[Tissue engineering of cartilagenous tissue. Vital cryopreservation]. / Ingeniería tisular de tejido cartilaginoso. Criopreservación vital.

The characteristics of in vitro synthesized cartilage tissue using tissue engineering techniques before and after cryopreservation are described. We determined cell survival, growth of extracellular matrix (collagen and proteoglycan) using a computer analysis system, and characterized the cell phenotype with a monoclonal antibody specific for collagen type II. Chondrocytes maintained a differentiated phenotype with collagen and proteoglycan synthesis before and after cryopreservation.

Tissue engineering of autologous cartilage transplants for rhinology.

In reconstructive surgery there is increasing demand for cartilage transplants to fill defects, especially nose and/or outer ear defects. Tissue engineering is one of the most modern pathways to generate autologous cartilage transplants. Isolated chondrocytes obtained from a tiny patient's biopsy were seeded on bioresorbable preshaped cell carriers to provide a 3-dimensional cell arrangement as in vivo. The combined use of these cell carriers in form of a non-woven mesh and a constant medium perfusion was performed to generate a cartilage-like cell-polymer-construct, which was finally subcutanously implanted in nude mice for full maturation. After explantation of 6 months, expression of cartilage specific extracellular matrix molecules was obvious by using histochemical and immunohistochemical methods. These data show that tissue engineering with isolated multiplied human chondrocytes from a tiny biopsy seeded on bioresorbable polymer is a promising system to generate autologous cartilage transplants for replacements in reconstructive surgery.

[Immunohistochemical studies with middle ear mucosal remnants in cholesteatoma]. / Immunhistochemische Untersuchungen von Mittelohrschleimhautresten im Cholesteatom.

The development of a middle ear cholesteatoma is usually associated with chronic inflammation and displacement of the mucosa present by the invading squamous epithelium. To analyze the clinically different behaviors of both epithelia, we used immunohistochemical methods to study the distribution and expression of interleukin-1 (Il-1), transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF), epidermal growth factor-receptor (EGF-R), the proliferation marker MIB 1, c-myc proto-oncogene product and activation marker 4F2. Results stromal that keratinocytes in a cholesteatoma exhibited a much higher activation and proliferation rate when compared to middle ear mucosa cells. Middle ear epithelial cells showed no immunoreactivity for TGF-alpha, EGF-R, Il-1 and c-myc in contrast to the markedly positive immunoreactivity found in cholesteatoma matrix. The local release of cytokines and growth factors, such as TGF-alpha, EGF and Il-1 by inflammatory cells seems to be an important factor for the hyper-proliferative behavior of cholesteatoma epithelium. Our findings could contribute to the pathogenesis of middle ear cholesteatoma and give a possible explanation for the sustained progression of its growth leading to displacement of the middle ear mucosa.

[Transplantation of in vitro cultured cartilage materials: characterization of matrix synthesis]. / Transplantation in vitro hergestellter Knorpelmaterialien: Charakterisierung der Matrixsynthese.

BACKGROUND: Recently a three-dimensional model for the formation of cartilage in vitro was developed. The aim of this study was to investigate the amount and quality of newly synthesized matrix after graftig in vitro engineered cartilage into athymic nude mice. MATERIAL AND METHODS: Group I received transplants consisting of human chondrocytes, agarose, and E 200 (a bioabsorbable polymer fleece that offers mechanical stability. Ethicon Inc). Group II received chondrocytes and agarose only. At intervals of six, 12, and 24 weeks after subcutaneous transplantation we used azan blue staining and antibodies against collagen type I, collagen type II, and chondroitin-4sulfate to characterize the matrix synthesis. A quantitative analysis was performed using the computer image analyzing software photoshop (Adobe Inc). RESULTS: In group I, the amounts of newly synthesized cartilage specific collagen type II and chondroitin-4 sulfate increased progressively. Twenty-four weeks after transplantation, these amounts were comparable to the original human cartilage from which the chondrocytes were derived. Collagen type I was detected only in small quantities in the periphery of the transplants. Gross examination revealed sufficient mechanical stability and unremarkable changes in size and form. In contrast to this, group II transplants showed markedly smaller amounts of cartilage specific matrix components as collagen type II and chondroitin-4 sulfate and at the same time greater amounts of collagen type I. It was found both in the periphery and in central parts of the transplants. There was a remarkable loss of volume in all transplants and mechanical stability was poor. CONCLUSIONS: The absorbable cell carrier E 200 not only offers mechanical stability to in vitro engineered cartilage but also had a positive effect on the development of cartilage in our experiments. In conclusion, in vitro engineered cartilage is a promising pathway for the replacement of cartilage defects.

