Long-term in vivo electromechanical reshaping for auricular reconstruction in the New Zealand white rabbit model.
ABSTRACT: To demonstrate the dosimetry effect of electromechanical reshaping (EMR) on cartilage shape change, structural integrity, cellular viability, and remodeling of grafts in an in vivo long-term animal model.Animal study.A subperichondrial cartilaginous defect was created within the base of the pinna of 31 New Zealand white rabbits. Autologous costal cartilage grafts were electromechanically reshaped to resemble the rabbit auricular base framework and mechanically secured into the pinna base defect. Forty-nine costal cartilage specimens (four control and 45 experimental) successfully underwent EMR using a paired set of voltage-time combinations and survived for 6 or 12 weeks. Shape change was measured, and specimens were analyzed using digital imaging, tissue histology, and confocal microscopy with LIVE-DEAD viability assays.Shape change was proportional to charge transfer in all experimental specimens (P?
Project description: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.
Project description:To investigate the feasibility of repairing osteochondral defects of critical size by performing mosaicplasty using multiple sliced costal cartilage grafts, which enables repair of extensively injured knees using grafts from a single rib.Critical osteochondral defects were prepared on the femoral groove of skeletally mature Japanese white rabbits. Costal cartilage grafts from a single rib were harvested and sliced into multiple segments (approximately 3-5 mm in length). The defects were left untreated or repaired by performing mosaicplasty using costal cartilage grafts (with or without a longitudinal cut along the middle). At 4 and 12 weeks after transplantation, International Cartilage Repair Society macroscopic and histological grading was performed.The macroscopic score and visual histological score were significantly higher in the repaired groups than in the untreated group at 4 and 12 weeks after surgery. Histological continuous integration between grafted costal cartilage and host bone was observed in both repaired groups.The findings suggest that costal cartilage might be a useful alternative source for chondral grafting. We were able to repair large osteochondral defects by performing mosaicplasty using multiple sliced costal cartilage grafts from a single rib.
Project description:Electromechanical reshaping (EMR) is a low-cost, needle-based, and simple means to shape cartilage tissue without the use of scalpels, sutures, or heat that can potentially be used in an outpatient setting to perform otoplasty.To demonstrate that EMR can alter the shape of intact pinnae in an in vivo animal model and to show that the amount of shape change and the limited cell injury are proportional to the dosimetry.In an academic research setting, intact ears of 18 New Zealand white rabbits underwent EMR using 6 different dosimetry parameters (4 V for 5 minutes, 4 V for 4 minutes, 5 V for 3 minutes, 5 V for 4 minutes, 6 V for 2 minutes, and 6 V for 3 minutes). A custom acrylic jig with 2 rows of platinum needle electrodes was used to bend ears at the middle of the pinna and to perform EMR. Treatment was repeated twice per pinna, in proximal and distal locations. Control pinnae were not subjected to current application when being bent and perforated within the jig. Pinnae were splinted for 3 months along the region of the bend using soft silicon sheeting and a cotton bolster.The ears were harvested the day after splints were removed and before euthanasia. Photographs of ears were obtained, and bend angles were measured. Tissue was sectioned for histologic examination and confocal microscopy to assess changes to microscopic structure and cellular viability.Treated pinnae were bent more and retained shape better than control pinnae. The mean (SD) bend angles in the 7 dosimetry groups were 55° (35°) for the control, 60° (15°) for 4 V for 4 minutes, 118° (15°) for 4 V for 5 minutes, 88° (26°) for 5 V for 3 minutes, 80° (17°) for 5 V for 4 minutes, 117° (21°) for 6 V for 2 minutes, and 125° (18°) for 6 V for 3 minutes. Shape change was proportional to electrical charge transfer, which increased with voltage and application time. Hematoxylin-eosin staining of the pinnae identified localized areas of cell injury and fibrosis in the cartilage and in the surrounding soft tissue where the needle electrodes were inserted. This circumferential zone of injury (range, 1.5-2.5 mm) corresponded to dead cells on cell viability assay, and the diameter of this region increased with total electrical charge transfer to a maximum of 2.5 mm at 6 V for 3 minutes.Electromechanical reshaping produced shape change in intact pinnae of rabbits in this expanded in vivo study. A short application of 4 to 6 V can achieve adequate reshaping of the pinnae. Tissue injury around the electrodes increases with the amount of total current transferred into the tissue and is modest in spatial distribution. This study is a critical step toward evaluation of EMR in clinical trials.NA.
