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.

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.

Subcutaneous Regeneration of Engineered Cartilage: A Comparison of Cell Sheets and Chondrocyte-Scaffold Constructs in a Porcine Model.

BACKGROUND: Stable cartilage regeneration in immunocompetent large animals remains a bottleneck problem that restricts clinical application. The inflammation elicited by degradation products of scaffolds has a decisive influence on cartilage formation. Although prolonged preculture in vitro could form mature engineered cartilage and allow sufficient degradation of scaffolds, the inflammatory reaction was still observed. This study explored the feasibility of using chondrocyte sheet technology to regenerate stable cartilage in the subcutaneous environment with a pig model. METHODS: Passage 1 chondrocytes were used to form cell sheets by high-density culture. As a control, chondrocytes were seeded onto polyglycolic acid/polylactic acid scaffolds for 6 and 12 weeks' in vitro preculture, respectively. Then, they were autologously implanted subcutaneously into pigs for 2, 8, and 24 weeks. Gross view, histologic staining, and biochemical and biomechanical characteristics were evaluated. RESULTS: With prolonged culture in vitro, relatively homogeneous engineered cartilages were formed with less scaffold residue. However, the chondrocyte-polyglycolic acid/polylactic acid group still encountered severe inflammation and inferior cartilage formation at 2 and 8 weeks in vivo. The engineered cartilage with cell sheet technique exhibited a relatively more stable and mature tissue structure without obvious inflammatory response at 24 weeks in vivo, which was similar to the native auricular cartilage. CONCLUSIONS: The chondrocyte sheet technique could successfully regenerate mature and stable engineered cartilages in pig models. It is possibly an effective method of repairing cartilage defects in the clinic that uses regenerated substitutes derived from autologous cell sheets.

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

OBJECTIVES/HYPOTHESIS: 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. STUDY DESIGN: This study compises an in vitro cell-culture part as well as an in vivo animal study with New Zealand White Rabbits. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. LEVEL OF EVIDENCE: N/A Laryngoscope, 131:E1624-E1632, 2021.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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.

Creating Cartilage in Tissue-Engineered Chamber Using Platelet-Rich Plasma Without Cell Culture.

Background: Clinically available cartilage, such as large-volume tissue-engineered cartilage, is urgently required for various clinical applications. Tissue engineering chamber (TEC) models are a promising organ-level strategy for efficient enlargement of cells or tissues within the chamber. The conventional TEC technology is not suitable for cartilage culture, because it lacks the necessary chondrogenic growth factor, which is present in platelet-rich plasma (PRP). In this study, we added autogenous auricular cartilage fragments mixed with PRP in a TEC to obtain a large amount of engineered cartilage. Experiment: To prove the efficacy of this method, 48 New Zealand white rabbits were randomly divided into 4 groups: PRP, vascularized (Ves), PRP, PRP+Ves, and control. Auricular cartilage was harvested from the rabbits, cut into fragments (2 mm), and then injected into TECs. Cartilage constructs were harvested at week 8, and construct volumes were measured. Histological morphology, immunochemical staining, and mechanical strength were evaluated. Results: At week 8, PRP+Ves constructs developed a white, cartilage-like appearance. The volume of cartilage increased by 600% the original volume from 0.30 to 1.8 ± 0.1789 mL. Histological staining showed proliferation of edge chondrocytes in the embedded cartilage in the PRP and PRP+Ves groups. Furthermore, the cartilage constructs in the PRP+Ves group show mechanical characteristics similar to those of normal cartilage. Conclusions: Auricular cartilage fragments mixed with PRP and vascularization of the TEC showed a significantly increased cartilage tissue volume after 8 weeks of incubation in rabbits. Impact Statement Repair of defects of ear cartilage tissue has always been a huge challenge to plastic surgeons. In this article, a new method is presented to produce within 8 weeks auricular cartilage in a tissue engineering chamber without cell culture. Having such a method is a valuable step toward creating a large volume of functional cartilage tissue, which may lead to successful construction of normal auricular structure with minimal donor-site morbidity.

Recellularization of auricular cartilage via elastase-generated channels.

