Xenogenic Implantation of Human Mesenchymal Stromal Cells Using a Novel 3D-Printed Scaffold of PLGA and Graphene Leads to a Significant Increase in Bone Mineralization in a Rat Segmental Femoral Bone Defect

Abstract

Tissue-engineering technologies have the potential to provide an effective approach to bone regeneration. Based on the published literature and data from our laboratory, two biomaterial inks containing PLGA and blended with graphene nanoparticles were fabricated. The biomaterial inks consisted of two forms of commercially available PLGA with varying ratios of LA:GA (65:35 and 75:25) and molecular weights of 30,000-107,000. Each of these forms of PLGA was blended with a form containing a 50:50 ratio of LA:GA, resulting in ratios of 50:65 and 50:75, which were subsequently mixed with a 0.05 wt% low-oxygen-functionalized derivative of graphene. Scanning electron microscopy showed interconnected pores in the lattice structures of each scaffold. The cytocompatibility of human ADMSCs transduced with a red fluorescent protein (RFP) was evaluated in vitro. The in vivo biocompatibility and the potential to repair bones were evaluated in a critically sized 5 mm mechanical load-bearing segmental femur defect model in rats. Bone repair was monitored by radiological, histological, and microcomputed tomography methods. The results showed that all of the constructs were biocompatible and did not exhibit any adverse effects. The constructs containing PLGA (50:75)/graphene alone and with hADMSCs demonstrated a significant increase in mineralized tissues within 60 days post-treatment. The percentage of bone volume to total volume from microCT analyses in the rats treated with the PLGA + cells construct showed a 50% new tissue formation, which matched that of a phantom. The microCT results were supported by Von Kossa staining.

Keywords: 3D bioprinting; PLGA blends; additive manufacturing; poly(lactic-co-glycolic acid); rat femur.

Figure 1

Biofabrication design. Slicer software showing (A) front, (B) bottom, and (C) oblique views of the printed scaffold.

Figure 2

Scanning electron micrograph images of (A) a blend of 50:50 and 65:35 poly(lactic-co-glycolic acid) (50:65 PLGA) with 0.05 wt% low-oxygen graphene (LOG) and (B) a blend of 50:50 and 75:25 poly(lactic-co-glycolic acid) (50:75 PLGA) with 0.05 wt% low-oxygen graphene (LOG).

Figure 3

Adhesion and proliferation of hADMSCs. Red fluorescent protein-transduced human ADMSCs adhering, proliferating, and clustering on discrete areas on (A) the scaffold of 50:50 and 65:35 poly(lactic-co-glycolic acid) (50:65 PLGA) with 0.05 wt% low-oxygen graphene (LOG) and (B) the scaffold of 50:50 and 75:25 poly(lactic-co-glycolic acid) (50:75 PLGA) with 0.05 wt% low-oxygen graphene (LOG) on day 1 (a), day 7 (b), day 14 (c), and day 21 (d) post-seeding. On day 1, adherence was noted, followed by proliferation (indicated by an increase in cell density) and cell-to-cell communication (indicated by cell clustering) on days 7, 14, and 21.

Figure 4

Radiography. Representative images of the rat femoral defects treated with the two scaffolds of poly(lactic-co-glycolic acid and low oxygen graphene (PLGA + LOG) alone ((A)—acellular) and with 1 × 106 hAD-MSCs ((B)—cellular) at days 1 and 60.The arrows show the defect at day 1, which is filled by day 60. The scaffolds are radiopaque and hence are not visible in the radiographs.

Figure 5

MicroCT data analysis. Graphical representation of percent bone volume/total volume (%BV/TV) from the microCT scans of all the rat femurs. Data is reported as a function of individua threshold Hounsfeld units. As described in the Section 3 these data are significant in the 105–255 unit range, which denotes 50% new bone and, which is closely matched to that of the phantom. Asterisks denote significant increase in the %BV/TV in rats treated with 50:75 + MSCs compared to those treated with 50:75 alone. Similarly, there is a significant increase in %BV/TV in rats treated with 50:75 + MSCs compared to those treated with 50:65 + MSCS.

Figure 6

Histological analyses. Representative images showing the Von Kossa staining of the region of interest (ROl) for a rat treated with (A) acellular i.e., PLGA 50:75 scaffold alone, and (B) PLGA 50:75 + 1 million hAD-Mscs. The RO is shown as a red box. Note the black staining indicating new mineralized tissue in the defect. Note that the medullary cavity as visualized with the screw marks. is filled with new bone in (B).