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Background: Current clinical approaches to regenerate temporomandibular joint (TMJ) articulating cartilage defects only treat the symptoms (i.e. pain and dysfunction) and do not seek to restore joint integrity for long-term relief. Therefore, we investigated a novel self-assembling tissue-engineered cartilage to overcome this significant clinical issue for TMJ regenerative purposes., Objectives: Examine the maturation of dynamic self-regenerating cartilage (dSRC) using auricular chondrocytes and evaluate a novel combinatorial approach with fractional laser treatment and dSRC implantation for TMJ cartilage repair., Materials and Methods: A suspension of 10 7 freshly harvested rabbit ear chondrocytes was cultured under a continuous reciprocating motion to form the dSRC. After 2, 4 and 8 weeks of culture, dSRC samples were stained with H&E, Safranin-O and Toluidine Blue. Immunohistochemistry (IHC) was performed for collagens type I and II. Channels (300-500 μm diameter and 1.2-1.5 mm depth) were created in six freshly harvested condyles using a fractional Erbium laser. Two groups were tested: dSRC in a laser-ablated lesion (experimental) and an empty laser-ablated channel (control). TMJ condyles were cultured for up to 8 weeks and analysed as described above., Results: H&E staining showed a high cell density in dSRC compared to native cartilage. All dSRC groups demonstrated intense Safranin-O staining, indicating high glycosaminoglycan (GAG) production and intense Toluidine Blue staining showed high proteoglycan content. IHC confirmed that dSRC consisted predominantly of collagen type II. The experimental group showed improved cartilage repair at both time points compared to the empty channels., Conclusion: dSRC viability and successful matrix formation were demonstrated in vitro. The combination of fractional laser ablation and dSRC implantation enhanced cartilage repair., (© 2024 John Wiley & Sons Ltd.)

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.

Reconstruction of the human auricle remains a formidable challenge for plastic surgeons. Autologous costal cartilage grafts and alloplastic implants are technically challenging, and aesthetic and/or tactile outcomes are frequently suboptimal. Using a small animal "bioreactor", we have bioengineered full-scale ears utilizing decellularized cartilage xenograft placed within a 3D-printed external auricular scaffold that mimics the size, shape, and biomechanical properties of the native human auricle. The full-scale polylactic acid ear scaffolds were 3D-printed based upon data acquired from 3D photogrammetry of an adult ear. Ovine costal cartilage was processed either through mincing (1 mm 3 ) or zesting (< 0.5 mm 3 ), and then fully decellularized and sterilized. At explantation, both the minced and zested neoears maintained the size and contour complexities of the scaffold topography with steady tissue ingrowth through 6 months in vivo. A mild inflammatory infiltrate at 3 months was replaced by homogenous fibrovascular tissue ingrowth enveloping individual cartilage pieces at 6 months. All ear constructs were pliable, and the elasticity was confirmed by biomechanical analysis. Longer-term studies of the neoears with faster degrading biomaterials will be warranted for future clinical application. STATEMENT OF SIGNIFICANCE: Accurate reconstruction of the human auricle has always been a formidable challenge to plastic surgeons. In this article, we have bioengineered full-scale ears utilizing decellularized cartilage xenograft placed within a 3D-printed external auricular scaffold that mimic the size, shape, and biomechanical properties of the native human auricle. Longer-term studies of the neoears with faster degrading biomaterials will be warranted for future clinical application., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

There are many physiologic and psychologic challenges associated with ear cartilage deformities which are incredibly distasteful to patients, particularly children. The development of regenerative medicine (RM) sciences has opened up a new window for the reconstruction of auricular cartilage because it allows the creation of a structure similar to the auricular in appearance and function. As part of this review, we discuss the role that each RM tool, including tissue engineering, cells, and biomolecules, plays in developing engineered auricular tissue. In previous studies, it was shown that the simultaneous use of natural and synthetic biomaterials as well as three-dimensional printing techniques could improve the biological and mechanical properties of this tissue. Another critical issue is using stem cells and differentiated cartilage cells to produce tissue-specific cellular structures and extracellular matrix. Also, the importance of choosing a suitable animal model in terms of handling and care facilities, physiologic similarities to humans, and breed uniformity in the preclinical assessments have been highlighted. Then, the clinical trials registered on the clinicaltrials.gov website, and the commercialized product, called AuriNovo, have been comprehensively explained. Overall, it is important to provide engineered auricular cartilage structures with acceptable safety and efficacy compared with standard methods, autologous cartilage transplantation, and prosthetic reconstruction in RM., Competing Interests: Disclosure: The authors have no conflicts of interest to report., (Copyright © ASAIO 2023.)

Designing clinical applicable polymeric composite scaffolds for auricular cartilage tissue engineering requires appropriate mechanical strength and biological characteristics. In this study, silk fiber-based scaffolds co-reinforced with poly-L-lactic acid porous microspheres (PLLA PMs) combined with either Bombyx mori (Bm) or Antheraea pernyi (Ap) silk fibers were fabricated as inspired by the "steel bars reinforced concrete" structure in architecture and their chondrogenic functions were also investigated. We found that the Ap silk fiber-based scaffolds reinforced by PLLA PMs (MAF) exhibited superior physical properties (the mechanical properties in particular) as compared to the Bm silk fiber-based scaffolds reinforced by PLLA PMs (MBF). Furthermore, in vitro evaluation of chondrogenic potential showed that the MAF provided better cell adhesion, viability, proliferation and GAG secretion than the MBF. Therefore, the MAF are promising in auricular cartilage tissue engineering and relevant plastic surgery-related applications., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright © 2020 by the American Society of Plastic Surgeons.)

