Retalho retroauricular ilhado para reconstrução parcial de orelha: relato de casos / Retroauricular island flap for partial ear reconstruction: case reports

Defeitos parciais de orelha podem ser tratados de diversas formas, dentre elas o fechamento primário, cicatrização por segunda intenção ou retalhos. Diversas opções técnicas foram descritas para a sua reconstrução de modo a manter o contorno natural da orelha, sem sacrificar tecido sadio ou alterar sua estética e função. Apresentamos neste artigo dois casos atendidos no Instituto do Câncer do Hospital de Base de São José do Rio Preto de reconstrução de defeitos condrocutâneos de orelha após ressecção de carcinoma basocelular em região central da orelha, com a confecção de retalho retroauricular ilhado transposto através de uma janela cartilaginosa e com o pedículo desepidermizado. Área doadora com fechamento primário. Tal procedimento constitui técnica segura, pois a região retroauricular é ricamente vascularizada, é de fácil execução, em único estágio e com resultado estético e funcional satisfatório.

Partial ear defects can be treated in several ways, including primary closure, healing by secondary intention, or flaps. Several surgical options have been described for reconstruction in order to maintain the natural contour of the ear, without sacrificing healthy tissues or changing the aesthetics and function. In this article, we present two cases of reconstruction of chondrocutaneous defects of the ear after resection of basal cell carcinoma in the central region of the ear, with the production of a retroauricular island flap transposed through a cartilaginous window with the de-epidermized pedicle. The donor area healed following a primary closure. This procedure can be performed in a single stage, yields satisfactory aesthetic and functional results, and is safe because the retroauricular region is richly vascularized.

Tissue engineering the human auricle by auricular chondrocyte-mesenchymal stem cell co-implantation.

Children suffering from microtia have few options for auricular reconstruction. Tissue engineering approaches attempt to replicate the complex anatomy and structure of the ear with autologous cartilage but have been limited by access to clinically accessible cell sources. Here we present a full-scale, patient-based human ear generated by implantation of human auricular chondrocytes and human mesenchymal stem cells in a 1:1 ratio. Additional disc construct surrogates were generated with 1:0, 1:1, and 0:1 combinations of auricular chondrocytes and mesenchymal stem cells. After 3 months in vivo, monocellular auricular chondrocyte discs and 1:1 disc and ear constructs displayed bundled collagen fibers in a perichondrial layer, rich proteoglycan deposition, and elastin fiber network formation similar to native human auricular cartilage, with the protein composition and mechanical stiffness of native tissue. Full ear constructs with a 1:1 cell combination maintained gross ear structure and developed a cartilaginous appearance following implantation. These studies demonstrate the successful engineering of a patient-specific human auricle using exclusively human cell sources without extensive in vitro tissue culture prior to implantation, a critical step towards the clinical application of tissue engineering for auricular reconstruction.

Ectopic bone formation during tissue-engineered cartilage repair using autologous chondrocytes and novel plasma-derived albumin scaffolds.

PURPOSE: The aims of this study were twofold: first, to evaluate the production of cartilaginous tissue in vitro and in vivo using a novel plasma-derived scaffold, and second, to test the repair of experimental defects made on ears of New Zealand rabbits (NZr) using this approach. MATERIALS AND METHODS: Scaffolds were seeded with chondrocytes and cultured in vitro for 3 months to check in vitro cartilage production. To evaluate in vivo cartilage production, a chondrocyte-seeded scaffold was transplanted subcutaneously to a nude mouse. To check in vivo repair, experimental defects made in the ears of five New Zealand rabbits (NZr) were filled with chondrocyte-seeded scaffolds. RESULTS: In vitro culture produced mature chondrocytes with no extracellular matrix (ECM). Histological examination of redifferentiated in vitro cultures showed differentiated chondrocytes adhered to scaffold pores. Subcutaneous transplantation of these constructs to a nude mouse produced cartilage, confirmed by histological study. Experimental cartilage repair in five NZr showed cartilaginous tissue repairing the defects, mixed with calcified areas of bone formation. CONCLUSION: It is possible to produce cartilaginous tissue in vivo and to repair experimental auricular defects by means of chondrocyte cultures and the novel plasma-derived scaffold. Further studies are needed to determine the significance of bone formation in the samples.