Decellularized tissue matrices are promising substrates for tissue generation by stem cells to replace poorly regenerating tissues such as cartilage. However, the dense matrix of decellularized cartilage impedes colonisation by stem cells. Here, we show that digestion of elastin fibre bundles traversing auricular cartilage creates channels through which cells can migrate into the matrix. Human chondrocytes and bone marrow-derived mesenchymal stromal cells efficiently colonise elastin-treated scaffolds through these channels, restoring a glycosaminoglycan-rich matrix and improving mechanical properties while maintaining size and shape of the restored tissue. The scaffolds are also rapidly colonised by endogenous cartilage-forming cells in a subcutaneously implanted osteochondral biopsy model. Creating channels for cells in tissue matrices may be a broadly applicable strategy for recellularization and restoration of tissue function.

Stable Tracheal Regeneration Using Organotypically Cultured Tissue Composed of Autologous Chondrocytes and Epithelial Cells in Beagles.

OBJECTIVES: In tracheal regeneration, the slow process of epithelialization is often a barrier to the stability and safety of the transplanted trachea. The aim of this study was to examine a new tracheal regeneration technique using organotypically cultured tissue composed of autologous cells. METHODS: Nine beagles were prepared. Chondrocytes from auricular cartilage and epithelial cells from buccal mucosa were isolated and cultured. Tissue-engineered cartilages were fabricated with chondrocytes at a density of 1 × 107 cells/mL (high-density group) and 1 × 106 cells/mL (low-density group). A fabricated epithelial cell sheet was laid on a poly(lactic-co-glycolic acid) block in atelocollagen gel containing the chondrocytes, and the organotypically cultured tissues were transplanted into a partially resected trachea. The control group had only the block transplanted. RESULTS: The tissue-engineered cartilages in the high-density group contained many viable chondrocytes and many cartilage matrices. The low-density group had abundant collagen fibers and no chondrocytes. Tracheal endoscopy revealed no deformation or atrophy at the transplant site in the high-density group. Histologically, partially hyaline cartilages covered with epithelium and lamina propria were found in the high-density group but not in the low-density and control groups. CONCLUSIONS: Stable tracheal regeneration was achieved using organotypically cultured tissue fabricated with autologous high-density chondrocytes and epithelial cells.

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.

Pore architecture effects on chondrogenic potential of patient-specific 3-dimensionally printed porous tissue bioscaffolds for auricular tissue engineering.

OBJECTIVE: This study aims to determine the effect of auricular scaffold microarchitecture on chondrogenic potential in an in vivo animal model. METHODS: DICOM computed tomography (CT) images of a human auricle were segmented to create an external anatomic envelope. Image-based design was used to generate 1) orthogonally interconnected spherical pores and 2) randomly interspersed pores, and each were repeated in three dimensions to fill the external auricular envelope. These auricular scaffolds were then 3D printed by laser sintering poly-l-caprolactone, seeded with primary porcine auricular chondrocytes in a hyaluronic acid/collagen hydrogel and cultured in a pro-chondrogenic medium. The auricular scaffolds were then implanted subcutaneously in rats and explanted after 4 weeks for analysis with Safranin O and Hematoxylin and Eosin staining. RESULTS: Auricular constructs with two micropore architectures were rapidly manufactured with high fidelity anatomic appearance. Subcutaneous implantation of the scaffolds resulted in excellent external appearance of both anterior and posterior auricular surfaces. Analysis on explantation showed that the defined, spherical micropore architecture yielded histologic evidence of more robust chondrogenic tissue formation as demonstrated by Safranin O and Hematoxylin and Eosin staining. CONCLUSIONS: Image-based computer-aided design and 3D printing offers an exciting new avenue for the tissue-engineered auricle. In early pilot work, creation of spherical micropores within the scaffold architecture appears to impart greater chondrogenicity of the bioscaffold. This advantage could be related to differences in permeability allowing greater cell migration and nutrient flow, differences in surface area allowing different cell aggregation, or a combination of both factors. The ability to design an anatomically correct scaffold that maintains its structural integrity while also promoting auricular cartilage growth represents an important step towards clinical applicability of this new technology.

