Auricular and laryngeal chondritis in nursery and finishing pigs.

This work aimed to characterize the clinic-pathological presentation of an outbreak of auricular and laryngeal chondritis in pigs. Visits were made to pig farms, where the clinical history was obtained, and clinical and postmortem examinations were performed. In those farms, 3% to 4% of pigs presented otohematomas, which started in the nursery and extended to the finishing phase. Moreover, some finishing pigs presented with respiratory distress, initially characterized as inspiratory dyspnea, associated by an uncommon respiratory stridor and culminating in death. Grossly, nursery piglets had enlarged ears, and on the cut surface, the cartilage was fragmented and associated with blood clots. In the finishing phase, in addition to auricular lesions, the epiglottis and arytenoid cartilages were thickened and distorted, which partially occluded the lumen. Microscopically, the laryngeal and auricular cartilages were fragmented, displayed a loss of matrix basophilia, and were surrounded by lymphohistiocytic inflammatory infiltrate, with occasional multinucleated giant cells and fibrosis. The lesions exclusively affected elastic cartilages. The disease in finishing pigs led to increased mortality and was a differential diagnosis to respiratory challenges. It was not possible to determine the factor that triggered this condition; however, a nutritional association is suspected. To the authors' knowledge, this is the first report of primary auricular and laryngeal chondritis in pigs.

Proximal ear hole injury heals by limited regeneration during the early postnatal phase in mice.

Ear pinna is a particular feature of mammals that shows several repair responses depending on age. Two millimeter hole made in the pinna of middle-aged female mice heals due to partial reconstitution of new tissues (limited regeneration), whereas a hole punched in the ear of young mice forms a scar tissue. In these studies, the injury is made in the center of the ear pinna, but little is known about the type of reparative response along the proximodistal polarity of the ear. This study evaluated the effect of pinna polarity, age, and sex in the ear hole-repairing response in Balb/c mice. Proximal injuries were repaired more efficiently by limited regeneration than wounds made in the middle region. Non-injured ear histological analysis revealed a higher presence of muscle, adipose tissue, cartilage, and larger blood vessels in the proximal ear area, which could influence ear hole closure by limited regeneration. To evaluate the healing response during ear growth, we punched a standard hole in the proximal area of the ear on postnatal day 21 and 8-month-old mice (adults). Thirty-five days after the wound, both groups reached the same wound closure, despite the greater proportional size of holes made in the younger mice. Ear growth also improved ear hole closure in male mice. These results suggest that ear growth accelerates hole closure, providing an example of enhanced regenerative abilities in growing structures. Finally, hole closure kinetics in the growing ear indicated an early re-differentiation phase exhibited at 14 days post-wound. In conclusion, ear topography and growth positively influenced the healing response to ear holes, making it a tractable model to study in mammals.

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

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

Remaining microtia tissue as a source for 3D bioprinted elastic cartilage tissue constructs, potential use for surgical microtia reconstruction.

The absence of ears in children is a global problem. An implant made of costal cartilage is the standard procedure for ear reconstruction; however, side effects such as pneumothorax, loss of thoracic cage shape, and respiratory complications have been documented. Three-dimensional (3D) printing allows the generation of biocompatible scaffolds that mimic the shape, mechanical strength, and architecture of the native extracellular matrix necessary to promote new elastic cartilage formation. We report the potential use of a 3D-bioprinted poly-ε-caprolactone (3D-PCL) auricle-shaped framework seeded with remaining human microtia chondrocytes for the development of elastic cartilage for autologous microtia ear reconstruction. An in vivo assay of the neo-tissue formed revealed the generation of a 3D pinna-shaped neo-tissue, and confirmed the formation of elastic cartilage by the presence of type II collagen and elastin with histological features and a protein composition consistent with normal elastic cartilage. According to our results, a combination of 3D-PCL auricle frameworks and autologous microtia remnant tissue generates a suitable pinna structure for autologous ear reconstruction.

Comparison of Prominent Ear Recurrence in Different Age Groups.