Introduction: In conditions like recurrent perforations, atelectatic tympanic membrane and poor eustachian tube function, temporalis fascia graft fails to give the desired result. In such cases cartilage is used for tympanoplasty. It was demonstrated that if the thickness of cartilage is reduced to around 0.5 mm, the sound conduction is comparable to that of normal tympanic membrane with excellent mechanical stability., Aim: To intra-operatively measure the mean thickness of tragal and conchal cartilage and compare it for age and sex variations., Material & Methods: A total of 114 tragal and conchal cartilage samples of 86 patients were included in the study. Thickness of cartilages was measured intra-operatively after removing the perichondrium from both sides., Results: Out of 58 tragal cartilages, 32 were from males and 26 from females. Mean thickness was 1.18 ± 0.11 mm among males and 1.12 ± 0.14 mm among females. Out of 56 conchal cartilage taken, 29 were from males and 27 females. Mean thickness among males were 1.38 ± 0.13 mm and 1.35 ± 0.08 mm in females. In 28 patients both tragal and conchal cartilage was taken. Mean thickness of both tragal (1.22 mm) and conchal cartilage (1.36 mm) increased with increase in age. Among 16 males in whom both cartilages were taken, mean thickness of tragal cartilage was 1.25 ± 0.11 mm and conchal cartilage was 1.41 ± 0.12 mm. Similarly among 12 females where both cartilages were taken, mean thickness of tragal cartilage was 1.20 ± 0.13 mm and conchal cartilage was 1.35 ± 0.07 mm., Conclusion: Sliced cartilage tympanoplasty is a relatively better technique. When using cartilage splitter to get sliced cartilage, ideally thickness of every graft should be known. As it is difficult to measure the exact thickness in every case, so knowing the mean for age and sex for cartilage thickness is important to have an idea of which plates to use for a successful outcome of slicing. We concluded that thickness of tragal cartilage is significantly less than the thickness of conchal cartilage. Also there is significant age related difference between mean thickness of cartilages, both for tragal and conchal cartilage. Surprisingly the difference between thickness in male and female is not statistically different., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: The etiology of osteoarthritis (OA) is unknown, however, there appears to be a significant contribution from genetics. We have identified recombinant inbred strains of mice derived from LG/J (large) and SM/J (small) strains that vary significantly in their ability to repair articular cartilage and susceptibility to post-traumatic OA due to their genetic composition. Here, we report cartilage repair phenotypes in the same strains of mice in which OA susceptibility was analyzed previously, and determine the genetic correlations between phenotypes., Design: We used 12 recombinant inbred strains, including the parental strains, to test three phenotypes: ear-wound healing (n = 263), knee articular cartilage repair (n = 131), and post-traumatic OA (n = 53) induced by the surgical destabilization of the medial meniscus (DMM). Genetic correlations between various traits were calculated as Pearson's correlation coefficients of strain means., Results: We found a significant positive correlation between ear-wound healing and articular cartilage regeneration (r = 0.71; P = 0.005). We observed a strong inverse correlation between articular cartilage regeneration and susceptibility to OA based on maximum (r = -0.54; P = 0.036) and summed Osteoarthritis Research Society International (OARSI) scores (r = -0.56; P = 0.028). Synovitis was not significantly correlated with articular cartilage regeneration but was significantly positively correlated with maximum (r = 0.63; P = 0.014) and summed (r = 0.70; P = 0.005) OARSI scores. Ectopic calcification was significantly positively correlated with articular cartilage regeneration (r = 0.59; P = 0.021)., Conclusions: Using recombinant inbred strains, our study allows, for the first time, the measurement of genetic correlations of regeneration phenotypes with degeneration phenotypes, characteristic of OA (cartilage degeneration, synovitis). We demonstrate that OA is positively correlated with synovitis and inversely correlated with the ability to repair cartilage. These results suggest an addition to the risk paradigm for OA from a focus on degeneration to regeneration., (Copyright © 2020 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Objective: To investigate the transcriptomic differences in chondrocytes obtained from LG/J (large, healer) and SM/J (small, non-healer) murine strains in an attempt to discern the molecular pathways implicated in cartilage regeneration and susceptibility to osteoarthritis (OA)., Design: We performed RNA-sequencing on chondrocytes derived from LG/J (n = 16) and SM/J (n = 16) mice. We validated the expression of candidate genes and compared single nucleotide polymorphisms (SNPs) between the two mouse strains. We also examined gene expression of positional candidates for ear pinna regeneration and long bone length quantitative trait loci (QTLs) that display differences in cartilaginous expression., Results: We observed a distinct genetic heterogeneity between cells derived from LG/J and SM/J mouse strains. We found that gene ontologies representing cell development, cartilage condensation, and regulation of cell differentiation were enriched in LG/J chondrocytes. In contrast, gene ontologies enriched in the SM/J chondrocytes were mainly related to inflammation and degeneration. Moreover, SNP analysis revealed that multiple validated genes vary in sequence between LG/J and SM/J in coding and highly conserved noncoding regions. Finally, we showed that most QTLs have 20-30% of their positional candidates displaying differential expression between the two mouse strains., Conclusions: While the enrichment of pathways related to cell differentiation, cartilage development and cartilage condensation infers superior healing potential of LG/J strain, the enrichment of pathways related to cytokine production, immune cell activation and inflammation entails greater susceptibility of SM/J strain to OA. These data provide novel insights into chondrocyte transcriptome and aid in identification of the quantitative trait genes and molecular differences underlying the phenotypic differences associated with individual QTLs., (Copyright © 2020 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

The effects of FM system fitted into the normal hearing ear (NHE) or a cartilage conduction hearing aid (CCHA) fitted into the affected ear (AE) on the speech recognition ability in noise were examined in children with unilateral congenital aural atresia (UCAA). In children with bilateral normal hearing (BNH), speech recognition score (SRS) was significantly decreased in the noisy environment of -5 dB signal-to-noise ratio (SNR), compared with those in quiet. In children with UCAA, SRS was significantly decreased in noisy environments of 0 and -5 dB SNR, compared with those in quiet. In noisy environments of 0 and -5 dB SNR, SRS in children with UCAA was significantly decreased, compared those in children with BNH. In the noisy environment of -5 dB SNR, SRS in UCAA children aided by FM system fitted into NHE was significantly better than those in unaided children in the same group. In the noisy environment of 0 dB SNR, SRS in UCAA children aided by CCHA into AE tended to be higher than those in unaided children in the same group. FM system and CCHA can be recommended as an audiological management for the improvement of speech recognition in children with UCHL in classrooms. J. Med. Invest. 67 : 134-138, February, 2020.

It has been long considered that air and bone are the two major mediators that conduct sounds to the inner ear. In 2004, Hosoi found that vibration of aural cartilage, generated by placing gently a transducer on it, could create audible sound with the same level of clarity as air- and bone-conduction sound. He thus proposed the term "cartilage conduction" for this concept. This research identified a third mediator for sound conduction to the inner ear. Hosoi also proposed the development of novel communication devices, such as hearing aids, telephones, etc. using his findings. For cartilage conduction, three sound pathways can be assumed. The transducer vibration may cause airborne sound which passes into the external auditory canal through the canal entrance (direct air pathway). Alternatively, the vibration at the cartilage may generate audible sound in the external auditory canal (cartilage-air pathway), or propagate directly to the inner ear through the skull bone (cartilage-bone pathway). A series of studies has illustrated that the cartilage-air pathway is dominant for hearing sensations in listeners with normal ears. The cartilage-bone pathway works for patients with bony aural atresia. A fourth pathway, the fibrotic-tissue pathway, is considered to act in the case of fibrotic aural atresia. In this review, we summarize this series of studies and discuss the nature of cartilage conduction., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Biomaterials currently in use for articular cartilage regeneration do not mimic the composition or architecture of hyaline cartilage, leading to the formation of repair tissue with inferior characteristics. In this study we demonstrate the use of "AuriScaff", an enzymatically perforated bovine auricular cartilage scaffold, as a novel biomaterial for repopulation with regenerative cells and for the formation of high-quality hyaline cartilage. AuriScaff features a traversing channel network, generated by selective depletion of elastic fibers, enabling uniform repopulation with therapeutic cells. The complex collagen type II matrix is left intact, as observed by immunohistochemistry, SEM and TEM. The compressive modulus is diminished, but three times higher than in the clinically used collagen type I/III scaffold that served as control. Seeding tests with human articular chondrocytes (hAC) alone and in co-culture with human adipose-derived stromal/stem cells (ASC) confirmed that the network enabled cell migration throughout the scaffold. It also guides collagen alignment along the channels and, due to the generally traverse channel alignment, newly deposited cartilage matrix corresponds with the orientation of collagen within articular cartilage. In an osteochondral plug model, AuriScaff filled the complete defect with compact collagen type II matrix and enabled chondrogenic differentiation inside the channels. Using adult articular chondrocytes from bovine origin (bAC), filling of even deep defects with high-quality hyaline-like cartilage was achieved after 6 weeks in vivo. With its composition and spatial organization, AuriScaff provides an optimal chondrogenic environment for therapeutic cells to treat cartilage defects and is expected to improve long-term outcome by channel-guided repopulation followed by matrix deposition and alignment. STATEMENT OF SIGNIFICANCE: After two decades of tissue engineering for cartilage regeneration, there is still no optimal strategy available to overcome problems such as inconsistent clinical outcome, early and late graft failures. Especially large defects are dependent on biomaterials and their scaffolding, guiding and protective function. Considering the currently used biomaterials, structure and mechanical properties appear to be insufficient to fulfill this task. The novel scaffold developed within this study is the first approach enabling the use of dense cartilage matrix, repopulate it via channels and provide the cells with a compact collagen type II environment. Due to its density, it also provides better mechanical properties than materials currently used in clinics. We therefore think, that the auricular cartilage scaffold (AuriScaff) has a high potential to improve future cartilage regeneration approaches., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Cell-based tissue engineering can promote cartilage tissue regeneration, but cell retention in the implant site post-delivery is problematic. Alginate microbeads containing adipose stem cells (ASCs) pretreated with chondrogenic media have been used successfully to regenerate hyaline cartilage in critical size defects in rat xiphoid suggesting that they may be used to treat defects in elastic cartilages such as the ear. To test this, we used microbeads made with low viscosity, high mannuronate medical grade alginate using a high electrostatic potential, and a calcium cross linking solution containing glucose. Microbeads containing rabbit ASCs (rbASCs) were implanted bilaterally in 3 mm critical size midcartilage ear defects of six skeletally mature male New Zealand White rabbits (empty defect; microbeads without cells; microbeads with cells; degradable microbeads with cells; and autograft). Twelve weeks post-implantation, regeneration was assessed by microCT and histology. Microencapsulated rbASCs cultured in chondrogenic media expressed mRNAs for aggrecan, Type II collagen, and Type X collagen. Histologically, empty defects contained fibrous tissue; microbeads without cells were still present in defects and were surrounded by fibrous tissue; nondegradable beads with rbASCs initiated cartilage regeneration; degradable microbeads with cells produced immature bone-like tissue, also demonstrated by microCT; and autografts appeared as normal auricular cartilage but were not fully integrated with the tissue surrounding the defect. Elastin, the hallmark of auricular cartilage, was not evident in the neocartilage. This delivery system offers the potential for regeneration of auricular cartilage, but vascularity of the treatment site and use of factors that induce elastin must be considered., (Copyright © 2018 John Wiley & Sons, Ltd.)

