Extracorporeal Shock Wave Combined with Teriparatide-Loaded Hydrogel Injection Promotes Segmental Bone Defects Healing in Osteoporosis

Abstract

Background: Osteoporosis is a systemic bone disease characterized by decreased bone density and deterioration of bone microstructure, leading to an increased probability of fragility fractures. Once segmental bone defect occurs, it is easy to cause delayed union and nonunion.

Methods: The aim of this study is to investigate the efficacy of extracorporeal shock wave (ESW) and teriparatide-loaded hydrogel (T-Gel) combined strategy on the cell activity and differentiation of osteoporosis derived bone marrow mesenchymal stem cells (OP-BMSCs) in vitro and bone regeneration in osteoporotic segmental bone defects in vivo.

Results: In vitro, the strategy of combining ESW and T-Gel significantly enhanced OP-BMSCs proliferation, survival, migration, and osteogenic differentiation by up-regulating the alkaline phosphatase activity, mineralization, and expression of runt-related transcription factor-2, type I collagen, osteocalcin, and osteopontin. In the segmental bone defect models of osteoporotic rabbits, Micro-CT evaluation and histological observation demonstrated this ESW-combined with T-Gel injection significantly induced bone healing by enhancing the osteogenic activity of the local microenvironment in osteoporotic defects.

Conclusion: In conclusion, ESW-combined with T-Gel injection could regulate the poor osteogenic microenvironment in osteoporotic defects and show potential for enhancing fragility fractures healing.

Keywords: Bone generation; Extracorporeal shock wave; Hydrogel; Osteoporosis; Teriparatide.

Fig. 1

A The optical images of the gelation progress. B The injectable and thermosensitive hydrogel rapidly form gel when injected into the hot plate at 37 °C. C Storage modulus (G′) and loss modulus (G′′) of hydrogel as a function of temperature. D Morphologies of hydrogel observed by SEM

Fig. 2

Release profiles of teriparatide in hydrogels

Fig. 3

A Effects of different treatment groups on cell proliferation. B Calcein-AM/PI staining indicated the live cells were stained with green and the dead cells were stained with red. C Quantitative analysis of cell survival rate by Calcein AM/PI staining. D The images of migration cells were stained with crystal violet by Transwell test. E Quantitative statistics of the number of migrating cells (*p < 0.05, **p < 0.01 compared with Con group; ^#p < 0.05, ^##p < 0.01 compared with ESW group; ^‡p < 0.05 compared with T-Gel group)

Fig. 4

A Images of ALP staining and alizarin red staining after 14 days of osteogenic induction. B statistical analysis of ALP activity. C Semi-quantitative analysis of alizarin red staining. D–G The mRNA expression levels of osteogenic-related genes, including Runx-2, Col-1, OCN, and OPN in OP-BMSCs after osteogenic induction for 14 days (*p < 0.05, **p < 0.01 compared with Con group; ^#p < 0.05, ^##p < 0.01 compared with ESW group; ^‡p < 0.05 compared with T-Gel group)

Fig. 5

A Serum estrogen (E2) levels at 10 months after OVX. B Reconstruction images of distal radius at 10 months after OVX. C Quantitative analysis of BV/TV according to Micro-CT examination. D Quantitative analysis of BMD according to Micro-CT examination (*p < 0.05, ***p < 0.001 compared with Sham group)

Fig. 6

A Tomographic images of radius defects by Micro-CT. B Quantitative analysis of BV/TV (%) according to Micro-CT at 12 weeks after treatment. C H and E staining to observe bone regeneration in segmental bone defects. D Representative images of calcein fluorescence double-labeling of regenerated bone in defects. E Quantitative statistics of MAR by calcein fluorescence double-labeling (*p < 0.05, **p < 0.01 compared with Con group; ^#p < 0.05 compared with ESW group; ^‡p < 0.05 compared with T-Gel group)

Fig. 7

A–D The mRNA expression levels of osteogenic-related genes, including Runx-2, Col-1, OCN, and OPN in regenerated bone tissue. E–I Immunofluorescence staining and quantitative analysis of Runx-2, Col-1, OCN, and OPN in regenerated bone tissue (*p < 0.05, **p < 0.01 compared with Con group; ^#p < 0.05, ^##p < 0.01 compared with ESW group; ^‡p < 0.05 compared with T-Gel group)