Histopathology and immunocytochemistry

Spinal cord sections from animals of all groups were prepared for histopathologic evaluation with hematoxylin & eosin (H&E) and solvent blue. A qualitative analysis confirmed the complete degradation of the PLGA scaffold in vivo, which confirmed that the PLGA scaffold was biocompatible and biodegradable. The scaffold degradation was further investigated in a subsequent experiment. In a quantitative assessment (Fig. 9A &B) of the tissue

sparing in 3 representative spinal cords from each group (i.e., tissue from SCI rats with BBB scores closest to the group mean) the group treated with scaffolded hMSCs showed the most extensive tissue sparing: the mean lesion volume was 8.76 mm³ in the lesion-alone group as compared to 3.99 mm³ in the scaffold+hMSC group, which represents a 54% reduction in tissue degeneration (p<0.05). The scaffold+hMSC treatment group also tended to show an increase in the spared white matter relative to the means from the three control groups (Fig. 9C), although this increase did not reach statistical significance. To examine the differential sparing and/or sprouting of motor neurons in the anterior horns of the peri-epicenter spinal cord tissues, we manually identified and counted the motor neurons in 1-mm intervals rostral and caudal to the injury epicenter in the anterior horns of representative spinal cords from each group (H&E and solvent blue staining). Although the difference did not reach statistical significance, the absolute number of motor neurons caudal to the injury epicenter was higher in the spinal cords treated with scaffolded hMSCs than in the other groups, which supports the observed functional motor improvement in the treated animals (Fig. 9D).

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Figure 9. Histopathological analyses: A) Hematoxylin & eosin and solvent blue staining of transverse sections from a representative spinal cord each study group.

B) Quantification of lesion volume and C) white matter sparing around the epicentre. The average total lesion volume in scaffold plus hMSCs group is significantly smaller than that in the other groups (p<o.o5, one way ANOVA). D) Quantification of motor neurons in the ventral horns of the injured spinal cord demonstrates that the average number of surviving motor neurons is higher in the scaffold plus hMSCs group than that in the other groups.

Immunocytochemistry was performed to characterize the molecular mechanisms potentially underlying the functional benefits of the scaffold and hMSC constructs. Endogenous stem cell proliferation was significantly increased (p<0.05) in the scaffold+hMSC spinal cords relative to all three control conditions as measured by a 314% semi-quantitative increase in doublecortin staining (DCX; Fig. 10A) in the scaffold-hMSC group relative to the lesion-alone group. The enhanced proliferation of endogenous neural stem cells was also supported by immunostaining for rat nestin (data not shown). The

activation of endogenous stem cells by hMSCs has been previously demonstrated¹³; however, the large difference compared with animals treated with hMSCs alone animals suggests that the hMSCs were held in place by the scaffold to effectively influence endogenous neural progenitors, rather than being washed away in the interstitial and cerebrospinal fluid. The hMSC+scaffold treatment also provided increased angiogenesis; staining for laminin, an angiogenic marker, was nearly four-fold higher (p<0.05) than in lesion-alone group (Fig. 10B). Improved angiogenesis within and surrounding the injury epicenter should enhance the healing of the spinal cord and may contribute to the overall functional benefit of the combined construct.

The inflammatory cascade following spinal cord injury is complex and both contribute to secondary neural damage as well as regeneration^14,15. Immunomodulation has become a centerpiece to approaches for neuronal regeneration after CNS injury. We used hMSCs inherent ability to home to sites of injury and engender an environment to limit the deleterious effects of obligatory inflammation after SCI. Immunohistochemistry was also used to test the hypothesis that secondary injury and tissue damage were decreased after treatment with scaffolded hMSCs. Inflammatory markers were evaluated in transverse sections at and adjacent to the injury epicenter to characterize the acute and chronic anti-inflammatory effects of the treatment. The immunoreactivity for GFAP, a marker of astroglial scarring, was lowest in tissue from animals treated with scaffold hMSCs (Fig. 10C). The immunoreactivity

for nitrotyrosine (Fig. 10D), which is a marker of protein nitration that indicates oxidative damage resulting from reactive nitrogen or oxygen species, was reduced in spinal cords from animals treated with scaffolded hMSCs. The immunoreactivity for CD68 (Fig. 10E) and CD11b (not shown), which are markers of activated microglia/macrophages, was decreased in spinal cords treated with scaffolded hMSCs (p<0.05 against all other conditions), thereby indicating an anti-inflammatory effect against cells that may be involved in the evolution of secondary neural loss and degeneration following SCI.