Purpose: The significant shortcomings associated with current autologous reconstructive options for auricular deformities have inspired great interest in a tissue engineering solution. A major obstacle in the engineering of human auricular cartilage is the availability of sufficient autologous human chondrocytes. A clinically obtainable amount of auricular cartilage tissue (ie, 1 g) only yields approximately 10 million cells, where 25 times this amount is needed for the fabrication of a full-scale pediatric ear. It is thought that repeated passaging of chondrocytes leads to dedifferentiation and loss of the chondrogenic potential. However, little to no data exist regarding the ideal number of times that human auricular chondrocytes (HAuCs) can be passaged in a manner that maximizes the cellular expansion while minimizing dedifferentiation., Methods: Human auricular chondrocytes were isolated from discarded otoplasty specimens. The HAuCs were then expanded, and cells from passages 3, 4, and 5 were encapsulated into discs 8 mm in diameter made from type I collagen hydrogels with a cell density of 25 million cells/mL. The constructs were implanted subcutaneously in the dorsa of nude mice and harvested after 1 and 3 months for analysis., Results: Constructs containing passages 3, 4, and 5 chondrocytes all maintained their original cylindrical geometry. After 3 months in vivo, the diameters of the P3, P4, and P5 discs were 69 ± 9%, 67 ± 10%, and 73 ± 15% of their initial diameter, respectively. Regardless of the passage number, all constructs developed a glossy white cartilaginous appearance, similar to native auricular cartilage. Histologic analysis demonstrated development of an organized perichondrium composed of collagen, a rich proteoglycan matrix, cellular lacunae, and a dense elastin fibrin network by Safranin-O and Verhoeff stain. Biochemical analysis confirmed similar amounts of proteoglycan and hydroxyproline content in late passage constructs when compared with native auricular cartilage., Conclusions: These data indicate that late passage HAuCs (up to passage 5) form elastic cartilage that is histologically, biochemically, and biomechanically similar to native human elastic cartilage and have the potential to be used for auricular cartilage engineering.

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.

Several methods for auricular cartilage engineering use tissue engineering techniques. However, an ideal method for engineering auricular cartilage has not been reported. To address this issue, we developed a strategy to engineer auricular cartilage using silk fibroin (SF) and polyvinyl alcohol (PVA) hydrogel. We constructed different hydrogels with various ratios of SF and PVA by using salt leaching, silicone mold casting, and freeze-thawing methods. We characterized each of the hydrogels in terms of the swelling ratio, tensile strength, pore size, thermal properties, morphologies, and chemical properties. Based on the cell viability results, we found a blended hydrogel composed of 50% PVA and 50% SF (P50/S50) to be the best hydrogel among the fabricated hydrogels. An intact 3D ear-shaped auricular cartilage formed six weeks after the subcutaneous implantation of a chondrocyte-seeded 3D ear-shaped P50/S50 hydrogel in rats. We observed mature cartilage with a typical lacunar structure both in vitro and in vivo via histological analysis. This study may have potential applications in auricular tissue engineering with a human ear-shaped hydrogel., Competing Interests: The authors declare no conflict of interest.

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.

Background . Currently, the major impediment to clinical translation of our previously described platform for the fabrication of high fidelity, patient-specific tissue engineered ears is the development of a clinically optimal cell sourcing strategy. A limited autologous auricular chondrocyte (AuC) supply in conjunction with rapid chondrocyte de-differentiation during in vitro expansion currently makes clinical translation more challenging. Mesenchymal stem cells (MSCs) offer significant promise due to their inherent chondrogenic potential, and large availability through minimally invasive procedures. Herein, we demonstrate the promise of AuC/MSC co-culture to fabricate elastic cartilage using 50% fewer AuC than standard approaches., Methods: Bovine auricular chondrocytes (bAuC) and bovine MSC (bMSC) were encapsulated within 10 mg ml -1 type I collagen hydrogels in ratios of bAuC:bMSC 100:0, 50:50, and 0:100 at a density of 25 million cells ml -1 hydrogel. One mm thick collagen sheet gels were fabricated, and thereafter, 8 mm diameter discs were extracted using a biopsy punch. Discs were implanted subcutaneously in the dorsa of nude mice (NU/NU) and harvested after 1 and 3 months., Results: Gross analysis of explanted discs revealed bAuC:bMSC co-culture discs maintained their size and shape, and exhibited native auricular cartilage-like elasticity after 1 and 3 months of implantation. Co-culture discs developed into auricular cartilage, with viable chondrocytes within lacunae, copious proteoglycan and elastic fiber deposition, and a distinct perichondrial layer. Biochemical analysis confirmed that co-culture discs deposited critical cartilage molecular components more readily than did both bAuC and bMSC discs after 1 and 3 months, and proteoglycan content significantly increased between 1 and 3 months., Conclusion: We have successfully demonstrated an innovative cell sourcing strategy that facilitates our efforts to achieve clinical translation of our high fidelity, patient-specific ears for auricular reconstruction utilizing only half of the requisite auricular chondrocytes to fabricate mature elastic cartilage.

Otoplasty is a commonly performed procedure to correct prominent ears. Many different otoplasty techniques have been described but there is no gold standard technique. As well, many different suture materials are used in otoplasty but studies directly comparing different sutures materials are lacking. An otoplasty outcome study with Nylon and Mersilene (2 of the most commonly used sutures in otoplasty) sutures was conducted using a rabbit model. Each rabbit ear was randomized to receive a Mustardé-type horizontal mattress suture with either 4-0 clear Nylon (N = 12 ears) or 4-0 Mersilene sutures (N = 12 ears). Two weeks after surgery, the auricular bend angle was measured with a finger goniometer and histologic analysis with hematoxylin and eosin staining was performed on the rabbit auricular cartilage. Overall, there was no significant difference in the mean bend angle between the 2 groups (Nylon: 135.8°, SD = 22.7° and Mersilene: 143.2°, SD = 19.7°; P = 0.559). Also, no qualitative difference was observed on histologic analysis between the 2 suture groups. In the current rabbit model study, both Nylon and Mersilene sutures performed well and no significant differences were noted.