Chondrocyte differentiation for auricular cartilage reconstruction using a chitosan based hydrogel.

Tissue engineering with the use of biodegradable and biocompatible scaffolds is an interesting option for ear repair. Chitosan-Polyvinyl alcohol-Epichlorohydrine hydrogel (CS-PVA-ECH) is biocompatible and displays appropriate mechanical properties to be used as a scaffold. The present work, studies the potential of CS-PVA-ECH scaffolds seeded with chondrocytes to develop elastic cartilage engineered-neotissues. Chondrocytes isolated from rabbit and swine elastic cartilage were independently cultured onto CS-PVA-ECH scaffolds for 20 days to form the appropriate constructs. Then, in vitro cell viability and morphology were evaluated by calcein AM and EthD-1 assays and Scanning Electron Microscopy (SEM) respectively, and the constructs were implanted in nu/nu mice for four months, in order to evaluate the neotissue formation. Histological analysis of the formed neotissues was performed by Safranin O, Toluidine blue (GAG's), Verhoeff-Van Gieson (elastic fibers), Masson's trichrome (collagen) and Von Kossa (Calcium salts) stains and SEM. Results indicate appropriate cell viability, seeded with rabbit or swine chondrocyte constructs; nevertheless, upon implantation the constructs developed neotissues with different characteristics depending on the animal species from which the seeded chondrocytes came from. Neotissues developed from swine chondrocytes were similar to auricular cartilage, while neotissues from rabbit chondrocytes were similar to hyaline cartilage and eventually they differentiate to bone. This result suggests that neotissue characteristics may be influenced by the animal species source of the chondrocytes isolated.

Role of insulin-transferrin-selenium in auricular chondrocyte proliferation and engineered cartilage formation in vitro.

The goal of this study is to determine the effects of Insulin-Transferrin-Selenium (ITS) on proliferation of auricular chondrocytes and formation of engineered cartilage in vitro. Pig auricular monolayer chondrocytes and chondrocyte pellets were cultured in media containing 1% ITS at different concentrations of fetal bovine serum (FBS, 10%, 6%, 2%, 0%), or 10% FBS alone as a control for four weeks. Parameters including cell proliferation in monolayer, wet weight, collagen type I/II/X (Col I, II, X) and glycosaminoglycan (GAG) expression, GAG content of pellets and gene expression associated with cartilage formation/dedifferentiation (lost cartilage phenotype)/hypertrophy within the chondrocyte pellets were assessed. The results showed that chondrocytes proliferation rates increased when FBS concentrations increased (2%, 6%, 10% FBS) in ITS supplemented groups. In addition, 1% ITS plus 10% FBS significantly promoted cell proliferation than 10% FBS alone. No chondrocytes grew in ITS alone medium. 1% ITS plus 10% FBS enhanced cartilage formation in terms of size, wet weight, cartilage specific matrices, and homogeneity, compared to 10% FBS alone group. Furthermore, ITS prevented engineered cartilage from dedifferentiation (i.e., higher index of Col II/Col I mRNA expression and expression of aggrecan) and hypertrophy (i.e., lower mRNA expression of Col X and MMP13). In conclusion, our results indicated that ITS efficiently enhanced auricular chondrocytes proliferation, retained chondrogenic phenotypes, and promoted engineered cartilage formation when combined with FBS, which is potentially used as key supplementation in auricular chondrocytes and engineered cartilage culture.

High-fidelity tissue engineering of patient-specific auricles for reconstruction of pediatric microtia and other auricular deformities.

