The aim of the study was to evaluate the effect of a cell-free hyaluronate/type I collagen/fibrin composite scaffold containing polyvinyl alcohol (PVA) nanofibers enriched with liposomes, basic fibroblast growth factor (bFGF) and insulin on the regeneration of osteochondral defects. A novel drug delivery system was developed on the basis of the intake effect of liposomes encapsulated in PVA nanofibers. Time-controlled release of insulin and bFGF improved MSC viability in vitro. Nanofibers functionalized with liposomes also improved the mechanical characteristics of the composite gel scaffold. In addition, time-controlled release of insulin and bFGF stimulated MSC recruitment from bone marrow in vivo. Cell-free composite scaffolds containing PVA nanofibers enriched with liposomes, bFGF, and insulin were implanted into seven osteochondral defects of miniature pigs. Control defects were left untreated. After 12 weeks, the composite scaffold had enhanced osteochondral regeneration towards hyaline cartilage and/or fibrocartilage compared with untreated defects that were filled predominantly with fibrous tissue. The cell-free composite scaffold containing PVA nanofibers, liposomes and growth factors enhanced migration of the cells into the defect, and their differentiation into chondrocytes; the scaffold was able to enhance the regeneration of osteochondral defects in minipigs.
- MeSH
- buněčná diferenciace MeSH
- buňky kostní dřeně cytologie MeSH
- chondrocyty cytologie MeSH
- fibrin chemie MeSH
- fibroblastový růstový faktor 2 aplikace a dávkování MeSH
- inzulin aplikace a dávkování MeSH
- kolagen typu I chemie MeSH
- kyselina hyaluronová chemie MeSH
- liposomy MeSH
- mezenchymální kmenové buňky cytologie MeSH
- miniaturní prasata MeSH
- modul pružnosti MeSH
- nanovlákna aplikace a dávkování chemie MeSH
- polyvinylalkohol chemie MeSH
- prasata MeSH
- regenerace kostí * MeSH
- tkáňové podpůrné struktury MeSH
- viabilita buněk MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In recent studies using a rat aortic balloon occlusion model, we have demonstrated that spinal grafting of rat or human neuronal precursors or human postmitotic hNT neurons leads to progressive amelioration of spasticity and rigidity and corresponding improvement in ambulatory function. In the present study, we characterized the optimal dosing regimen and safety profile of human spinal stem cells (HSSC) when grafted into the lumbar spinal cord segments of naive immunosuppressed minipigs. Gottingen-Minnesota minipigs (18-23 kg) were anesthetized with halothane, mounted into a spine-immobilization apparatus, and received five bilateral injections of HSSC delivered in 2, 4, 6, 8, or 10 μl of media targeted into L2-L5 central gray matter (lamina VII). The total number of delivered cells ranged between 2,500 and 100,000 per injection. Animals were immunosuppressed with Prograf® for the duration of study. After cell grafting, ambulatory function was monitored daily using a Tarlov's score. Sensory functions were assessed by mechanically evoked skin twitch test. Animals survived for 6-7 weeks. Three days before sacrifice animals received daily injections of bromodeoxyuridine (100 mg/kg; IV) and were then transcardially perfused with 4% paraformaldehyde. Th12-L6 spinal column was then dissected; the spinal cord was removed and scanned with MRI. Lumbar transverse spinal cord sections were then cut and stained with a combination of human-specific (hNUMA, hMOC, hNSE, hSYN) or nonspecific (DCX, MAP2, GABA, CHAT) antibodies. The total number of surviving cells was estimated using stereological quantification. During the first 12-24 h after cell grafting, a modest motor weakness was observed in three of eight animals but was no longer present at 4 days to 7 weeks. No sensory dysfunction was seen at any time point. Postmortem MRI scans revealed the presence of the individual grafts in the targeted spinal cord areas. Histological examination of spinal cord sections revealed the presence of hNUMA-immunoreactive grafted cells distributed between the base of the dorsal horn and the ventral horn. In all grafts intense hMOC, DCX, and hSYN immunoreactivity in grafted cells was seen. In addition, a rich axodendritic network of DCX-positive processes was identified extending 300-700 μm from the grafts. On average, 45% of hNUMA-positive neurons were GABA immunoreactive. Stereological analysis of hNUMA-positive cells showed an average of 2.5- to 3-fold increase in number of surviving cells compared with the number of injected cells. Analysis of spinal structural morphology showed that in animals injected with more than 50,000 cells/injection or volumes of injectate higher than 6 μl/injection there was tissue expansion and disruption of the local axodendritic network. Based on these data the safe total number of injected cells and volume of injectate were determined to be 30,000 cells delivered in ≤6 μl of media. These data demonstrate that highly reproducible delivery of a potential cell therapeutic candidate into spinal parenchyma can be achieved across a wide range of cell doses by direct intraspinal injections. The resulting grafts uniformly showed robust cell survival and progressive neuronal maturation.