[Realistic imaging of cell systems using confocal laser scanning microscopy exemplified by 3-dimensional chondrocyte culture]. / Die realitätsgetreue Darstellung von Zellsystemen mit Hilfe der Konfokalen-Laser-Scanning-Mikroskopie am Beispiel dreidimensional kultivierter Chondrozyten.

BACKGROUND: We have developed a three-dimensional model for tissue engineering of cartilage. Chondrocytes were isolated and first multiplied in conventional monolayer cultures. Then the cells are seeded with or without agarose on special absorbable scaffolds that provided stability and enabled three-dimensional cell distribution of the tissue-engineered cartilage. The aim of the study was to investigate the possibility of avoiding agarose in tissue engineering because of the potential risk of causing an inflammatory process in later human implantation. METHOD: For the first time we investigated cell distribution combined with vitality directly in the cell carrier under the conditions described by using confocal laser-scanning microscopy. Working with unfixed cells, this method enables the reconstruction of three-dimensional cell cultures with suitable cell markers that closely simulates the physiologic situation, thereby exceeding each other method. RESULTS: It was evident that agarose had no positive effect on cell distribution and vitality. CONCLUSION: Further experiments concerning the effect of agarose on synthesis of cartilage-specific matrix are in progress.

[Proliferative potential of nasal septum chondrocytes for in vitro culture of cartilage transplants]. / Untersuchungen zum Vermehrungspotential von Nasenseptum-Chondrozyten für die In-vitro-Züchtung von Knorpeltransplantaten.

BACKGROUND: Recent developments in the field of tissue engineering provide novel approaches in tissue repair and reconstructive surgery using the patients own cells. Isolated chondrocytes form new cartilage when seeded in appropriate scaffolds. Usually the number of cells from a cartilage biopsy is not sufficient. The present study investigates the potential of cell amplification of human nasal chondrocytes in monolayer culture. METHODS: Nasal cartilage cells from seven healthy patients with age between 16 and 60 years were enzymatically isolated with collagenase and hyaluronidase. Subsequently, cells were seeded in 75 cm2 culture flasks. After confluency, cultures were trypsinized, counted, and again seeded at a concentration of 5 x 10(4) cells/ml. Dulbecco's MEM supplemented with 10% FCS was used as culture medium. RESULTS: After enzymatic digest, an average of 5 x 10(5) cells per patient were isolated. At least 85% of the cells were vital. Within four to eight weeks, the cells number was increased 10(3) to 10(5) fold. No correlation between the proliferative activity and the age of the patient was observed in this study. DISCUSSION: The observed increase in cell number resembles about 10 to 20 cell doublings. Although the doubling time appears to be longer during the second month, no definite limit of proliferative activity was seen during the time of study. Proliferating chondrocytes in monolayer lose their tissue-specific phenotype. For the de novo formation of cartilage transplants, redifferentiation of the expanded cells has to be stimulated. CONCLUSION: This study shows that human nasal chondrocytes can be expanded sufficiently in monolayer for the engineering of autologous cartilage transplants.

[Determination of intercellular adhesion molecule-1 (ICAM-1) in nasal secretions of patients with rhinitis]. / Determinación de la molécula de adhesión intercelular-1 (ICAM-1) en la secreción nasal en pacientes con rinitis.

Intercellular adhesion molecule-1 (ICAM-1) is an integrin involved in the regulation of cell migration and activation in various tissues. ICAM-1 expression has been shown to be associated closely with cell infiltration in allergic diseases. We analyzed the possible participation of soluble ICAM-1 (sICAM-1) in the pathogenesis of various diseases of the nasal mucosa by measuring sICAM-1 levels in nasal secretions using sandwich ELISA. Soluble ICAM-1 levels in patients with allergic rhinitis and nasal polyposis did not increase with respect to healthy controls, but patients with chronic sinusitis had higher sICAM-1 levels. Soluble ICAM-1 levels also increased in patients with allergic rhinitis after a provocation test with the specific allergen. These results show that ICAM-1 is involved in the pathogenesis of chronic sinusitis and allergic rhinitis.

