Construction of a 3D Degradable PLLA/β-TCP/CS Scaffold for Establishing an Induced Membrane Inspired by the Modified Single-Stage Masquelet Technique.

Abstract

Although the Masquelet-induced membrane technique (MIMT) is now employed worldwide for bone defects, it often needs to be repeated and autogenous bone graft. This study aims to investigate the theoretical feasibility of replacing PMMA (poly(methyl methacrylate)) bone cement with PLLA (poly-l-lactic acid)/β -TCP (beta-tricalcium phosphate)/CS (calcium sulfate) scaffold for single-stage bone defect reconstruction, which evoke the induced membrane (IM) formation in the early stage and directly acts as the implantation in the second stage to reconstruct the bone defect. We constructed a corn-like PLLA/β -TCP/CS scaffold by the fused deposition 3D printing method. The characterizations of the scaffolds were investigated systematically. The P/T15/S15 scaffolds (the PLLA/β -TCP/CS scaffold with a 15% mass fraction of β-TCP and 15% mass fraction of CS) were filled into the large-segmental radius bone defects of white rabbits to evoke the formation of IMs. HE (hematoxylin-eosin) and VG (van gieson) staining, along with immunofluorescent staining, were performed to analyze the architecture and cellularity, the expression of BMP-2 (bone morphogenetic protein-2), VEGF (vascular endothelial growth factor), and TGF-β1 (transforming growth factor-β1) was evaluated by IHC (immunohistochemistry) and WB (western-blot) respectively, the ALP (alkaline phosphatase) and ARS (alizarin red S) staining was applied to assess the osteogenic potential. The corn-like PLLA/β-TCP/CS scaffolds with excellent physicochemical properties are successfully constructed using the fused deposition 3D printing technique. The HE and VG staining, along with immunofluorescent staining, suggested that the P/T15/S15 scaffold effectively mediated the formation of IM after 6 weeks of placement. A significant presence of M2 macrophages was observed in IM. The results of IHC and WB demonstrated that the IMs derived from the P/T15/S15 scaffolds exhibited elevated levels of VEGF, BMP-2, and TGF-β1, all of which promote the osteogenic differentiation of BMSCs. The results of cellular immunofluorescence, flow cytometry, and WB indicate that P/T15/S15 regulates the phenotypic polarization of M0 macrophages toward the M2 phenotype via the PI3K/AKT/β-Catenin pathway. These findings suggest that the biodegradable PLLA/β-TCP/CS scaffold may serve as a viable alternative to PMMA bone cement for single-stage bone defect reconstruction, owing to its unique ability to stimulate IM formation and promote the polarization of macrophages toward the M2 phenotype. This work presents innovative materials and strategies for the management of bone defects.