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In this study, the aligned (A) and randomly oriented (R) polycaprolactone (PCL-A and PCL-R) and PCL/collagen (PCL/Col-A and PCL/Col-R) nanofibers were electrospun onto smooth PCL membranes (PCLMs) prepared by solvent casting. In order to investigate the effects of chemical composition and nanotopography of fibrous surfaces on proliferation and on neural differentiation of mesenchymal stem cells (MSCs), adipose and bone marrow-derived rat MSCs (AdMSCs and BMSCs) were cultivated in suitable media i.e. inducing medium containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), and cell maintenance medium (CMM). BMSCs adhered and proliferated on all nanofibrous membranes more efficiently than AdMSCs. PCL/Col-A was found as the most convenient surface supporting proliferation in both cell types. Immunofluorescence staining indicated that BMSCs and AdMSCs are prone for differentiation to oligodendrocytes more than they differentiate to other neuronal cell types. PCL-A nanofibrous membranes supported differentiation of MSCs to O4(+) (an oligodendrocytes surface antigen) cells in both culture media. The intensity of immunoreactivity of O4(+) cells differentiated from BMSCs on PCL-A was highest when compared with the other groups (p < 0.001). Some BIII-T signed neural cells were investigated on PCL-A nanofibrous membranes, but the intensity of immunoreactivity was lower than that of O4(+) cells. In conclusion, this study can be evaluated to establish the cell therapy strategies in neurodegenerative disorders, which are relevant to oligodendrocyte abstinence using BMSCs or AdMSCs on aligned nanofibrous membranes.

We investigated the effects of different implant biomaterials on cultured canine bone marrow stromal cells (BMSC) undergoing differentiation into osteoblasts (dBMSC). BMSC were isolated from canine humerus by marrow aspiration, cultured and differentiated on calcium phosphate scaffold (CPS), hydroxyapatite, hydroxyapatite in gel form and titanium mesh. We used the MTT method to determine the effects of osteogenic media on proliferation. The characteristics of dBMSC were assessed using alizarin red (AR), immunocytochemistry and osteoblastic markers including alkaline phosphatase/von Kossa (ALP/VK), osteocalcin (OC) and osteonectin (ON), and ELISA. The morphology of dBMSC on the biomaterials was investigated using inverted phase contrast microscopy and scanning electron microscopy. We detected expression of ALP/VK, AR, OC and ON by day 7 of culture; expression increased from day 14 until day 21. CPS supported the best adhesion, cell spreading, proliferation and differentiation of BMSCs. The effects of the biomaterials depended on their surface properties. Expression of osteoblastic markers showed that canine dBMSCs became functional osteoblasts. Tissue engineered stem cells can be useful clinically for autologous implants for treating bone wounds.

Bone marrow contains mesenchymal stem cells that form many tissues. Various scaffolds are available for bone reconstruction by tissue engineering. Osteoblastic differentiated bone marrow stromal cells (BMSC) promote osteogenesis on scaffolds and stimulate bone regeneration. We investigated the use of cultured autologous BMSC on different scaffolds for healing defects in tibias of adult male canines. BMSC were isolated from canine humerus bone marrow, differentiated into osteoblasts in culture and loaded onto porous ceramic scaffolds including hydroxyapatite 1, hydroxyapatite gel and calcium phosphate. Osteoblast differentiation was verified by osteonectine and osteocalcine immunocytochemistry. The scaffolds with stromal cells were implanted in the tibial defect. Scaffolds without stromal cells were used as controls. Sections from the defects were processed for histological, ultrastructural, immunohistochemical and histomorphometric analyses to analyze the healing of the defects. BMSC were spread, allowed to proliferate and differentiate to osteoblasts as shown by alizarin red histochemistry, and osteocalcine and osteonectine immunostaining. Scanning electron microscopy showed that BMSC on the scaffolds were more active and adhesive to the calcium phosphate scaffold compared to the others. Macroscopic bone formation was observed in all groups, but scaffolds with stromal cells produced significantly better results. Bone healing occurred earlier and faster with stromal cells on the calcium phosphate scaffold and produced more callus compared to other scaffolds. Tissue healing and osteoblastic marker expression also were better with stromal cells on the scaffolds. Increased trabecula formation, cell density and decreased fibrosis were observed in the calcium phosphate scaffold with stromal cells. Autologous cultured stromal cells on the scaffolds were useful for healing of canine tibial bone defects. The calcium phosphate scaffold was the best for both cell differentiation in vitro and bone regeneration in vivo. It may be possible to improve healing of bone defects in humans using stem cells from bone marrow.

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