Current techniques for autologous auricular reconstruction produce substandard ear morphologies with high levels of donor-site morbidity, whereas alloplastic implants demonstrate poor biocompatibility. Tissue engineering, in combination with noninvasive digital photogrammetry and computer-assisted design/computer-aided manufacturing technology, offers an alternative method of auricular reconstruction. Using this method, patient-specific ears composed of collagen scaffolds and auricular chondrocytes have generated auricular cartilage with great fidelity following 3 months of subcutaneous implantation, however, this short time frame may not portend long-term tissue stability. We hypothesized that constructs developed using this technique would undergo continued auricular cartilage maturation without degradation during long-term (6 month) implantation. Full-sized, juvenile human ear constructs were injection molded from high-density collagen hydrogels encapsulating juvenile bovine auricular chondrocytes and implanted subcutaneously on the backs of nude rats for 6 months. Upon explantation, constructs retained overall patient morphology and displayed no evidence of tissue necrosis. Limited contraction occurred in vivo, however, no significant change in size was observed beyond 1 month. Constructs at 6 months showed distinct auricular cartilage microstructure, featuring a self-assembled perichondrial layer, a proteoglycan-rich bulk, and rounded cellular lacunae. Verhoeff's staining also revealed a developing elastin network comparable to native tissue. Biochemical measurements for DNA, glycosaminoglycan, and hydroxyproline content and mechanical properties of aggregate modulus and hydraulic permeability showed engineered tissue to be similar to native cartilage at 6 months. Patient-specific auricular constructs demonstrated long-term stability and increased cartilage tissue development during extended implantation, and offer a potential tissue-engineered solution for the future of auricular reconstructions.

It is well-accepted that articular (ART) cartilage composition and tissue architecture are intimately related to mechanical properties. On the other hand, very little information about other cartilage tissues is available, such as elastin-rich auricular (AUR) cartilage. While thorough investigation of ART cartilage has enhanced osteoarthritis research, ear cartilage reconstruction and tissue engineering (TE) could benefit in a similar way from in-depth analysis of AUR cartilage properties. This study aims to explore the constituent-function relationships of AUR cartilage, and how elastin influences mechanical behavior. Stress-relaxation indentation and tensile tests were performed on bovine ART and AUR cartilage. Elastase incubation was performed to simultaneously deplete elastin and sulfated glycosaminoglycans (sGAG), while hyaluronidase incubation was used to deplete sGAG-only, in order to systematically investigate matrix components in material behavior. ART and AUR cartilages showed different viscoelastic behaviors, with AUR cartilage exhibiting a more elastic behavior. Higher equilibrium properties and limited viscous dissipation of strain energy were observed in AUR cartilage, while ART cartilage exhibited a rapid viscous response and high resistance to instantaneous loading. In conclusion, loss of sGAG had no effect on auricular mechanics in contrast to articular cartilage where GAG loss clearly correlated with mechanical properties. Auricular cartilage without elastin lost all compressive mechanical integrity, whereas in articular cartilage this was provided by collagen. This work shows for the first time the involvement of elastin in the mechanical behavior of ear cartilage. In future, this data can be used in AUR cartilage TE efforts to support reproduction of tissue-specific mechanical properties., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Total auricular reconstruction is still a challenge, and autologous cartilage transplant is the main therapy so far. Tissue engineering provides a promising method for auricular cartilage reconstruction. However, although degradable framework demonstrated excellent initial cosmetic details, it is difficult to maintain the auricular contour over time and the metabolites tended to be harmful to human body. In this study, biocompatible and safe nondegradable elastic polyurethane was used to make porous scaffold in specific details by rapid prototyping technology. Platelet-rich plasma contains fibrin and abundant autologous growth factors, which was used as cell carriers for in vitro expanded cells. When crosslinking polyurethane framework, platelet-rich plasma and cells together, we successfully made polyurethane/platelet-rich plasma/cell composites, and implanted them into dorsal subcutaneous space of nude mice. The results showed that this method resulted in more even cell distribution and higher cell density, promoted chondrocyte proliferation, induced higher level expressions of aggrecan and type II collagen gene, increased content of newly developed glycosaminoglycans, and produced high-quality cartilaginous tissue. This kind of cartilage tissue engineering approach may be a potential promising alternative for external ear reconstruction., (© The Author(s) 2015.)

Recent advances in regenerative medicine place us in a unique position to improve the quality of engineered tissue. We use auricular cartilage as an exemplar to illustrate how the use of tissue-specific adult stem cells, assembly through additive manufacturing and improved understanding of postnatal tissue maturation will allow us to more accurately replicate native tissue anisotropy. This review highlights the limitations of autologous auricular reconstruction, including donor site morbidity, technical considerations and long-term complications. Current tissue-engineered auricular constructs implanted into immune-competent animal models have been observed to undergo inflammation, fibrosis, foreign body reaction, calcification and degradation. Combining biomimetic regenerative medicine strategies will allow us to improve tissue-engineered auricular cartilage with respect to biochemical composition and functionality, as well as microstructural organization and overall shape. Creating functional and durable tissue has the potential to shift the paradigm in reconstructive surgery by obviating the need for donor sites.

Objectives/hypothesis: The mechanical properties of normal auricular cartilage provide a benchmark against which to characterize changes in auricular structure/function due to genetic defects creating phenotypic abnormalities in collagen subtypes. Such properties also provide inputs/targets for auricular reconstruction scaffold design. Several studies report the biomechanical properties for septal, costal, and articular cartilage. However, analogous data for auricular cartilage are lacking. Therefore, our aim in this study was to characterize both whole-ear and auricular cartilage mechanics by mechanically testing specimens and fitting the results to nonlinear constitutive models., Study Design: Mechanical testing of whole ears and auricular cartilage punch biopsies., Methods: Whole human cadaveric ear and auricular cartilage punch biopsies from both porcine and human cartilage were subjected to whole-ear helix-down compression and quasistatic unconfined compression tests. Common hyperelastic constitutive laws (widely used to characterize soft tissue mechanics) were evaluated for their ability to represent the stress-strain behavior of auricular cartilage., Results: Load displacement curves for whole ear testing exhibited compliant linear behavior until after significant displacement where nonlinear stiffening occurred. All five commonly used two-term hyperelastic soft tissue constitutive models successfully fit both human and porcine nonlinear elastic behavior (mean R(2) fit >0.95)., Conclusions: Auricular cartilage exhibits nonlinear strain-stiffening elastic behavior that is similar to other soft tissues in the body. The whole ear exhibits compliant behavior with strain stiffening at high displacement. The constants from the hyperelastic model fits provide quantitative baselines for both human and porcine (a commonly used animal model for auricular tissue engineering) auricular mechanics., Level of Evidence: NA, (© 2015 The American Laryngological, Rhinological and Otological Society, Inc.)

Auricular malformations, which impose a significant social and psychological burden, are currently treated using ear prostheses, synthetic implants or autologous implants derived from rib cartilage. Advances in the field of regenerative medicine and biofabrication provide the possibility to engineer functional cartilage with intricate architectures and complex shapes using patient-derived or donor cells. However, the development of a successful auricular cartilage implant still faces a number of challenges. These challenges include the generation of a functional biochemical matrix, the fabrication of a customized anatomical shape, and maintenance of that shape. Biofabrication technologies may have the potential to overcome these challenges due to their ability to reproducibly deposit multiple materials in complex geometries in a highly controllable manner. This topical review summarizes this potential of biofabrication technologies for the generation of implants for auricular reconstruction. In particular, it aims to discuss how biofabrication technologies, although still in pre-clinical phase, could overcome the challenges of generating and maintaining the desired auricular shapes. Finally, remaining bottlenecks and future directions are discussed.