- MeSH
- buňky - růstové procesy fyziologie MeSH
- imunosupresivní léčba MeSH
- lidé MeSH
- mícha cytologie chirurgie MeSH
- miniaturní prasata MeSH
- myši MeSH
- nervové kmenové buňky cytologie transplantace MeSH
- prasata MeSH
- přežívání štěpu imunologie fyziologie MeSH
- reprodukovatelnost výsledků MeSH
- transplantace kmenových buněk metody MeSH
- viabilita buněk imunologie fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Background and Objectives Two-dimensional SDS PAGE (sodium dodecyl sulfate polyacrylamidegel electrophoresis) coupled with mass spectrometry is still a mainstream approach to analysing multiple protein expression levels. The requirement for some sophisticated devices and the lack of quantitative measurements for low-abundant proteins (e.g. cytokines) greatly limit its broad application. Cytokines present in the pg/ml levels in non-stimulated biological samples are traditionally detected by ELISA. We used a cytokine antibody array, a highly sensitive protein chip, for simultaneous detection of multiple cytokine expression levels in rat sarcoma lysates and serum samples . Material and methods We present here an optimized protocol for preparation and handling of tumour tissue lysates in protein chip detection. The sarcoma samples were processed at low temperatures to prevent cytokine degradation. Tumour cryosections (8?10 mm) were used for extraction of cytokines. The addition of NaN3 destroyed a high endogenous peroxidase activity, which may interfere with protein chip assay and decrease the signal/noise ratio. The data for the protein matrix effect from sandwich ELISA can also affect the protein chip detection. The optimal dilution of samples must be found to prevent pitfalls due to the non-optimal signal-to-noise ratio. This also enables recovery of low amounts of cytokines from difficult samples. Results We report optimized procedures for extraction, sample handling, inhibition of endogenous peroxidase activity and prevention of the protein matrix effect in serum and tumour lysates by detection of cytokine expression using the cytokine antibody array protein chip.
- MeSH
- aktivace makrofágů MeSH
- antigeny nádorové MeSH
- dendritické buňky imunologie MeSH
- financování organizované MeSH
- fúze buněk MeSH
- melanom imunologie terapie MeSH
- modely nemocí na zvířatech MeSH
- mutantní kmeny potkanů MeSH
- nádorové buňky kultivované imunologie MeSH
- prasata MeSH
- protinádorové vakcíny MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
The potential of novel scaffold containing sodium hyaluronate, type I collagen, and fibrin was investigated in the regeneration of osteochondral defects in miniature pigs. Both autologous chondrocyte-seeded scaffolds and non-seeded scaffolds were implanted into two defects located in the non-weight-bearing zone of the femoral trochlea (defect A was located more distally and medially, defect B was located more proximally and laterally). Control defects were left untreated. Twelve weeks after the operation, the knees were evaluated in vivo using MRI. Six months after the implantation, the defects were analyzed using MRI, histological, and immunohistochemical analysis. In the A defects of chondrocyte-seeded scaffold group, hyaline cartilage and fibrocartilage was formed, containing type II collagen, acidic and neutral glycosaminoglycans while the non-seeded scaffold group was predominantly filled with fibrocartilage. Defects in the control group were predominantly filled with fibrous tissue. Histomorphometric analysis of photomicrographs revealed a significantly higher amount of hyaline cartilage in the cell-seeded scaffold group in A defects than in other groups. Both scaffold groups in A defects showed significantly less fibrous tissue than cell-seeded defects B and the control group. Both histological and MRI analysis proved that the novel composite scaffold has a potential to regenerate osteochondral defects within six months.