Cultivation of auricular chondrocytes in poly(ethylene glycol)/poly(ε-caprolactone) hydrogel for tracheal cartilage tissue engineering in a rabbit model.

Tissue engineering has the potential to overcome the limitations of tracheal reconstruction. To tissue-engineer a tracheal cartilage, auricular chondrocytes were encapsulated in a photocurable poly(ethylene glycol)/poly(ε-caprolactone) (PEG/PCL) hydrogel. Chondrogenic genes, including Sox9, Acan and Col2a1, were up-regulated in auricular chondrocytes after 2 weeks of in vitro cultivation in the PEG/PCL hydrogel. Co-cultivation of 70 % auricular chondrocytes and 30 % bone marrow mesenchymal stem cells (BMSCs) accelerated the chondrogenic genes' expression in the PEG/PCL hydrogel. Cartilaginous matrix markers, including proteoglycans and collagen type II, were detected in the chondrocytes-encapsulated PEG/PCL hydrogel after 4 weeks of in vitro cultivation. The higher expression level of cartilaginous matrix markers was observed in the PEG/PCL hydrogel with co-cultivation of 70 % chondrocytes and 30 % BMSCs. After 4 weeks of ectopic cultivation in rabbits, the cylindrical PEG/PCL structure was sustained with the use of a luminal silicon stent. However, without the stent, the construct collapsed under a compression force. No fibrosis or vessel ingrowth were found in the PEG/PCL hydrogel after 4 weeks of ectopic cultivation, whereas the auricular chondrocytes showed proteoglycans' accumulation and collagen type II production. Rabbit auricular chondrocytes could survive and retain chondrogenic ability in the PEG/PCL hydrogel under both in vitro and in vivo conditions. While the PEG/PCL hydrogel did not show sufficient mechanical properties for supporting the cylindrical shape of the construct, the high chondrogenesis level of chondrocytes in the PEG/PCL hydrogel displayed the potential of this material for tracheal tissue engineering.

Progenitor cells in auricular cartilage demonstrate cartilage-forming capacity in 3D hydrogel culture.

Paramount for the generation of auricular structures of clinically-relevant size is the acquisition of a large number of cells maintaining an elastic cartilage phenotype, which is the key in producing a tissue capable of withstanding forces subjected to the auricle. Current regenerative medicine strategies utilize chondrocytes from various locations or mesenchymal stromal cells (MSCs). However, the quality of neo-tissues resulting from these cell types is inadequate due to inefficient chondrogenic differentiation and endochondral ossification, respectively. Recently, a subpopulation of stem/progenitor cells has been identified within the auricular cartilage tissue, with similarities to MSCs in terms of proliferative capacity and cell surface biomarkers, but their potential for tissue engineering has not yet been explored. This study compared the in vitro cartilage-forming ability of equine auricular cartilage progenitor cells (AuCPCs), bone marrow-derived MSCs and auricular chondrocytes in gelatin methacryloyl (gelMA)-based hydrogels over a period of 56 d, by assessing their ability to undergo chondrogenic differentiation. Neocartilage formation was assessed through gene expression profiling, compression testing, biochemical composition and histology. Similar to MSCs and chondrocytes, AuCPCs displayed a marked ability to generate cartilaginous matrix, although, under the applied culture conditions, MSCs outperformed both cartilage-derived cell types in terms of matrix production and mechanical properties. AuCPCs demonstrated upregulated mRNA expression of elastin, low expression of collagen type X and similar levels of proteoglycan production and mechanical properties as compared to chondrocytes. These results underscored the AuCPCs' tissue-specific differentiation potential, making them an interesting cell source for the next generation of elastic cartilage tissue-engineered constructs.

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.

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.

Long-Term Comparison between Human Normal Conchal and Microtia Chondrocytes Regenerated by Tissue Engineering on Nanofiber Polyglycolic Acid Scaffolds.