INTRODUCTION: Autologous techniques for the reconstruction of pediatric microtia often result in suboptimal aesthetic outcomes and morbidity at the costal cartilage donor site. We therefore sought to combine digital photogrammetry with CAD/CAM techniques to develop collagen type I hydrogel scaffolds and their respective molds that would precisely mimic the normal anatomy of the patient-specific external ear as well as recapitulate the complex biomechanical properties of native auricular elastic cartilage while avoiding the morbidity of traditional autologous reconstructions. METHODS: Three-dimensional structures of normal pediatric ears were digitized and converted to virtual solids for mold design. Image-based synthetic reconstructions of these ears were fabricated from collagen type I hydrogels. Half were seeded with bovine auricular chondrocytes. Cellular and acellular constructs were implanted subcutaneously in the dorsa of nude rats and harvested after 1 and 3 months. RESULTS: Gross inspection revealed that acellular implants had significantly decreased in size by 1 month. Cellular constructs retained their contour/projection from the animals' dorsa, even after 3 months. Post-harvest weight of cellular constructs was significantly greater than that of acellular constructs after 1 and 3 months. Safranin O-staining revealed that cellular constructs demonstrated evidence of a self-assembled perichondrial layer and copious neocartilage deposition. Verhoeff staining of 1 month cellular constructs revealed de novo elastic cartilage deposition, which was even more extensive and robust after 3 months. The equilibrium modulus and hydraulic permeability of cellular constructs were not significantly different from native bovine auricular cartilage after 3 months. CONCLUSIONS: We have developed high-fidelity, biocompatible, patient-specific tissue-engineered constructs for auricular reconstruction which largely mimic the native auricle both biomechanically and histologically, even after an extended period of implantation. This strategy holds immense potential for durable patient-specific tissue-engineered anatomically proper auricular reconstructions in the future.

Fetal development and variations in the cartilages surrounding the human external acoustic meatus.

In contrast to the osseus part that develops from the tympanic ring of the squamous part of the temporal bone after birth, there is little information on fetal development of the cartilages surrounding the human external acoustic meatus. Using routine histology and immunohistochemistry, we examine sections of 22 fetuses (CRL 100-270mm) to study the development of these cartilages. Early external ear cartilages are composed of three groups: (1) a ring-like cartilage at the putative tragus on the anterior side of the meatus, (2) two or three bar-like cartilages along the inferior wall of the meatus, and (3) a plate-like cartilage in a skin fold for the putative helix on the posterior side. In contrast to the first and second pharyngeal arch cartilages, all the external ear cartilages express glial fibrillary acidic protein. Notably, the bar-like cartilages along the meatus are connected with a fascia-like structure to the second pharyngeal arch cartilage. Later, with considerable individual variation, new cartilage bars extend from the inferior cartilages to the superior side of the meatus. Thus, via an intermediate stage showing a chain of triangular elastic cartilages, a chain of bar-like cartilages on the inferior side appears to change into a complex of H-shaped cartilages. Numerous ceruminous glands are seen in the thick subcutaneous tissue overlying the cartilaginous part of the meatus. However, they do not insert into the cartilage. The external ear cartilages develop much earlier than, and independently of, the osseus part.

Differentiation of adipose-derived stem cells into ear auricle cartilage in rabbits.

BACKGROUND: Adipose-derived stem cells have been reported as a novel candidate for the repair of cartilage injuries in vivo. METHODS: In order to assess their differentiation ability, adipose-derived stem cells isolated from rabbit fat tissue were injected into the midportion of a surgically created rabbit ear auricle cartilage defect. After several months, the auricles were resected, histopathologically assessed and compared with a control group. RESULTS: Histopathological examination of auricles removed three, four and five months after injection showed islands of new cartilage formation at the site of the surgically induced defect. Six months after injection, we observed well-formed, mature cartilaginous plates that completely filled the defect in the native cartilage. In the control group, there was no significant growth of new cartilage. CONCLUSION: The results of this study suggest the great potential of adipose-derived stem cells to repair damaged cartilage tissue in vivo.

Tissue engineering a model for the human ear: assessment of size, shape, morphology, and gene expression following seeding of different chondrocytes.

