Sustained-release of sclerostin single-chain antibody fragments using poly(lactic-co-glycolic acid) microspheres for osteoporotic fracture repair

Abstract

Osteoporotic fracture is one of the most common bone diseases in middle and old age, as the most serious consequence of osteoporosis. Sclerostin single-chain antibody fragments (SCL-scFv) have been proven to promote bone formation by binding to scleroprotein, a natural antagonist of the Wnt pathway, but it is difficult to rule alone due to the weak permeability and immunogenicity. Herein, we prepared poly(lactic-co-glycolic acid) microspheres as a sustained-release vehicle to prolong the activity of SCL-scFv. The morphology of microspheres were uniform and nearly sphere, loading efficiency and encapsulation efficiency of SCL-scFv microspheres were 6.28 ± 1.04% and 48.37 ± 8.11%, respectively. Approximately 90% of the SCL-scFvs were released from the microspheres over 28 days with initial burst releasing (38%) in the first 4 days. Sustained-release of active SCL-scFv from microspheres promoted bone marrow mesenchymal stem cells osteogenic differentiation in vitro and enhanced fracture healing in ovariectomized rats by improving bone mass and bone formation in the fracture region. All these findings demonstrate that the microspheres are able to simultaneously achieve localized long-term SCL-scFv controlled release and effectively promote bone formation, which provides a promising approach for osteoporotic fracture.

Keywords: fracture healing; microspheres; osteoporosis; protein release; sclerostin-scFv.

Figure 1

(a and b) SEM images of the microspheres

Figure 2

Microsphere release curve

Figure 3

BMSC growth curves. Proliferation was evaluated by a CCK‐8 assay. All experiments were performed at least three times with duplication within each individual experiment

Figure 4

(a) ALP staining of BMSCs in the different groups (×40); (b) Alizarin red staining of BMSCs in the different groups (×40)

Figure 5

(a) Real‐time PCR detection of osteogenic‐differentiation‐related genes (COL‐I, ALP, OCN) in the different groups of BMSCs. Osteogenic differentiation was induced for 14 days. (b) A western blot assay for evaluating the amounts of COL‐I and β‐actin in the different groups; (c) Expression of OCN in BMSCs in the different groups. Each bar represents the mean of the experiments ± SD. *p < 0.05, **p < 0.01. NS, not significant. All experiments were performed at least three times with duplication within each individual experiment

Figure 6

SCL‐scFv microspheres increased bone mass in the fracture region after 12 weeks of treatment. (a) Radiograph of the fracture region at week 12 in the two groups. (b) 3D reconstruction images of the fracture region at week 12 in the two groups. (d) Histological analysis of femoral bone tissue regeneration at week 12 in the two groups. (c) Analysis of BMD, BV/TV, and Tb.Th of the fracture region at 12 weeks in the two groups. Each bar represents the mean ± SD. *p < 0.05, **p < 0.01