The Masquelet induced membrane technique with PRP-FG-nHA/PA66 scaffold can heal a rat large femoral bone defect

Abstract

Background: Masquelet membrane induction technology is one of the treatment strategies for large bone defect (LBD). However, the angiogenesis ability of induced membrane decreases with time and autologous bone grafting is associated with donor site morbidity. This study investigates if the PRP-FG-nHA/PA66 scaffold can be used as a spacer instead of PMMA to improve the angiogenesis ability of induced membrane and reduce the amount of autologous bone graft.

Methods: Platelet rich plasma (PRP) was prepared and PRP-FG-nHA/PA66 scaffold was synthesized and observed. The sustained release of VEGFA and porosity of the scaffold were analyzed. We established a femur LBD model in male SD rats. 55 rats were randomly divided into four groups depending on the spacer filled in the defect area. "Defect only" group (n = 10), "PMMA" group (n = 15), "PRP-nHA/PA66" group (n = 15) and "PRP-FG-nHA/PA66" group (n = 15 ). At 6 weeks, the spacers were removed and the defects were grafted. The induced membrane and bone were collected and stained. The bone formation was detected by micro-CT and the callus union was scored on a three point system.

Results: The PRP-FG-nHA/PA66 scaffold was porosity and could maintain a high concentration of VEGFA after 30 days of preparation. The induced membrane in PRP-FG-nHA/PA66 group was thinner than PMMA, but the vessel density was higher.The weight of autogenous bone grafted in PRP-FG-nHA/PA66 group was significantly smaller than that of PMMA group. In PRP-FG-nHA/PA66 group, the bone defect was morphologically repaired.

Conclusion: The study showed that PRP-FG-nHA/PA66 scaffold can significantly reduce the amount of autologous bone graft, and can achieve similar bone defect repair effect as PMMA. Our findings provide some reference and theoretical support for the treatment of large segmental bone defects in humans.

Keywords: Masquelet membrane induction technology; angiogenesis; bone regeneration; large bone defect; platelet rich plasma.

Fig. 1

A, the morphology of nHA/PA66, PRP-nHA/PA66 and PRP-FG-nHA/PA66. After the addition of fibrinogen and lyophilization, the PRP-gel had a crystalline shape and attached to the walls of the pores. B, ELISA assay for concentration of VEGFA after 30 days of preparation. Data in mean ± SD, n = 5

Fig. 2

A, HE staining results of the induced membrane formed in the defect area after 6 weeks of first-stage operation (scale bar: 100 μm, black arrows indicate microvessels). B Quantitative results for the thickness of outer layers of the induced membrane. C Quantitative results of vessel density in induced membrane. n = 5, mean ± SD, ^*p < 0.05, ^**p < 0.01, versus PMMA group, ^###p < 0.001, versus PRP-FG-nHA/PA66 group

Fig. 3

A - F illustrated the procedure of the establishment of induced membrane in femoral defect site. A Bone defect site of 5 mm in length in the right femur of the rats was prepared in the first-stage surgery. (B) PMMA and (C) PRP-FG-nHA/PA66 were prepared into 5 × 5 × 5 mm³ small pieces and implanted in the defect site. The induced membrane formed by (D) PMMA and (E) PRP-FG-nHA/PA66 after 6 weeks of first-stage surgery. F The autogenous bone was planted in graft bed in PRP-FG-nHA/PA66 group. G The weight of autogenous bone planted in graft bed after the removement of PMMA and PRP-FG-nHA/PA66, n = 5, mean ± SD, ^*p < 0.05

Fig. 4

Representative images of masson’s staining from different groups. F: fibrous tissue, NB: new bone, IM: induced membrane

Fig. 5

Representative images of micro CT from different groups. The top images show high magnifications of the defect areas, and the bottom images show low magnification overview of the whole femurs after 6 weeks of second-stage surgery (The straight line in the femoral marrow cavity is the K-wire)

Fig. 6

Bone volume (BV, mm³), bone volume density (BV/TV, %) and bone mineral density (g/cm³) of the three groups, n = 5, mean ± SD, ^***p < 0.001, versus defect only group; ^###p < 0.001, versus PRP-nHA/PA66 group