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Objective: To investigate the effects of the misshapen auricular chondrocytes from microtia in inducing., Methods: Human ADSCs at passage 3 and misshapen chondrogenesis of human adipose derived stem cells (ADSCs) in vitro. auricular chondrocytes at passage 2 were harvested and mixed at a ratio of 7:3 as experimental group (group A, 1.0 x 10(6) mixed cells). Misshapen auricular chondrocytes or ADSCs at the same cell number served as control groups (groups B and C, respectively). All samples were incubated in the centrifuge tubes. At 28 days after incubation, the morphological examination was done and the wet weight was measured; the content of glycosaminoglycan (GAG) was detected by Alcian blue colorimetry; the expressions of collagen type II and Aggrecan were determined with RT-PCR; and HE staining, toluidine blue staining, Safranin O staining of GAG, and collagen type II immunohistochemical staining were used for histological and immunohistochemical observations., Results: At 28 days after incubation, all specimens formed disc tissue that was translucent and white with smooth surface and good elasticity in groups A and B; the specimens shrank into yellow spherical tissue without elasticity in group C. The wet weight and GAG content of specimens in groups A and B were significantly higher than those in group C (P < 0.05), but no significant difference was found between groups A and B in the wet weight (t = 1.8203, P = 0.0687) and in GAG content (t = 1.8614, P = 0.0627). In groups A and B, obvious expressions of collagen type II and Aggrecan mRNA could be detected by RT-PCR, but no obvious expressions were observed in group C; the expressions in groups A and B were significantly higher than those in group C (P < 0.05), but no significant difference was found between groups A and B in collagen type II mRNA expression (t = 1.4576, P = 0.1449) and Aggrecan mRNA expression (t = 1.5195, P = 0.1286). Mature cartilage lacunas and different degrees of dyeing for the extracellular matrix could be observed in groups A and B; no mature cartilage lacunas or collagen type II could be observed in group C. The expression of collagen type II around cartilage lacuna was observed in groups A and B, but no expression in group C; the gray values of groups A and B were significantly lower than that of group C (P < 0.01), but no significant difference was found between groups A and B (t = 1.6615, P = 0.0970)., Conclusion: Misshapen auricular chondrocytes from microtia can induce chondrogenic differentiation of human ADSCs in vitro.

Introduction: We have designed an engineered graft fabricated from a biodegradable scaffold using chondrocytes and applied this construct to augment repair of tracheal stenosis. This study investigated the feasibility of using such tissue-engineered airways with autologous chondrocytes in a rabbit model., Material and Methods: Chondrocytes were isolated and expanded from the auricular cartilage of New Zealand white rabbits, then seeded onto composite 3-layer scaffolds consisting of a collagen sheet, a polyglycolic acid mesh, and a copolymer (l-lactide/epsilon-caprolactone) coarse mesh. The engineered grafts were implanted into a 0.5 x 0.8-cm defect created in the midventral portion of the cervical trachea. Gelatin sponges that slowly released basic fibroblast growth factor (b-FGF) were then placed on the constructs, which were retrieved 1 or 3 months after implantation., Results: The biodegradable scaffold seeded with chondrocytes could maintain airway structure up to 3 months after implantation. Tracheal epithelial regeneration occurred in the internal lumen of this composite scaffold. Three months after implantation, staining of the sections showed cartilage accumulation in the engineered tracheal wall., Conclusion: This composite biodegradable scaffold may be useful for developing engineered trachea. A gelatin sponge slowly releasing b-FGF might enhance chondrogenesis.

