24 weeks. The chondrocyte membrane, 8- and 24-week implants were analyzed by safranin O, toluidine blue, elastica van Gieson, and collagen type II immunohistochemistry stains and quantitative measurement of glycosaminoglycan and total collagen compared with native cartilage. Mechanical strength was compared by compressive Young's modulus. RESULTS: Results showed that the chondrocyte membrane was durable and nonfragile and easily manipulated by forceps. The composite of chondrocyte membrane and titanium alloy maintained the stable ear-like shape after 8 and 24 weeks of subcutaneous implantation. Histologic examination verified that the newly formed tissue at the implant construct was elastic cartilage at both 8 and 24 weeks by safranin O, toluidine blue, elastica van Gieson, and collagen type II immunohistochemistry stains. The Young's modulus was only half of and similar to normal cartilage in 8- and 24-week implants, respectively. CONCLUSION: This study demonstrated that an ear-shaped elastic cartilage could be regenerated by a scaffold-free chondrocyte membrane shaped by a prefabricated, three-dimensional, ear-shaped titanium mold.

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.

Cell-engineered human elastic chondrocytes regenerate natural scaffold in vitro and neocartilage with neoperichondrium in the human body post-transplantation.

We have developed a unique method that allows us to culture large volumes of chondrocyte expansion from a small piece of human elastic cartilage. The characteristic features of our culturing method are that fibroblast growth factor-2 (FGF2), which promotes proliferation of elastic chondrocytes, is added to a culture medium, and that cell-engineering techniques are adopted in the multilayered culture system that we have developed. We have subsequently discovered that once multilayered chondrocytes are transplanted into a human body, differentiation induction that makes use of surrounding tissue occurs in situ, and a large cartilage block is obtained through cartinogenesis and matrix formation. We have named this method two-stage transplantation. We have clinically applied this transplantation method to the congenital ear defect, microtia, and reported successful ear reconstruction. In our present study, we demonstrated that when FGF2 was added to elastic chondrocytes, the cell count increased and the level of hyaluronic acid, which is a major extracellular matrix (ECM) component, increased. We also demonstrated that these biochemical changes are reflected in the morphology, with the elastic chondrocytes themselves producing a matrix and fibers in vitro to form a natural scaffold. We then demonstrated that inside the natural scaffold thus formed, the cells overlap, connect intercellularly to each other, and reconstruct a cartilage-like three-dimensional structure in vitro. We further demonstrated by immunohistochemical analysis and electron microscopic analysis that when the multilayered chondrocytes are subsequently transplanted into a living body (abdominal subcutaneous region) in the two-stage transplantation process, neocartilage and neoperichondrium of elastic cartilage origin are regenerated 6 months after transplantation. Further, evaluation by dynamic mechanical analysis showed the regenerated neocartilage to have the same viscoelasticity as normal auricular cartilage. Using our multilayered culture system supplemented with FGF2, elastic chondrocytes produce an ECM and also exhibit an intercellular network; therefore, they are able to maintain tissue integrity post-transplantation. These findings realized a clinical application for generative cartilage surgery.

FGF2 and dexamethasone increase the production of hyaluronan in two-dimensional culture of elastic cartilage-derived cells: in vitro analyses and in vivo cartilage formation.

Elastic cartilage-derived cells cultured two-dimensionally with FGF2 and corticosteroid produce gel-type masses that become mature cartilage when injected into a subcutaneous pocket. This unique method has previously been clinically applied for treatments of nasal augmentation. However, the components of the gel-type mass and the mechanism of its synthesis remain unknown. Here, we have investigated the components of the gel-type mass produced by elastic cartilage-derived cells, and whether this gel-type mass can be produced by using other cell sources or other media. Human elastic cartilage-derived cells from auricular cartilage, hyaline cartilage-derived cells from articular cartilage, and mesenchymal stem cells from synovium were cultured in three media: "redifferentiation medium" containing FGF2 and dexamethasone; "chondrogenic medium" containing bone morphogenetic protein-2, transforming growth factor-beta3, and dexamethasone specific for in vitro chondrogenesis of mesenchymal stem cells; control medium. The elastic cartilage-derived cells cultured in redifferentiation medium produced a gelatinous matrix positive for Alcian blue. During culture, the amount of chondroitin 4-sulfate, chondroitin 6-sulfate, and especially hyaluronan increased. However, the expression of RNAs for most chondrogenic genes did not increase. We also reproduced cartilage tissue formation by the injection of elastic cartilage-derived cells with the gelatinous mass into the subcutaneous space of the nude mouse. The synthesis of gelatinous matrix in vitro and the formation of cartilage tissue in vivo could be obtained only for the combination of elastic cartilage-derived cells with redifferentiation medium.

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.

Immunolocalization of matrix proteins in different human cartilage subtypes.

Cartilage exerts many functions in different tissues and parts of the body. Specific requirements presumably also account for a specific biochemical composition. In this study, we investigated the presence and distribution pattern of matrix components, in particular collagen types in the major human cartilages (hyaline, fibrous, and elastic cartilage) by histochemical and immunohistochemical means. Macroscopically normal articular cartilages, menisci, disci (lumbar spine), epiglottal, and tracheal tissues were obtained from donors at autopsy. Aurical and nasal cartilages were part of routine biopsy samples from tumor resection specimens. Conventional histology and immunohistochemical stainings with collagen types I, II, III, IV, V, VI, and X and S-100 protein antibodies were performed on paraformaldehyde-fixed and paraffin-embedded specimens. The extracellular matrix is the functional component of all cartilages as indicated by the low cell densities. In particular major scaffold forming collagen types I (in fibrous cartilage) and II (in hyaline and elastic cartilages) as well as collagen type X (in the calcified layer of articular cartilages, the inner part of tracheal clips, and epiglottis cartilage) showed a specific distribution. In contrast, the "minor" collagen types III, V, and VI were found in all, collagen type IV in none of the cartilage subtypes. In this study, we present a biochemical profile of the major cartilage types of the human body which is important for understanding the physiology and the pathophysiology of cartilages.