CD34+ enriched cell products intended for autologous transendocardial CD34+ cell transplantation release significant amounts of angiopoietin-1.

OBJECTIVES: Cell-based therapy has emerged as a promising strategy for the treatment of patients with heart failure. Increasing evidence supports the hypothesis that paracrine mechanisms mediated by soluble factors released by the cells play a predominate role in reparative processes. The aim of our study was to analyze which cytokines are released by CD34+ enriched cell products intended for autologous transendocardial CD34+ cell transplantation in patients with cardiomyopathy. MATERIAL AND METHODS: The peripheral blood CD34+ cells from 12 patients were mobilized with granulocyte colony-stimulating factor, collected via apheresis and enriched by immunoselection. RESULTS: In CD34+ enriched cell population, hematopoietic, but not mesenchymal or endothelial, progenitors were detected. Except for angiopoietin-1, other measured cytokines (FGF1, FGF2, VEGF, PDGF, IL-6, HGH, SDF-1α/CXCL12, NRG1) were not released by CD34+ cells. The average concentration of angiopoietin-1 released by 5×106 CD34+ cells grown in neutral DMEM medium was 213.6±130.0pg/mL (range: 74-448pg/mL). Angiopoietin-1 secretion correlated well with CD34+ cell's capacity for generating colonies derived from hematopoietic progenitors (Pearson's correlation=0.964; P<0.001). CONCLUSION: Our study presents angiopoietin-1 as an interesting candidate and suggests future studies to explore how its release by CD34+ cells might impact the success of autologous CD34+ cell transplantation.

Evaluation of a flow cytometric assay for the confirmation of heparin-induced thrombocytopenia.

INTRODUCTION: Heparin-induced thrombocytopenia (HIT) is an immune-mediated thrombocytopenia associated with heparin therapy. The diagnosis consists of a combination of pretest probability and laboratory testing. The routinely available laboratory antigen binding assays for the detection of specific antibodies have a low HIT-positive predictive value; therefore, to exclude false-positive results, one of the functional assays should be performed. Functional assays evaluate the ability of heparin/PF4 antibodies to activate the platelets. The aim of our study was to validate the flow cytometric functional assay, based on the use of anti-CD61 and anti-CD62 antibodies, as a suitable diagnostic test for HIT. METHODS: Sera from patients with a clinical suspicion of HIT were previously analyzed with screening IgG-specific ELISA, and 41 of those which were positive were selected for the functional assay. RESULTS: Our results were compared to another functional assay - the HIPA (heparin-induced platelet aggregation assay). The diagnostic specificity of the flow cytometric assay was calculated based on HIPA results and was 83%. CONCLUSION: Performing this functional test after the screening assay could significantly improve the specificity of HIT testing as heparin/PF4 antibodies are often not clinically significant.

Cartilage from the edge of a debrided articular defect is inferior to that from a standard donor site when used for autologous chondrocyte cultivation.

The aim of this study was to evaluate the cultivation potential of cartilage taken from the debrided edge of a chronic lesion of the articular surface. A total of 14 patients underwent arthroscopy of the knee for a chronic lesion on the femoral condyles or trochlea. In addition to the routine cartilage biopsy, a second biopsy of cartilage was taken from the edge of the lesion. The cells isolated from both sources underwent parallel cultivation as monolayer and three-dimensional (3D) alginate culture. The cell yield, viability, capacity for proliferation, morphology and the expressions of typical cartilage genes (collagen I, COL1; collagen II, COL2; aggrecan, AGR; and versican, VER) were assessed. The cartilage differentiation indices (COL2/COL1, AGR/VER) were calculated. The control biopsies revealed a higher mean cell yield (1346 cells/mg vs 341 cells/mg), but similar cell proliferation, viability and morphology compared with the cells from the edge of the lesion. The cartilage differentiation indices were superior in control cells: COL2/COL1 (threefold in biopsies (non-significant)); sixfold in monolayer cultures (p = 0.012), and 7.5-fold in hydrogels (non-significant), AGR/VER (sevenfold in biopsies (p = 0.04), threefold (p = 0.003) in primary cultures and 3.5-fold in hydrogels (non-significant)). Our results suggest that the cultivation of chondrocytes solely from the edges of the lesion cannot be recommended for use in autologous chondrocyte implantation.

