Repopulation of an auricular cartilage scaffold, AuriScaff, perforated with an enzyme combination.

Biomaterials currently in use for articular cartilage regeneration do not mimic the composition or architecture of hyaline cartilage, leading to the formation of repair tissue with inferior characteristics. In this study we demonstrate the use of "AuriScaff", an enzymatically perforated bovine auricular cartilage scaffold, as a novel biomaterial for repopulation with regenerative cells and for the formation of high-quality hyaline cartilage. AuriScaff features a traversing channel network, generated by selective depletion of elastic fibers, enabling uniform repopulation with therapeutic cells. The complex collagen type II matrix is left intact, as observed by immunohistochemistry, SEM and TEM. The compressive modulus is diminished, but three times higher than in the clinically used collagen type I/III scaffold that served as control. Seeding tests with human articular chondrocytes (hAC) alone and in co-culture with human adipose-derived stromal/stem cells (ASC) confirmed that the network enabled cell migration throughout the scaffold. It also guides collagen alignment along the channels and, due to the generally traverse channel alignment, newly deposited cartilage matrix corresponds with the orientation of collagen within articular cartilage. In an osteochondral plug model, AuriScaff filled the complete defect with compact collagen type II matrix and enabled chondrogenic differentiation inside the channels. Using adult articular chondrocytes from bovine origin (bAC), filling of even deep defects with high-quality hyaline-like cartilage was achieved after 6 weeks in vivo. With its composition and spatial organization, AuriScaff provides an optimal chondrogenic environment for therapeutic cells to treat cartilage defects and is expected to improve long-term outcome by channel-guided repopulation followed by matrix deposition and alignment. STATEMENT OF SIGNIFICANCE: After two decades of tissue engineering for cartilage regeneration, there is still no optimal strategy available to overcome problems such as inconsistent clinical outcome, early and late graft failures. Especially large defects are dependent on biomaterials and their scaffolding, guiding and protective function. Considering the currently used biomaterials, structure and mechanical properties appear to be insufficient to fulfill this task. The novel scaffold developed within this study is the first approach enabling the use of dense cartilage matrix, repopulate it via channels and provide the cells with a compact collagen type II environment. Due to its density, it also provides better mechanical properties than materials currently used in clinics. We therefore think, that the auricular cartilage scaffold (AuriScaff) has a high potential to improve future cartilage regeneration approaches.

The Ex Vivo Time of Fresh Autologous Cartilage Before Transplantation and Cartilage Absorption Degree.

OBJECTIVE: This study aims to determine the relationship between the time of autogenous cartilage in vitro and the degree of absorption in animal experiments. METHODS: New Zealand white rabbits were randomly divided into 3 groups according to the time of cartilage in vitro: 1-hour group, 2-hour group, and 3-hour group. A volume of ear cartilage was taken and transplanted into the back, according to the group. After 1 month, the volume was taken out and remeasured. Then, these were compared by scanning electron microscopy and hematoxylin and eosin staining. RESULTS: The cartilage bulk absorption level of different groups is different (P < 0.05). There was statistical significance when the 3-hour group was compared with the other 2 groups (P < 0.05). This shows that cartilage volume absorption level becomes higher after 3 hours. Scanning electron microscopy revealed that before and after transplantation, the arrangement of collagen fibers and the gap between these fibers changed. Hematoxylin and eosin staining revealed that there were some morphological changes in chondrocytes, and the degree of chondrocyte apoptosis increased with time, which was accompanied by granulation tissue formation. In addition, the cartilage tissue survived after transplantation. CONCLUSION: The change in cartilage volume was more obvious after 3 hours of autogenous fresh cartilage transplantation, when compared with that of the first 2 hours. The longer the time of light microscopy was, the longer the apoptosis of cartilage cells, the more serious the destruction of collagen fibers and the cartilage matrix, and the greater the absorption of cartilage and the new chondrocytes.

Long-Term Comparison of Rib and Ear Cartilage Grafts in Autologous and Allogenic Fascia Lata: An Experimental Study in a White Rabbit Model.