AIM: Although many prominent ear deformity (PED) surgery techniques have been described to date, there have been few comprehensive studies evaluating the recurrence rates in different age groups. Previous studies have focused either on the young or the elderly. The present clinical study compares recurrence rates among patients of different age groups undergoing PED repair and discusses cartilage morphology. PATIENTS AND METHODS: A total of 380 patients with a mean age of 24.2 years underwent PED repair surgery between 2001 and 2019. The patients were divided into five subgroups according to age. Group I (5-10 years) was composed of 44 patients, Group II (10-20 years) was composed of 47 patients, Group III (20-30 years) was composed of 166 patients, Group IV (30-40 years) was composed of 90 patients, and Group V (over 40 years) was composed of 33 patients. The cephaloauricular angle (CAA) and the distance between the helix (upper, middle, lower) and mastoid were measured and recorded prior to surgery. The patients were all treated with three concha-mastoid sutures to achieve concha reduction and to narrow the cephaloauricular angle. The anterior aspect of the cartilage was thinned with a rasp, and an antihelix was created using non-absorbable sutures in patients with an inadequate antihelix. RESULTS: The CAA and the upper pole-mastoid distance were measured immediately after surgery and at 15 days, 3 and 6 months after surgery. Recurrence was observed in a total of 18 patients, with PED recurring in one patient in Group I (2.3%), three patients in Group II (6.38%), 10 patients in Group III (6.8%), three patients in Group IV (3.3%) and one patient in Group V (3.03%). Although the difference in the recurrence rate among the age groups would appear to be clinically significant, the difference was not significant, statistically. Clinically, the differences among the groups could be attributed to intragroup numerical differences. CONCLUSIONS: In the present study, no significant relationship was identified between the patient age and recurrence rate. Although PED repair is recommended in the preschool period, prominent ear repairs can be carried out in any age group, although the degree of cartilage scoring should differ depending on the age group. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. 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 .

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

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

Angiogenesis after administration of basic fibroblast growth factor induces proliferation and differentiation of mesenchymal stem cells in elastic perichondrium in an in vivo model: mini review of three sequential republication-abridged reports.

To date, studies on mesenchymal tissue stem cells (MSCs) in the perichondrium have focused on in vitro analysis, and the dynamics of cartilage regeneration from the perichondrium in vivo remain largely unknown. We have attempted to apply cell and tissue engineering methodology for ear reconstruction using cultured chondrocytes. We hypothesized that by inducing angiogenesis with basic fibroblast growth factor (bFGF), MSCs or cartilage precursor cells would proliferate and differentiate into cartilage in vivo and that the regenerated cartilage would maintain its morphology over an extended period. As a result of a single administration of bFGF to the perichondrium, cartilage tissue formed and proliferated while maintaining its morphology for at least 3 months. By day 3 post bFGF treatment, inflammatory cells, primarily comprising mononuclear cells, migrated to the perichondrial region, and the proliferation of matrix metalloproteinase 1 positive cells peaked. During week 1, the perichondrium thickened and proliferation of vascular endothelial cells was noted, along with an increase in the number of CD44-positive and CD90-positive cartilage MSCs/progenitor cells. Neocartilage was formed after 2 weeks, and hypertrophied mature cartilage was formed and maintained after 3 months. Proliferation of the perichondrium and cartilage was bFGF concentration-dependent and was inhibited by neutralizing antibodies. Angiogenesis induction by bFGF was blocked by the administration of an angiogenesis inhibitor, preventing perichondrium proliferation and neocartilage formation. These results suggested that angiogenesis may be important for the induction and differentiation of MSCs/cartilage precursor cells in vivo, and that morphological changes, once occurring, are maintained.

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

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

Auricular ossification: A newly recognized feature of osteoprotegerin-deficiency juvenile Paget disease.