Growth and differentiation of alveolar bone cells in tissue-engineered constructs and monolayer cultures.

The ability to enhance bone regeneration by implanting autologous osteoblasts in combination with an appropriate scaffold would be of great clinical interest. The aim of our study was to compare the growth and differentiation of alveolar bone cells in tissue-engineered constructs and in monolayer cultures, as the basis for developing procedures for routine preparation of bone-like tissue constructs. Alveolar bone tissue was obtained from four human donors and explant cultures of the cells were established. Expanded cells were seeded on macroporous hydroxyapatite granules, and cultured in medium supplemented with osteogenic differentiation factors for up to 3 weeks. Control monolayer cultures were established in parallel, and cultured in media with or without osteogenic supplements. Cell proliferation, alkaline phosphatase (AP) activity and gene expression of AP, osteopontin and osteocalcin were determined under different culture conditions at weekly intervals. Cells in tissue constructs exhibited growth patterns similar to those in control monolayer cultures: enhanced proliferation was noted during the first 2 weeks of cultivation, followed by a decrease in cell numbers. AP activity at 3 weeks was higher in all cultures in osteogenic medium than in control medium. Gene expression levels were stable in monolayer cultures in both types of media whereas, in tissue constructs, they exhibited patterns of osteogenic differentiation. Light and scanning electron microscopy examination of the cell-seeded constructs showed uniform cell distribution, as well as cell attachment and growth into the interior region of the hydroxyapatite granules. Our results show that bone-like constructs with viable cells exhibiting differentiated phenotype can be prepared by cultivation of alveolar-bone cells on the tested hydroxyapatite granules.

Evaluation of rabbit auricular chondrocyte isolation and growth parameters in cell culture.

Auricular cartilage is an attractive potential source of cells for many tissue engineering applications. However, there are several requirements that have to be fulfilled in order to develop a suitable tissue engineered implant. Animal experiments serve as important tools for validating novel concepts of cartilage regeneration; therefore rabbit auricular chondrocytes were studied. Various parameters including isolation procedures, passage number, rate of proliferation and gene expression profile for major extracellular matrix components were evaluated in order to assess the potential use of elastic chondrocytes for tissue engineering. Chondrocytes were isolated from rabbit ear cartilage and grown in monolayer cultures over four passages. Yields of harvested cells and proliferation were analysed from the digestion step to the fourth passage, and changes in phenotype were monitored. The proliferation capacity of cell cultures decreased during cultivation and was accompanied by enlargement of cells, this phenomenon being especially evident in the third and fourth passages. The expression of cartilage specific genes for collagen type II, aggrecan and cartilage non-specific collagen type I was determined. The mRNA levels for all three genes were obviously lower in the primo culture than immediately after isolation. During subsequent cultivation the expression of collagen type II decreased further, while there were only slight changes in expression of aggrecan and collagen type I. This study provides a valuable basis for testing of different tissue engineering applications in rabbit model, where auricular chondrocytes are considered as cell source.

Immunogenicity of dendritic cells pulsed with cartilage derived soluble alloantigens.

Investigation of allo- and autoreactivity of cartilage components is useful not only for the potential allogeneneic transplantation but also for the treatment of tissue specific degenerative autoimmune diseases. We generated dendritic cells (DCs) to study articular cartilage immunogenicity, by using human peripheral blood mononuclear cells. Dendritic cells are extremely efficient antigen-presenting cells that initiate and modulate T-cell dependant immune responses. After isolation by density centrifugation, GM-CSF and IL-4 were added to adherent mononuclear cells. After 7 days of cultivation the cells in cell culture were: HLA-DR+, CD86+, CD14-, CD80-, CD83, CD19-, CD56-, CD3-. Dendritic cells were then pulsed with a supernatant taken from primary monolayer cell cultures of human allogeneic chondrocytes. The stimulating capacity of antigen-pulsed DCs was measured in a classical proliferation assay using tritium labelled thymidine as a marker of mitosis. Proliferative responses were significantly strong but became much stronger when DCs were concomitantly incubated with soluble antigens and human recombinant TNF-alpha and used subsequently as stimulators. Although TNF-alpha positively influenced the stimulating capacity of pulsed DCs, we were unable to detect major differences in expression of specific DCs' phenotype markers assessed by flow cytometry in the absence or presence of this cytokine.