BACKGROUND: Diced cartilage in fascia has become the graft material of choice for dorsal grafts in rhinoplasty. Allogenic fascia lata has not yet been investigated as an isolated fascial graft or as a combined graft with ear and rib cartilage, especially in comparison with autologous fascia and over a long implantation period. METHODS: Ten different grafts were built from either autologous or allogenic fascia lata alone or as diced cartilage in fascia grafts with diced costal or ear cartilage and implanted into the dorsal skin of 15 rabbits. After 3 or 9 months, the grafts were explanted and analyzed histologically. Chondrocytes and cartilage matrix characteristics, including calcification, ossification, formation of bone marrow, fibrosis ingrowth and fibrotic transformation, the presence of immune reactions, vascular ingrowth, regenerative capacity, and capsule formation, were examined in a semiquantitative manner. RESULTS: All grafts were vital and without inflammatory response. The cartilage showed regular nuclei, a normal matrix, and regenerative capacity. A higher grade of calcification and ossification was observed in the fascia/cartilage grafts than in isolated cartilage grafts, and was more pronounced for costal cartilage. Both types of fascia were shown to be equally stable and without degradation. There were no significant differences in the diced cartilage in fascia grafts built with autologous compared to allogenic fascia. CONCLUSIONS: This study shows the equivalency of diced cartilage in fascia grafts and isolated fascial grafts using allogenic fascia lata compared to autologous fascia. The type of cartilage used accounts for different long-term characteristics of diced cartilage in fascia grafts.

Development of scaffold-free elastic cartilaginous constructs with structural similarities to auricular cartilage.

External ear reconstruction with autologous cartilage still remains one of the most difficult problems in the fields of plastic and reconstructive surgery. As the absence of tissue vascularization limits the ability to stimulate new tissue growth, relatively few surgical approaches are currently available (alloplastic implants or sculpted autologous cartilage grafts) to repair or reconstruct the auricle (or pinna) as a result of traumatic loss or congenital absence (e.g., microtia). Alternatively, tissue engineering can offer the potential to grow autogenous cartilage suitable for implantation. While tissue-engineered auricle cartilage constructs can be created, a substantial number of cells are required to generate sufficient quantities of tissue for reconstruction. Similarly, as routine cell expansion can elicit negative effects on chondrocyte function, we have developed an approach to generate large-sized engineered auricle constructs (≥3 cm(2)) directly from a small population of donor cells (20,000-40,000 cells/construct). Using rabbit donor cells, the developed bioreactor-cultivated constructs adopted structural-like characteristics similar to native auricular cartilage, including the development of distinct cartilaginous and perichondrium-like regions. Both alterations in media composition and seeding density had profound effects on the formation of engineered elastic tissue constructs in terms of cellularity, extracellular matrix accumulation, and tissue structure. Higher seeding densities and media containing sodium bicarbonate produced tissue constructs that were closer to the native tissue in terms of structure and composition. Future studies will be aimed at improving the accumulation of specific tissue constituents and determining the clinical effectiveness of this approach using a reconstructive animal model.

In vitro enzymatic treatment and carbon dioxide laser beam irradiation of morphologic cartilage specimens.

OBJECTIVES: To determine the role of the main cartilage components in the internal system of interlocked stresses and to clarify the effect of laser beam irradiation on cartilage. DESIGN: Control and experimental series. SUBJECTS: Rabbit ear cartilage. INTERVENTION: Rabbit ear cartilage strips incubated in collagenase and hyaluronidase enzyme solutions for specific periods were examined, and the observed changes in shape, strength, and elasticity were recorded, as well as the effect of carbon dioxide laser irradiation. Laser-pretreated cartilage strips were also incubated in the enzyme solutions to determine whether the laser-provoked changes were susceptible to enzymatic action. All cartilage pieces were examined by light and electron microscopy. RESULTS: Collagenase-treated cartilage strips gradually lost their interlocked stresses, while hyaluronidase-treated strips mostly maintained their shape and their physical characteristics. Hyaluronidase-incubated cartilage strips altered their shape when they were laser treated. Collagenase-treated cartilages did not modify their shape when they were laser treated. Laser-pretreated cartilage pieces lost their new form in collagenase solutions but kept their laser-evoked shape when put in hyaluronidase solutions. CONCLUSION: The macroscopic observations combined with light and electron microscopy findings argue for the distinct role of the collagen network in morphologic cartilage shape and tensile strength preservation and provide a probable mechanism of cartilage transformation owing to carbon dioxide laser irradiation.

