Nicole Rotter

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.

Cartilage reconstruction in head and neck surgery: Comparison of resorbable polymer scaffolds for tissue engineering of human septal cartilage

New cell culture techniques raise the possibility of creating cartilage in vitro with the help of tissue engineering. In this study, we compared two resorbable nonwoven cell scaffolds, a polyglycolic acid/poly-L-lactic acid (PGA/PLLA) (90/10) copolymer (Ethisorb) and pure PLLA (V 7-2), with different degradation characteristics in their aptitude for cartilage reconstruction. Chondrocytes were isolated enzymatically from human septal cartilage. The single cells were resuspended in agarose and transferred into the polymer scaffolds to create mechanical stability and retain the chondrocyte-specific phenotype. The cell-polymer constructs were then kept in perfusion culture for 1 week prior to subcutaneous transplantation into thymusaplastic nude mice. After 6, 12, and 24 weeks, the specimens were explanted and analyzed histochemically on the presence of collagen (azan staining), proteoglycans (Alcian blue staining), and calcification areas (von Kossa staining). Furthermore, different collagen types (collagen type I, which is found in most tissues, but not in hyaline cartilage matrix; and collagen type II, which is cartilage specific) were differentiated immunohistochemically by the indirect immunoperoxidase technique. Vascular ingrowth was investigated by a factor VIII antibody, which is a endothelial marker. Quantification of several matrix components was performed using the software Photoshop. Significant differences were found between both nonwoven structures concerning matrix synthesis and matrix quality as well as vascular ingrowth. Ethisorb, with a degradation time of approximately 3 weeks in vitro, showed no significant differences from normal human septal cartilage in the amount of collagen types I and II 24 weeks after transplantation. Thin fibrous tissue layers containing blood vessels encapsulated the transplants. V 7-2 constructs, which did not show strong signs of degradation even 24 weeks after transplantation, contained remarkably smaller amounts of cartilage-specific matrix components. At the same time, there was vascular ingrowth even in central parts of the transplants. In conclusion, polymer scaffolds with a short degradation time are suitable materials for the development of cartilage matrix products, while longer stability seems to inhibit matrix synthesis. Thus, in vitro engineering of human cartilage can result in a cartilage-like tissue when appropriate nonwovens are used. Therefore, this method could be the ideal cartilage replacement method without the risk of infection and with the possibility of reconstructing large defects with different configurations.

Age dependence of biochemical and biomechanical properties of tissue-engineered human septal cartilage.

The aim of this study was to determine whether the biomechanical and biochemical properties of tissue-engineered human septal cartilage vary with donor age and in vitro culture time. Chondrocytes were isolated from human septal cartilage of patients from 15 to 60 year old and maintained in primary monolayer culture for 14 days. Cells were seeded onto 0.5% PLA coated PGA disks and kept in stationary three-dimensional culture for either 1 day or 3 weeks. Specimens were then implanted subcutaneously into athymic nude mice and harvested after either 4 or 8 weeks. Upon harvest, the equilibrium confined compression modulus was measured as to quantify mechanical properties, and the glycosaminoglycan, hydroxyproline, and DNA contents were determined as measures of tissue proteoglycans, collagen, and cell density. This study demonstrated that native nasal cartilage showed distinct changes in these parameters with age, but cartilage engineered using the cells of these specimens showed no significant dependence on the age of the donor. There was little difference in quality of cartilage between samples cultured for 3 weeks in vitro and those implanted directly after seeding. Together, the results of this study suggest that the process of extracellular matrix assembly by chondrocytes on three-dimensional scaffolds may be independent of in vivo conditions experienced by the tissue prior to harvest.

Age-related changes in the composition and mechanical properties of human nasal cartilage.

Nasal cartilage is widely used in reconstructive surgery for the replacement of soft tissue defects and nasal reconstruction procedures. The ability to shape harvested tissue and the performance in the transplant site are related to the mechanical properties of nasal cartilage. Several studies have documented changes in composition and mechanical properties of other cartilages with age, but little is known about these processes in nasal cartilage. In this study, 45 human nasal septum specimens were gathered from patients 15-60 years of age after reconstructive surgery. Samples were cut to 6 mm in diameter and tested in confined compression to determine equilibrium modulus and hydraulic permeability and analyzed for glycosaminoglycan and hydroxyproline content. Equilibrium modulus decreased significantly with increasing donor age (P<0.01) while hydraulic permeability increased significantly (P<0.02). Glycosaminoglycan (GAG) content decreased significantly with age (P<0.05), while hydroxyproline content showed a slight, but not significant, increase with age (P>0.2). These trends are qualitatively similar to those observed in articular cartilage, suggesting the existence of a systemic process of cartilage degradation that is independent of mechanical loading. Further, the relationships between biochemical composition and mechanical properties were age-dependent, with cartilage from patients less than 30 years of age showing greater dependence of equilibrium modulus and hydraulic permeability on GAG and hydroxyproline content. This suggests that changes in matrix organization may accompany changes in tissue composition.