Protein synthesis of human articular chondrocytes cultured in vitro for autologous transplantation.

Chondrocytes in hyaline cartilage produce typical matrix proteins, the most abundant of them being collagen type II and aggrecan. Chondrocytes in monolayer cell culture dedifferentiate and gain fibroblastic phenotype. The cells gradually start to produce collagen type I while the production of collagen type II and aggrecan decreases. Transplantation of autologous chondrocytes cultured in vitro is used for treatment of aseptic articular cartilage lesions. For this purpose, cartilage biopsy is taken and isolated cells are subsequently proliferated in a monolayer cell culture. When implanted, the cells start to produce specific cartilaginous matrix that fills the defect. Prior to surgical procedure the cells can also be cryopreserved for longer periods of time after reaching appropriate numbers. We tested the influence of cultivation time and number of continuous culture passages as well as the influence of cryopreservation on the matrix protein synthesis of human articular chondrocytes. The ability of dedifferentiated chondrocytes to redifferentiate has been monitored by measuring matrix protein synthesis of the cells, re-seeded in agarose suspension culture. The results obtained show progressive dedifferentiation during monolayer cell culture procedures, facilitated by cryopreservation. Successful redifferentiation of cells re-seeded in suspension cultures was observed regardless of the previous level of chondrocyte dediferentiation.

Determination of optimal transport conditions for biopsies of human articular cartilage as well as for suspensions of cultured chondrocytes used in autologous transplantation.

Autologous transplantation of chondrocytes is currently being promoted as a novel approach for the treatment of deep cartilage lesions. Briefly, the method involves enzyme-mediated release of chondrocytes from cartilage biopsies, the expansion of cells by in vitro cultivation and their re-implantation into the defect. The success of this technique depends on many factors including transport conditions for both, cartilage biopsies from the operating hall to the laboratory and the return transport of final suspension of cultured chondrocytes. To determine the extent of cellular damage in biopsies, chondrocytes were enzymatically isolated following a few days of tissue preservation in different tissue culture media. The proportion of dead cells was assessed by Trypan blue staining and counting. The viability was not dependant of the type of the medium used and remained approximately 50% in all samples, even after 72 h. To develop optimal conditions for transport of final chondrocyte suspension, isolated cells were firstly grown in monolayer cultures. Cell suspensions in media with different additives were injected into special glass containers used for the transport and left at 4 degrees C or 25 degrees C for up to 3 days. During this period every 24 h the samples were taken and viability as well as apoptosis levels were assessed. Viability of cells in suspensions at 25 degrees C decreased significantly and became inadequate already after 48 h. In contrast to that, the proportion of viable cells at 4 degrees C remained above 80% even after 48 h. In the majority of the samples, culture medium containing serum and vitamin C provided the best conditions for long-term preservation of chondrocytes.

Vitamin C induced apoptosis in human articular chondrocytes.

Chondrocytes present in articular cartilage survive as a resident cell population throughout the lifespan of the individual organism. However, articular chondrocytes as other cells also undergo apoptosis and there is an ever increasing list of diverse stimuli that can induce this phenomenon in vitro. Our main interest was to investigate potential cytotoxic effects of vitamin C (L-ascorbic acid) on human articular chondrocytes. The present study suggests that vitamin C can induce apoptosis in a cell culture of chondrocytes after 18 h of cultivation. Apoptosis-inducing activity of L-ascorbic acid is dose dependent and significantly affected by the presence of serum. The increased number of vitamin C induced apoptotic cells was associated with DNA fragmentation and morphological changes of the cells.