Estudio de la apoptosis en la coxartrosis: influencia de lascargas biomecánicas locales / Study of apoptosis in hip-osteoarthritis: influence of local biomechanical loading

Objetivo: Cuantificar el número de condrocitos apoptóticos en el cartílago articular de la cabeza femoral artrósica en las zonas de carga y no carga. Material y métodos: Se obtuvieron muestras de cartílago a partir de cabezas femorales de pacientes sometidos a artroplastia de cadera por coxartrosis primaria (n = 10). Se estudió la apoptosis en los polos superior e inferior de dicha cabeza femoral mediante la técnica de TUNEL y microscopia electrónica (ME). Los resultados se expresaron como media aritmética de los porcentajes ñ desviación estándar. Resultados: El estudio histológico utilizando la técnica de TUNEL demostró que el 11,9 ñ 17,6 por ciento de las células fueron positivas en los cartílagos procedentes del polo superior y el 10,6 ñ 9,1 por ciento en el polo inferior. El estudio ultraestructural de estos cartílagos reflejó que el 13,6 ñ 7,7 por ciento de las células presentaban cambios apoptóticos en el polo superior, mientras que el 16,7 ñ 12,0 por ciento presentaban apoptosis en el polo inferior. Conclusión: Los resultados de nuestro estudio no apoyan la existencia de una relación directa entre el porcentaje de condrocitos apoptóticos y las zonas de máxima y mínima carga de la articulación coxofemoral de pacientes artrósicos (AU)

In vitro ear cartilage shaping with carbon dioxide laser: an experimental study.

Deformed cartilage remodeling appears to be a challenge, as cartilage tends to keep its initial shape because of internal stresses. Cartilage can be remodeled with heat, and this report describes the use of a CO2 laser beam for in vitro cartilaginous tissue remodeling. Straight cartilage samples were removed from the ears of 21 rabbits deprived of perichondrium and remodeled with the CO2 laser at an output power of 3 W, a spot diameter of 2 mm, and an exposure time of 0.5 second. The remodeled cartilages and control samples were implanted into the rabbits' backs and retrieved 6 to 12 months later. Histologic and morphological analysis showed that the irradiated cartilages retained both their shape and their viability. This may be a useful clinical technique for in situ remodeling of a deformed cartilage in operations such as septoplasty.

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

Relapsing poly(peri)chondritis diagnosed by biopsy during inflammatory free interval: destructive polychondritis versus fibrosing perichondritis.

Relapsing poly(peri)chondritis (RP) is a connective tissue disorder characterized by recurrent inflammatory episodes of cartilaginous structures and the involvement of special sense organs. The diagnostic criteria of McAdam 1976 include at least three of the following criteria: a) bilateral auricular chondritis, b) nonerosive sero-negative inflammatory polyarthritis, c) nasal chondritis, d) ocular inflammation, e) respiratory tract chondritis, f) audiovestibular chondritis. A cartilage biopsy according to these criteria is not mandatory. Nevertheless, unclear cases still remain as there is a broad spectrum of differential diagnosis. In these individuals it is important to obtain a biopsy from the affected cartilage. Although up to 89% develop auricular inflammation, only few electron microscope studies are performed on cartilage specimens. The purpose of this study is to report on a patient with a history of recurrent swelling of both ears, where the diagnosis could only be established by ear biopsy which was studied by light and electron microscopy. Differential diagnosis is discussed and a review of the literature is given.

Three-dimensional visualization of the aorta and elastic cartilage after removal of extracellular ground substance with a modified NaOH maceration method.

A modified NaOH maceration method for removing extracellular ground substance was applied to scanning electron microscopic (SEM) studies of the mammalian aorta and elastic cartilage. Fixed tissues were freeze-fractured in liquid nitrogen and then immersed in 2N NaOH solution for 4-6 hr at room temperature. Since this method selectively digested proteoglycans, cellular and fibrous elements were observable clearly under the SEM. The elastic laminae of smooth muscle cells of the aortic tunica media were concealed by fine collagen fibrils, while chondrocytes in the elastic cartilage were encapsulated by a dense network of fine collagen fibrils and branching elastin fibers.

[Relapsing polychondritis]. / La policondrite recidivante.

