Bone-targeted lipoplex-loaded three-dimensional bioprinting bilayer scaffold enhanced bone regeneration

Abstract

Clinical bone-morphogenetic protein 2 (BMP2) treatment for bone regeneration, often resulting in complications like soft tissue inflammation and ectopic ossification due to high dosages and non-specific delivery systems, necessitates research into improved biomaterials for better BMP2 stability and retention. To tackle this challenge, we introduced a groundbreaking bone-targeted, lipoplex-loaded, three-dimensional bioprinted bilayer scaffold, termed the polycaprolactone-bioink-nanoparticle (PBN) scaffold, aimed at boosting bone regeneration. We encapsulated BMP2 within the fibroin nanoparticle based lipoplex (Fibroplex) and functionalized it with DSS₆ for bone tissue-specific targeting. 3D printing technology enables customized, porous PCL scaffolds for bone healing and soft tissue growth, with a two-step bioprinting process creating a cellular lattice structure and a bioink grid using gelatin-alginate hydrogel and DSS₆-Fibroplex, shown to support effective nutrient exchange and cell growth at specific pore sizes. The PBN scaffold is predicted through in silico analysis to exhibit biased BMP2 release between bone and soft tissue, a finding validated by in vitro osteogenic differentiation assays. The PBN scaffold was evaluated for critical calvarial defects, focusing on sustained BMP2 delivery, prevention of soft tissue cell infiltration and controlled fiber membrane pore size in vivo. The PBN scaffold demonstrated a more than eight times longer BMP2 release time than that of the collagen sponge, promoting osteogenic differentiation and bone regeneration in a calvarial defect animal. Our findings suggest that the PBN scaffold enhanced the local concentration of BMP2 in bone defects through sustained release and improved the spatial arrangement of bone formation, thereby reducing the risk of heterotopic ossification.

Keywords: 3D bioprinting; BMP2; bone regeneration; bone-targeting lipoplex.

Graphical abstract

Figure 1.

Schematic description and fabrication of bone-targeting lipoplex-loaded 3D-printed scaffolds. (A) Schematic of the PBN scaffold. The PCL scaffold faces the soft tissue side, and the bioink containing the lipoplex is positioned toward the bone defect area. (B) Fabrication process and structure of the PBN scaffold. 3D, three-dimensional; PBN, polycaprolactone-bioink-nanoparticle; PCL, polycaprolactone.

Figure 2.

Characterization of bone-targeting functionalized fibroin particle encapsulated cationic lipid complex. (A) Schematic representation of the surface functionalization structure of the Fibroplex. (B) MALDI-TOF analysis results of DSS₆ for Fibroplex functionalization. (C) Concentration optimization of BMP2 introduced into the Fibroplex. Each scanning electron microscopic image is magnified ×5000 (enlarged right corners = ×10 000). BMP2, bone morphogenetic protein 2; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight.

Figure 3.

Structure of PCL-bioink-nanoparticle scaffold and its biocompatibility. (A) Scanning electron microscope (SEM) image of PCL scaffold. Scale = 100 μm. (B) SEM image of bioink. Scale = 200 μm. (C) SEM image of PBN scaffold (left) and confocal microscope image of GFP-loaded Fibroplex dispersed in bioink (right). (D) SEM images for observing the attachment and growth of NH3T3 cells on PBN scaffolds with various filling densities (10–30%). White arrows indicate individual cells. (E) Cytotoxicity of each component of the PBN scaffold. PCL, polycaprolactone; PBN, polycaprolactone-bioink-nanoparticle; GFP, green fluorescence protein.

Figure 4.

In silico assessment of BMP2 release modeling. (A) ELISA assay measuring BMP2 release rate for each component of the PBN scaffold. (B) Predicted release patterns of BMP2 dispersed in the bioink. Bar = densitometer reflecting the predicted concentration. (C) Predicted BMP2 release pattern, assuming a high-density boundary (soft tissue phase) and a low-density boundary (bone defect phase). (D) Tissue release pattern curve predicted through modeling. BMP2, bone morphogenetic protein 2; PBN, polycaprolactone-bioink-nanoparticle; ELISA, enzyme-linked immunosorbent assay.

Figure 5.

Osteogenic inductive efficacy of BMP2 from the PBN scaffold. (A) Strategy for the acquisition of BMP2 released from the PBN scaffold over time. (B) ALP staining and Alizarin Red S (ARS) staining images of MC3T3-E1 cells treated with BMP2 collected at each time point after 4 days and 21 days, respectively. (C) ALP assay results of MC3T3-E1 cells treated with BMP2 collected at each time point after 4 days. (D) qPCR results of osteogenic markers in MC3T3-E1 cells treated with BMP2 collected at each time point after 4 and 7 days. P < 0.05, all biological replicates are N = 4. BMP2, bone morphogenetic protein 2; PBN, polycaprolactone-bioink-nanoparticle; ALP, alkaline phosphatase; qPCR, quantitative polymerase chain reaction.

Figure 6.

Cavarial bone defect histology and defect regeneration analysis. (A) Formation of calvarial defects and experimental group implantation strategy. (B) H&E and Masson’s trichrome (MT) staining images 2 weeks after PBN scaffold and control group implantation. In the H&E images, the white box area corresponds to the site of MT staining. Scale = 1 mm. (C) MicroCT images 4 and 8 weeks after PBN scaffold and control group implantation. (D) Analysis of the defect area ratio shown in microCT. * P < 0.05, ** P < 0.01, N = 4. (E) Quantitative analysis of microCT of PBN scaffold implanted group. ** P < 0.01. PBN, polycaprolactone-bioink-nanoparticle; H&E, hematoxylin and eosin; microCT, microcomputed tomography.