Sustained delivery of calcium and orthophosphate ions from amorphous calcium phosphate and poly(L-lactic acid)-based electrospinning nanofibrous scaffold

Abstract

The purpose of this study is to investigate electrospinning poly(L-lactic acid) (PLLA) nanofibrous scaffold with different contents of amorphous calcium phosphate (ACP), which is suitable for using in bone regeneration through sustained release of calcium and orthophosphate ions. Three groups of nanofibrous scaffolds, ACP-free PLLA, ACP-5 wt%/PLLA and ACP-10 wt%/PLLA, are developed and characterized by scanning electron microscopy and gel permeation chromatography. Calcium and phosphate colorimetric assay kits are used to test ions released from scaffold during hydrolytic degradation. The results show ACP-5 wt%/PLLA and ACP-10 wt%/PLLA scaffolds have relatively high degradation rates than ACP-free PLLA group. The bioactivity evaluation further reveals that ACP-5 wt%/PLLA scaffold presents more biocompatible feature with pre-osteoblast cells and significant osteogenesis ability of calvarial bone defect. Due to the facile preparation method, sustained calcium and orthophosphate release behavior, and excellent osteogenesis capacity, the presented ACP/PLLA nanofibrous scaffold has potential applications in bone tissue engineering.

Figure 1. SEM micrographs of electrospinning ACP-free PLLA nanofibrous scaffold with different PLLA concentrations.

(a) 5%, (b) 7%, (c) 9%, (d) 11%. The corresponding average diameter of nanofibers was evaluated using Image J software (n = 10) (e).

Figure 2. SEM micrographs of PLLA-based electrospinning nanofibrous scaffold with different contents of ACP particles.

(a–c) Morphology of ACP-free PLLA group. (d–f) Morphology of ACP-5 wt%/PLLA group. (g–i) Morphology of ACP-10 wt%/PLLA group.

Figure 3. Time dependence of number average molecular weight decrease for ACP/PLLA electrospinning nanofibrous scaffolds with different contents of ACP particles during 24 weeks of in vitro hydrolytic degradation.

Figure 4. Kinetic release of calcium and orthophosphate ions from ACP/PLLA electrospinning nanofibrous scaffold during 24 weeks of in vitro hydrolytic degradation.

(a) ACP-5 wt%/PLLA scaffold. (b) ACP-10 wt%/PLLA scaffold.

Figure 5. SEM micrographs of ACP/PLLA electrospinning nanofibrous scaffold with various ACP contents during 12 and 24 weeks of in vitro hydrolytic degradation.

(a–b) ACP-free PLLA scaffold; (c–d) ACP-5 wt%/PLLA scaffold; (e–f) ACP-10 wt%/PLLA scaffold.

Figure 6. In vitro biocompatibility evaluation of ACP/PLLA electrospinning nanofibrous scaffolds with various ACP contents.

(a) The proliferation of MC3T3-E1 cells cultured with ACP-free PLLA, ACP-5 wt%/PLLA and ACP-10 wt%/PLLA scaffold for 1, 4 and 7 days, respectively. (b) ALP activity to show the differentiation of MC3T3-E1 cells cultured with ACP-free PLLA, ACP-5 wt%/PLLA and ACP-10 wt%/PLLA scaffold for 7 and 14 days, respectively. *p < 0.05 was considered to be statistically significant.

Figure 7. Implantation of cell-free ACP-5 wt%/PLLA scaffold improved bone formation in calvarial defect of rats after 8 weeks.

The results were investigated by micro-CT and H&E staining. (a,b) Micro-CT images and quantitative micro-CT analysis (new bone volume) showed the regenerated bone volumes. The neo bone volume in ACP-5 wt%/PLLA group was significantly increased than the ACP-free PLLA or HA-5 wt%/PLLA group. (c–h) H&E staining showed the calvarial defect and middle area. (c and f) ACP-free PLLA group almost had no regenerated bone in bone defect area. (d and g) In HA-5 wt%/PLLA group, a part of bone defect was filled with neo bone. (e and h) With implantation of ACP-5 wt%/PLLA scaffold, almost total bone repairing was observed in calvarial defect area.