Osteogenically differentiated cell sheet techniques using mesenchymal stem cells (MSCs) are available to stimulate bone regeneration. The advantage of the cell sheet technique is delivering live cells effectively into the focal region. We developed a novel osteogenic cell sheet technique by adding gelatin to osteogenic cell medium. Gelatin-induced osteogenic cell sheets (GCSs) were compared to conventional osteogenic cell sheets (OCSs). Undifferentiated MSCs (UCs) were used as a control. The morphology of these cell sheets was evaluated microscopically and histologically. The time-dependent cell proliferation rate was estimated by DNA quantification. The expression of osteogenic gene markers and the number of calcium depositions were assessed by quantitative real-time polymerase chain reaction and Alizarin red S (ARS) staining, respectively. GCSs were thicker and stronger than OCSs. GCSs showed a significantly higher cell proliferation rate compared to OCSs (p < 0.05). GCSs exhibited significantly higher upregulation of BMP-7 mRNA compared to OCSs (p < 0.05). Both GCSs and OCSs showed negative ARS reactivity on day 10, but only GCSs showed positive ARS reactivity on day 21. With this technique, we observed active cell proliferation with abundant ECM and upregulation of osteogenic bone markers, and our results suggest that GCSs could be promising for therapeutic applications in bone regeneration.

Heme oxygenase-1 (HO-1) is a stress-responsive enzyme that modulates the immune response and oxidative stress associated with spinal cord injury (SCI). This study aimed to investigate neuronal regeneration via transplantation of mesenchymal stromal cells (MSCs) overexpressing HO-1. Canine MSCs overexpressing HO-1 were generated by using a lentivirus packaging protocol. Eight beagle dogs with experimentally-induced SCI were divided into GFP-labeled MSC (MSC-GFP) and HO-1-overexpressing MSC (MSC-HO-1) groups. MSCs (1 × 107 cells) were transplanted at 1 week after SCI. Spinal cords were harvested 8 weeks after transplantation, after which histopathological, immunofluorescence, and western blot analyses were performed. The MSC-HO-1 group showed significantly improved functional recovery at 7 weeks after transplantation. Histopathological results showed fibrotic changes and microglial cell infiltration were significantly decreased in the MSC-HO-1 group. Immunohistochemical (IHC) results showed significantly increased expression levels of HO-1 and neuronal markers in the MSC-HO-1 group. Western blot results showed significantly decreased expression of tumor necrosis factor-alpha, interleukin-6, cycloogygenase 2, phosphorylated-signal transducer and activator of transcription 3, and galactosylceramidase in the MSC-HO-1 group, while expression levels of glial fibrillary acidic protein, β3-tubulin, neurofilament medium, and neuronal nuclear antigen were similar to those observed in IHC results. Our results demonstrate that functional recovery after SCI can be promoted to a greater extent by transplantation of HO-1-overexpressing MSCs than by normal MSCs.

Abundant expression of proinflammatory cytokines after a spinal cord injury (SCI) creates an inhibitory microenvironment for neuroregeneration. The mesenchymal stem cells help to mitigate the inflammation and improve neural growth and survival. For this purpose, we potentiated the function of adipose-derived mesenchymal stem cells (Ad-MSCs) by transfecting them with brain-derived neurotrophic factor (BDNF) and heme oxygenase-1 (HO-1), through a lentivirus, to produce BDNF overexpressed Ad-MSCs (BDNF-MSCs), and HO-1 overexpressed Ad-MSCs (HO-1-MSCs). Sixteen SCI beagle dogs were randomly assigned into four treatment groups. We injected both HO-1 and BDNF-overexpressed MSCs as a combination group, to selectively control inflammation and induce neuroregeneration in SCI dogs, and compared this with BDNF-MSCs, HO-1-MSCs, and GFP-MSCs injected dogs. The groups were compared in terms of improvement in canine Basso, Beattie, and Bresnahan (cBBB) score during 8 weeks of experimentation. After 8 weeks, spinal cords were harvested and subjected to western blot analysis, immunofluorescent staining, and hematoxylin and eosin (H&E) staining. The combination group showed a significant improvement in hindlimb functions, with a higher BBB score, and a robust increase in neuroregeneration, depicted by a higher expression of Tuj-1, NF-M, and GAP-43 due to a decreased expression of the inflammatory markers interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), and an increased expression of interleukin-10 (IL-10) (P ≤ 0.05). H&E staining showed more reduced intraparenchymal fibrosis in the combination group than in other groups (P ≤ 0.05). It was thus suggested that the cotransplantation of HO-1 and BDNF-MSCs is more effective in promoting the healing of SCI. HO-1-MSCs reduce inflammation, which favors BDNF-induced neuroregeneration in SCI of dogs.

