Auricular Cartilage Tissue Engineering Research Articles

Co-culture of RhoA-overexpressed microtia chondrocytes and adipose-derived stem cells in the construction of tissue-engineered ear-shaped cartilage.

Microtia is a congenital auricle dysplasia with a high incidence and tissue engineering technology provides a promising strategy to reconstruct auricles. We previously described that the engineered cartilage constructed from microtia chondrocytes exhibited inferior levels of biochemical and biomechanical properties, which was proposed to be resulted of the decreased migration ability of microtia chondrocytes. In the current study, we found that Rho GTPase members were deficient in microtia chondrocytes. By overexpressing RhoA, Rac1, and CDC42, respectively, we further demonstrated that RhoA took great responsibility for the decreased migration ability of microtia chondrocytes. Moreover, we constructed PGA/PLA scaffold-based cartilages to verify the chondrogenic ability of RhoA overexpressed microtia chondrocytes, and the results showed that overexpressing RhoA was of limited help in improving the quality of microtia chondrocyte engineered cartilage. However, coculture of adipose-derived stem cells (ADSCs) significantly improved the biochemical and biomechanical properties of engineered cartilage. Especially, coculture of RhoA overexpressed microtia chondrocytes and ADSCs produced an excellent effect on the wet weight, cartilage-specific extracellular matrix, and biomechanical property of engineered cartilage. Furthermore, we presented that coculture of RhoA overexpressed microtia chondrocytes and ADSCs combined with human ear-shaped PGA/PLA scaffold and titanium alloy stent fabricated by CAD/CAM and 3D printing technology effectively constructed and maintained auricle structure in vivo. Collectively, our results provide evidence for the essential role of RhoA in microtia chondrocytes and a developed strategy for the construction of patient-specific tissue-engineered auricular cartilage.

Human adult, pediatric and microtia auricular cartilage harbor fibronectin-adhering progenitor cells with regenerative ear reconstruction potential

SummaryRemaining challenges in auricular cartilage tissue engineering include acquiring sufficient amounts of regeneration-competent cells and subsequent production of high-quality neocartilage. Progenitor cells are a resident subpopulation of native cartilage, displaying a high proliferative and cartilage-forming capacity, yet their potential for regenerative medicine is vastly understudied. In this study, human auricular cartilage progenitor cells were newly identified in healthy cartilage and, importantly, in microtia-impaired chondral remnants. Their cartilage repair potential was assessed via in vitro 3D culture upon encapsulation in a gelatin-based hydrogel, and subsequent biochemical, mechanical, and histological analyses. Auricular cartilage progenitor cells demonstrate a potent ability to proliferate without losing their multipotent differentiation ability and to produce cartilage-like matrix in 3D culture. As these cells can be easily obtained through a non-deforming biopsy of the healthy ear or from the otherwise redundant microtia remnant, they can provide an important solution for long-existing challenges in auricular cartilage tissue engineering.

Cadherin-11 promotes the mechanical strength of engineered elastic cartilage by enhancing extracellular matrix synthesis and microstructure.

Limitations of current treatments for auricular cartilage defects have prompted the field of auricular cartilage tissue engineering. To date, inducing the formation of cartilaginous constructs with biochemical and biomechanical properties of native tissue is the final aim. Through hematoxylin-eosin and immunohistochemistry staining, Cadherin-11(CDH11) was confirmed highly expressed in the auricular cartilage tissue and chondrocytes. In vitro, by knockdown and overexpression of CDH11 in chondrocytes, CDH11 was demonstrated to promote the expression of collagen type II (COL2A), elastin (ELN), aggrecan (ACAN), and cartilage oligomeric matrix protein (COMP). In addition, the CDH11 overexpressed chondrocytes promoted neo-cartilage formation and its biomechanical property by increasing the key transcription factor of chondrogenesis SOX9 expression and cartilage extracellular matrix (ECM) production. The young's modulus and yield stress of the neo-cartilage in CDH11 overexpression group were about 1.7 times (p=0.0152) and 2 times (p=0.0428) higher than those in control group, respectively. Then, the immunohistochemistry staining, qRT-PCR and western blot examination results showed that the expression of COL2A and ELN were significantly increased. Notably, the electron microscopy results showed that the collagen and elastic fibers of the neo-cartilage in CDH11-OV group arranged in bunches and were more uniform and compact compared to the control group. Furthermore, CDH11 promoted elastic fiber assembly by increasing lysyl oxidase (LOX), fibrillin-1 (FBN1) expression. Taken together, our results demonstrated that CDH11 improves the mechanical strength of tissue-engineered elastic cartilage by promoting ECM synthesis and elastic fiber assembly.