Immunohistochemistry was also used to evaluate the expression of potential molecular mediators of hMSC-derived effects. BDNF expression was increased by two to three-fold (p<0.05) in the spinal cords of the animals receiving scaffolded hMSCs, and co-staining for DAPI and HSP27 confirmed that the BDNF expression was co-localized with the remaining implanted hMSCs.

Expression of IL-10, which is a cytokine that is primarily thought to have anti-inflammatory effects, was significantly higher in the scaffold + hMSC condition (p<0.05) than in any of the other conditions. Co-staining with DAPI and CD90, which is another hMSC marker), confirmed that the IL-10 was expressed by the remaining hMSCs (Fig. 10F).

The findings of the present study collectively demonstrate the effectiveness of the PLGA scaffold+hMSC construct in a rat model of spinal cord injury; however, this therapy would not be clinically feasible if the scaffolded cells eventually differentiate into a terminal mesenchymal lineage

such as collagen, lipid or bone. To support our early in vitro results, we immunohistochemically evaluated the expression of Col1 and Col2 (Fig. 10G) to determine the mesenchymal differentiation status of the implanted cells in the spinal cords after implantation. The lack of observed collagen expression suggests that the cells did not undergo differentiation to a terminal mesenchymal lineage, and this finding was confirmed with confocal microscopy.

To ensure that the construct did not develop into bone or fat, the lack of alkaline phosphatase (ALP) activity was confirmed immunohistochemically (Fig. H), and the lack of lipid accumulation was confirmed by Oil Red O staining (data not shown). Furthermore, analysis of donor hMSC survival was performed with co-staining for DAPI and either CD 90 or HSP (Fig. 10I). Cells were manually counted at each millimeter away from the injury epicenter. The trend of surviving CD 90 positive and HSP positive cells were consistent; the highest concentration remained at the epicenter, with a sharp decrease within 1-2 mm on either side. Interestingly, there was slightly better survival rostral to the epicenter than caudally.

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Figure 10. Immunohistochemistry. A) The activation of endogenous neural stem cells is detected by doublecortin (DCX) immunostaining. There appears to be more DCX-positive cells in the scaffold plus hMSCs treated spinal cords compared with the other groups (scale bar =50µm, p<0.05, one-way ANOVA).

B)Representative images of immunohistochemical staining using a laminin antibody show that there is an overall increase in angiogenic activity in tissue

area adjacent to the epicentre following the scaffold plus hMSCs compared with the other groups (scale bar=200µm, p<0.05, one-way ANOVA). C-E) The immunoreactivity of GFAP (C, glial fibrillary acidic protein, a marker for reactive astrogliosis), nitrotyrosine (D, a marker for secondary oxidative damage), and CD68 (E, a marker for inflammatory cell inflitratration) in the injured spinal cord stumps was reduced in the scaffold plus hMSCs group compared with the other groups, which indicates that the scaffold plus hMSCs, as an implant, is most effective for reducing the inflammatory response following spinal cord injury (scale bar=100µm, p<0.05, one-way ANOVA). F)hMSCs seeded in PLGA scaffold shows a higher level of immunoreactivity to antibody against BDNF, compared with hMSCs only. G) The expression of collagen I and II and alkaline phosphatise was not found in the scaffold plus hMSCs. I) Quantification of survived cells shows the highest concentration of cells in the scaffold plus hMSCs.

The lack of corticospinal tract neuronal regeneration is supported by data displayed in Figure 11 with BDA tracing and ICC analysis. We also injected lipophilic retrograde tracers into the cervical musculature (DiI) and thoracic intercostals musculature (Fast Blue, FB) to confirm synaptophysin activation around the propriospinal interneurons (Fig. 11). Furthermore, we performed synaptophysin ICC semiquantification at the cervical and lumbar levels of the treatment and lesion control spinal cords. At both levels, there was increased signal in the scaffold+hMSC treated animals, suggesting increased sprouting of propriospinal neurons both above and below the injury (Fig. 11). There is increased tracer uptake in the scaffold+hMSC group relative to the lesion

control group in lumbar spinal cord and they could represent areas of central pattern generator (CPG), which mechanistically supports the improved function in the scaffold+hMSC animals.

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Figure 11. Neural tracing. A) Anterograde tracer biotinylated dextran amine (BDA) injections 4 weeks after initial surgery. B) In both group (lesion alone and scaffold plus hMSCs treated group), no corticospinal tract regeneration was not found. C) Retrograde tracer DiI, fast blue, and fluorogold for identification of propriospinal interneurons. The immunoreactivity for synaptophysin (syn) was higher around protpriospinal interneurons in the scaffold plus hMSCs treated group compared with the lesion alone group in all the spinal cord.