[Treatment of nasal polyposis with systemic and local corticoids]. / Tratamiento de la poliposis nasal mediante aplicación sistémica y local de corticosteroides.

Surgical or medical treatment of nasal polyposis still is a challenge in otorhinolaryngology. A study was made of the effectiveness of combined local and systemic, steroid treatment. Thirty-seven patients with different grades of nasal polyposis received a combination of systemic (methylprednisolone oral 10 days) and local (inhaled budesonide, day 8 to day 28) steroids. Therapeutic effects were measured using nasal endoscopy with a four-stage system for grading nasal fossa involvement and a symptomatic scale of none, mild, moderate, and severe. The mean endoscopic score decreased from 1.14 on day 1 to 0.14 on day 28 and the symptom score from 2.8 to 1.2. The results show than nasal polyposis can be treated successfully with combined local and systemic steroid treatment.

[Acoustic rhinometry: determination of early and late-phase reactions in allergic rhinitis]. / Rinometría acústica: determinación de la fase temprana y tardía en la rinitis alérgica.

Allergic rhinitis is characterized by morphological changes in the nasal cavity that produce airway obstruction. Acoustic rhinometry (AR) was used to analyze changes in the nasal airways of 10 patients with allergic rhinitis who underwent a nasal provocation test with 1000 biological units of specific allergen. Acoustic rhinometry was carried out 10, 20, 30, 45, and 60 minutes, and 2 and 8 hours after exposure to allergen. The parameters analyzed were minimal cross-sectional area (MCA) and a new parameter, volume A (VA), obtained by integration of the distance/area graph surrounding the anterior part of the lower turbinate. The early-phase reaction after exposure to allergen was a 10-30% reduction of MCA and VA compared with baseline value. The late-phase reaction was one-third as strong. The contralateral MCA and VA were affected only minimally in the early-phase reaction, but a symmetrical response was observed in the late-phase reaction. VA changes were more evident than MCA changes. The results show that acoustic rhinometry is suitable for measuring local changes following exposure to nasal antigens, particularly if both VA and MCA are evaluated.

Enhanced epithelial proliferation due to elevated levels of interleukin-1 receptors in middle ear cholesteatomas.

Middle ear cholesteatoma epithelium is a rich source of interleukin-1-alpha (IL-1-alpha), being involved in both keratinocyte hyperproliferation and bone destruction. IL-1-alpha exerts its effects by binding to two distinct IL-1 receptors (IL-1-R). In this study, we have examined the expression of IL-1-R type II (IL-1-R-II) in cholesteatoma samples and have quantified these levels with computer-assisted image analysis. Normal aural skin was used as control. Immunostaining demonstrated the presence of IL-1-R-II in both epidermis and cholesteatoma keratinocytes. The receptors were 3 times higher than those in normal epidermis. The presence of IL-1-alpha in cholesteatoma epithelium coupled with the induced expression of IL-1-R-II indicates the existence of a highly regulated system of autocrine stimulation of cholesteatoma keratinocytes by IL-1.

Basement membrane in middle ear cholesteatoma. Immunohistochemical and ultrastructural observations.

We investigated the distribution of basement membrane zone (BMZ) components collagen type IV, collagen type VII, and fibronectin in human middle ear cholesteatoma, auditory meatal skin, and middle ear mucosa using both immunohistochemical and ultrastructural methods. Collagen type IV immunoreactivity of skin and middle ear mucosa is continuous in the BMZ, whereas cholesteatoma frequently showed absent immunoreactivity or focal discontinuities. Collagen type VII immunoreactivity is detected similarly within the BMZ of cholesteatoma and skin. Fibronectin immunoreactivity is observed within the dermoepithelial junction of skin and middle ear mucosa. In cholesteatoma, however, fibronectin immunoreactivity is markedly increased within the extrinsic BMZ and the subepithelial connective tissue. The ultrastructural arrangement of the BMZ of cholesteatoma is like that of skin; however, it exhibits distinct alterations of the lamina fibroreticularis and lamina densa. Our results outline cholesteatoma as a disease with disturbed cell matrix interactions analogous to those of wound reepithelialization.