Owing to the antioxidant and anti-inflammatory functions of hemeoxygenase-1 (HO-1), HO-1-expressing canine adipose-derived mesenchymal stem cells (Ad-MSCs) could be efficacious in treating spinal cord injury (SCI). Further, frozen thawed HO-1 Ad-MSCs could be instantly available as an emergency treatment for SCI. We compared the effects of intravenous treatment with freshly cultured HO-1 Ad-MSCs (HO-1 MSCs), only green fluorescent protein-expressing Ad-MSCs (GFP MSCs), and frozen thawed HO-1 Ad-MSCs (FT-HO-1 MSCs) in dogs with acute SCI. For four weeks, dogs were evaluated for improvement in hind limb locomotion using a canine Basso Beattie Bresnahan (cBBB) score. Upon completion of the study, injured spinal cord segments were harvested and used for western blot and histopathological analyses. All cell types had migrated to the injured spinal cord segment. The group that received HO-1 MSCs showed significant improvement in the cBBB score within four weeks. This group also showed significantly higher expression of NF-M and reduced astrogliosis. There was reduced expression of proinflammatory cytokines (IL6, TNF-α, and IL-1β) and increased expression of anti-inflammatory markers (IL-10, HO-1) in the HO-1 MSC group. Histopathological assessment revealed decreased fibrosis at the epicenter of the lesion and increased myelination in the HO-1 MSC group. Together, these data suggest that HO-1 MSCs could improve hind limb function by increasing the anti-inflammatory reaction, leading to neural sparing. Further, we found similar results between GFP MSCs and FT-HO-1 MSCs, which suggest that FT-HO-1 MSCs could be used as an emergency treatment for SCI.

Cell sheets technology is being available for fracture healing. This study was performed to clarify bone healing mechanism of undifferentiated (UCS) and osteogenic (OCS) differentiated mesenchymal stromal cell (MSC) sheets in the fracture model of dogs. UCS and OCS were harvested at 10 days of culture. Transverse fractures at the radius of six beagle dogs were assigned into three groups (n = 4 in each group) i.e. UCS, OCS and control. The fractures were fixed with a 2.7 mm locking plate and six screws. Cell sheets were wrapped around the fracture site. Bones were harvested 8 weeks after operation, then scanned by micro-computed tomography (micro-CT) and analyzed histopathologically. The micro-CT revealed different aspects of bone regeneration among the groups. The percentages of external callus volume out of total bone volume in control, UCS, and OCS groups were 42.1, 13.0 and 4.9% (p \ 0.05) respectively. However, the percentages of limbs having connectivity of gaps were 25, 12.5 and 75% respectively. In histopathological assessments, OCS group showed well organized and mature woven bone with peripheral cartilage at the fracture site, whereas control group showed cartilage formation without bone maturation or ossification at the fracture site. Meanwhile, fracture site was only filled with fibrous connective tissue without endochondral ossification and bone formation in UCS group. It was suggested that the MSC sheets reduced the quantity of external callus, and OCS induced the primary bone healing.