We report auricular ossification (AO) affecting the elastic cartilage of the ear as a newly recognized feature of osteoprotegerin (OPG)-deficiency juvenile Paget disease (JPD). AO and auricular calcification refer interchangeably to rigid pinnae, sparing the ear lobe, from various etiologies. JPD is a rare Mendelian disorder characterized by elevated serum alkaline phosphatase activity accompanied by skeletal pain and deformity from rapid bone turnover. Autosomal recessive transmission of loss-of-function mutations within TNFRSF11B encoding OPG accounts for most JPD (JPD1). JPD2 results from heterozygous constitutive activation of TNFRSF11A encoding RANK. Other causes of JPD remain unknown. In 2007, we reported a 60-year-old man with JPD1 who described hardening of his external ears at age 45 years, after 4 years of treatment with bisphosphonates (BPs). Subsequently, we noted rigid pinnae in a 17-year-old boy and 14-year-old girl, yet pliable pinnae in a 12-year-old boy, each with JPD1 and several years of BP treatment. Cranial imaging indicated cortical bone within the pinnae of both teenagers. Radiologic studies of our three JPD patients without mutations in TNFRSF11B showed normal auricles. Review of the JPD literature revealed possible AO in several reports. Two of our JPD1 patients had experienced difficult tracheal intubation, raising concern for mineralization of laryngeal elastic cartilage. Thus, AO is a newly recognized feature of JPD1, possibly exacerbated by BP treatment. Elastic cartilage at other sites in JPD1 might also ossify, and warrants investigation.

Characterization of pediatric microtia cartilage: a reservoir of chondrocytes for auricular reconstruction using tissue engineering strategies.

The external ear is composed of elastic cartilage. Microtia is a congenital malformation of the external ear that involves a small reduction in size or a complete absence. The aim of tissue engineering is to regenerate tissues and organs clinically implantable based on the utilization of cells and biomaterials. Remnants from microtia represent a source of cells for auricular reconstruction using tissue engineering. To examine the macromolecular architecture of microtia cartilage and behavior of chondrocytes, in order to enrich the knowledge of this type of cartilage as a cell reservoir. Auricular cartilage remnants were obtained from pediatric patients with microtia undergoing reconstructive procedures. Extracellular matrix composition was characterized using immunofluorescence and histological staining methods. Chondrocytes were isolated and expanded in vitro using a mechanical-enzymatic protocol. Chondrocyte phenotype was analyzed using qualitative PCR. Microtia cartilage preserves structural organization similar to healthy elastic cartilage. Extracellular matrix is composed of typical cartilage proteins such as type II collagen, elastin and proteoglycans. Chondrocytes displayed morphological features similar to chondrocytes derived from healthy cartilage, expressing SOX9, COL2 and ELN, thus preserving chondral phenotype. Cell viability was 94.6 % during in vitro expansion. Elastic cartilage from microtia has similar characteristics, both architectural and biochemical to healthy cartilage. We confirmed the suitability of microtia remnant as a reservoir of chondrocytes with potential to be expanded in vitro, maintaining phenotypical features and viability. Microtia remnants are an accessible source of autologous cells for auricular reconstruction using tissue engineering strategies.

Differentiating the extent of cartilage repair in rabbit ears using nonlinear optical microscopy.

Nonlinear optical microscopy (NLOM) was used as a noninvasive and label-free tool to detect and quantify the extent of the cartilage recovery. Two cartilage injury models were established in the outer ears of rabbits that created a different extent of cartilage recovery based on the presence or absence of the perichondrium. High-resolution NLOM images were used to measure cartilage repair, specifically through spectral analysis and image texture. In contrast to a wound lacking a perichondrium, wounds with intact perichondria demonstrated significantly larger TPEF signals from cells and matrix, coarser texture indicating the more deposition of type I collagen. Spectral analysis of cells and matrix can reveal the matrix properties and cell growth. In addition, texture analysis of NLOM images showed significant differences in the distribution of cells and matrix of repaired tissues with or without perichondrium. Specifically, the decay length of autocorrelation coefficient based on TPEF images is 11.2 ± 1.1 in Wound 2 (with perichondrium) and 7.5 ± 2.0 in Wound 1 (without perichondrium), indicating coarser image texture and faster growth of cells in repaired tissues with perichondrium (p < 0.05). Moreover, the decay length of autocorrelation coefficient based on collagen SHG images also showed significant difference between Wound 2 and 1 (16.2 ± 1.2 vs. 12.2 ± 2.1, p < 0.05), indicating coarser image texture and faster deposition of collagen in repaired tissues with perichondrium (Wound 2). These findings suggest that NLOM is an ideal tool for studying cartilage repair, with potential applications in clinical medicine. NLOM can capture macromolecular details and distinguish between different extents of cartilage repair without the need for labelling agents.

