Incorporation of Calcium Sulfate Dihydrate into a Mesoporous Calcium Silicate/Poly-ε-Caprolactone Scaffold to Regulate the Release of Bone Morphogenetic Protein-2 and Accelerate Bone Regeneration

Abstract

Tissue engineering and scaffolds play an important role in tissue regeneration by supporting cell adhesion, proliferation, and differentiation. The design of a scaffold is critical in determining its feasibility, and it is critical to note that each tissue is unique in terms of its morphology and composition. However, calcium-silicate-based scaffolds are undegradable, which severely limits their application in bone regeneration. In this study, we developed a biodegradable mesoporous calcium silicate (MS)/calcium sulfate (CS)/poly-ε-caprolactone (PCL) composite and fabricated a composite scaffold with 3D printing technologies. In addition, we were able to load bone morphogenetic protein-2 (BMP-2) into MS powder via a one-step immersion procedure. The results demonstrated that the MS/CS scaffold gradually degraded within 3 months. More importantly, the scaffold exhibited a gradual release of BMP-2 throughout the test period. The adhesion and proliferation of human dental pulp stem cells on the MS/CS/BMP-2 (MS/CS/B) scaffold were significantly greater than that on the MS/CS scaffold. It was also found that cells cultured on the MS/CS/B scaffold had significantly higher levels of alkaline phosphatase activity and angiogenic-related protein expression. The MS/CS/B scaffold promoted the growth of new blood vessels and bone regeneration within 4 weeks of implantation in rabbits with induced critical-sized femoral defects. Therefore, it is hypothesized that the 3D-printed MS/CS/B scaffold can act both as a conventional BMP-2 delivery system and as an ideal osteoinductive biomaterial for bone regeneration.

Keywords: 3D printing; bone morphogenetic protein-2; calcium silicate; calcium sulfate; osteogenesis.

Figure 1

The microstructure and composition of mesoporous calcium silicate (MS) and calcium sulfate (CS) powders and composites. The scale bar of the SEM photograph is 1 µm.

Figure 2

(A) Field emission–scanning electron microscopy (FE-SEM) photographs and (B) X-ray diffractometry (XRD) of an MS/CS scaffold after soaking in SBF. The scale bar of the SEM photograph is 1 µm.

Figure 3

The weight loss of MS/CS scaffolds after soaking in SBF for various time periods.

Figure 4

Stress–strain curve of the MS/CS scaffold after immersion in SBF for 7, 30, and 180 days.

Figure 5

Bone morphogenetic protein-2 (BMP-2) release from an MS/CS/B and MS/B scaffold after immersion in Dulbecco’s modified Eagle’s medium (DMEM) at 37 °C for 6 months.

Figure 6

(A) The proliferation of the human dental pulp cells (hDPSCs) cultured on scaffolds for 1, 3, 7, and 14 days. * indicates a significant difference (p < 0.05) when compared to MS/CS. (B) F-actin filament (green) staining of hDPSCs cultured on different scaffolds for 3 and 7 days. The scale bar of the photograph is 100 µm.

Figure 7

(A) Immunodetection of anti-bone morphogenetic protein receptor type II (BMP2R), anti-extracellular signal-regulated kinase 1/2 (ERK 1/2), anti-phospho extracellular signal-regulated kinase 1/2 (p-ERK 1/2), alkaline phosphatase (ALP), osteocalcin (OC), and β-actin protein expression in hDPSCs cultured on scaffolds for 3 days. Quantification of (B) BMP2R, (C) anti-phospho SMAD1/5/8 (p-SMAD1/5/8), (D) p-ERK 1/2, (E) ALP, and (F) OC. * indicates a significant difference (p < 0.05) when compared to MS/CS.

Figure 8

(A) Western blotting of knockdown by pairs of BMPR2-specific siRNAs in hDPSCs. (B) The effects on siRNA–BMPR2 transfection of proliferation in hDPSCs cultured on scaffolds for 1 and (B) 7 days. * indicates a significant difference (p < 0.05) when compared to MS/CS.

Figure 9

The osteogenic and angiogenic protein expression of (A) the hDPSCs and (B) the siRNA–BMPR2 transfected hDPSCs on scaffolds for 7 days. * indicates a significant difference (p < 0.05) when compared to MS/CS.

Figure 10

(A) Alizarin red S staining and (B) quantification of hDPSC calcium mineral deposits cultured on specimens for 7, 10, and 14 days. * indicates a significant difference (p < 0.05) when compared to MS/CS. The scale bar of the Optical microscope photograph is 400 µm.

Figure 11

(A) Micro-CT image showing the morphology of bone growth for a fixed-sized critical lesion after it underwent 8-week regeneration with scaffolds; (B) data analysis of relative bone mass volume (bone volume/tissue volume (BV/TV) ratio) for a fixed-sized critical lesion after it underwent 8 weeks of regeneration with scaffolds. * indicates a significant difference (p < 0.05) when compared to MS/CS.

Figure 12

Histological analysis of new bone regeneration around and within the scaffolds in the rabbit femoral defect model. Left, hematoxylin and eosin (HE) stain; middle, Masson’s trichrome (MT) stain; and right, von Kossa (VK) stain of regenerated bone mass after 8 weeks of regeneration in the in vivo experiment. Purple: tissue morphology. Blue: collagen and bone matrix. Brown: bone mineralization. The scale bar of the histological photograph is 400 µm.