Contribution of perichondrium to the mechanical properties of auricular cartilage

The poor mechanical strength of tissue-engineered auricular cartilage which is possibly due to the lack of perichondrium limits its application in auricular reconstruction surgery. However, there is insufficient research and no reliable data to support this. This study aims to investigate the contribution of perichondrium to the mechanical strength of auricular cartilage under loading. Rabbit auricular cartilage was harvested and classified into two groups. The perichondrium was removed in Group A and was left intact in Group B. Young’s modulus, stress relaxation slope, and relaxation amout were analyzed through tensile and compressive tests using a material testing machine. Group B exhibited significantly higher Young’s modulus in both the tensile and compressive tests (p < 0.05), lower relaxation slope (p < 0.05 in tensile test, p = 0.65 in compressive test), and lower relaxation amout (p < 0.05 in tensile test, p < 0.01 in compressive test). Our results showed that the perichondrium has a definite contribution to the mechanical properties of ear cartilage. This study may provide new insights to researchers focusing on improving the mechanical strength of tissue-engineered auricular cartilage.

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.

PLLA Porous Microsphere-Reinforced Silk-Based Scaffolds for Auricular Cartilage Regeneration.

Microtia, frequently encountered in plastic surgery practice, is usually corrected by auricular reconstruction with prostheses or autologous cartilages. In recent decades, however, cartilage tissue engineering has been emerging as a promising alternative for its minimal invasion and low immunogenicity. As a critical factor for tissue engineering, scaffolds are expected to be sufficiently porous and stiff to facilitate chondrogenesis. In this work, we introduce novel poly-l-lactic acid (PLLA) porous microsphere-reinforced silk-based hybrid (SBH) scaffolds with a multihierarchical porous structure. The scaffolds are fabricated by embedding PLLA porous microspheres (PMs) into a blending matrix of silk fibroin (SF) and gelatin solution, followed by mixing with a degummed silk fiber mesh and freeze-drying process. Through adjusting the amount of PLLA PMs, the mechanical strength approximates to natural cartilage and also balanced physical properties were realized. Biological evaluations of SBH scaffolds, both in vitro and in vivo, were conducted and PM-free plain silk-based (PSB) scaffolds were applied as control. Overall, it suggests that the incorporation of PLLA PMs remarkably improves mechanical properties and the capability to promote chondrogenesis of SBH scaffolds, and that SBH scaffolds appear to be a promising construct for potential applications in auricular cartilage tissue engineering and relevant fields.

A photo-crosslinkable cartilage-derived extracellular matrix bioink for auricular cartilage tissue engineering

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.

Transforming Growth Factor Beta 3 Induced Human Adipose-derived Stem Cells for Auricular Chondrogenesis

The limitation of self-repair and proliferation capacity of chondrocytes in cartilage reconstruction lead to alternative search of cell source that can improve the auricular regeneration. Human adipose-derived stem cells (HADSC) are an alternative cell source that have unique characteristics to self-renew and differentiate into various tissues making it suitable for cell therapy and tissue engineering. This study aimed to examine the chondrogenic differentiation potential of (HADSC) in monolayer culture by the presence of different transforming growth factor beta’s, TFG-β1, -β2 and -β3. HADSC at passage 3 (1.5 × 105 cell/mL) were cultured in chondrogenic medium containing 5 ng/mL of different transforming growth factor beta’s, TFG-β1, -β2 and -β3 for 7, 14 and 21 days. Data analysis was evaluated based on the growth rate of cells, cells morphological changed, production of collagen type II and glycosaminoglycan sulphate (sGAG). The quantitative RT-PCR was carried out to determine the chondrogenic, fibrogenic and hypertrophic gene expression levels. Differentiation of HADSC into chondrocytes using TFG-β indicates the occurrence of the chondrogenesis process. The best chondrogenic differentiation was observed in HADSC induced by TFG-β3 through the chondrocytes-like cells morphology with cells aggregation and high production of proteoglycan matrices compared to other TGF-βs groups. Additionally, the expression of chondrocytes-specific genes such as Type II collagen, Aggrecan core protein, Elastin and Sox 9 was high. In conclusion, this study has showed that TGF-β3 is the potential growth factor in producing chondrogenic cells for auricular cartilage tissue engineering.