Background: The repair of a craniofacial or nose deformity requires a large volume of reconstructive material. A conventional cartilage graft does not provide a sufficient volume of reconstructive material. Therefore, augmentation of the facial form to the defect shape is quite difficult. The authors developed a new treatment method that provides a sufficiently large volume of reconstructive material and enables an easier reconstruction of the original shape., Methods: Ages of the patients ranged between 9 and 63 years. Approximately 1 cm of auricular cartilage was collected from the auricular concha. Isolated chondrocytes were cultured with autologous serum that accelerates cell proliferation. The cells were subcultured and formed a gel-form mass. This mass, together with autologous serum, was grafted (injected) on the periosteum and into the subcutaneous pocket. The volume of grafted cultured chondrocytes ranged from 1.7 to 40 cc (1 to 5 x 10(7) cells/cc). The lesion changed from soft gel form into hard cartilage tissues within 2 to 3 weeks and stabilized., Results: Excellent or good satisfactory results were obtained in all patients and have been maintained for periods ranging from 3 to 34 months. No patient experienced absorption of cultured chondrocytes. Biopsy of the newly formed tissues showed that it was an elastic cartilage derived from the original tissue., Conclusions: A small number of chondrocytes obtained from a 1-cm auricular cartilage are successfully cultured into a large number of cells in a gel form. Those autologous auricular chondrocytes in a gel form allow for the repair of complicated shapes of the defect area. This technique is applicable to various treatments for craniofacial or nose deformity.

Objectives/hypothesis: Current surgical techniques for the correction of microtia are challenging. Research in the field of tissue engineering is providing insight into chondrocyte behavior for a possible future treatment of microtia. The authors wished to evaluate the biological potential of chondrocytes isolated from microtia cartilage as compared with normal auricular cartilage as a source of tissue-engineered cartilage., Study Design: A comparative research design to study the potential of microtia cartilage chondrocytes with normal auricular chondrocytes as a source of tissue-engineered cartilage., Methods: Cartilage specimens from 12 pediatric patients (six normal auricular specimens and six auricular specimens with microtia) were obtained. The chondrocytes were isolated and cultured in vitro; chondrocyte number was increased by passaging. Each type of cell was implanted in nude mice to generate tissue-engineered cartilage. Eight weeks after implantation the specimens were dissected and removed. Results were compared between the normal auricular and microtia specimens in regard to cell number expansion in vitro and generation of tissue-engineered cartilage in vivo., Results: An initial mean cell number of 150,000 cells in each group (normal and microtia) increased to an average cell number of 120 million cells/mL in the normal and 130 million cells in the microtia subgroups, respectively, at the end of the second passage. Histologically, both types of chondrocytes generated normal elastic cartilage., Conclusion: The study demonstrated the potential of cells isolated from microtia cartilage to generate tissue-engineered cartilage. Microtia cartilage represents an important additional donor source for the possible generation of a human tissue-engineered auricle.

To date, there is no optimal way to reconstruct an external ear in cases of microtia or after trauma or burns damaging the external ear. However, success in the area of tissue engineering has indicated that autologous elastic cartilage produced in vitro might be of great importance in the future treatment of these patients. In the present study we have engineered human, elastic cartilage in vitro by culturing chondrocytes in fibrin glue. Furthermore, the engineered elastic cartilage was seeded with human keratinocytes to investigate the possibility of combining these two tissues into one integrated structure. Histological analysis and immunohistochemistry were done every second week for 10 weeks. The elastic chondrocytes were shown to grow well in the matrix and proliferated in a dense pattern. After 10 weeks a matrix containing elastin was shown by staining with orcein, indicating that an elastic cartilage had been formed. The seeded keratinocytes adhered to the cartilage, proliferated, and formed a stratified epidermal layer, which was shown by routine histological staining and immunohistochemistry. This study shows that human elastic chondrocytes can be cultured in fibrin glue and that human keratinocytes can be cocultured with this engineered cartilage, which might be of great importance in future reconstruction of ears.