Relapsing polychondritis is a rare disease of unknown origin consisting in recurrent inflammatory episodes of cartilaginous structures as well as other organs such as the eye, the inner ear and the kidney. Symptoms of autoimmune or rheumatic disorders frequently precede those typical of relapsing polychondritis and thus the contemporaneous presence of these diseases is a common observation. Consequently some diagnostic concerns connected to the great variability of the early symptoms and also to the lack of a semiotic test do exist. It is widely recognized that a serious involvement of the tracheo-bronchial cartilage the lumen diameter with a consequent heavy dyspnoea which reduces occurs in relapsing polychondritis. In these cases the study of the thorax area by means of CT was of fundamental importance. Furthermore, histological analysis of bioptic specimens from tissues only apparently not affected, such as the pinna of the ears, better defines the nature of the disease. The Authors present a case report, characterized by the presence of a saddle-nose deformity as well as a dyspnoea caused by a lumen stenosis related to tracheo-bronchial cartilage chondritis.

[Alkaptonuria-ochronosis]. / Alkaptonuria-ochronosis.

The authors describe the case of a 40-year old female patient. Since her childhood she realised of her urine the black discoloration of the underwear. For about a year, without subjective complaints, blue-black color of the skin involved the axillae and pinnae. For a year appeared the increased pain of thoracal and lumbal spine column and the limitation of motion of these parts. The examination of urine, histological and electron microscopical findings, the X-ray photograph of the spinal column confirmed the diagnosis of alkaptonuria or rather congenital ochronosis.

[Antigenicity of the auricular cartilage and its modification by vital preservation. 1. Xenogeneic transplantation of human auricular concha cartilage to the rabbit]. / Die Antigenität des Ohrknorpels und ihre Beeinflussung durch vitale Konservierung. Teil 1: Xenotransplantation humanen Ohrmuschelknorpels an Kaninchen.

By means of xenotransplantation of human auricular cartilage to rabbits tests were carried out to see, if antigenicity is reduced through storing elastic cartilage either in a nutrient solution (Ham F 12) at 4 degrees C above zero or in a tissue culture (Ham F 12 + 10% serum of a calf-foetus) at 37 degrees C above zero, and if vital preservation prevents degeneration- and resorption processes of cartilage. The results were compared to transplants of fresh, not treated cartilages, and those preserved with cialit and merthiolate. It turned out that these vital preservation methods seem to cause an extensive loss of the cartilages characteristic qualities and that they keep degeneration- and resorption processes at a minimal level. Big differences between storage in nutrient solution and tissue culture could not be observed.

[Structuro-functional organization of human elastic cartilage]. / Strukturno-funktsional'naia organizatsiia élasticheskogo khriashcha cheloveka.

The light- and electron-microscopic investigation performed in order to study the elastic cartilage ultrastructure in the human concha auriculae has demonstrated that a peculiar feature of the later is abundance of elastic fibers both in its perichondrium and in the cartilagenous tissue. In the perichondrium the fibrillar elements (collagenous and elastic fibers) are oriented in parallel with the cartilagenous plate surface. There is not any definite regularity in the arrangement of the fibers of the cartilagenous tissue. As demonstrates the light microscopy, homogeneity of the ground substance is connected with a very compact arrangement of its fibrillar elements. Spaces between the fibers and fibrillae are filled with an amorphous ruthenium-positive substance of the proteoglycane nature.

X-ray microanalysis of cultured chondrocytes.

Methodological aspects of X-ray microanalysis of cultured chondrocytes from rabbit auricular cartilage were investigated. The cells were grown on a Formvar-film covered carbon specimen holder with a hole, which allowed examination in STEM. Several different fluids for washing away the culture medium from the cells were used. The effect of these fluids on the elemental composition of the cells was studied. The composition of cultured chondrocytes was compared with that of auricular chondrocytes in situ. Washing with 0.3 M sucrose gave the most satisfactory results. Subsequently, the specimen holders could be frozen in liquid nitrogen and the cells freeze-dried. The effects of human growth hormone on rabbit chondrocytes were investigated by X-ray microanalysis and transmission electron microscopy. Although no ultrastructural effects of hormone treatment could be observed, marked changes in the elemental concentrations in nucleus and cytoplasm of the chondrocytes were detected.

Secretory features of chondrocytes related to morphogenesis of the elastic elements in the auricular cartilage.