Noninvasive Measurement of Ear Cartilage Elasticity on the Cellular Level: A New Method to Provide Biomechanical Information for Tissue Engineering.

Background:An important feature of auricular cartilage is its stiffness. To tissue engineer new cartilage, we need objective tools to provide us with the essential biomechanical information to mimic optimal conditions for chondrogenesis and extracellular matrix (ECM) development. In this study, we used an optomechanical sensor to investigate the elasticity of auricular cartilage ECM and tested whether sensitivity and measurement reproducibility of the sensor would be sufficient to accurately detect (subtle) differences in matrix compositions in healthy, diseased, or regenerated cartilage.Methods:As a surrogate model to different cartilage ECM compositions, goat ears (n = 9) were subjected to different degradation processes to remove the matrix components elastin and glycosaminoglycans. Individual ear samples were cut and divided into 3 groups. Group 1 served as control and was measured within 2 hours after animal death and at 24 and 48 hours, and groups 2 and 3 were measured after 24- and 48-h hyaluronidase or elastase digestion. Per sample, 9 consecutive measurements were taken ±300 μm apart.Results:Good reproducibility was seen between consecutive measurements with an overall interclass correlation coefficient average of 0.9 (0.81–0.98). Although degradation led to variable results, overall, a significant difference was seen between treatment groups after 48 hours (control, 4.2 MPa [±0.5] vs hyaluronidase, 2.0 MPa [±0.3], and elastase, 3.0 MPa [±0.4]; both P < 0.001).Conclusions:The optomechanical sensor system we used provided a fast and reliable method to perform measurements of cartilage ECM in a reverse tissue-engineering model. In future applications, this method seems feasible for the monitoring of changes in stiffness during the development of tissue-engineered auricular cartilage.

T2 and Apparent Diffusion Coefficient of MRI Reflect Maturation of Tissue-Engineered Auricular Cartilage Subcutaneously Transplanted in Rats.

In cartilage regenerative medicine, autologous chondrocyte implantation (ACI) has been applied clinically for partial defects of joint cartilage or nasal augmentation. To make treatment with ACI more effective and prevalent, modalities to evaluate the quality of transplanted constructs noninvasively are necessary. In this study, we compared the efficacy of several noninvasive modalities for evaluating the maturation of tissue-engineered auricular cartilage containing a biodegradable polymer scaffold. We first transplanted tissue-engineered cartilage consisting of human auricular chondrocytes, atelocollagen gel, and a poly-l-lactic acid (PLLA) porous scaffold subcutaneously into the back of athymic nude rats. Eight weeks after transplantation, the rats were examined by magnetic resonance imaging (MRI), X-ray, and ultrasound as noninvasive modalities. Then, the excised constructs were examined by histological and biochemical analysis including toluidine blue (TB) staining, glycosaminoglycans content, and enzyme-linked immunosorbent assay of type II collagen. Among the modalities examined, transverse relaxation time (T2) and apparent diffusion coefficient of MRI showed quite a high correlation with histological and biochemical results, suggesting that these can effectively detect the maturation of tissue-engineered auricular cartilage. Since these noninvasive modalities would realize time-course analysis of the maturation of tissue-engineered auricular cartilage, this study provides a substantial insight for improving the quality of tissue-engineered cartilage, leading to improvement of the quality and technique in cartilage regenerative medicine.