It is key for successful auricular (AUR) cartilage tissue-engineering (TE) to ensure that the engineered cartilage mimics the mechanics of the native tissue. This study provides a spatial map of the mechanical and biochemical properties of human auricular cartilage, thus establishing a benchmark for the evaluation of functional competency in AUR cartilage TE. Stress-relaxation indentation (instantaneous modulus, Ein; maximum stress, σmax; equilibrium modulus, Eeq; relaxation half-life time, t1/2; thickness, h) and biochemical parameters (content of DNA; sulfated-glycosaminoglycan, sGAG; hydroxyproline, HYP; elastin, ELN) of fresh human AUR cartilage were evaluated. Samples were categorized into age groups and according to their harvesting region in the human auricle (for AUR cartilage only). AUR cartilage displayed significantly lower Ein, σmax, Eeq, sGAG content; and significantly higher t1/2, and DNA content than NAS cartilage. Large amounts of ELN were measured in AUR cartilage (>15% ELN content per sample wet mass). No effect of gender was observed for either auricular or nasoseptal samples. For auricular samples, significant differences between age groups for h, sGAG and HYP, and significant regional variations for Ein, σmax, Eeq, t1/2, h, DNA and sGAG were measured. However, only low correlations between mechanical and biochemical parameters were seen (R<0.44). In conclusion, this study established the first comprehensive mechanical and biochemical map of human auricular cartilage. Regional variations in mechanical and biochemical properties were demonstrated in the auricle. This finding highlights the importance of focusing future research on efforts to produce cartilage grafts with spatially tunable mechanics., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objective: Auricular cartilage is used as a surgical implant in the management of orbital floor fractures. However, no specific parameters exist regarding the use/limitations of this potential graft. In order to determine the mechanical efficacy of adult auricular cartilage grafts, a mechanical model was developed and studied for structural threshold size limits., Methods: Thirty-seven cadaveric auricular cartilage specimens were tested in a laboratory. A plexiglass baseplate was created with four different sized holes, defined as 1.0×, 1.2×, 1.4×, and 1.6× the mean minor axis of the specimens. Each specimen was used to bridge one hole under increasing loads until mechanical failure. Structural stiffness at three different loading stages, structural failure strength, and percent failure of the entire system for each defect size was calculated., Results: Specimens tested on 1.0×, 1.2×, 1.4× and 1.6× defects demonstrated 0%, 0%, 20%, and 60% system failure rates, respectively. Structural stiffness curves showed a similar trend, with ANOVA demonstrating a significant difference in mechanical properties between defect sizes (p = 0.03). The curve representing 1.6 × defect size demonstrated significantly reduced structural stiffness relative to 1.0×, 1.2×, and 1.4× curves. There was no statistical difference between 1.2× and 1.4× testing sets (p = 0.09)., Conclusion: A clinically significant biomechanical and functional threshold exists between 1.2×and 1.4× defect sizes. Given a mean minor axis of 2.06 cm, orbital blow-out defects <2.4 cm (1.2 × 2.06 cm) are suitable for auricular cartilage grafts; fractures >2.4 cm may require a more rigid material. Cartilage grafts that allow failure, however, may better protect the globe in subsequent injury.

To compare the cellular viability of diced, crushed, and morselized cartilage used in nasal surgeries. In this study, cartilage was extracted from the ears of seven New Zealand rabbits and was subsequently either diced, crushed or morselized to an amorphous state, or left unmodified. The four types of grafts were then implanted in the back regions of the rabbits. After 3 months, the cellular viability from four groups was compared to a control group using confocal microscopy. Analysis of the data obtained from the enumeration of live cells showed no statistically significant difference between the unmodified graft group and the control group. The diced, crushed, and morselized cartilage groups did show a statistically significant difference in terms of live cell count with the highest number of live cells in diced cartilage group. A statistically significant decrease in live cell count was detected in crushed cartilage group. Our study shows that the viability of cells in diced cartilage grafts is greater than those in crushed or morselized cartilage grafts.

Objective: By attaching a transducer to the aural cartilage, a relatively loud sound is audible even with a negligibly small fixation force. Previous study has identified several pathways for sound transmission by means of cartilage conduction. This investigation focused on the relative contribution of direct vibration of the aural cartilage to sound transmission in an open and in an occluded ear., Methods: Thresholds with and without an earplug were compared for three experimental conditions: the transducer being placed on the tragus, pretragus, and mastoid. Eight volunteers with normal hearing participated., Results: The thresholds increased with distance of the transducer from the ear canal (tragus, pretragus, mastoid, in that order). The differences were statistically significant for all conditions except for the occluded ear at 4 kHz. With the earplug inserted, the thresholds for the tragus condition were most sensitive below 2 kHz, indicating a significant contribution of direct vibration of the aural cartilage., Conclusion: Direct vibration of the aural cartilage can enhance sound transmission. At low frequencies, cartilage conduction can deliver sound efficiently across a blockage in the ear canal. Stray airborne sound radiating from the transducer dominates cartilage conduction in the open ear at high frequencies., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Scaffolds are widely used to reconstruct cartilage. Yet, the fabrication of a scaffold with a highly organized microenvironment that closely resembles native cartilage remains a major challenge. Scaffolds derived from acellular extracellular matrices are able to provide such a microenvironment. Currently, no report specifically on decellularization of full thickness ear cartilage has been published. In this study, decellularized ear cartilage scaffolds were prepared and extensively characterized. Cartilage decellularization was optimized to remove cells and cell remnants from elastic cartilage. Following removal of nuclear material, the obtained scaffolds retained their native collagen and elastin contents as well as their architecture and shape. High magnification scanning electron microscopy showed no obvious difference in matrix density after decellularization. However, glycosaminoglycan content was significantly reduced, resulting in a loss of viscoelastic properties. Additionally, in contact with the scaffolds, human bone-marrow-derived mesenchymal stem cells remained viable and are able to differentiate toward the chondrogenic lineage when cultured in vitro. These results, including the ability to decellularize whole human ears, highlight the clinical potential of decellularization as an improved cartilage reconstruction strategy.

Unlabelled: Cartilage grafts are the most commonly used grafts for structural and aesthetic purposes. This study aimed to compare the viability of diced cartilage grafts wrapped in fascia with diced cartilage grafts wrapped in perichondrium. Approximately 2 × 2 cm cartilage grafts were harvested from the ears of seven New Zealand rabbits, diced to approximately 1-mm cubes, and then wrapped in perichondrium harvested from the ears or muscle fascia harvested from the right rear leg of the same rabbits. The wrapped grafts were then weighed and implanted into two paravertebral subcutaneous cavities created on the shaved backs of the donor rabbits. After 3 months, the rabbits were sacrificed and the grafts were removed, weighed and examined histopathologically. We found no statistically significant difference in the weights of the two graft types before and after embedding. The mean chondrocyte viability was 87.14 % in the perichondrium-wrapped cartilage grafts and 41.43 % in the fascia-wrapped grafts, which was determined to be statistically significant. Overall, our findings show that cartilage grafts wrapped in perichondrium led to higher chondrocyte viability and graft survival rates as compared with grafts wrapped in fascia. This method may be used as an alternative in clinical practice to provide patients requiring cartilage grafts with positive long-term effects, lower morbidity and lower costs associated with the procedure., No Level Assigned: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

