Tissue engineering and autologous transplant formation: practical approaches with resorbable biomaterials and new cell culture techniques.

The engineering of living tissues in vivo requires new concepts in cell culture technology. In contrast to conventional cell cultures, the development of tissues depends on a three-dimensional arrangement of cells and the formation or synthesis of an appropriate extracellular matrix. Special emphasis is given to the major role of the extracellular matrix and cell differentiation in an artificial tissue. New technical approaches of in vitro tissue engineering are compared to the natural development of tissues in vivo. Current methods using resorbable biomaterials, tissue encapsulation and perfusion culture are discussed. Major consideration is given to scaffold structures of biomaterials that define a three-dimensional shape of a tissue or guide matrix formation. The different goals of tissue engineering such as in vitro models and transplant production are taken into account in the described techniques. Practical concepts comprising cell multiplication and differentiation in subsequent steps for future clinical applications are outlined.

Engineering of cartilage tissue using bioresorbable polymer carriers in perfusion culture.

Bioresorbable polymer fleeces with a high internal surface area were used as temporary matrices to establish three-dimensional cultures of isolated human articular chondrocytes. The polymer surface was coated with poly-L-lysine to support cell attachment. The resulting cell-polymer tissues were cultured in perfusion culture chambers to achieve a constant supply of nutrients by diffusion. Retention and accumulation of extracellular matrix components synthesized by the chondrocytes were improved by encapsulation of the cell-polymer integrate in agarose gel. The cell-polymer tissues formed abundant collagen fibrils in vitro with a typical cross-triation clearly visible in electron microscopy analysis. Chondrocytes and intercellular matrix stained positively with monoclonal antibodies specific for differentiated chondrocytes and type II collagen. Synthesis of proteoglycans and collagen was also evident by further analysis with alcian blue and azan staining of cell-polymer tissue sections. The presented experimental tissue culture technique offers a novel concept for the in vitro formation of vital cartilage implants for reconstructive surgery or treatment of destructive joint diseases and possibly for the in vitro engineering of human tissues in general, with applications in drug testing and replacement of animal experiments.

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.

A comparison of class II antigenicity of human tracheal allografts stored in cialit and in merthiolate.

This report deals with some of the immunological aspects of the transplantation and preservation of human tracheal grafts. The immunological behavior of the transplanted graft depends largely on the interaction of the preservatives with the tissue proteins. Using monoclonal antibodies and the immunoperoxidase staining technique we have investigated the effect of Merthiolate, Cialit, formaldehyde/Merthiolate, and formaldehyde/Cialit preservation techniques on monomorphic determinants of class II transplantation antigens in human tracheal allografts. Unpreserved grafts were found to express class II antigens. These antigens were totally destroyed after 7 days in formaldehyde and 42 days in Cialit and Merthiolate. Preservation in Cialit and Merthiolate showed a gradual disintegration of the histological structures. In contrast, buffered formaldehyde did not appear to alter the histological structure of the tracheal graft. Irrespective of the mode of preservation, the cartilaginous tissue appeared to persist virtually unaffected. No essential differences were observed between the immunological staining of tracheal grafts preserved in Cialit and Merthiolate.

Laryngeal reconstruction using allogeneic cartilages.

Preserved allogeneic cartilage has been used to reconstruct laryngeal defects. The most important problem with this approach has been graft resorption, which seems to be caused by devitalization of the grafts as a consequence of preservation. In this study, the authors compared the in vivo behavior of vital and nonvital preserved cartilage used to reconstruct the larynx of New Zealand white rabbits. The vital cartilage grafts were stored using organ culture procedures, and the nonvital grafts were stored in formaldehyde. While the formaldehyde-preserved cartilage showed inflammatory changes, the transplanted vital cartilage was well accepted and showed no evidence of immune cell infiltrations. The authors concluded that viable cartilage grafts are preferable to grafts of chemically preserved cartilage.

Quantitative analysis of interleukin-1-alpha gene expression in middle ear cholesteatoma.