Immunobiological peculiarities of cholesteatoma in children: quantification of epithelial proliferation by MIB1.

Cholesteatoma in children is characterized by a more extensive and rapid growth in the middle ear and mastoid cavities. The growth characteristics of the cholesteatoma in 20 children were studied using the monoclonal antibody MIB 1, which recognizes a nuclear antigen expressed by cells in the G1, S, and G2/M phases. Specimens of normal adult auditory meatal skin (n = 15) and adult cholesteatoma (n = 15) served as controls. The tissue specimens were prepared for immunohistochemical examination using the alkaline phosphatase-antialkaline phosphatase method and an automatic image analyzer. Specimens of normal skin revealed an average MIB 1 score of 9.2 +/- 3.10%. Child and adult cholesteatomas showed higher values. The average MIB 1 score was higher in child cholesteatoma (42 +/- 9.4%) than in adult cholesteatoma (28.2 +/- 6%). This difference was statistically significant (P<.01). Our results confirm a significant increase of the proliferative rate of cholesteatoma keratinocytes in children, giving an explanation for the more aggressive clinical behavior observed in these patients.

[Cellular viability of cryopreserved cartilage grafts]. / Viabilidad celular de injertos de cartílago crioconservados.

Transplantation of preserved cartilage has an important role in reconstructive surgery. Opinions vary with regard to the performance of cryopreserved cartilage. We studied the functional state of chondrocytes after cryopreservation. Cellular survival was studied using the trypan blue dye exclusion test, a functional assay for cell adhesion, and transmission electron microscopy. Most chondrocytes were irreversibly damaged by cryopreservation and the cartilage could not originate new cartilage. Therefore, cryopreserved cartilage tends to generate fibrosis and resorption and is not practical for reconstructing skeletal parts exposed to mechanical stress.

[A quantitative study of the presence of interleukin-1 and interleukin-6 in cholesteatoma of the middle ear]. / Estudio cuantitativo de la presencia de interleucina-1 e interleucina-6 en el colesteatoma de oído medio.

Cholesteatoma of the middle ear is an inflammatory disease characterized by the presence of a keratinized squamous layer that leads to bone destruction. The process may be mediated by various factors (interleukins) produced by an activated macrophages and keratinocytes. Interleukin-1 (IL-1) and interleukin-6 (IL-6) concentrations were measured in extracts of cholesteatoma and normal tissue using the enzyme immunoassay technique (ELISA) after protein concentrations were determined. IL-1 alpha and IL-6 had higher concentrations in cholesteatoma than in normal skin and played a prominent role in bone resorption.

Monoclonal antibodies against human chondrocytes.

Cell-specific antigens are mainly found in cells or membrane surfaces rather than in the surrounding matrix. However, until now it was not possible to produce antibodies specific for cellular structures of chondrocytes. In 1989, Lance (Immunol. Lett. 21:63-73; 1989) first established specific monoclonal antibodies for human articular chondrocytes tested only by immunofluorescence. Studies describing the specificity of these five antibodies (HUMC 1-5) and their relevance for immunohistological analysis of cartilage tissue were not available until now. Therefore, the aim of the following study was to investigate the distribution of HUMC 1, 2, 3, 4, and 5 in mesenchymal cells in vivo and in vitro immunohistochemically. Further investigations concentrate on the localization of chondrocyte specific antigens using immunoelectron microscopy. Immunohistological studies showed positive immunostainings with all five antibodies in human chondrocytes in vivo and in vitro. A cross-reaction with human fibroblasts and osteoblasts for the antibodies HUMC 2 and HUMC 5 was observed. Furthermore, a parallel loss of immunoreactivity for HUMC 1, HUMC 3, and HUMC 4 was observed in cultured chondrocytes indicating that the specific antigens vanish during differentiation observed in vitro. Subsequent immunoblot analysis employing collagens as antigens did not show any reactivity. Using immunoelectron microscopy, gold particle labeling was observed in intracytoplasmatic vesicles of isolated chondrocytes. Our results indicate that HUMC 1, HUMC 3, and HUMC 4 are specific for cartilage cells and might be suitable for immunohistological analysis of different cartilage tissues and pathologically altered chondrocytes.