This study examines the tissue engineering of a human ear model through use of bovine chondrocytes isolated from four different cartilaginous sites (nasoseptal, articular, costal, and auricular) and seeded onto biodegradable poly(l-lactic acid) and poly(L-lactide-epsilon-caprolactone) (50 : 50) polymer ear-shaped scaffolds. After implantation in athymic mice for up to 40 weeks, cell/scaffold constructs were harvested and analyzed in terms of size, shape, histology, and gene expression. Gross morphology revealed that all the tissue-engineered cartilages retained the initial human auricular shape through 40 weeks of implantation. Scaffolds alone lost significant size and shape over the same period. Quantitative reverse transcription-polymerase chain reaction demonstrated that the engineered chondrocyte/scaffolds yielded unique expression patterns for type II collagen, aggrecan, and bone sialoprotein mRNA. Histological analysis showed type II collagen and proteoglycan to be the predominant extracellular matrix components of the various constructs sampled at different implantation times. Elastin was also present but it was found only in constructs seeded with auricular chondrocytes. By 40 weeks of implantation, tissue-engineered cartilage of costal origin became calcified, marked by a notably high relative gene expression level of bone sialoprotein and the presence of rigid, nodular protrusions formed by mineralizing rudimentary cartilaginous growth plates. The collective data suggest that nasoseptal, articular, and auricular cartilages represent harvest sites suitable for development of tissue-engineered human ear models with retention over time of three-dimensional construct architecture, gene expression, and extracellular matrix composition comparable to normal, nonmineralizing cartilages. Calcification of constructs of costal chondrocyte origin clearly shows that chondrocytes from different tissue sources are not identical and retain distinct characteristics and that these specific cells are inappropriate for use in engineering a flexible ear model.

Comparison of different chondrocytes for use in tissue engineering of cartilage model structures.

This study compares bovine chondrocytes harvested from four different animal locations--nasoseptal, articular, costal, and auricular--for tissue-engineered cartilage modeling. While the work serves as a preliminary investigation for fabricating a human ear model, the results are important to tissue- engineered cartilage in general. Chondrocytes were cultured and examined to determine relative cell proliferation rates, type II collagen and aggrecan gene expression, and extracellular matrix production. Respective chondrocytes were then seeded onto biodegradable poly(L-lactide-epsilon-caprolactone) disc-shaped scaffolds. Cell-copolymer constructs were cultured and subsequently implanted in the subcutaneous space of athymic mice for up to 20 weeks. Neocartilage development in harvested constructs was assessed by molecular and histological means. Cell culture followed over periods of up to 4 weeks showed chondrocyte proliferation from the tissue sources varied, as did levels of type II collagen and aggrecan gene expression. For both genes, highest expression was found for costal chondrocytes, followed by nasoseptal, articular, and auricular cells. Retrieval of 20-week discs from mice revealed changes in construct dimensions with different chondrocytes. Greatest disc diameter was found for scaffolds seeded with auricular chondrocytes, followed by those with costal, nasoseptal, and articular cells. Greatest disc thickness was measured for scaffolds containing costal chondrocytes, followed by those with nasoseptal, auricular, and articular cells. Retrieved copolymer alone was smallest in diameter and thickness. Only auricular scaffolds developed elastic fibers after 20 weeks of implantation. Type II collagen and aggrecan were detected with differing expression levels on quantitative RT-PCR of discs implanted for 20 weeks. These data demonstrate that bovine chondrocytes obtained from different cartilaginous sites in an animal may elicit distinct responses during their respective development of a tissue-engineered neocartilage. Thus, each chondrocyte type establishes or maintains its particular developmental characteristics, and this observation is critical in the design and elaboration of any tissue-engineered cartilage model.

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.

Cell yield, proliferation, and postexpansion differentiation capacity of human ear, nasal, and rib chondrocytes.

