Evaluation of 3D-Printed Polycaprolactone Scaffolds Coated with Freeze-Dried Platelet-Rich Plasma for Bone Regeneration

Abstract

Three-dimensional printing is one of the most promising techniques for the manufacturing of scaffolds for bone tissue engineering. However, a pure scaffold is limited by its biological properties. Platelet-rich plasma (PRP) has been shown to have the potential to improve the osteogenic effect. In this study, we improved the biological properties of scaffolds by coating 3D-printed polycaprolactone (PCL) scaffolds with freeze-dried and traditionally prepared PRP, and we evaluated these scaffolds through in vitro and in vivo experiments. In vitro, we evaluated the interaction between dental pulp stem cells (DPSCs) and the scaffolds by measuring cell proliferation, alkaline phosphatase (ALP) activity, and osteogenic differentiation. The results showed that freeze-dried PRP significantly enhanced ALP activity and the mRNA expression levels of osteogenic genes (ALP, RUNX2 (runt-related gene-2), OCN (osteocalcin), OPN (osteopontin)) of DPSCs (p < 0.05). In vivo, 5 mm calvarial defects were created, and the PRP-PCL scaffolds were implanted. The data showed that compared with traditional PRP-PCL scaffolds or bare PCL scaffolds, the freeze-dried PRP-PCL scaffolds induced significantly greater bone formation (p < 0.05). All these data suggest that coating 3D-printed PCL scaffolds with freeze-dried PRP can promote greater osteogenic differentiation of DPSCs and induce more bone formation, which may have great potential in future clinical applications.

Keywords: 3D-printed scaffold; bone regeneration; platelet-rich plasma; polycaprolactone.

Figure 1

An overview of the in vivo bone regeneration of the calvarial defects.

Figure 2

Characterization of 3D-printed polycaprolactone (PCL) scaffolds before (a,c,e) and after (b,d,f) treatment with ethanolic sodium hydroxide. At a higher magnification, we can see the roughness and micro-size pores (d,f). The magnification levels were ×100 (a,b), ×500 (c,d) and ×30,000 (e,f).

Figure 3

Scanning electron microscope (SEM) microphotographs of freeze-dried platelet-rich plasma polycaprolactone (PRP-PCL) scaffolds (a,d,g), traditional PRP-PCL scaffolds (b,e,h), and bare PCL scaffolds (c,f,i) at ×200, ×800, and ×3000 magnification. PRP could be seen after coating with freeze-dried PRP-PCL scaffolds (a,d,g) or traditional PRP-PCL scaffolds (b,e,h). Randomly distributed PRP are visible around the surface of the scaffolds, while no PRP are visible on the bare PCL scaffolds (c,f,i).

Figure 4

Cell attachment on the freeze-dried PRP-PCL scaffolds (a–c); traditional PRP-PCL scaffolds (d–f); bare PCL scaffolds (g–i) after three days of seeding; all images shown at ×50 magnification (* p < 0.05, ** p < 0.01) and (j) The number of the cell attachment.

Figure 5

Number of migrated cells in the (a) freeze-dried PRP-PCL scaffold group; (b) traditional PRP-PCL scaffold group; (c) bare PCL scaffold group after 12 h of seeding; all images shown at ×50 magnification (** p < 0.01) and (d) The number of the cell migration.

Figure 6

(a) Cell proliferation on the freeze-dried PRP-PCL scaffolds, traditional PRP-PCL scaffolds and bare PCL scaffolds 1, 3, 5, and 7 days after seeding; (b) ALP activity of the freeze-dried PRP-PCL scaffolds, traditional PRP-PCL scaffolds and bare PCL scaffolds seven and 14 days after seeding (* p < 0.05, ** p < 0.01).

Figure 7

Expression of bone-specific genes, RUNX2, ALP, OPN, and OCN on the freeze-dried PRP-PCL scaffold, traditional PRP-PCL scaffold and bare PCL scaffold (* p < 0.05, ** p < 0.01).

Figure 8

Evaluation of bone formation in calvarial defects via micro-CT. Representative micro-CT images of calvarial defects showing mineralized bone formation after treatment with freeze-dried PRP-PCL scaffolds (a,d,g,j), traditional PRP-PCL scaffolds (b,e,h,k) and bare PCL scaffolds (c,f,i,l). The bone tissue in the circle represents the regenerated bone. (m) Regenerated bone formation rate at 2, 4, 8 and 12 weeks after scaffold implantation (* p < 0.05, ** p < 0.01).

Figure 9

Histological sections stained with H&E showing calvarial defects treated with freeze-dried PRP-PCL scaffold (a1–c1,a2–c2,a3–c3), traditional PRP-PCL scaffold (d1–f1,d2–f2,d3–f3) and bare PCL scaffold (g1–i1,g2–i2,g3–i3) at 4, 8, and 12 weeks after implantation. The bone-like tissues between the dashed lines are newly formed bone. Empty circles are the scaffold locations. (j) New bone formation rate at 4, 8, and 12 weeks after scaffold implantation (* p < 0.05, ** p < 0.01).