Bone regeneration in Ds-Red pig calvarial defect using allogenic transplantation of EGFP-pMSCs – A comparison of host cells and seeding cells in the scaffold

Abstract

Background Cells, scaffolds, and factors are the triad of regenerative engineering; however, it is difficult to distinguish whether cells in the regenerative construct are from the seeded cells or host cells via the host blood supply. We performed a novel in vivo study to transplant enhanced green fluorescent pig mesenchymal stem cells (EGFP-pMSCs) into calvarial defect of DsRed pigs. The cell distribution and proportion were distinguished by the different fluorescent colors through the whole regenerative period. Method/Results Eight adult domestic Ds-Red pigs were treated with five modalities: empty defects without scaffold (group 1); defects filled only with scaffold (group 2); defects filled with osteoinduction medium-loaded scaffold (group 3); defects filled with 5 x 10 3 cells/scaffold (group 4); and defects filled with 5 x 10 4 cells/scaffold (group 5). The in vitro cell distribution, morphology, osteogenic differentiation, and fluorescence images of groups 4 and 5 were analyzed. Two animals were sacrificed at 1, 2, 3, and 4 weeks after transplantation. The in vivo fluorescence imaging and quantification data showed that EGFP-pMSCs were represented in the scaffolds in groups 4 and 5 throughout the whole regenerative period. A higher seeded cell density resulted in more sustained seeded cells in bone regeneration compared to a lower seeded cell density. Host cells were recruited by seeded cells if enough space was available in the scaffold. Host cells in groups 1 to 3 did not change from the 1st week to 4th week, which indicates that the scaffold without seeded cells cannot recruit host cells even when enough space is available for cell ingrowth. The histological and immunohistochemical data showed that more cells were involved in osteogenesis in scaffolds with seeded cells. Conclusion Our in vivo results showed that more seeded cells recruit more host cells and that both cell types participate in osteogenesis. These results suggest that scaffolds without seeded cells may not be effective in bone transplantation.

Osteogenic and Fluorescent Characterization of Pig-Derived Mesenchymal Stem Cells Between eGFP Transgenesis and Non-viral Plasmid Transfection

“…These have been proven to exhibit homogeneous surface epitopes and possess classic trilineage differentiation potential into osteogenic, adipogenic, and chondrogenic lineages, and with maintenance of robust eGFP expression in all differentiated progeny (10). Increasing green uorescence was observed from day 3 to day 28 after cells were seeded to the scaffolds, and following transplantation to the pig's calvarial defect (19).…”

“…The OIM medium was composed of complete MEM-α medium enriched with 10 −7 M dexamethasone (Sigma), 10 mM β-glycerophosphate (Sigma) and 50 μg/mL ascorbic acid (Sigma). (19) (20) The OIM medium was replenished every two or three days for a total of 7 days.…”

“…The quantitative distribution of the green uorescent cells was analyzed using NIH Image J software (National Institutes of Health, USA) and presented as the modi ed integrated density (Modi ed IntDen). The modi ed integrated density was calculated based on the following equation: [Selected Integrated Density -(Area of Selected Cell × Mean Fluorescence of Background Beadings]) (19). The images merged form light microscope and uorescent microscope samples were also investigated.…”

“…A hemostatic gelatin sponge, Spongostan TM (Ferrosan Medical Device, MS0003, thickness 0.1 cm), was used as the 3D scaffold. (19) To perform transplantation, the scaffolds were cut into disks with a diameter of 0.8 cm, sterilized by 75% (v/v) ethanol and washed three times with phosphate-buffered saline. The sterile scaffold disks were then immersed in Opti-MEM medium (Gibco) before use.…”

“…The OIM medium was composed of complete MEM-α medium enriched with 10 -7 M dexamethasone (Sigma), 10 mM βglycerophosphate (Sigma) and 50 μg/mL ascorbic acid (Sigma). (19) The OIM medium was replenished every two or three days for a total of 7 days. The constructs in each time point were washed twice with phosphate buffered saline (PBS; GIBCO™, Invitrogen Corp., Carlsbad, CA) and fixed in 1.5% v/v glutaraldehyde in 0.14M sodium cacodylate (pH 7.4) for 30 mins at room temperature.…”