BACKGROUND: Previous regeneration studies of auricle-shaped cartilage by tissue engineering leave unresolved whether the chondrocyte phenotype from human auricular chondrocytes seeded onto polymeric scaffolds is retained over the long term and whether microtia remnants may be a viable cell source for auricular reconstruction. METHODS: Chondrocytes were isolated from human ears, either normal conchal ear or microtia cartilage remnants, expanded in vitro, and seeded onto nanoscale-diameter polyglycolic acid sheets. These tissue-engineered constructs were implanted into athymic mice for up to 40 weeks. At harvest times of 5, 10, 20, and 40 weeks, samples were documented by gross morphology, histology, and reverse transcription-quantitative polymerase chain reaction analysis. RESULTS: Neocartilages generated from the two types of surgical tissues were similar in appearance of their extracellular matrices and positive staining for elastin and proteoglycans. In the 5- to 40-week time interval, there was an increasing trend in gene expression for type II collagen, elastin, and sex determining region Y box 5, important to normal cartilage phenotype, and a decreasing trend in gene expression for type III collagen, a fibroblast and dedifferentiation marker. Over 40 weeks of implantation, the original nanoscale-diameter polyglycolic acid scaffold dimensions (1 cm × 1 cm × 80 µm) were generally maintained in tissue-engineered cartilage length and width, and thickness was statistically significantly increased. CONCLUSIONS: Auricular cartilage can be regenerated over the long term (40 weeks) from surgical remnants by tissue-engineering techniques incorporating nanoscale-diameter polyglycolic acid scaffolds. Based on the present assays, microtia neocartilage very closely resembles tissue-engineered cartilage regenerated from chondrocytes isolated from normal conchal cartilage.

Three-Dimensional Cell Printing of Large-Volume Tissues: Application to Ear Regeneration.

The three-dimensional (3D) printing of large-volume cells, printed in a clinically relevant size, is one of the most important challenges in the field of tissue engineering. However, few studies have reported the fabrication of large-volume cell-printed constructs (LCCs). To create LCCs, appropriate fabrication conditions should be established: Factors involved include fabrication time, residence time, and temperature control of the cell-laden hydrogel in the syringe to ensure high cell viability and functionality. The prolonged time required for 3D printing of LCCs can reduce cell viability and result in insufficient functionality of the construct, because the cells are exposed to a harsh environment during the printing process. In this regard, we present an advanced 3D cell-printing system composed of a clean air workstation, a humidifier, and a Peltier system, which provides a suitable printing environment for the production of LCCs with high cell viability. We confirmed that the advanced 3D cell-printing system was capable of providing enhanced printability of hydrogels and fabricating an ear-shaped LCC with high cell viability. In vivo results for the ear-shaped LCC also showed that printed chondrocytes proliferated sufficiently and differentiated into cartilage tissue. Thus, we conclude that the advanced 3D cell-printing system is a versatile tool to create cell-printed constructs for the generation of large-volume tissues.

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.

Bioengineering pediatric scaffold-free auricular cartilaginous constructs.

OBJECTIVE: The use of exogenous materials as scaffolds in cartilage tissue engineering has limited the clinical application of resultant constructs due to the risk of postoperative complications. In an effort to minimize such complications, we aim to generate human, scaffold-free auricular cartilaginous constructs. STUDY DESIGN: Laboratory study using pediatric auricular cartilage. METHODS: Remnant, normal pediatric auricular cartilage samples that would have otherwise been discarded were collected and digested to free cells. Harvested cells were cultured and expanded in vitro for two passages and plated as micromass cultures. The culture medium was replaced with a chemically defined chondrogenic medium, and cellular monolayers surrounding micromass cultures were continuously scraped off. Constructs were allowed to mature for a period of 8 weeks. RESULTS: Micromass constructs showed mechanical stability and structurally resembled native auricular tissue, with a perichondrium-like layer of cells surrounding the inner cartilaginous zone. Constructs accumulated equivalent sulphated glycosaminoglycan and 50% of collagen content compared to native auricular cartilage by mass, while displaying 156% more cellularity. CONCLUSIONS: High-density micromass cultures of pediatric auricular chondrocytes can generate stable cartilaginous constructs following prolonged chondrogenic inductions in vitro. This technique is an essential step toward the development of three-dimensional constructs to recreate clinically applicable auricular cartilaginous constructs. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:E153-E158, 2017.

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.