The study aims to investigate the feasibility of repairing cartilaginous defects with chondrocytes induced from allogenic bone marrow mesenchymal stem cells (BMMSC) in rabbits' ear. BMMSCs were isolated and purified from New Zealand rabbits, in vitro amplified, and cultured in chondrocyte induction medium in order to acquire chondrocytes. After 3 weeks of induction, their phenotypes were confirmed as chondrocytes, then they were implanted onto novel polymeric scaffolds made from Poly (dl-lactide-co-glycolide) (PLGA) embedded with chitosan nonwoven cloth. The experimental group was transplanted with tissue engineering cartilaginous grafts composed of chondrogenetic BMMSC/scaffolds; the scaffold group was treated with scaffolds without cells, while in the control group, nothing was implanted. Specimens were taken at 6, 12, and 18 weeks after implantation, and the healing condition was observed by hematoxylin-eosin staining and toluidine blue staining. The right and left ears with cartilage defects of eighteen rabbits were randomly divided into three groups. In the experimental group, after 18 weeks of transplantation, the gross observation indicated that the cartilaginous defects were completely repaired by chondrocytes with smooth surface and similar color with the surrounding tissue. Hematoxylin-eosin staining and toluidine blue staining suggested that the defective area was filled with mature cartilage cells with obvious lacunae but without obvious boundaries with the normal cartilage tissue, and that the new cartilage cells were evenly distributed with homogeneously dyed cytoplasm and smaller in size. The chondrocyte induced from allogenic BMMSC can be used to repair cartilage defects in rabbit's ear.

Multiphoton microscopy (MPM) was applied for imaging and quantifying the elastic cartilage regeneration tissue in a rabbit ear model without using labeling agents. Morphology of cells and collagen matrix were analysis, showing significant difference between regenerated and intact cartilage in cellular size and collagen distribution. The results demonstrate that high resolution images provide by MPM are consistent with the histological results, and show additional biological behavior which is not visible in standard histology. Advantages in instrumentation may lead to the application of MPM for intravital detection and treatment., (© 2014 Wiley Periodicals, Inc.)

Bacterial nanocellulose (BNC), synthesized by the bacterium Gluconacetobacter xylinus, is composed of highly hydrated fibrils (99 % water) with high mechanical strength. These exceptional material properties make BNC a novel biomaterial for many potential medical and tissue engineering applications. Recently, BNC with cellulose content of 15 % has been proposed as an implant material for auricular cartilage replacement, since it matches the mechanical requirements of human auricular cartilage. This study investigates the biocompatibility of BNC with increased cellulose content (17 %) to evaluate its response in vitro and in vivo. Cylindrical BNC structures (Ø48 × 20 mm) were produced, purified in a built-in house perfusion system, and compressed to increase the cellulose content in BNC hydrogels. The reduction of endotoxicity of the material was quantified by bacterial endotoxin analysis throughout the purification process. Afterward, the biocompatibility of the purified BNC hydrogels with cellulose content of 17 % was assessed in vitro and in vivo, according to standards set forth in ISO 10993. The endotoxin content in non-purified BNC (2,390 endotoxin units (EU)/ml) was reduced to 0.10 EU/ml after the purification process, level well below the endotoxin threshold set for medical devices. Furthermore, the biocompatibility tests demonstrated that densified BNC hydrogels are non-cytotoxic and cause a minimal foreign body response. In support with our previous findings, this study concludes that BNC with increased cellulose content of 17 % is a promising non-resorbable biomaterial for auricular cartilage tissue engineering, due to its similarity with auricular cartilage in terms of mechanical strength and host tissue response.

Background: In conventional studies on the regeneration of auricle-shaped cartilage in autogenous models using large animals, there were problems with the cartilage regeneration induction capacity and long-term retention of geometric shape. In this study, the authors sought to improve on outcome in these regards: a nonwoven fabric of polyglycolic acid was developed through nanotechnology, and the effect of nanofiber diameter on in vitro cell-seeding efficiency and the in vivo response after implantation in an autogenous large-animal model were evaluated., Methods: Canine chondrocytes were isolated and seeded onto polyglycolic acid fabric ranging from 0.5 to 20 μm in average diameter. Cell seeding efficiency was highest for mid-range polyglycolic acid fibers (average diameter, 0.8, 3.0, and 7.0 μm). Flat and auricle-shaped scaffolds were constructed using polypropylene structural support, sandwiching a nonwoven polyglycolic acid fabric that contained autogenous chondrocytes together with basic fibroblast growth factor-laden particles and an exterior fibrin sealant. Scaffolds were then implanted autogenously and evaluated at 5 and 20 weeks., Results: Biomechanical strength was optimal for polyglycolic acid fiber diameters of 0.8 to 3.0 μm. Optimal cell maintenance and neocartilage response were seen with polyglycolic acid fiber diameters in the same mid-range for nanofiber constructs., Conclusion: These findings demonstrate the potential for nanoscale modulation of auricle-shaped cartilage regeneration in a large-animal model.

Although the regeneration process for injured cartilage requires an intact perichondrium, few studies have addressed the importance of the intact perichondrial layer in the regeneration of damaged cartilage. In this study, we evaluated the role of the perichondrium on regenerative activities in injured cartilage according to different degrees of perichondrial injury. Auricular cartilage harvested from six New Zealand white rabbits was irradiated with a 1,460-nm diode laser at two different power settings (0.3 or 0.5 W). Irradiated cartilage was reimplanted into a subperichondrial pocket under three different conditions: non-injured perichondrium (NPI), unilaterally injured perichondrium (UPI), or bilaterally injured perichondrium (BPI). Rabbits were sacrificed at 1, 2, and 4 weeks after reimplantation and the auricular cartilage was reharvested. A histopathological study using hematoxylin and eosin staining, a live/dead viability assay, and immunohistochemical staining for proliferating cell nuclear antigen were performed to evaluate structural changes and regenerative and proliferative activities of the injured chondrocytes. A modified array and restored boundary of chondrocytes were observed in the NPI and UPI groups. Regeneration of chondrocytes was prominent in the NPI and UPI groups, but was not observed in the BPI group. Proliferative activity of chondrocytes was observed only when the perichondrium was preserved in the NPI and UPI groups. In contrast, proliferative activity was not observed until 4 weeks in the BPI group. The degree of perichondrial injury affected proliferation and regeneration in injured elastic cartilage. In the case of unilateral perichondrial injury, the surgeon should be careful to avoid damaging the other side of the perichondrium, because at least a unilateral perichondrial layer is needed for the regeneration of elastic cartilage.

Objectives/hypothesis: The aim of this study was to establish the sound transmission characteristics of cartilage conduction proposed by Hosoi (2004), which is available by a vibration signal delivered to the aural cartilage from a transducer., Study Design: Experimental study., Method: Eight volunteers with normal hearing participated. Thresholds at frequencies of 0.5, 1, 2, and 4 kHz for air conduction, bone, and cartilage conductions were measured with and without an earplug. The sound pressure levels on the eardrum at the threshold estimated with a Head and Torso Simulator were compared between air and cartilage conductions. The force levels calibrated with an artificial mastoid at the threshold were compared between bone and cartilage conductions., Results: The difference in the estimated sound pressure levels on the eardrum at the thresholds between air and cartilage conductions were within 10 dB. In contrast, the force levels at the thresholds for cartilage conduction were remarkably lower than those for bone conduction. These findings suggested that sounds were probably transmitted via the eardrum for cartilage conduction. The threshold shifts by an earplug showed no significant difference between bone and cartilage conductions at 0.5 kHz. At 1 and 2 kHz, the threshold-shifts increased significantly in the order of bone, cartilage, and air conductions. These results suggested that airborne sound induced by the vibration of the cartilaginous portion of the ear canal played a significant role in sound transmission for cartilage conduction., Conclusions: Cartilage conduction has different characteristics from conventional air and bone conductions., (© 2013 The American Laryngological, Rhinological and Otological Society, Inc.)

