ABSTRACT
Degeneration of the intervertebral disc (IVD) is a progressive and chronic process, and the high incidence of discogenic disorders calls for new therapeutic approaches, such as cell-based therapies using three dimensional cultures and mesenchymal stem cells (MSC), which can differentiate to chondrogenic- and IVD-lineages. Here, we investigated the growth and differentiation of human MSC culture on biodegradable collagen scaffolds in order to obtain an injectable suspension. Commercially available wound dressings were downsized to dimensions between 100 and 1500 µm and seeded with freshly isolated or early passages MSC. Proliferation rate and chondrogenic differentiation potential was tested at oxygenation levels of 2%, 5%, 10% and 21% in static and dynamic cultures. Evaluation methods included cell viability test, disc marker genes expression (aggrecan, collagen type I and type II), histological detection of proteoglycans and immunohistochemical analysis. On microcarriers, freshly isolated MSC had lower proliferation rate and chondrogenic differentiation potential compared with early passages MSC. Proliferation of MSC was significantly increased 1.7-fold at 5% oxygen level and in combination with dynamic culture was further increased to 2.3-fold, with respect to normoxia. Chondrogenesis was positively affected by 2% and 5% hypoxia, as shown by increased transcription levels and protein expression of collagen type II and proteoglycan accumulation in static cultures, while it was inhibited in dynamic cultures. Collagen type I and aggrecan expression were not affected by hypoxia. In conclusion, collagen based microcarriers are a suitable support for in vitro MSC growth and chondrogenesis especially when cultured at 5% oxygen level.
Subject(s)
Cartilage/cytology , Intervertebral Disc Degeneration/therapy , Mesenchymal Stem Cells/cytology , Tissue Engineering/methods , Adult , Aggrecans/genetics , Aggrecans/metabolism , Cartilage/metabolism , Cell Culture Techniques/instrumentation , Cell Culture Techniques/methods , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Chondrocytes/cytology , Chondrocytes/metabolism , Chondrogenesis/drug effects , Collagen Type I/chemistry , Collagen Type I/genetics , Collagen Type I/metabolism , Collagen Type II/genetics , Collagen Type II/metabolism , Dose-Response Relationship, Drug , Gene Expression/drug effects , Humans , Immunohistochemistry , Injections , Mesenchymal Stem Cell Transplantation/instrumentation , Mesenchymal Stem Cell Transplantation/methods , Mesenchymal Stem Cells/metabolism , Middle Aged , Oxygen/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Tissue Scaffolds/chemistryABSTRACT
Degeneration of intervertebral discs (IVDs) is associated with back pain and elevated levels of inflammatory cells. It has been hypothesised that discogenic pain is a direct result of vascular and neural ingrowth along annulus fissures, which may expose the avascular nucleus pulposus (NP) to the systemic circulation and induce an autoimmune reaction. In this study, we confirmed our previous observation of antibodies in human degenerated and post-traumatic IVDs cultured in vitro. We hypothesised that the presence of antibodies was due to an autoimmune reaction against specific proteins of the disc. Furthermore we identified antigens which possibly trigger an autoimmune response in degenerative disc diseases. We demonstrated that degenerated and post-traumatic IVDs contain IgG antibodies against typical extracellular proteins of the disc, particularly proteins of the NP. We identified IgGs against collagen type II and aggrecan, confirming an autoimmune reaction against the normally immune privileged NP. We also found specific IgGs against collagens types I and V, but not against collagen type III. In conclusion, this study confirmed the association between disc degeneration and autoimmunity, and may open the avenue for future studies on developing prognostic, diagnostic and therapy-monitoring markers for degenerative disc diseases.
Subject(s)
Autoantibodies/immunology , Extracellular Matrix Proteins/immunology , Intervertebral Disc Degeneration/immunology , Intervertebral Disc/immunology , Adult , Aged , Cells, Cultured , Female , Humans , Immunoglobulin G/immunology , Male , Middle AgedABSTRACT
Lsc (the murine homolog of human p115 Rho GEF) is a member of the Dbl-homology family of GTP exchange factors and is a specific activator of Rho. Lsc is activated by the G alpha(13) subunit of heterotrimeric G proteins and contains a regulator of G protein signaling domain that downmodulates G alpha(12) and G alpha(13). Lsc is expressed primarily in the hematopoietic system and links the activation of G alpha(12) and G alpha(13)-coupled receptors to actin polymerization in B and T cells. Lsc is essential for marginal zone B (MZB) cell homeostasis and for the generation of immune responses. Although Lsc-deficient lymphocytes show reduced basal motility, MZB cells show enhanced migration after serum activation. Thus, Lsc is a critical regulator of MZB cells and immune functions.
Subject(s)
B-Lymphocytes/immunology , Chemotaxis, Leukocyte , Guanine Nucleotide Exchange Factors , Proto-Oncogene Proteins/physiology , Animals , Cells, Cultured , DNA-Binding Proteins/metabolism , GTP-Binding Protein alpha Subunits, G12-G13 , Gene Targeting , Hematopoietic Stem Cells/immunology , Heterotrimeric GTP-Binding Proteins/metabolism , Immunoglobulins/biosynthesis , Lymphocyte Activation , Lymphocyte Subsets/classification , Lymphoid Tissue/immunology , Mice , Mice, Knockout , Platelet Aggregation , Proto-Oncogene Proteins/genetics , Recombination, Genetic , Rho Guanine Nucleotide Exchange Factors , Tissue DistributionABSTRACT
A standardised questionnaire and personality test were used to study whether women after breast cancer treatment, or women treated for genital cancer need psychosocial counselling more frequently. The interview was given to 308 women during the author's tumor after-care consultations. 1. Repercussion on their marital lives were reported three times more often by women treated for genital cancer than by women after mastectomy (p less than 0.01). There was not significant difference with regard to their family status. 2. As epidemiologically expected -- mastectomy patients had born less children than women treated for genital cancer (p less than 0.05). 3. A reduced self-confidence was reported more often by patients treated for breast cancer than by the other patient group (p less than 0.01). 4. No decrease in working performance was observed by 50% of the women treated for genital cancers as against one third of the masectomy patients. 5. Work resumption was reported more often to be strenuous for masectomy patients than for women treated for genital cancer (p less than 0.05). 6. Woman after mastectomy experiences 50% less sexual repercussions than genital cancer patients (p less than 0.05).