BMP signaling maintains auricular chondrocyte identity and prevents microtia development by inhibiting protein kinase A.

Elastic cartilage constitutes a major component of the external ear, which functions to guide sound to the middle and inner ears. Defects in auricle development cause congenital microtia, which affects hearing and appearance in patients. Mutations in several genes have been implicated in microtia development, yet, the pathogenesis of this disorder remains incompletely understood. Here, we show that Prrx1 genetically marks auricular chondrocytes in adult mice. Interestingly, BMP-Smad1/5/9 signaling in chondrocytes is increasingly activated from the proximal to distal segments of the ear, which is associated with a decrease in chondrocyte regenerative activity. Ablation of Bmpr1a in auricular chondrocytes led to chondrocyte atrophy and microtia development at the distal part. Transcriptome analysis revealed that Bmpr1a deficiency caused a switch from the chondrogenic program to the osteogenic program, accompanied by enhanced protein kinase A activation, likely through increased expression of Adcy5/8. Inhibition of PKA blocked chondrocyte-to-osteoblast transformation and microtia development. Moreover, analysis of single-cell RNA-seq of human microtia samples uncovered enriched gene expression in the PKA pathway and chondrocyte-to-osteoblast transformation process. These findings suggest that auricle cartilage is actively maintained by BMP signaling, which maintains chondrocyte identity by suppressing osteogenic differentiation.

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

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

Monitoring wound healing of elastic cartilage using multiphoton microscopy.

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

Epimorphic Regeneration of Elastic Cartilage: Morphological Study into the Role of Cellular Senescence.

Control over endogenous reparative mechanisms is the future of regenerative medicine. The rabbit ear defect is a rare model which allows the observation of the epimorphic regeneration of elastic cartilage. However, the mechanisms of phenotypical restoration of this highly differentiated tissue have not been studied. We modelled circular ear defects of different sizes (4, 6, and 8 mm in diameter) in 12 laboratory rabbits, and observed them during 30, 60, 90, and 120 day periods. Excised tissues were processed and analyzed by standard histological methods and special histochemical reactions for senescence associated-ß-galactosidase and lectin markers. We demonstrated that larger defects caused significant elevation of senescence associated-ß-galactosidase in chondrocytes. The fullness of epimorphic regeneration of elastic cartilage depended on the activation of cellular senescence and synthesis of elastic fibers. Further investigation into the role of cells with senescence-associated secretory phenotype in damaged tissues can present new targets for controlled tissue regeneration.

Characterization of a decellularized rat larynx: comparison between microscopy techniques.

BACKGROUND: No effective method has yet been developed to efficiently reconstruct the larynx and restore its function. Decellularization has recently been tested for this purpose with very promising results. The goal of decellularization is to remove cells leaving an intact scaffold made of an extracellular matrix (ECM). Although the use of hematoxylin/eosin and Masson trichrome stains is widely accepted to highlight tissue structure, the methods based on evaluation of collagen and elastin are considered highly variable. The aim of this study was to develop a whole organ decellularization protocol and compare the qualitative and quantitative efficiency of some microscopy techniques for collagen and elastin detection in paraffin-embedded tissues. METHODS: H&E, Masson Trichrome and DAPI staining as well as DNA quantification were used to evaluate decellularization efficiency. Van Gieson stain, Picrosirius Red stain (PRS) and multiphoton laser scanning microscopy (MPM) were carried out for collagen detection and quantitative assessment. Polarized PRS was used to investigate collagen network, and Weigert stain and MPM were used to detect and estimate elastin content. RESULTS: The decellularization process removed the cellular components without affecting glycosaminoglycan, collagen and elastin content. Concerning collagen quantification, Van Gieson stain underestimated collagen content, while PRS, apparently less fading, did not reach reliable results when used as quantitative method. MPM effectively quantified collagen content. Collagen fibers were visualized much better under polarized light microscopy, allowing to underline that decellularization process affects the homogeneity of 3D collagen network. Concerning elastin detection, Weigert stain and MPM produced overlapping results. CONCLUSIONS: An efficient protocol to decellularize the whole larynx was developed, allowing the removal of cells without affecting ECM integrity. The results supported the use of non-polarized PRS to highlight collagen, even the thin fibers, second harmonic generation for major fibrillar collagens and polarized PRS for 3D collagen network. Concerning elastin, Weigert stain and MPM showed similar results, thus the use of MPM, rather than that of the Weigert stain, may be suitable to avoid the additional time and costs of a histological staining.