- MeSH
- biokompatibilní materiály * MeSH
- časové faktory MeSH
- chondrocyty metabolismus transplantace MeSH
- fibrin chemie MeSH
- glykosaminoglykany metabolismus MeSH
- hyalinní chrupavka metabolismus patologie chirurgie MeSH
- imunohistochemie MeSH
- kolagen typ II metabolismus MeSH
- kolagen typu I chemie MeSH
- kolenní kloub u koně, psa metabolismus patologie patofyziologie chirurgie MeSH
- kultivované buňky MeSH
- kyselina hyaluronová chemie MeSH
- magnetická rezonanční tomografie MeSH
- miniaturní prasata MeSH
- modely nemocí na zvířatech MeSH
- nemoci chrupavky metabolismus patologie patofyziologie chirurgie MeSH
- prasata MeSH
- tkáňové inženýrství MeSH
- tkáňové podpůrné struktury * MeSH
- vazivová chrupavka metabolismus patologie chirurgie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Computed tomography (CT) is an effective diagnostic modality for three-dimensional imaging of bone structures, including the geometry of their defects. The aim of the study was to create and optimize 3D geometrical and real plastic models of the distal femoral component of the knee with joint surface defects. Input data included CT images of stifle joints in twenty miniature pigs with iatrogenic osteochondrosis-like lesions in medial femoral condyle of the left knee. The animals were examined eight and sixteen weeks after surgery. Philips MX 8000 MX and View workstation were used for scanning parallel plane cross section slices and Cartesian discrete volume creation. On the average, 100 slices were performed in each stifle joint. Slice matrices size was 512 x 512 with slice thickness of 1 mm. Pixel (voxel) size in the slice plane was 0.5 mm (with average accuracy of +/-0.5 mm and typical volume size 512 x 512 x 100 voxels). Three-dimensional processing of CT data and 3D geometrical modelling, using interactive computer graphic system MediTools formerly developed here, consisted of tissue segmentation (raster based method combination and 5 % of manual correction), vectorization by the marching-cubes method, smoothing and decimation. Stifle- joint CT images of three individuals of different body size (small, medium and large) were selected to make the real plastic models of their distal femurs from plaster composite using rapid prototyping technology of Zcorporation. Accuracy of the modeling was +/- 0.5 mm. The real plastic models of distal femurs can be used as a template for developing custom made press and fit scaffold implants seeded with mesenchymal stem cells that might be subsequently implanted into iatrogenic joint surface defects for articular cartilage-repair enhancement.
- MeSH
- anatomické modely * MeSH
- design s pomocí počítače MeSH
- femur radiografie MeSH
- kolenní kloub u koně, psa radiografie MeSH
- kultivované buňky MeSH
- mezenchymální kmenové buňky * MeSH
- miniaturní prasata MeSH
- modely nemocí na zvířatech MeSH
- osteochondritida radiografie MeSH
- počítačová rentgenová tomografie * MeSH
- prasata MeSH
- protézy - design MeSH
- rentgenový obraz - interpretace počítačová MeSH
- tkáňové inženýrství * MeSH
- tkáňové podpůrné struktury * MeSH
- zobrazování trojrozměrné * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The aim of our study was to examine in vivo and in vitro cytokines produced by Lewis ratderived R5-28 sarcoma cells. These cells produce rapidly growing tumours in approximately two weeks after subcutaneous inoculation. However, spontaneous tumour regression was noted in about 40% of animals. For an explanation of this phenomenon, we evaluated the profile of 19 cytokines during tumour growth and spontaneous regression by the use of "antibody array". To detect cytokines directly originated by the sarcoma, the R5-28 cells were cultivated in vitro and then both the supernatants and the cell lysates were analysed. Our experiments showed three cytokines (MCP-1, TIMP-1 and VEGF) to be produced by R5-28 cells in vitro. Moreover, in vivo, another three cytokines (TNF-alpha, beta-NGF and LIX) were detected both in blood sera and tumour lysates, probably produced by immune and stromal cells during tumour growth. Changes in their expression after spontaneous regression are discussed.
Corneal allograft rejection is frequently studied in small rodent or rabbit models. To study mechanisms of rejection in a model that more closely mimics transplantation in humans, we performed orthotopic corneal transplantation in the miniature pig using a 7-mm diameter donor graft. Four groups of recipients were studied: 1) untreated naive, 2) untreated vascularized (high risk), 3) high-risk grafts treated by topical application of prednisolone, or 4) high-risk grafts treated with a combined systemic immunosuppression regime of oral prednisone, cyclosporine A, and mycophenolate mofetil. Both the clinical features and histological assessment of corneal graft rejection showed close similarities to graft rejection in humans. Interestingly, preliminary results indicated that topical steroid treatment was superior to systemic immunosuppression in significantly promoting graft survival. Thus, corneal transplantation in the pig represents an animal model most closely resembling corneal grafting in humans, and offers possibilities for testing various clinically applicable immunosuppressive treatments.
- MeSH
- financování organizované MeSH
- imunosupresiva terapeutické užití MeSH
- kombinovaná farmakoterapie MeSH
- miniaturní prasata MeSH
- modely u zvířat MeSH
- prasata MeSH
- přežívání štěpu účinky léků MeSH
- rejekce štěpu prevence a kontrola MeSH
- transplantace rohovky imunologie metody MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