Influence of chemical allograft preservation procedures on the human immunodeficiency virus.

Since chemically preserved allogenic transplants have an established place in reconstructive procedures, the possibility of transferring the human immunodeficiency virus (HIV) with these transplants has been intensively discussed. In this study the authors obtained brain and spleen samples from six HIV-infected cadavers and preserved them with Merthiolate, Cialit, and formaldehyde. After preservation, the tissues were examined for proviral HIV-1 DNA (gag, pol, env) using the polymerase chain reaction. Proviral sequences were clearly demonstrated after the preservation procedure. The results of this study indicate that HIV remains in tissues that have been treated with Merthiolate, formaldehyde, or Cialit. Further investigations are necessary to determine if the virus is in an inactivated or activated form. It can be concluded that, because of the possible transmission of HIV by chemically preserved homografts, serologic screening of donors should be mandatory.

Soluble interleukin 2 receptors in patients with Bell's palsy.

The etiology and pathogenesis of Bell's palsy are still an enigma. Some studies have reported about the presence of cellular and humoral immune dysfunction in this disease. Recently, determination of soluble interleukin 2 receptor has proven to be a valuable clinical tool to detect dysregulation of T lymphocyte function. The concentrations of soluble interleukin 2 receptor alpha was determined in serum samples from 11 patients with Bell's palsy by an enzyme linked immunosorbent assay (ELISA). Concomitantly 8 age- and sex-matched healthy blood donor as well as six patients with dermatitis herpetiformis served as negative and positive controls, respectively. The concentration of soluble interleukin 2 receptor in serum samples from these patients was similar to that in samples from normal subjects. In contrast to this, patients with dermatitis herpetiformis showed higher values of interleukin 2 receptor. Our results showed that Bell's palsy is not accompanied by a massive activation of T cells.

[Angiogenesis in cholesteatoma of the middle ear]. / Angiogénesis en el colesteatoma de oído medio.

Middle ear cholesteatoma is characterized by the presence of a keratinizing squamous epithelium with hyperproliferative features. Such growth can only be supported by abundant blood vessels. The presence and distribution of blood vessels in cholesteatoma was studied to determine the mechanisms responsible for its origin and maintenance. Cholesteatoma (n = 30) and retroauricular skin samples (n = 30) were studied with indirect immunoperoxidase and alkaline phosphatase anti-alkaline phosphatase methods. Antibodies were used to recognize endothelial cells (von Willebrand Factor VII), vascular basal membrane components (type VI collagen), intercellular adhesion molecules (ICAM-1, 1CAM-2), human histocompatibility antigen (HLA-II) as a marker for cellular activation, angiogenetic growth factors and their receptors (TGF-alpha and VEGF), lymphocytes (CD3), and macrophages (KiM8). The cholesteatoma stroma had numerous vessels with intact basal membrane. The vessel concentration was higher in regions with abundant macrophage infiltration. Perivascular cell infiltrates were positive for antibodies against angiogenetic factors and HLA-II. Endothelial cells had increased expression of intercellular adhesion molecules and angiogenetic growth factor receptors. These results confirm the presence of increased vascularization in cholesteatoma, which may play an important role in sustaining continuous abnormal growth.

Epidermal growth factor receptor (EGF-R) in human middle ear cholesteatoma: an analysis of protein production and gene expression.

Previous studies have shown an altered epithelial cell proliferation in middle ear cholesteatoma, reporting an aberrant expression of epidermal growth factor receptor (EGF-R) glycoprotein by immunohistochemistry. In this study, we quantified the presence of EGF-R using enzyme-linked immunosorbent assays (ELISAs) on tissue extracts, as well as the EGF-R gene expression by in situ hybridization on frozen sections. Human skin obtained from the external ear canal was used as control. The amounts of EGF-R glycoprotein in cholesteatoma were very similar to those in human skin. Human skin showed EGF-R messenger RNA (mRNA) only in the basal layer. A higher percentage of cells hybridized for the anti-sense probes EGF-R was found in cholesteatoma epithelium. Furthermore, we could find suprabasal cells with EGF-R mRNA. Our results confirm that the abnormal growth of cholesteatoma epithelium is reflected in an aberrant expression of EGF receptor.