Intrinsic sphincter deficiency (ISD) is frequently treated with collagen bulking at the bladder neck. The standard material used, Contigen, biodegrades over 3-19 months requiring repeated injections to maintain efficacy. The study objective was to evaluate use of autologous ear chondrocytes for treatment of ISD. Women with documented ISD had harvest of auricular cartilage. Chondrocytes were isolated from the cartilage and expanded in culture and formulated with calcium alginate to form an injectable gel. Thirty-two patients received a single outpatient injection just distal to the bladder neck. Outcome measures included voiding diary, quality-of-life scores, incontinence severity grading, and pad weight testing. Incontinence grading indicated 16 patients dry, and 10 improved at 12 months for a total of 26 of 32 (81.3%) dry and improved after one treatment. Only four patients had a 12-month pad weight test over 2.2 g. Quality-of-life scores improved significantly after treatment. There was a decrease in incontinence impact scores in all categories. The urogenital distress inventory declined for all categories except bladder emptying and lower abdominal pain. Endoscopic treatment of ISD with autologous chondrocytes is safe, effective, and durable with 50 % of patients dry 12 months after one injection. Twenty-six of 32 patients dry or improved at 3 months after the injection maintained the effect at the 12-month visit., (Copyright 2001 Wiley-Liss, Inc.)

Recently proposed procedures for in vitro generation of new cartilage may be difficult to perform in humans because so many chondrocytes are needed for tissue engineering. In this study the authors investigated new, efficient, low-cost techniques for the isolation and culture of chondrocytes from the ear cartilage of the rabbit. They performed a low-density monolayer culture with a low concentration (0.5%, 1%) of human platelet supernatant and observed cell proliferation (seeding efficiency, deoxyribonucleic acid synthesis), matrix synthesis (glycosaminoglycan synthesis), and the expression of type I and type II collagen (reverse transcriptase polymerase chain reaction). Seeding efficiency was increased in 1% of platelet supernatant-treated cultures by two to three times compared with untreated controls. One percent platelet supernatant had increased the incorporation of [3H]-thymidine by 1.9 to 2.5 times at 72 hours compared with controls. Glycosaminoglycan synthesis was increased in platelet supernatant-treated chondrocytes at 96 hours compared with controls. Chondrocytes treated with 1% platelet supernatant showed a decreased expression of the type II collagen gene. Supplementation with a high concentration (10%) of the platelet supernatant provided the conditions for in vitro chondrocyte mass formation. These results indicate that proliferation and matrix synthesis of auricular chondrocytes is stimulated by a low concentration of platelet supernatant. On the other hand, chondrocytes were immobilized by a high concentration of platelet supernatant. Platelet supernatant may be useful as an inexpensive autologous source of multiple growth factors to enhance chondrocyte proliferation, and also may play the role of scaffold for chondrocytes. Additional investigation is underway to generate culture conditions that promote the differentiation as well as the proliferation of chondrocytes.

To date, many efforts to engineer cartilage have focused on matrix construction with the goal of producing a durable construct as cartilage replaces the resorbing matrix. However, the importance of matrix construction is at least matched by the challenge of efficient chondrocyte extraction, culture expansion, and prevention of dedifferentiation. This challenge is underscored by the large number of chondrocytes needed for a clinically significant construct such as an ear. Because human rib provides a large, readily available source of hyaline cartilage, the authors evaluated human rib chondrocyte extraction and found that maximum viable cell yield occurred after a 6-hour digestion. They also evaluated human microtic auricular remnant chondrocyte extraction and identified fibroblast contamination as a shortcoming of this potential source of chondrocytes. Initially, rib chondrocytes proliferated in vitro with a doubling time of approximately 1 week. As the cells were passaged, proliferation decreased such that the cells stopped proliferating and adopted a large, spindle-shaped morphology by passage 6. Interestingly, no increase in proliferation was noted when rib chondrocytes were stimulated with transforming growth factor beta 1, bone morphogenetic protein 2, and basic fibroblast growth factor. The major obstacles to the use of autologous rib chondrocytes in matrix construction are the low cell yield from a small piece of rib and the limited proliferation that these cells will undergo in vitro. Further investigation of culture systems and mitogenic cytokines may help resolve these limitations.