Regardless of its origin, cholesteatoma is characterized by the presence of a keratinizing epithelium with an hyperproliferative behavior leading to a very important bone resorption. Previous studies have demonstrated overexpression of interleukin-1 (IL-1 protein in middle ear cholesteatoma by immunohistochemistry and enzyme-linked immunosorbent assay, suggesting a significant role for IL-1-alpha. In this study, the presence of IL-1-alpha messenger ribonucleic acid (mRNA) was quantified by in situ hybridization on frozen sections (n = 10) and by computer-assisted image analysis. Human skin obtained from the external ear canal (n = 10) was used as the control. A higher percentage of cells hybridized for the antisense probes IL-1-alpha mRNA was found in cholesteatoma epithelium. Furthermore, keratinocytes of the suprabasal cell layers were also found to contain specific hybridizations. Some cells in cholesteatoma stroma also contained IL-1-alpha mRNA transcripts. The results of this study confirm the central role of IL-1-alpha in the epithelium hyperproliferation and bone resorption observed in middle ear cholesteatoma.

Expression of a cell-cycle-associated nuclear antigen (MIB 1) in cholesteatoma and auditory meatal skin.

Middle ear cholesteatoma is often invasive with consequent bone destruction. Inflammatory stimulation of the underlying connective tissue, as well as an autocrine mechanism, may be responsible for the dysregulation and abnormal proliferative features of the keratinocytes in cholesteatoma. Comparative investigations were performed to assess the epithelial cell kinetics of cholesteatoma and normal auditory meatal skin. Monoclonal antibody MIB 1 immunostaining (which recognizes a nuclear antigen expressed by dividing cells) was applied using the alkaline phosphatase antialkaline phosphatase immunolabeling method. Specimens of normal auditory meatal skin (n = 7) revealed an average MIB 1 score (quotient of the MIB 1-positive cells and the total number of cells) of 7.6 +/- 2.2%. Cholesteatoma samples (n = 13) showed an average MIB 1 score of 17.4 +/- 8.9% and a heterogeneity of proliferating epithelial areas. Epithelial cones growing toward the underlying stroma exhibited high mitotic activity. Statistically, the results of this study confirm a highly significant increase in the proliferation rate of cholesteatoma keratinocytes, which had an MIB 1 score that was 2.3 times higher than the score for keratinocytes of normal external auditory meatal skin.

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.

Aberrant expression of epidermal growth factor receptor in aural cholesteatoma.

A monoclonal antibody recognizing an epitope of the external domain of the human epidermal growth factor (EGF) receptor was used in an alkaline phosphatase-antialkaline phosphatase (APAAP) technique to compare the distribution of this protein in normal human skin and aural cholesteatoma. EGF receptors appear to be highly expressed on the basal layer of the epidermis, in hair follicle apocrine sweat glands, and in the capillary system of normal skin. Cholesteatoma epithelium showed increased positive reactions in the suprabasal layers. A heterogeneity in the expression was found in different parts of the cholesteatoma. These results suggest the presence of an aberrant regulation and persistence of EGF receptors in cholesteatoma and confirm the hyperproliferative character of the cholesteatoma epithelium.

Expression of ICAM-1 on intact cartilage and isolated chondrocytes.

A major factor in cellular cytotoxicity is the interaction between LFA-1 on leukocytes and ICAM-1 on targets. Because several inflammatory cartilage diseases are characterized by the presence of leukocyte infiltrates, the expression of ICAM-1 on human cartilage, cultured chondrocytes, and transplanted cartilage was investigated using monoclonal antibodies. Frozen tissue sections, chondrocytes in suspension, as well as total cellular mRNA were prepared from human cartilage samples. ICAM-1 expression was studied with two different monoclonal antibodies directed against ICAM-1 by immunohistochemical APAAP-staining and additional flow cytometric analyses. The expression of ICAM-1-mRNA in cartilage tissue was analyzed using the northern blot hybridization technique. Furthermore, chondrocytes were treated in culture with interleukin-1 (IL-1) and gamma-interferon (gamma-IFN). ICAM-1 expression after culture was quantified using flow cytometric analysis. We could detect ICAM-1 mRNA in cartilage tissue, however, the immunostaining of tissue sections using monoclonal antibodies did not give clear positive reactions. Isolated chondrocytes showed strongly positive staining patterns in comparison with adequate negative controls as assessed by flow cytometry. A dose-dependent increase of the expression of ICAM-1 on chondrocytes was observed when stimulated with IL-1 and gamma-IFN. Finally, two of the three studied transplanted autologous cartilage samples with advanced resorption showed the presence of ICAM-1 molecules as assessed by immunohistochemistry. This expression of ICAM-1 suggests that the molecule plays a role in severe cartilage inflammatory processes, where tissue damage leads to the exposure of chondrocyte surfaces.

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.

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.