The well-developed secreting vacuolar system is a characteristic feature of all chondrocytes (CH) during prenatal and early postnatal ontogenesis as well as of the CH near the perichondrium in the adult rat's auricular cartilage. Two kinds of secretion vacuoles exist, dense vacuole (DV) and electron lucent vacuoles (LV). The DV are heterogeneous in size, matrix appearance and origin. The factors (organ necessities and chemical maturation) responsible for the DV matrix heterogeneity are discussed. The presence of dilated rough endoplasmic cisternae with local parts of their cisternal surfaces without bound ribosomes suggest the existence of a direct secretory pathway which bypasses the Golgi complex. During prenatal ontogenesis the extracellular matrix of the auricular cartilage consists of bundles of elastic microfibrils and primary elastic fibres. The elastic microfibrils are tubular structures with a wall composed of 3-5 subunits. After birth the primary elastic fibres are joined to form intercellular septae. The presence of matrix vesicles is discussed with regard to the fine differentiation of the auricular elastic system by their lysosomal activity.

Light and electron microscopic study of ear cartilage in a case of relapsing polychondritis evolving under corticoid treatment.

Light and electron microscope studies of the ear cartilage in a patient with relapsing polychondritis (RP) under corticoid treatment are reported. Unilateral auricular deformation evolved without inflammatory epidoses and the lesions consisted mainly of marginal erosions filled with fine collagen fibrils and containing degenerating perichondrial cells in their basal parts. Degenerative cells were scattered throughout the perichondrium, but cartilage erosions only occurred when numerous cells were affected in a same area. Cartilage outside the eroded zones did not seem to be modified. Cartilage lesions thus appear to be a result of a chondrocyte renewal defect leading to loss of proteoglycans and elastic fibers, with only collagen remaining. These data suggest that inflammation is probably not the initial pathogenic process responsible for cartilage injury in RP, but that a metabolic defect in perichondrial cells might be involved.

Ultrastructure of elastic cartilage in the rat external ear.

The structure of elastic cartilage in the external ear of the rat was investigated by transmission and scanning electron microscopy. The narrow subperichondrial, boundary zone contains predominantly ovoid cells rich in cell organelles: mitochondria, Golgi complex, granular endoplasmic reticulum and small (40--100 nm) vesicles. Scarce glycogen granules and bundles of 6--7 nm cytoplasmic filaments are also present. Deeper in the boundary zone, one or more cytoplasmic lipid droplets appear and cytofilaments become more abundant. Fully differentiated chondrocytes in the central zone of the cartilage plate resemble white adipose cells. They are globular and contain a single, large cytoplasmic lipid droplet. The cytoplasm is reduced to a thin peripheral rim; it contains a flattened nucleus, few cytoplasmic organelles and abundant, densely packed, cytoplasmic filaments. The intercellular matrix is very sparse. The pericellular ring consists of collagen fibrils about 20 nm in diameter and a proteoglycan cartilage matrix in the form of a "stellate reticulum". The complex of these two structures appears in the scanning electron micrographs as a a network of randomly oriented, ca 100 nm thick fibrils. Spaces between pericellular rings of matrix also contain thick elastic fibers or plates, apparently devoid of microfibrils. In scanning electron micrographs elastic fibers could be detected only in a few areas, in which they were not obscured by other constituents of the matrix. Immature forms of elastic fibers, oxytalan (pre-elastic) and elaunin fibers, were found in the perichondrial and boundary zones.

An ultrastructural study on the ear cartilage of rabbits after the administration of papain. Appearance of cross-striated collagen segments of an atypical FLS-type.

Crude papain was administered intravenously to young rabbits and the cartilage of the collapsed ear was examined electron-microscopically. Degeneration and recovery of chondrocytes, and decrease in and recovery of the electron-density of elastic fibers, were observed during the collapse and restoration of the ear. Some samples were stained with ruthenium red. In the collapsed ear, with a marked decrease of proteoglycan in the cartilage, loss of ruthenium red-positive granules was observed in the extracellular matrix. Collagen fibrils in the cartilage appeared to be somewhat increased in number, some of their diameters became slightly greater, and a part were assembled into bundles, occasionally accompanied by periodic cross-striation. Decrease of proteoglycan in the cartilage matrix probably brought about the unmasking and the assembly of collagen fibrils. In one of the experimental animals, collagen fibrous segments of an atypical fibrous long spacing (FLS-)type with symmetrical cross-striation were found around the chondrocytes in the ear cartilage, during the period of recovery. Some kind of the endogenous sulfated carbohydrate may have acted to affect the arrangement of type II collagen or procollagen molecules newly produced by the recovering chondrocytes.