Human ear, nasal, and rib chondrocytes were compared with respect to their suitability to generate autologous cartilage grafts for nonarticular reconstructive surgery. Cells were expanded for two passages in medium containing 10% fetal bovine serum without (control) or with transforming growth factor beta(1) (TGF-beta(1)), fibroblast growth factor 2 (FGF-2), and platelet-derived growth factor bb (PDGF-bb) (TFP). Expanded cells were cultured as three-dimensional pellets in chondrogenic serum-free medium containing insulin, dexamethasone, and TGF-beta(1). Chondrocytes from all three sources were successfully isolated, increased their proliferation rate in response to TFP, and dedifferentiated during passaging. Redifferentiation by ear and nasal, but not rib, chondrocytes was enhanced after TFP expansion, as assessed by the significant increase in glycosaminoglycan (GAG)/DNA content and collagen type II mRNA expression in the resulting pellets. TFP-expanded ear and nasal chondrocytes generated pellets of better quality than rib chondrocytes, as assessed by the significantly higher GAG/DNA content and collagen type II mRNA expression, and by the relative stain intensities for GAG and collagen types I and II. In conclusion, postexpansion cell yields suggest that all three sources investigated could be used to generate autologous grafts of clinically relevant size. However, ear and nasal chondrocytes, if expanded with TFP, display superior postexpansion chondrogenic potential and may be a preferred cell source for cartilage tissue engineering.

Tissue engineering auricular reconstruction: in vitro and in vivo studies.

Although investigators have demonstrated that neocartilage can be constituted in a predetermined shape and in complex three-dimensional structures, such as a human ear, by using cell transplantation on polymer constructs, many unsolved problems still remain. The crucial issues for auricular tissue engineering consisted of optimal cell culture environment, choice of polymers, behavior of chondrocytes, study of cell-polymer constructs in an acceptable animal model, and long-term structural integrity. Here we describe our tissue engineering approaches for auricular reconstruction including auricular scaffold fabrication, in vitro chondrogenesis, in vivo immunocompromized xenograft and immunocompetent autologous animal models, and long-term follow-up. Though many current obstacles regarding auricular tissue engineering still exist, we demonstrate techniques of auricular scaffold fabrication with promising in vitro and in vivo neocartilage formation, optimal selection and application of animal models, and, to the best of our knowledge, the first report of different biodegradable biomaterial trials and the longest in vivo results (10 months) for auricular tissue engineering.

Otoplastia: revisão de uma casuística / Ear correction surgery: revision of cases

Concorre à cirurgia plástica, a remoção cirúrgica da orelha proeminente, restituindo a harmonia da forma e reintegrando o indivíduo ao convívio social. Referenciamos a técnica de Stenstrõn, defendida em 1963, que modificou o conceito do comportamento da cartilagem conchal quando escarificada, conceito este fundamentado em Gibson. Relatamos uma seqüëncia de quarenta e sete (47) casos realizados no Hospital Cristo Redentor, com técnica mista (Stenstrõn associada a rotação conchal), com obtenção de excelentes resultados.

Plastic surgery is credited with the correction of the protruding ear, making beneficial changes on patient’s self – image. We make reference to the Stenstrõn technique that was described in 1963 and brought a new concept on conchal cartilage’s physical and phisiological changes. We also consider the use of this technique associated to conchal rotation procedures. Fourty seven (47) cases have been treated by this method at Hospital Cristo redentor, wich is described herein.

The cartilage-sparing versus the cartilage-cutting technique: a retrospective quality control comparison of the Francesconi and Converse otoplasties.