3D bioprinting of human chondrocyte-laden nanocellulose hydrogels for patient-specific auricular cartilage regeneration

Auricular cartilage tissue engineering (TE) aims to provide an effective treatment for patients with acquired or congenital auricular defects. Bioprinting has gained attention in several TE strategies for its ability to spatially control the placement of cells, biomaterials and biological molecules. Although considerable advances have been made to bioprint complex 3D tissue analogues, the development of hydrogel bioinks with good printability and bioactive properties must improve in order to advance the translation of 3D bioprinting into the clinic. In this study, the biological functionality of a bioink composed of nanofibrillated cellulose and alginate (NFC-A) is extensively evaluated for auricular cartilage TE. 3D bioprinted auricular constructs laden with human nasal chondrocytes (hNC) are cultured for up to 28 days and the redifferentiation capacity of hNCs in NFC-A is studied on gene expression as well as on protein levels. 3D bioprinting with NFC-A bioink facilitates the biofabrication of cell-laden, patient-specific auricular constructs with an open inner structure, high cell density and homogenous cell distribution. The cell-laden NFC-A constructs exhibit an excellent shape and size stability as well as an increase in cell viability and proliferation during in vitro culture. Furthermore, NFC-A bioink supports the redifferentiation of hNCs and neo-synthesis of cartilage-specific extracellular matrix components. This demonstrated that NFC-A bioink supports redifferentiation of hNCs while offering proper printability in a biologically relevant aqueous 3D environment, making it a promising tool for auricular cartilage TE and many other biomedical applications.

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.

Novel bilayer bacterial nanocellulose scaffold supports neocartilage formation in vitro and in vivo

Tissue engineering provides a promising alternative therapy to the complex surgical reconstruction of auricular cartilage by using ear-shaped autologous costal cartilage. Bacterial nanocellulose (BNC) is proposed as a promising scaffold material for auricular cartilage reconstruction, as it exhibits excellent biocompatibility and secures tissue integration. Thus, this study evaluates a novel bilayer BNC scaffold for auricular cartilage tissue engineering. Bilayer BNC scaffolds, composed of a dense nanocellulose layer joined with a macroporous composite layer of nanocellulose and alginate, were seeded with human nasoseptal chondrocytes (NC) and cultured in vitro for up to 6 weeks. To scale up for clinical translation, bilayer BNC scaffolds were seeded with a low number of freshly isolated (uncultured) human NCs combined with freshly isolated human mononuclear cells (MNC) from bone marrow in alginate and subcutaneously implanted in nude mice for 8 weeks. 3D morphometric analysis showed that bilayer BNC scaffolds have a porosity of 75% and mean pore size of 50 ± 25 μm. Furthermore, endotoxin analysis and in vitro cytotoxicity testing revealed that the produced bilayer BNC scaffolds were non-pyrogenic (0.15 ± 0.09 EU/ml) and non-cytotoxic (cell viability: 97.8 ± 4.7%). This study demonstrates that bilayer BNC scaffolds offer a good mechanical stability and maintain a structural integrity while providing a porous architecture that supports cell ingrowth. Moreover, bilayer BNC scaffolds provide a suitable environment for culture-expanded NCs as well as a combination of freshly isolated NCs and MNCs to form cartilage in vitro and in vivo as demonstrated by immunohistochemistry, biochemical and biomechanical analyses.

Response to letter to the editor concerning "Quantitative evaluation of mechanical properties in tissue-engineered auricular cartilage".

Response to letter to the editor concerning "Quantitative evaluation of mechanical properties in tissue-engineered auricular cartilage".

Biocompatibility evaluation of densified bacterial nanocellulose hydrogel as an implant material for auricular cartilage regeneration.

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.

Quantitative Evaluation of Mechanical Properties in Tissue-Engineered Auricular Cartilage

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.

Advancing nasal reconstructive surgery: the application of tissue engineering technology

Cartilage tissue engineering is a rapidly progressing area of regenerative medicine with advances in cell biology and scaffold engineering constantly being investigated. Many groups are now capable of making neocartilage constructs with some level of morphological, biochemical, and histological likeness to native human cartilage tissues. The application of this useful technology in articular cartilage repair is well described in the literature; however, few studies have evaluated its application in head and neck reconstruction. Although there are many studies on auricular cartilage tissue engineering, there are few studies regarding cartilage tissue engineering for complex nasal reconstruction. This study therefore highlighted the challenges involved with nasal reconstruction, with special focus on nasal cartilage tissue, and examined how advancements made in cartilage tissue engineering research could be applied to improve the clinical outcomes of total nasal reconstructive surgery.