Cartilage and bone tissue engineering for reconstructive head and neck surgery

The loss of cartilage and bone because of congential defects, trauma and after tumor resection is a major clinical problem in head and neck surgery. The most prevalent methods of tissue repair are through autologous grafting or using implants. Tissue engineering applies the principles of engineering and life sciences in order to create bioartificial cartilage and bone. Most strategies for cartilage tissue engineering are based on resorbable biomaterials as temporary scaffolds for chondrocytes or precursor cells. Clinical application of tissue-engineered cartilage for reconstructive head and neck surgery as opposed to orthopedic applications has not been well established. While in orthopedic and trauma surgery engineered constructs or autologous chondrocytes are placed in the immunoprivileged region of joints, the subcutaneous transplant site in the head and neck can lead to strong inflammatory reactions and resorption of the bioartificial cartilage. Encapsulation of the engineered cartilage and modulation of the local immune response are potential strategies to overcome these limitations. In bone tissue engineering the combination of osteoconductive matrices, osteoinductive proteins such as bone morphogenetic proteins and osteogenic progenitor cells from the bone marrow or osteoblasts from bone biopsies offer a variety of tools for bone reconstruction in the craniofacial area. The utility of each technique is site dependent. Osteoconductive approaches are limited in that they merely create a favorable environment for bone formation, but do not play an active role in the recruitment of cells to the defect. Delivery of inductive signals from a scaffold can incite cells to migrate into a defect and control the progression of bone formation. Rapid osteoid matrix production in the defect site is best accomplished by using osteoblasts or progenitor cells.

The characterisation of human respiratory epithelial cells cultured on resorbable scaffolds: first steps towards a tissue engineered tracheal replacement

In this study we have used lectin histochemistry and scanning electron microscopy (SEM) to assess the growth and characterise the differentiation of human respiratory epithelial cells (REC) cultured on two biomaterial scaffolds. The first scaffold, based on a hyaluronic acid derivative, was observed to be non-adhesive for REC. This lack of adhesion was found to be unrelated to the presence of the hyaluronic acid binding domain on the surface of isolated REC. The other scaffold, consisting of equine collagen. was observed to encourage REC spreading and adhesion. Positive Ulex Europaeus agglutinin (UEA) lectin staining of this preparation indicated the presence of ciliated REC on the scaffold surface. However, the marked decrease in peanut agglutinin (PNA) positive staining, relative to that of control cultures and native tissue, indicates a dedifferentiation of the secretory cells of the REC monolayer. SEM analysis of REC cultured on the collagen scaffold confirmed the presence of ciliated cells thereby validating the UEA positive staining. The presence of both established and developing cilia was also verified. This study indicates that collagen biomaterials are appropriate for the tissue engineering of REC. Furthermore, that UEA and PNA staining is a useful tool in the characterisation of cells cultured on biomaterials, therefore helpful in identifying biomaterials that are suitable for specific tissue engineering purposes.

Human Nasal Mucosa Contains Tissue-Resident Immunologically Responsive Mesenchymal Stromal Cells

Multipotent mesenchymal stromal cells (MSC) are present in bone marrow and other tissues such as adipose tissue, muscle, pancreas, liver, and so on. Recent evidence suggests that MSC migrate to sites of infection, inflammation, and cancer, and interact with different immune cell subsets. Here, we report for the first time on the isolation and characterization of multipotent nasal mucosa-derived mesenchymal stromal cells (nm-MSC). nm-MSC showed a plastic adherent and fibroblast-like morphology and were able to form colonies. They expressed the typical bone marrow MSC marker antigens CD29, CD44, CD73, CD90, and CD105 and were able to differentiate along the adipogenic, chondrogenic, and osteogenic pathways. nm-MSC produced a set of inflammatory cytokines, expressed chemokine receptors, and were responsive to stimulation with cytokines, chemokines, and TLR4 ligand LPS. Thus, these cells may serve as an alternative adult stromal cell resource for regenerative tissue repair and may represent important regulators of local mucosal immunity.