From a total of 281 patients with protruding ears who underwent a bilateral otoplasty between 1990 and 2001, a group of 28 (10%) was selected for a retrospective quality control study. The goal was to compare two methods of otoplasty, the Francesconi, a cartilage-sparing technique, and the Converse, a cartilage-cutting technique, in terms of objectively measurable and subjectively discernable differences in results. Objective parameters included measurement of the three cephaloauricular distances and the conchoscapal angle. An independent plastic surgeon performed the evaluation by means of a systematic evaluation system for rating cosmetic surgical procedures and a 5-point visual analog scale for rating satisfaction. The patients' subjective rate of satisfaction also was investigated using the 5-point scale. The mean medial and inferior cephaloauricular distances were significantly smaller in the Francesconi group. The concoscaphal angle was 90 degrees, or less in all the patients of the Francesconi group, but more than 90 degrees in eight patients (57%) of the Converse group (p = 0.041). Accordingly, the independent surgeon found adequate correction of protrusion in 86% of the Francesconi group and 50% of the Converse group (p = 0.050). His satisfaction rate was significantly in favor of the Francesconi technique (p = 0.006). Not unexpectedly, the patients' satisfaction rate was comparably high in both groups, and there was no statistical difference between them. In conclusion, the quality control led to a clear preference of the Francesconi over the Converse otoplasty. In addition, the assessment of the postoperative results with the systematic evaluation system offered an excellent information base by which to judge the results of otoplasty. Consequent use of this evaluation system will lead to progress in the surgical procedure.

The fate of fresh, layered, nonsutured and sutured, autogenous cartilage in the rabbit model.

OBJECTIVE: To compare the thickness, area, and volume of sutured and nonsutured multilayered cartilage grafts in a rabbit population. DESIGN: Autogenous rabbit cartilage grafts were harvested, layered, and placed in the contralateral auricle. Half the grafts were sutured; the other half were nonsutured. Graft thickness, area, and volume were measured before implantation, after 90 days in vivo, and after explantation. RESULTS: The area and volume of the cartilage grafts increased during the 90-day period. Histologically, this was caused by increased fibrous tissue around the cartilage grafts. Minimal cartilage resorption was observed. No differences were noted between sutured and nonsutured grafts. CONCLUSIONS: Autogenous, fresh, uncrushed, layered nonsutured or sutured cartilage grafts are well tolerated. Statistically significant increases in the area and volume of autogenous, fresh, uncrushed, layered cartilage grafts occurred primarily because of fibrous tissue formation at the margins of the layered grafts. Suturing had no effect on the postoperative volume retention of these layered grafts. This information will be helpful to the facial plastic surgeon when using fresh-layered autogenous cartilage grafts during cosmetic or reconstructive procedures. Arch Facial Plast Surg. 2000;2:256-259

Long-term growth of a vascularized auricular perichondrocutaneous flap in laryngotracheal reconstruction.

This study addresses the potential for ongoing cartilage proliferation after repair of laryngotracheal stenosis with vascularized perichondrium. We randomly assigned 32 New Zealand white rabbits to 1 of 3 groups: group 1 (early cartilage growth, n = 10), group 2 (long-term cartilage growth after pedicle ligation, n = 11), and group 3 (long-term cartilage growth without pedicle ligation, n = 11). Bilateral auricular perichondrocutaneous flaps were elevated and transposed into full-thickness anterior tracheal wall or anterior cricothyroid membrane defects. Six weeks after elevation of the flap, animals were randomly assigned to undergo ligation of either the right or left vascular pedicle (group 2), with the contralateral auricular flap used as a matched control (group 3). Neochondrogenesis was present at 6 weeks in group 1 (0.74 +/- 0.14 mm, n = 12 ears). Cartilage thickness did not differ between groups 2 and 3 one year after ligation of the vascular pedicle: group 2 (0.48 +/- 0.24 mm, n = 18) versus group 3 (0.42 +/- 0.12 mm); P > 0.05. We conclude that in the rabbit model, chondrogenesis did not appear to be ongoing and did not result in late stenosis of the reconstructed airway. Furthermore, delayed ligation of the vascular pedicle neither inhibited nor stimulated cartilage proliferation.

The role of trabecular demineralized bone in combination with perichondrium in the generation of cartilage grafts.

