Bioengineering Full-scale auricles using 3D-printed external scaffolds and decellularized cartilage xenograft.

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 mm3) or zesting (< 0.5 mm3), 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.

The Promise of Regenerative Medicine in the Reconstruction of Auricular Cartilage Deformities.

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.

Non-mulberry silk fiber-based scaffolds reinforced by PLLA porous microspheres for auricular cartilage: An in vitro study.

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.

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.

Intraoperative tragal and conchal cartilage thickness: Comparative study for cartilage tympanoplasty.

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.

Genetic correlations between cartilage regeneration and degeneration reveal an inverse relationship.

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.

Effects of FM system fitted into the normal hearing ear orcartilage conduction hearing aid fitted into the affected ear on speech-in-noise recognition in Japanese children with unilateral congenital aural atresia.

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.

RNA-seq analysis of chondrocyte transcriptome reveals genetic heterogeneity in LG/J and SM/J murine strains.

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.

Cartilage conduction as the third pathway for sound transmission.

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.

Repopulation of an auricular cartilage scaffold, AuriScaff, perforated with an enzyme combination.

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.

Microencapsulated rabbit adipose stem cells initiate tissue regeneration in a rabbit ear defect model.

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.

Tissue Engineering Auricular Cartilage Using Late Passage Human Auricular Chondrocytes.

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.

Comparisons of Auricular Cartilage Tissues from Different Species.

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

Artificial Auricular Cartilage Using Silk Fibroin and Polyvinyl Alcohol Hydrogel.

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.

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.

Optimizing cell sourcing for clinical translation of tissue engineered ears.

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 Outcomes With Different Suture Materials in a Rabbit Model.

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.

Long-Term Morphological and Microarchitectural Stability of Tissue-Engineered, Patient-Specific Auricles In Vivo.

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.

Combining regenerative medicine strategies to provide durable reconstructive options: auricular cartilage tissue engineering.

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.

Tissue composition regulates distinct viscoelastic responses in auricular and articular cartilage.

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.