Tracheal remodeling: comparison of different composite cultures consisting of human respiratory epithelial cells and human chondrocytes.

The reconstruction of extensive tracheal defects is still an unsolved challenge for thoracic surgery. Tissue engineering is a promising possibility to solve this problem through the generation of an autologous tracheal replacement from patients’ own tissue. Therefore, this study investigated the potential of three different coculture systems, combining human respiratory epithelial cells and human chondrocytes. The coculture systems were analyzed by histological staining with alcian blue, immunohistochemical staining with the antibodies, 34betaE12 and CD44v6, and scanning electron microscopy. The first composite culture consisted of human respiratory epithelial cells seeded on human high-density chondrocyte pellets. For the second system, we used native articular cartilage chips as base for the respiratory epithelial cells. The third system consisted of a collagen membrane, seeded with respiratory epithelial cells and human chondrocytes onto different sides of the membrane, which achieved the most promising results. In combination with an air–liquid interface system and fibroblast-conditioned medium, an extended epithelial multilayer with differentiated epithelial cells could be generated. Our results suggest that at least three factors are necessary for the development towards a tracheal replacement: (1) a basal lamina equivalent, consisting of collagen fibers for cell–cell interaction and cell polarization, (2) extracellular factors of mesenchymal fibroblasts, and (3) the presence of an air–liquid interface system for proliferation and differentiation of the epithelial cells.

The Oral Serine Protease Inhibitor WX-671 – First Experience in Patients with Advanced Head and Neck Carcinoma

Tumour invasion and metastasis depend on the capacity of tumour cells to coordinate various biological processes such as detachment of cells from their original localisation, cancer cell migration, invasion of cancer cells into surrounding tissue, access to blood and lymphatic vessels and adhesion to and invasion through the endothelium, allowing colonisation at distant sites of the organism. This complex scenario requires the concerted and regulated expression of extracellular proteolytic systems, integrins and adhesion proteins. Degradation of proteins in basement membranes and extracellular matrix is the prerequisite for the invasion of cells and the formation of metastases. This is mediated by various extracellular proteolytic enzymes including serine proteinases, metalloproteinases and cysteine proteinases [1]. There is abundant experimental evidence that the plasminogen activator system plays an essential role in these processes [1,2,3,4,5,6,7,8]. It consists of two serine proteinases, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), the cell surface uPA receptor (uPAR) and the plasminogen activator inhibitors 1 and 2 (PAI-1, PAI-2). uPA is agreed to be the enzyme with the major influence on cancer-related processes, whereas the primary role of tPA is the generation of plasmin for fibrinolysis of blood vessels [9]. Besides its proteolytic activity, uPA in concert with uPAR also mediates mitogenic, adhesive and migratory processes [10]. Clinical studies have demonstrated the relevance of uPA, uPAR and PAI-1 in malignant tumours such as breast, ovary, cervical, upper urinary, gastrointestinal, lung, prostate and other cancers. Elevated levels of these factors correlate with increased malignancy and poor patient outcome [1, 7, 11, 12]. For head and neck squamous cell carcinoma (HNSCC) the role of the uPA system has been extensively investigated [13,14,15,16,17]. In tumour lysates, it has been demonstrated that both uPA and PAI-1 may play a specific role in the process of invasion and metastasis and might also be of prognostic value in this carcinoma [18, 19]. Recently, Wilex developed a serine protease inhibitor of the uPA system, the intravenous drug WX-UK1, and its new orally administered pro-drug WX-671 to treat cancer. Once WX-671 is absorbed, the hydroxyamidino function is reductively converted to the amidino function, thereby generating pharmacologically active WX-UK1. In rat, dog and monkey, WX-671 is effectively absorbed, although with varying rates between species, and readily metabolised to WX-UK1. In rat models, WX-671 has been shown to be efficacious at inhibiting tumour growth and spread. For clinical testing in man, hard gelatine capsules are available that contain WX-671 hydrogen sulphate equivalent to 50 mg and 200 mg WX-671 (free base). The aim of this study was to evaluate the pharmacokinetics of both the oral pro-drug WX-671 and the metabolite WX-UK1 in human tissue and plasma of patients with HNSCC. In addition, the safety and tolerability as well as the effects on the uPA system of WX-671 were assessed.

Expression of ICAM-1 on intact cartilage and isolated chondrocytes

SummaryA 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.