Heterologous epidermal growth factor receptor (EGF-R) expression in larynx cancer cell lines: evidence for the existence of structurally modified receptors.

Overexpression of epidermal growth factor receptor (EGF-R) is a common characteristic of epidermoid tumors and its expression in head and neck squamous cell carcinoma biopsies and cell lines has been reported previously by several authors. With the aim to provide more structural and functional details about the protein overexpression of EGF-R in head and neck squamous cell carcinomas, the expression of this molecule was studied in four larynx carcinoma cell lines (HLaC-78, HLaC-79, UM-SCC-17A and UM-SCC-17B). The results were compared with those from a spontaneously immortalized aneuploid human keratinocyte cell line (HaCaT) and cultured fresh skin keratinocytes. The EGF-R identification was performed using two well characterized monoclonal antibodies (MoAb) which recognize antigen determinants located on the extracellular domain of the receptor: EGF-R I and 29.1.1. Cytocentrifuge smears and cell suspensions were prepared for flow cytometric analysis. With the monoclonal antibody EGF-R I the highest EGF-R expression was obtained in the cell line UM-SCC-17A, whereas the cell line with the highest EGF-R overexpression was the HLa-C-78 if the monoclonal antibody 29.1.1 was used. The cultured keratinocytes always showed a histogram similar to the control sample (cells were incubated with the second antibody alone). These results could be explained by the existence of structurally modified EGF-receptors. Further studies including possible differences in both the autophosphorylization and the kinase activities are in progress to clarify the functional repercussions of these observed structural defects.

Tracheal transplantation: demonstration of HLA class II subregion gene products on human trachea.

The present morphological study was designed to investigate the expression of HLA class II subregion gene products on human trachea. Frozen sections from the tracheas of 20 cadavers not suffering from ear, nose or throat diseases were studied immunohistochemically using monoclonal antibodies which recognize monomorphic determinants of HLA-DR, -DP and -DQ molecules. Positive reactions could be detected in the airway epithelium and mixed glands. HLA-DR and -DP showed a stronger presence than HLA-DQ. Our results indicate that tracheal mucosa may be the major antigenic structure of the trachea and therefore responsible for the immunogenic action of allogenic tracheal transplants.

Effect of growth factors on cell proliferation by human nasal septal chondrocytes cultured in monolayer.

In the field of reconstructive surgery, autologous cartilage grafting is commonly performed to reconstruct skeletal defects. Because of the limited supply of fresh autologous cartilage many investigators concentrate on in vitro production of cartilage tissue. Several growth factors regulate the metabolism and activation of cartilage cells. In order to enhance the culture conditions for cartilage cells, the aim of our investigations was to characterize the influence of transforming growth factor (TGF)-beta, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on the proliferation of differentiated human nasal septal chondrocytes. The isolated cells were cultured in monolayer using DMEM with and without 10% FCS. The cell proliferation was assessed using tritiated thymidine. We measured an increase of the proliferation rates when the different growth factors were added. The most important stimulatory effect was due to bFGF and the less to EGF. If all growth factors were added together a fivefold increase in the proliferative activity of the cells was achieved. The effects were further enhanced by factors present in fetal calf serum. We conclude that the culture conditions for cell expansion for cartilage engineering can be optimized employing growth factors.

Expression of ICAM-1 on isolated human nasal, auricular and costal chondrocytes.

Expression of intercellular adhesion molecule-1 (ICAM-1) on targets has been reported to be a relevant factor for leukocyte migration, adhesion and function. Because stimulated chondrocytes have been shown to express molecules of immunological import (like HLA class II antigens) and because rejected or resorbed cartilage grafts used in the field of ENT are often characterized by adjacent infiltrating leukocytes, the presence of ICAM-1 on human nasal, auricular and costal cartilage was investigated. For this study, cartilage tissue sections and chondrocytes in suspension as well as cultured chondrocytes were prepared. Specific monoclonal antibodies (mAb) were used for immunocyto- and immunohistochemical Alkaline-Phosphatase-anti-Alkaline-Phosphatase staining (APAAP staining) as well as for flow cytometry analysis. ICAM-1 on healthy cartilage tissue sections was not found. On the other hand, both chondrocytes freed from matrix and cultured chondrocytes showed strongly positive staining patterns for ICAM-1. This result was obtained for chondrocytes from nasal, auricular as well as costal cartilage. This observed expression of ICAM-1 on chondrocytes with defective extracellular matrix demonstrates that cartilage cells are able to synthesize ICAM-1 without any paracrine stimulus from non-chondrocyte cells. It suggests that ICAM-1 plays a role in processes where tissue damage leads to the exposure of chondrocyte surfaces. Therefore, ICAM-1 expression on chondrocytes may also be a factor in destructive cartilage graft resorption.

