Damage of cartilage structures in the head and neck region as well as in orthopedic sites are frequently caused by trauma, tumor resection, or congenital defects. Despite a high demand in many clinical fields, until today, no adequate cartilage replacement matrix is available for these fields of application. Materials that are clinically applied for joint cartilage repair still need optimization due to difficult intraoperative handling and risk of early mechanical damage. We have developed and applied a novel chemical process to completely decellularize and sterilize human and porcine cartilage tissues (meniscus cartilage and nasal septum) to generate a new type of bioimplant matrix. To characterize this matrix and to determine the effect of the decellularization process, the content of denatured collagen (w(D)) and the content of glycosaminoglycans (GAGs) (w(G)) were determined. Possible cytotoxic effects and cellular compatibility of the matrix in vitro have been examined by seeding processed cartilage biomatrices with human primary chondrocytes as well as murine fibroblasts (L929). Vitality and state of metabolism of cells were measured using MTS assays. Both cell types adhered to scaffold surfaces and proliferated. No areas of growth inhibition or cytotoxic effects were detected. New synthesis of cartilage-specific extracellular matrix was observed. By histological staining, electron microscopy, and μCT analysis, an increase of matrix porosity, complete cell elimination, and high GAG removal were demonstrated. Being from natural-origin, processed xenogenic and allogeneic cartilage biomatrices are highly versatile with regard to shape, size, and biomechanics, making them promising candidates for various biomedical applications.

The aim of this study was to examine the influence of matrix elasticity on the maintenance of the chondrogenic phenotype of chondrocytes cultured in monolayer. We used a two-dimensional culturing system in which polyacrylamide gels with different concentrations of bis-acrylamide were coated with collagen type I. Matrices with a Young's modulus of 4, 10, 40, and 100 kPa were produced, as determined by atomic force microscopy. Porcine chondrocytes were cultivated on these matrices at a low density for 7 days. The proliferation of cells was analyzed by 5-Bromo-2′-deoxy-uridine incorporation. Maintenance of the chondrogenic phenotype was analyzed by measuring collagen type I, type II, and aggrecan gene expression, immunofluorescence staining for collagen type II, and phalloidin staining for actin filaments. Cellular proliferation and actin organization were decreased on matrices of 4 kPa compared with stiffer substrates. The differentiated phenotype of the chondrocytes grown on matrices of 4 kPa was stabilized, indicated by higher collagen type II and aggrecan, and lower collagen type I expression. These findings indicate that chondrocytes sense the elasticity of the matrix and might be used for the design of scaffolds with mechanical properties specifically tailored to support the chondrogenic phenotype in tissue engineering applications.

Currently, adult stem cells are attracting significant interest in regenerative medicine and tissue engineering. These cells have been isolated from various tissue sources; however, in most cases, adult stem cells useful for tissue engineering and regeneration are present at a low frequency. High numbers of stem cells with an effective and reliable potential for differentiation are needed for clinical applications. Thus, the identification of new stem cell sources and the establishment of optimized cell culture conditions that allow for the amplification of stem cells are of utmost relevance. In addition, the isolation procedure should ideally be minimally invasive and possibly be performed under local anesthesia. We report here for the first time on the identification of adult stem cells with mesenchymal characteristics in human parotid gland tissue. Cells were isolated from freshly resected specimens of parotid glands using enzymatic digestion and plastic adhesion protocols. Following an initial proliferation period and short-term culture for four passages, immunophenotyping revealed the presence of mesenchymal stem cell markers. In the presence of tissue-specific induction medium, stem cells could be differentiated into adipogenic, osteogenic, and chondrogenic cell types. Tissue-specific differentiation was confirmed by histochemical and immunocytochemical staining as well as by RT-PCR for defined marker genes. This study is, to the best of our knowledge, the first report on the isolation and differentiation of stem cells from adult human parotid glands. Although isolated from an endodermal tissue source, these stem cells share many characteristics with MSCs. Easy accessibility and a high differentiation potential make salivary gland-derived stem cells a promising source for future applications in regenerative medicine.