Sound information is known to travel to the cochlea via either air or bone conduction. However, a vibration signal, delivered to the aural cartilage via a transducer, can also produce a clearly audible sound. This type of conduction has been termed "cartilage conduction." The aural cartilage forms the outer ear and is distributed around the exterior half of the external auditory canal. In cartilage conduction, the cartilage and transducer play the roles of a diaphragm and voice coil of a loudspeaker, respectively. There is a large gap between the impedances of cartilage and skull bone, such that cartilage vibrations are not easily transmitted through bone. Thus, these methods of conduction are distinct. In this study, force was used to apply a transducer to aural cartilage, and it was found that the sound in the auditory canal was amplified, especially for frequencies below 2 kHz. This effect was most pronounced at an application force of 1 N, which is low enough to ensure comfort in the design of hearing aids. The possibility of using force adjustments to vary amplification may also have applications for cell phone design.

External ear reconstruction with autologous cartilage still remains one of the most difficult problems in the fields of plastic and reconstructive surgery. As the absence of tissue vascularization limits the ability to stimulate new tissue growth, relatively few surgical approaches are currently available (alloplastic implants or sculpted autologous cartilage grafts) to repair or reconstruct the auricle (or pinna) as a result of traumatic loss or congenital absence (e.g., microtia). Alternatively, tissue engineering can offer the potential to grow autogenous cartilage suitable for implantation. While tissue-engineered auricle cartilage constructs can be created, a substantial number of cells are required to generate sufficient quantities of tissue for reconstruction. Similarly, as routine cell expansion can elicit negative effects on chondrocyte function, we have developed an approach to generate large-sized engineered auricle constructs (≥3 cm(2)) directly from a small population of donor cells (20,000-40,000 cells/construct). Using rabbit donor cells, the developed bioreactor-cultivated constructs adopted structural-like characteristics similar to native auricular cartilage, including the development of distinct cartilaginous and perichondrium-like regions. Both alterations in media composition and seeding density had profound effects on the formation of engineered elastic tissue constructs in terms of cellularity, extracellular matrix accumulation, and tissue structure. Higher seeding densities and media containing sodium bicarbonate produced tissue constructs that were closer to the native tissue in terms of structure and composition. Future studies will be aimed at improving the accumulation of specific tissue constituents and determining the clinical effectiveness of this approach using a reconstructive animal model.

Tissue-engineering (TE) efforts for ear reconstruction often fail due to mechanical incompetency. It is therefore key for successful auricular cartilage (AUC) TE to ensure functional competency, that is, to mimic the mechanical properties of the native ear tissue. A review of past attempts to engineer AUC shows unsatisfactory functional outcomes with various cell-seeded biodegradable polymeric scaffolds in immunocompetent animal models. However, promising improvements to construct stability were reported with either mechanically reinforced scaffolds or novel two-stage implantation techniques. Nonetheless, quantitative mechanical evaluation of the constructs is usually overlooked, and such an evaluation of TE constructs alongside a benchmark of native AUC would allow real-time monitoring and improve functional outcomes of auricular TE strategies. Although quantitative mechanical evaluation techniques are readily available for cartilage, these techniques are designed to characterize the main functional components of hyaline and fibrous cartilage such as the collagen matrix or the glycosaminoglycan network, but they overlook the functional role of elastin, which is a major constituent of AUC. Hence, for monitoring AUC TE, novel evaluation techniques need to be designed. These should include a characterization of the specific composition and architecture of AUC, as well as mechanical evaluation of all functional components. Therefore, this article reviews the existing literature on AUC TE as well as cartilage mechanical evaluation and proposes recommendations for designing a mechanical evaluation protocol specific for AUC, and establishing a benchmark for native AUC to be used for quantitative evaluation of TE AUC.

Tissue-engineered cartilage has historically been an attractive alternative treatment option for auricular reconstruction. However, the ability to reliably generate autologous auricular neocartilage in an immunocompetent preclinical model should first be established. The objectives of this study were to demonstrate engineered autologous auricular cartilage in the immunologically aggressive subcutaneous environment of an immunocompetent animal model, and to determine the impact of in vitro culture duration of chondrocyte-seeded constructs on the quality of neocartilage maturation in vivo. Auricular cartilage was harvested from eight adult sheep; chondrocytes were isolated, expanded in vitro, and seeded onto fibrous collagen scaffolds. Constructs were cultured in vitro for 2, 6, and 12 weeks, and then implanted autologously in sheep and in control nude mice for 6 and 12 weeks. Explanted tissue was stained with hematoxylin and eosin, safranin O, toluidine blue, collagen type II, and elastin. DNA and glycosaminoglycans (GAGs) were quantified. The quality of cartilage engineered in sheep decreased with prolonged in vitro culture time. Superior cartilage formation was demonstrated after 2 weeks of in vitro culture; the neocartilage quality improved with increased implantation time. In nude mice, neocartilage resembled native sheep auricular cartilage regardless of the in vitro culture length, with the exception of elastin expression. The DNA quantification was similar in all engineered and native cartilage (p>0.1). All cartilage engineered in sheep had significantly less GAG than native cartilage (p<0.02); significantly more GAG was observed with increased implantation time (p<0.02). In mice, the GAG content was similar to that of native cartilage and became significantly higher with increased in vitro or in vivo durations (p<0.02). Autologous auricular cartilage was successfully engineered in the subcutaneous environment of an ovine model using expanded chondrocytes seeded on a fibrous collagen scaffold after a 2-week in vitro culture period.

The reconstruction of an auricle for congenital deformity or following trauma remains one of the greatest challenges in reconstructive surgery. Tissue-engineered (TE) three-dimensional (3D) cartilage constructs have proven to be a promising option, but problems remain with regard to cell vitality in large cell constructs. The supply of nutrients and oxygen is limited because cultured cartilage is not vascular integrated due to missing perichondrium. The consequence is necrosis and thus a loss of form stability. The micro-surgical implantation of an arteriovenous loop represents a reliable technology for neovascularization, and thus vascular integration, of three-dimensional (3D) cultivated cell constructs. Auricular cartilage biopsies were obtained from 15 rabbits and seeded in 3D scaffolds made from polycaprolactone-based polyurethane in the shape and size of a human auricle. These cartilage cell constructs were implanted subcutaneously into a skin flap (15 × 8 cm) and neovascularized by means of vascular loops implanted micro-surgically. They were then totally enhanced as 3D tissue and freely re-implanted in-situ through microsurgery. Neovascularization in the prefabricated flap and cultured cartilage construct was analyzed by microangiography. After explantation, the specimens were examined by histological and immunohistochemical methods. Cultivated 3D cartilage cell constructs with implanted vascular pedicle promoted the formation of engineered cartilaginous tissue within the scaffold in vivo. The auricles contained cartilage-specific extracellular matrix (ECM) components, such as GAGs and collagen even in the center oft the constructs. In contrast, in cultivated 3D cartilage cell constructs without vascular pedicle, ECM distribution was only detectable on the surface compared to constructs with vascular pedicle. We demonstrated, that the 3D flaps could be freely transplanted. On a microangiographic level it was evident that all the skin flaps and the implanted cultivated constructs were well neovascularized. The presented method is suggested as a promising alternative towards clinical application of engineered cartilaginous tissue for plastic and reconstructive surgery.