In vitro elastic cartilage reconstruction using human auricular perichondrial chondroprogenitor cell-derived micro 3D spheroids.

Morphologically stable scaffold-free elastic cartilage tissue is crucial for treating external ear abnormalities. However, establishing adequate mechanical strength is challenging, owing to the difficulty of achieving chondrogenic differentiation in vitro; thus, cartilage reconstruction is a complex task. Auricular perichondrial chondroprogenitor cells exhibit high proliferation potential and can be obtained with minimal invasion. Therefore, these cells are an ideal resource for elastic cartilage reconstruction. In this study, we aimed to develop a novel in vitro scaffold-free method for elastic cartilage reconstruction, using human auricular perichondrial chondroprogenitor cells. Inducing chondrogenesis by using microscopic spheroids similar to auricular hillocks significantly increased the chondrogenic potential. The size and elasticity of the tissue were maintained after craniofacial transplantation in immunodeficient mice, suggesting that the reconstructed tissue was morphologically stable. Our novel tissue reconstruction method may facilitate the development of future treatments for external ear abnormalities.

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

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

Microstructure and Thermal Property of Designed Alginate-Based Polymeric Composite Foam Materials Containing Biomimetic Decellularized Elastic Cartilage Microscaffolds.

This study presents a designed alginate-based polymeric composite foam material containing decellularized elastic cartilage microscaffolds from porcine elastic cartilage by using supercritical fluid and papain treatment for medical scaffold biomaterials. The microstructure and thermal property of the designed alginate-based polymeric composite foam materials with various controlled ratios of alginate molecules and decellularized elastic cartilage microscaffolds were studied and characterized by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and differential thermal gravimetric analysis (TGA/DTG). The microstructure and thermal property of the composite foam materials were affected by the introduction of decellularized elastic cartilage microscaffolds. The designed alginate-based polymeric composite foam materials containing decellularized elastic cartilage microscaffolds were ionically cross-linked with calcium ions by soaking the polymeric composite foam materials in a solution of calcium chloride. Additional calcium ions further improved the microstructure and thermal stability of the resulting ionic cross-linked alginate-based polymeric composite foam materials. Furthermore, the effect of crosslinking functionality on microstructures and thermal properties of the resulting polymeric composite foam materials were studied to build up useful information for 3D substrates for cultivating and growing cartilage cells and/or cartilage tissue engineering.

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

OBJECTIVE: A major obstacle in the clinical translation of engineered auricular scaffolds is the significant contraction and loss of topography that occur during maturation of the soft collagen-chondrocyte matrix into elastic cartilage. We hypothesized that 3-dimensional-printed, biocompatible scaffolds would "protect" maturing hydrogel constructs from contraction and loss of topography. DESIGN: External disc-shaped and "ridged" scaffolds were designed and 3D-printed using polylactic acid (PLA). Acellular type I collagen constructs were cultured in vitro for up to 3 months. Collagen constructs seeded with bovine auricular chondrocytes (BAuCs) were prepared in 3 groups and implanted subcutaneously in vivo for 3 months: preformed discs with ("Scaffolded/S") or without ("Naked/N") an external scaffold and discs that were formed within an external scaffold via injection molding ("Injection Molded/SInj"). RESULTS: The presence of an external scaffold or use of injection molding methodology did not affect the acellular construct volume or base area loss. In vivo, the presence of an external scaffold significantly improved preservation of volume and base area at 3 months compared to the naked group (P < 0.05). Construct contraction was mitigated even further in the injection molded group, and topography of the ridged constructs was maintained with greater fidelity (P < 0.05). Histology verified the development of mature auricular cartilage in the constructs within external scaffolds after 3 months. CONCLUSION: Custom-designed, 3D-printed, biocompatible external scaffolds significantly mitigate BAuC-seeded construct contraction and maintain complex topography. Further refinement and scaling of this approach in conjunction with construct fabrication utilizing injection molding may aid in the development of full-scale auricular scaffolds.