Project description:Peroxisome proliferator-activated receptor beta/delta (PPARD) is an important determinant of multiple biological processes. Our previous studies identified a missense mutation in the PPARD gene that significantly reduces its transcription activity, and consequently causes enlarged external ears in pigs. However, the mechanisms underlying the causality has remained largely unknown. Here, we show that PPARD retards the development of auricular cartilage by accelerating the apoptosis of cartilage stem/progenitor cells (CSPCs), the terminal differentiation of cartilage cells and the degradation of cartilage extracellular matrix in the auricle. At the transcription level, PPARD upregulates a set of genes that are associated with CSPCs apoptosis and chondrogenic differentiation, chondroblast differentiation and extracellular matrix degradation. ChIP-seq identified direct target genes of PPARD, including a well-documented gene for cartilage development: PPARG. We further show that compared to wild-type PPARD, the G32E mutant up-regulates the expression of PPARG and subsequently leads to the downregulation of critical genes that inhibit cartilage growth. These findings allow us to conclude that PPARD is an inhibitor of auricular cartilage growth in pigs. The causative mutation (G32E) in the PPARD gene attenuates the PPARD-mediated retardation of cartilage growth in the auricle, contributing to enlarged ears in pigs. The findings advance our understanding of the mechanisms underlying auricular development in mammals, and shed insight into the studies of innate pinna disorders and cartilage regeneration medicine in humans.
Project description:Total hip arthroplasty is a common surgical technique, yet it has severe complications, such as loosening and repeated revision. Thus, hip-preserving surgical options should be considered first to treat cartilage defects in the femoral head, especially for younger patients. Current surgical options for chondral repair of the femoral head include microfracture, trapdoor procedure, transplantation of osteochondral allografts and autografts, and autologous chondrocyte implantation. Each of these techniques has unique advantages and limitations; however, none of them have been consented as the best practice for cartilage defects. In this review article, we also introduced a novel technique for repairing osteochondral defects of the femoral head using autologous costal cartilage grafts that may have good translational potential for cost-effective and safe applications. The translational potential of this article:This review updates current surgical options for reparing articular cartilage defects in the femoral head. We also introduce a novel technique for repairing osteochondral defects of the femoral head using autologous costal cartilage grafts.
Project description:The prominent ear is a common external ear anomaly that is usually corrected through surgery. Electromechanical reshaping (EMR) may provide the means to reshape cartilage through the use of direct current (in milliamperes) applied percutaneously with needle electrodes and thus to reduce reliance on open surgery.To determine the long-term outcomes (shape change, cell viability, and histology) of a more refined EMR voltage and time settings for reshaping rabbit auricle.The intact ears of 14 New Zealand white rabbits were divided into 2 groups. Group 1 received 4 V for 5 minutes (5 ears), 5 V for 4 minutes (5 ears), or no voltage for 5 minutes (control; 4 ears). Group 2 received an adjusted treatment of 4 V for 4 minutes (7 ears) or 5 V for 3 minutes (7 ears). A custom mold with platinum electrodes was used to bend the pinna and to perform EMR. Pinnae were splinted for 6 months along the region of the bend. Rabbits were killed humanely and the ears were harvested the day after splint removal. Data were collected from March 14, 2013, to July 8, 2014, and analyzed from August 29, 2013, to March 1, 2015.Bend angle and mechanical behavior via palpation were recorded through photography and videography. Tissue was sectioned for histologic examination and confocal microscopy to assess changes to microscopic structure and cell viability.Rabbits ranged in age from 6 to 8 months and weighed 3.8 to 4.0 g. The mean (SD) bend angles were 81° (45°) for the controls and, in the 5 EMR groups, 72° (29°) for 4 V for 4 minutes, 101° (19°) for 4 V for 5 minutes, 78° (18°) for 5 V for 3 minutes, and 126° (21°) for 5 V for 4 minutes. At 5 V, an increase in application time from 3 to 4 minutes provided significant shape change (78° [18°] and 126° [21°], respectively; P?=?.003). Pinnae stained with hematoxylin-eosin displayed localized areas of cell injury and fibrosis in and around electrode insertion sites. This circumferential zone of injury (range, 1.3-2.1 mm) corresponded to absence of red florescence on the cell viability assay.In this in vivo study, EMR produces shape changes in the intact pinnae of rabbits. A short application of 4 V or 5 V can achieve adequate reshaping of the pinnae. Tissue injury around the electrodes is modest in spatial distribution. This study provides a more optimal set of EMR variables and a critical step toward evaluation of EMR in clinical trials.NA.