In this study, we investigated the temporal post-mortem limits, within which there will be guarantees of obtaining living cells from several tissues of sheep and cattle and the effect of vitrification on the ability of cells from tissue stored at different times. Muscle tissue and auricular cartilage were stored at 4°C for 5, 48, 72, 96 and 216 h post-mortem (hpm). Tissue samples were sorted into two groups: one group was in vitro cultured immediately after storage and the other was vitrified after storage and then in vitro cultured. In cattle and sheep, no differences in subconfluence rates were observed between the two experimental groups. At the same time, no significant differences were observed in the number of days required in culture to reach confluence between non-vitrified and vitrified groups when tissues were stored at 4°C for different times. In sheep, while the population doubling times (PDT) were similar in cartilage cells from vitrified and non-vitrified tissues and stored at 4°C for 5 and 216 hpm, PDT of muscle cells were longer in 216 hpm stored groups than in 5 hpm stored groups. In bovine, although the PDT of muscle cells were similar for 5 and 216 hpm and both vitrified and non-vitrified tissues and the PDT were longer in cartilage cells from vitrified than from non-vitrified tissues. In conclusion, although storage times and vitrification have different effects on tissues from cattle and sheep, this study showed that living cells could be obtained from all groups. Therefore, cartilage and muscle tissues can be stored at 4°C for 216 hpm and used for cyrobanking.

The focus of this study was to compare the role of nerve growth factor (NGF) and vascular endothelial growth factor (VEGF) in the regeneration of experimental skin and cartilage trauma. The role of VEGF in this process is known since decade; the NGF participation on this process has been first discussed within the spinal cord injury repair. We hypothesized that both VEGF and NGF induce angiogenesis and take part on the repair process. The angiogenesis response and the cartilage regeneration after phVEGF(165) plasmid and rat pcNGF plasmid administration were investigated using BALB/c mice. PhVEGF(165) and pcNFG were injected into the right mice ear and plain vector injection into the left ear the day before trauma. The next day, all mice were ear-punched, resulting in 2-mm diameter puncture through the center of both pinnae. In BALB/c mouse strain, a significantly faster cartilage repair was observed after phVEGF(165) and pcNGF injection into punched ear area in comparison to the control group. It has been shown that the healing process is after VEGF and NGF injection driven differentially. In case of VEGF is the cartilage wound repaired by induction of new chondrocytes differentiation. In the case of NGF, the regeneration is supported by immature leukocytes attracted into the punched area. The leukocytes induct angiogenesis so far indirectly by inflammation. The NGF-induced inflammation environment may be a part of mosaic creating the complete picture of regeneration.

Unlabelled: Round window's movability measurements with helping of LDV in evaluation of ossicular chain functioning., Aim of Study: Quantitive evaluation of round window movability in normal conditions and after malleus stapes assembly reconstruction were aims of the study., Methods and Materials: In the experiment there were taken 10 non-frozen temporal bones harvested within 48 hours. Temporal bones specimens were prepared like in closed technique with antromastoidectomy and large posterior tympanotomy. Hearing system before and after MSA reconstruction were evaluated by measurement of round window movement. Measurements were performed at four frequencies: 1000 Hz, 2000 Hz, 4000 Hz, 8000 Hz., Results: In the normal ossicular chain the biggest movability were stated at frequency of 1 kHz. After reconstruction at all frequencies measurements were significantly worse. In reconstructed ears the highest movabilities were stated at frequencies 2000 Hz and 4000 Hz., Conclusions: Round window movability could be measured by Laser Doppler Vibrometry in posterior tympanotomy approach. Before reconstruction the biggest movability were evaluated at 1000 Hz and after MSA at 2000 Hz

Due to the lack of appropriate scaffolds, the in vitro engineering of cartilage tissue with a sophisticated structure, such as a human ear, remains a great challenge. Although polyglycolic acid (PGA) has become one of the most successful scaffolds for cartilage regeneration, how to overcome its limitations in achieving desirable mechanical strength and accurate control over shape remains an unsolved problem. In this study, the mechanical strength of PGA scaffold was enhanced by coating with polylactic acid (PLA). The content of PLA was optimized by balancing the scaffold's biocompatibility and mechanical strength. The PLA/PGA scaffold was then fabricated into a human ear-shape mirror-symmetrical to a normal ear by pressing the scaffold in the ear negative molds, which were fabricated by the computer aided design and manufacturing (CAD/CAM) technique according to the CT scan data from the normal ear. The ear-shaped scaffold reached a similarity level of over 97% compared to the positive ear mold by the shape analysis using a 3D laser scan system. Most importantly, after chondrocyte seeding, the constructs largely retained the original shape during culture with a similarity level of over 84%. Furthermore, the constructs formed ear-shaped cartilage-like tissues at 12 weeks, which revealed a tissue structure with abundant cartilage extracellular matrices and mature lacuna. Additionally, the ear-shaped cartilage at 12 weeks also exhibited fine elasticity and good mechanical strength. These results may provide a useful strategy for reconstructing cartilage tissue with complicated shapes such as a human ear by an in vitro engineering approach., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

Round holes in the ears of MRL mice tend to close with characteristics of regeneration believed to be absent in other mouse strains (e.g., C57BL/6). We evaluated the kinetics and the histopathology of ear wound closure in young (8 weeks old) C57BL/6 and BALB/c mice. We also used middle-aged (40 weeks old) C57BL/6 mice to evaluate the influence of aging on this process. A circular through-and-through hole was made in the ear, photographs were taken at different times after injury and wound area was measured with digital analysis software. The percentages of closed area measured on day 100 were: 23.57 +/- 8.66% for young BALB/c mice, 56.47 +/- 7.39% for young C57BL/6 mice, and 75.31 +/- 23.65% for middle-aged C57BL/6 mice. Mice were sacrificed on days 1, 3, 5, 25, 44, and 100 for histological evaluation with hematoxylin and eosin, Gomori's trichrome, periodic acid-Schiff, or picrosirius red staining. In young mice of both strains, healing included re-epithelialization, chondrogenesis, myogenesis, and collagen deposition. Young C57BL/6 and BALB/c mice differed in the organization of collagen fibers visualized using picrosirius-polarization. Sebaceous glands and hair follicles regenerated and chondrogenesis was greater in young C57BL/6 mice. In middle-aged C57BL/6 mice all aspects of regeneration were depressed. The characteristics of regeneration were present during ear wound healing in both young BALB/c and young C57BL/6 mice although they differed in intensity and pattern. Greater ear wound closure in middle-aged C57BL/6 mice was not correlated with regeneration.

Goals: To assess the effects of thickness and position of cartilage used to reconstruct the tympanic membrane (TM) using a novel technique, time-averaged laser holography., Background: Cartilage is commonly used in TM reconstruction to prevent formation of retraction pockets. The thickness, position, and shape of the cartilage graft may adversely affect TM motion and hearing. We sought to systematically investigate these parameters in an experimental setting., Methods: Computer-assisted optoelectronic laser holography was used in 4 human cadaveric temporal bones to study sound-induced TM motion for 500 Hz to 8 kHz. Stapes velocity was measured with a laser Doppler vibrometer. Baseline (control) measurements were made with the TM intact. Measurements were repeated after a 0.5- or 1.0-mm-thick oval piece of conchal cartilage was placed on the medial TM surface in the posterior-superior quadrant. The cartilage was rotated so that it was either in contact with the bony tympanic rim and manubrium or not., Results: At frequencies less than 4 kHz, the cartilage graft had only minor effects on the overall TM fringe patterns. The different conditions had no effects on stapes velocity. Greater than 4 kHz, TM motion was reduced over the grafted TM, both with 0.5- and 1.0-mm-thick grafts. No significant differences in stapes velocity were seen with the 2 different thicknesses of cartilage compared with control., Conclusion: Computer-assisted optoelectronic laser holography is a promising technique to investigate middle ear mechanics after tympanoplasty. Such positioning may prevent postoperative TM retraction. These findings and conclusions apply to cartilage placed in the posterior-superior TM quadrant.