The Tissue-Engineered Auricle: Past, Present, and Future

The reconstruction, repair, and regeneration of the external auricular framework continue to be one of the greatest challenges in the field of tissue engineering. To replace like with like, we should emulate the native structure and composition of auricular cartilage by combining a suitable chondrogenic cell source with an appropriate scaffold under optimal in vitro and in vivo conditions. Due to the fact that a suitable and reliable substitute for auricular cartilage has yet to be engineered, hand-carved autologous costal cartilage grafts and ear-shaped porous polyethylene implants are the current treatment modalities for auricular reconstruction. However, over the last decade, significant advances have been made in the field of regenerative medicine and tissue engineering. A variety of scaffolds and innovative approaches have been investigated as alternatives to using autologous carved costal cartilage or porous polyethylene implants. A review of recent developments and the current state of the art and science is presented, focusing on scaffolds, cell sources, seeding densities, and mechanical characteristics of tissue-engineered auricular cartilage.

Tissue engineering of an auricular cartilage model utilizing cultured chondrocyte-poly(L-lactide-epsilon-caprolactone) scaffolds.

To determine the potential development in vivo of tissue-engineered auricular cartilage, chondrocytes from articular cartilage of bovine forelimb joints were seeded on poly(L-lactic acid-epsilon-caprolactone) copolymer scaffolds molded into the shape of a human ear. Copolymer scaffolds alone in the same shape were studied for comparison. Chondrocyte-seeded copolymer constructs and scaffolds alone were each implanted in dorsal skin flaps of athymic mice for up to 40 weeks. Retrieved specimens were examined by histological and molecular techniques. After 10 weeks of implantation, cell-seeded constructs developed cartilage as assessed by toluidine blue and safranin-O red staining; a vascular, perichondrium-like capsule enveloped these constructs; and tissue formation resembled the auricular shape molded originally. Cartilage matrix formation increased, the capsule persisted, and initial auricular configuration was maintained through implantation for 40 weeks. The presence of cartilage production was correlated with RT-PCR analysis, which showed expression of bovine-specific type II collagen and aggrecan mRNA in cell-seeded specimens at 20 and 40 weeks. Copolymer scaffolds monitored only for 40 weeks failed to develop cartilage or a defined capsule and expressed no mRNA. Extensive vascularization led to scaffold erosion, decrease in original size, and loss of contour and shape. These results demonstrate that poly(L-lactic acid-epsilon-caprolactone) copolymer seeded with articular chondrocytes supports development and maintenance of cartilage in a human ear shape over periods to 40 weeks in this implantation model.

Normal features of tissue-engineered auricular cartilage by flow cytometry and histology: patient safety

Background Cytokinetic abnormalities in DNA content, such as aneuploidy, haploidy, and tetraploidy, have been found to occur in human cartilaginous tumors. The high number of chondrocytes needed for tissue-engineered cartilaginous implants requires the cells to be passaged repeatedly. The theoretical risk of changes in the normal diploid state of these cells during their growth in vitro and after generation of tissue-engineered cartilage in vivo is not known. Materials and methods Auricular chondrocytes were obtained from 6 patients and cultured in vitro. Chondrocyte number was increased by repeated passaging. The passaged cells were implanted in nude mice for 8 weeks to generate tissue-engineered cartilage. Fresh control chondrocytes along with the passaged cells and cells obtained from the tissue-engineered constructs were collected and compared for DNA content by flow cytometry. Results Flow cytometry demonstrated 100% diploidy with no evidence of aneuploidy, haploidy, or tetraploidy in all groups of cells. Histology of the tissue-engineered cartilage also showed no evidence of cellular atypia. Conclusion The number of human auricular chondrocytes can be increased by repeated passaging and passaged chondrocytes can be safely used for implantation to generate tissue-engineered constructs without a change in the normal diploid state of the cells. Histology of the cartilage generated showed normal features without atypia.