Project description:Autologous cartilage grafting during open airway reconstruction is a complex skill instrumental to the success of the operation. Most trainees lack adequate opportunities to develop proficiency in this skill. We hypothesized that 3-dimensional (3D) printing and computer-aided design can be used to create a high-fidelity simulator for developing skills carving costal cartilage grafts for airway reconstruction. The rapid manufacturing and low cost of the simulator allow deployment in locations lacking expert instructors or cadaveric dissection, such as medical missions and Third World countries. In this blinded, prospective observational study, resident trainees completed a physical simulator exercise using a 3D-printed costal cartilage grafting tool. Participant assessment was performed using a Likert scale questionnaire, and airway grafts were assessed by a blinded expert surgeon. Most participants found this to be a very relevant training tool and highly rated the level of realism of the simulation tool.
Project description:The reconstruction of the external ear to correct congenital deformities or repair following trauma remains a significant challenge in reconstructive surgery. Previously, we have developed a novel approach to create scaffold-free, tissue engineering elastic cartilage constructs directly from a small population of donor cells. Although the developed constructs appeared to adopt the structural appearance of native auricular cartilage, the constructs displayed limited expression and poor localization of elastin. In the present study, the effect of growth factor supplementation (insulin, IGF-1, or TGF-?1) was investigated to stimulate elastogenesis as well as to improve overall tissue formation. Using rabbit auricular chondrocytes, bioreactor-cultivated constructs supplemented with either insulin or IGF-1 displayed increased deposition of cartilaginous ECM, improved mechanical properties, and thicknesses comparable to native auricular cartilage after 4 weeks of growth. Similarly, growth factor supplementation resulted in increased expression and improved localization of elastin, primarily restricted within the cartilaginous region of the tissue construct. Additional studies were conducted to determine whether scaffold-free engineered auricular cartilage constructs could be developed in the 3D shape of the external ear. Isolated auricular chondrocytes were grown in rapid-prototyped tissue culture molds with additional insulin or IGF-1 supplementation during bioreactor cultivation. Using this approach, the developed tissue constructs were flexible and had a 3D shape in very good agreement to the culture mold (average error <400 µm). While scaffold-free, engineered auricular cartilage constructs can be created with both the appropriate tissue structure and 3D shape of the external ear, future studies will be aimed assessing potential changes in construct shape and properties after subcutaneous implantation.
Project description:Microtia is a congenital external ear malformation that can seriously influence the psychological and physiological well-being of affected children. The successful regeneration of human ear-shaped cartilage using a tissue engineering approach in a nude mouse represents a promising approach for auricular reconstruction. However, owing to technical issues in cell source, shape control, mechanical strength, biosafety, and long-term stability of the regenerated cartilage, human tissue engineered ear-shaped cartilage is yet to be applied clinically. Using expanded microtia chondrocytes, compound biodegradable scaffold, and in vitro culture technique, we engineered patient-specific ear-shaped cartilage in vitro. Moreover, the cartilage was used for auricle reconstruction of five microtia patients and achieved satisfactory aesthetical outcome with mature cartilage formation during 2.5years follow-up in the first conducted case. Different surgical procedures were also employed to find the optimal approach for handling tissue engineered grafts. In conclusion, the results represent a significant breakthrough in clinical translation of tissue engineered human ear-shaped cartilage given the established in vitro engineering technique and suitable surgical procedure. This study was registered in Chinese Clinical Trial Registry (ChiCTR-ICN-14005469).
Project description:Three-dimensional (3D) bioprinting of patient-specific auricular cartilage constructs could aid in the reconstruction process of traumatically injured or congenitally deformed ear cartilage. To achieve this, a hydrogel-based bioink is required that recapitulates the complex cartilage microenvironment. Tissue-derived decellularized extracellular matrix (dECM)-based hydrogels have been used as bioinks for cell-based 3D bioprinting because they contain tissue-specific ECM components that play a vital role in cell adhesion, growth, and differentiation. In this study, porcine auricular cartilage tissues were isolated and decellularized, and the decellularized cartilage tissues were characterized by histology, biochemical assay, and proteomics. This cartilage-derived dECM (cdECM) was subsequently processed into a photo-crosslinkable hydrogel using methacrylation (cdECMMA) and mixed with chondrocytes to create a printable bioink. The rheological properties, printability, and in vitro biological properties of the cdECMMA bioink were examined. The results showed cdECM was obtained with complete removal of cellular components while preserving major ECM proteins. After methacrylation, the cdECMMA bioinks were printed in anatomical ear shape and exhibited adequate mechanical properties and structural integrity. Specifically, auricular chondrocytes in the printed cdECMMA hydrogel constructs maintained their viability and proliferation capacity and eventually produced cartilage ECM components, including collagen and glycosaminoglycans (GAGs). The potential of cell-based bioprinting using this cartilage-specific dECMMA bioink is demonstrated as an alternative option for auricular cartilage reconstruction.