Hyperproliferation-associated keratin expression in human middle ear cholesteatoma.

Cholesteatoma is characterized by the presence of a squamous epithelium invading the middle ear altering its growth properties. This epithelium is believed to have hyperproliferative properties. Keratin 16 is accepted as a molecular marker for hyperproliferative epithelia. Two monoclonal antibodies K8.12 (directed against keratin 13) and KS.1A3 (directed against keratin 13 and 16) were used in an alkaline phosphatase anti-alkaline-phosphatase (APAAP)-technique to compare the expression of both keratin 13 and keratin 16 in normal human skin and aural cholesteatoma. Furthermore, the cytokeratin expression was compared to that of normal skin and palatine tonsil using one-dimensional gel electrophoresis. For both monoclonal antibodies, normal ear skin was stained only in the basal layer. In contrast, in the cholesteatoma samples the immunostaining of the antibody KS-1A3 was done not only in the basal cell layer but also in the suprabasal cells of the stratum spinosum and stratum granulosum. Using gel-electrophoresis, the presence of cytokeratin 16 was demonstrated in the cholesteatoma samples only. These results support the hyperproliferative character of cholesteatoma epithelium.

Engineering of cartilage tissue using bioresorbable polymer fleeces and perfusion culture.

Replacement of injured or diseased skeletal tissues by either autograft or allograft cartilage has increased steadily during recent decades. The ideal method is to use autologous cartilage; however, this is extremely limited due to the scarcity of donor sites. We present a new approach to the in vitro formation of cartilage grafts for autologous grafting in reconstructive surgery. Bioresorbable polymer fleeces of polylactic acid were used as temporary cell carrier matrices to establish three-dimensional cultures of human chondrocytes. The polymer surface was coated with poly-L-lysine before cell integration. These cell-polymer tissue constructs were encapsulated with low melting point agarose and then placed in perfusion culture chambers to provide a constant supply of nutrients into the cultures. The culture medium consisted of Ham's F12 supplemented with 2% fetal calf serum and 50 micrograms/ml ascorbic acid. The cell-polymer tissues were harvested and frozen for toloudine and alcian blue staining as well as electron microscopic examination after different periods of time in culture. A monoclonal antibody specific for collagen type II was used to characterize the cell phenotype. With this culture procedure chondrocytes maintained a differentiated phenotype with synthesis of collagen and proteoglycan. Collagen fibrils with clear cross-striation were evident in electron microscopic images. The results show that our organotypic cell culture method allows the in vitro production of bioartificial cartilage for transplantation.

Antigen presenting cell function of class II positive human nasal chondrocytes.

It is postulated that class II positive chondrocytes may be actively involved in the destruction or rejection of vital transplanted cartilage grafts. To investigate whether human nasal chondrocytes may also function as accessory cells in ongoing immune reactions with cartilage destruction, mixed leukocyte-chondrocyte cultures and antigen presentation assays were performed. Freshly isolated HLA class II antigen negative chondrocytes obtained from nasal septa were not stimulatory to autologous resting T lymphocytes. HLA class II positive chondrocytes treated with gamma-interferon were able to present antigens to autologous activated T cells derived from an antigen (tetanus) specific T cell line. Upon incubation with activated T cells, initially class II negative changed their phenotype resulting in the expression of class II antigens and enabling them to effectively present antigen. These results suggest an active role of chondrocytes in the rejection of cartilage grafts.

[New immunobiologic trends concerning etiopathogenicity of cholesteatoma]. / Nuevos aspectos inmunobiológicos sobre la etiopatogenia del colesteatoma.

After an epoch in which was pretended to explain the etiopathogenetic phenomena observed in Cholesteatoma through enzymatic studies, nowadays other investigations focus the topic in the possible presence of immunobiologic alterations at cellular level, so the research work is directed to the occurrence, distribution and activity of several growth factors and leukins. In this paper the AA. made a perusal of the new acquisitions and devote themselves to two important aspects of the cholesteatoma: the biologic behaviour of the squamous cell epithelia with an uncontrolled growth and to the immunobiologic mechanisms responsible for the bone resorption.