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...

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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.

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.

Gál P., A. Neãas, L. Plánka, H. Kecová, L. Kfien, P. Krupa, J. Hluãilová, D. Usvald: Chondrocytic Potential of Allogenic Mesenchymal Stem Cells Transplanted without Immunosuppression to Regenerate Physeal Defect in Rabbits. Acta Vet. Brno 2007, 76: 265-275.Mesenchymal stem cells (MSCs) from bone marrow are multipotent cells capable of forming cartilage, bone, and other connective tissues. The objective of this study was to determine whether the use of allogenic mesenchymal stem cells could functionally heal a defect in the distal femoral physis in rabbits without the use of immunosuppressive therapy.A iatrogenic defect was created in the lateral femoral condyle of thirty-two New Zealand white rabbits, 7 weeks old, weighing 2.25 ± 0.24 kg. Each defect, 3.5 mm in width and 12 mm in length, in the right distal femoral physis was treated with allogenic mesenchymal stem cells in new composite hyaluronate/collagen type I/fibrin scaffold. The healing response was evaluated radiographically, by MRI (three weeks and four months after implantation) and also histologically, by Pearl's reaction and with immunofluorescence (four months after implantation). The results were compared with the data for the control defects (without stem cell implantation) in left distal femoral physes.On average, right femurs with a damaged distal physis and transplanted MSCs grew more in length (0.55 ± 0.21 cm) compared with left femurs with a physeal defect without stem cell transplantation (0.46 ± 0.23 cm). Valgus deformity of right femurs with a physeal defect and transplanted MSCs was mild (0.2 ± 0.1 °). On the contrary, left femurs with a physeal defect without transplanted MSCs showed a significant valgus deformity (2.7 ± 1.6 °). For defects treated with allogenic mesenchymal stem cell implants, no adverse immune response and implant rejection were detected in this model. Histologically, no lymphocytic infiltration occurred. At four months after transplantation, hyaline cartilage had formed throughout the defects treated with allogenic MSCs. Labelled mesenchymal stem cells/differentiated chondrocytes were detected in the physeal defects based on magnetic resonance imaging and immunofluorescence.The results of this study demonstrated that allogenic mesenchymal stem cells in a new composite hyaluronate/collagen type I/fibrin scaffold repaired iatrogenic defects in the distal femoral physes in rabbits without the use of immunosuppressive therapy. The use of allogenic mesenchymal stem cells for the repair of physeal defects may be an alternative to autologous MSCs transplantation. An allogenic approach would enable mesenchymal stem cells to be isolated from any donor, providing a readily available source of cells for cartilage tissue repair. Growth plate injury, bone bridge, limb deformity, physeal repair, rejectionTrauma is a typical cause of defect in growth cartilage and the subsequent bone bridge formation. The standard surgical treatment method for bone growth defects due to closure of physeal growth cartilage consists of the res...