The use of a composite graft of bovine trabecular demineralized bone matrix (DBM) and perichondrium has been found a reliable method for in vivo generation of cartilage. In the present study, the mechanism whereby this commercially available matrix increases cartilage formation was investigated. First, the time course of cartilage formation in vivo, in the combined implant of perichondrium and DBM in the rabbit ear was studied, with special focus on tissue reactions to DBM. DBM was colonized by macrophages from day 3 post-operatively, reaching a maximum after 2 weeks. Only a minimal number of neutrophils was found. After 3 weeks the DBM appeared to be resorbed. In the first week the DBM was invaded with chondroblasts, and chondrogenesis occurred between the first and second week of implantation. After 3 weeks, the initially formed islets of cartilage had fused. Next, the chondrogenic capacity of DBM itself was investigated by implantation of DBM without perichondrium. This never resulted in cartilage formation. Immunohistochemistry showed only a faint staining of the DBM for growth factors. This indicates a minimal chondrogenic effect of DBM alone and the requirement of perichondrium as cell provider. In order to define the conditions which cause chondrogenesis in composites of perichondrium and DBM, a series of in vitro culture experiments was performed in which the in vivo situation was mimicked step by step. The basic condition was perichondrium cultured in medium with 10% FCS. In this condition, cartilage formation was variable. Because in the in vivo situation both DBM and macrophages can release growth factors, the effect of IGF1, TGFbeta2 or OP1 added to the culture medium was tested. Neither the incidence nor the amount of cartilage formation was stimulated by addition of growth factors. Perichondrium wrapped around DBM in vitro gave cartilage formation in the perichondrium but the incidence and amount were not significantly stimulated compared to cultures of perichondrium without DBM. However, cartilage-like cells were found in the DBM suggesting an effect of DBM on perichondrium-derived cells. Finally, macrophages and/or blood were added to the composite DBM-perichondrium to mimic the in vivo situation as close as possible. However, no effect of this treatment was found. In conclusion, this study indicates that DBM itself has few chondrogenic qualities but functions merely as a spacer for cell ingrowth. The fast resorption of DBM by macrophages in vivo seems of importance for the cartilage forming process, but in vitro the presence of macrophages (in combination with blood) could not enhance chondrogenesis.

Role of FGF3 in otic capsule chondrogenesis in vitro: an antisense oligonucleotide approach.

Initiation of otic capsule chondrogenesis depends on interactions between the otocyst and surrounding periotic mesenchyme. We previously reported localization of endogenous basic fibroblast growth factor (FGF2) to the epithelium of the mouse otocyst, and initiation of chondrogenesis in cultured periotic mesenchyme by this epithelial-derived signaling molecule. We now report that FGF3, related to FGF2, can also initiate otic capsule chondrogenesis. We show localization of endogenous FGF3 to the otocyst, and suppression of chondrogenesis by antisense oligonucleotides complementary to different regions of the murine FGF3 gene. Our results support a role for FGF3 in otic capsule formation in situ.

Transplantation of chondrocytes utilizing a polymer-cell construct to produce tissue-engineered cartilage in the shape of a human ear.

This study evaluates the feasibility of growing tissue-engineered cartilage in the shape of a human ear using chondrocytes seeded onto a synthetic biodegradable polymer fashioned in the shape of a 3-year-old child's auricle. A polymer template was formed in the shape of a human auricle using a nonwoven mesh of polyglycolic acid molded after being immersed in a 1% solution of polylactic acid. Each polyglycolic acid-polylactic acid template was seeded with chondrocytes isolated from bovine articular cartilage and then implanted into subcutaneous pockets on the dorsa of 10 athymic mice. The three-dimensional structure was well maintained after removal of an external stent that had been applied for 4 weeks. Specimens harvested 12 weeks after implantation and subjected to gross morphologic and histologic analysis demonstrated new cartilage formation. The overall geometry of the experimental specimens closely resembled the complex structure of the child's auricle. These findings demonstrate that polyglycolic acid-polylactic acid constructs can be fabricated in a very intricate configuration and seeded with chondrocytes to generate new cartilage that would be useful in plastic and reconstructive surgery.