A novel open-porous magnesium scaffold with controllable microstructures and properties for bone regeneration

Abstract

The traditional production methods of porous magnesium scaffolds are difficult to accurately control the pore morphologies and simultaneously obtain appropriate mechanical properties. In this work, two open-porous magnesium scaffolds with different pore size but in the nearly same porosity are successfully fabricated with high-purity Mg ingots through the titanium wire space holder (TWSH) method. The porosity and pore size can be easily, precisely and individually controlled, as well as the mechanical properties also can be regulated to be within the range of human cancellous bone by changing the orientation of pores without sacrifice the requisite porous structures. In vitro cell tests indicate that the scaffolds have good cytocompatibility and osteoblastic differentiation properties. In vivo findings demonstrate that both scaffolds exhibit acceptable inflammatory responses and can be almost fully degraded and replaced by newly formed bone. More importantly, under the same porosity, the scaffolds with larger pore size can promote early vascularization and up-regulate collagen type 1 and OPN expression, leading to higher bone mass and more mature bone formation. In conclusion, a new method is introduced to develop an open-porous magnesium scaffold with controllable microstructures and mechanical properties, which has great potential clinical application for bone reconstruction in the future.

Figure 1. Illustration of preparation process of the open-porous magnesium scaffold and in vivo animal model.

Step1: the 3D entangled titanium wire material (a,e) was prepared with Ti wires. Step2: the Ti-Mg composite (b,f) was prepared with high purity Mg melts. Step3: Ti wires were removed by HF solution and open-porous magnesium scaffold(c,g) was successful manufactured.Step4:open-porous magnesium scaffolds were implanted into the lateral epicondyle of rabbits(d).

Figure 2. The microstructures and chemical compositions of open-porous magnesium scaffolds.

(a) SEM and EDS results of 250-PMg and 400-PMg scaffolds; (b) 2D and 3D images of 250-PMg and 400-PMg scaffolds acquired from micro-CT scanning.

Figure 3. The results of in vitro immersion tests of 250-PMg and 400-PMg scaffold.

(a) Magnesium ions concentration of the immersion extract from 250-PMg and 400-PMg scaffold; (b) PH values of the immersion extract from 250-PMg and 400-PMg scaffold; (c) Total weight lost from 250-PMg and 400-PMg scaffold; (d) The corrosion rates of 250-PMg and 400-PMg scaffolds after immersed for 2 weeks. *Denotes a significant difference between 250-PMg and 400-PMg scaffold (p < 0.05).

Figure 4

(a) Cell morphology on 250-PMg and 400-PMg scaffold after 3 days of incubation displayed by SEM; (b) Viability of MG63 osteoblasts in 250-PMg and 400-PMg scaffolds extraction using live/dead assay; (c) Viability of MG63 osteoblasts in 250-PMg and 400-PMg scaffold extraction using MTT assay; (d) ALP activity of MG63 osteoblasts after 7 and 14 days; (e) Osteogenic differentiation of MG63 osteoblasts by measuring the mRNA expression level of Col1a1,ALP, OPN and RUNX-2 after 7 days. ^#Denotes a significant difference compared to the control (p < 0.05).

Figure 5

(a) H&E staining of subcutaneous and muscular tissue in contact with 250-PMg and 400-PMg scaffold, the pentagrams represent the fibrous tissues formed around the scaffolds; (b) Percentage changes in serum magnesium levels before and after implantation; (c) Representative sections from important internal organs of the rabbits after implantation with 250-PMg and 400-PMg scaffold for 16 weeks.

Figure 6. Characterization of scaffolds and the newly formed bone by Micro-CT.

(a) Micro-CT 2D (The red arrows refer to the newly formed bone) and 3D reconstruction models showing the status of new bone (white in color) response 16 weeks after surgery; (b) Quantitative analysis of bone volume fraction (BV/TV),trabecular number (TN) and trabecular thickness. *Denotes a significant difference compared to the 250-PMg scaffold (p < 0.05).

Figure 7. Histological and immunohistological analysis of bone formation at 8 weeks after the implantation.

(a) Representative histological analysis (H&E staining) of bone formation in the 250-PMg and 400-PMg scaffold group. The lower images are higher magnifications of the areas within the black boxes. In the 250-PMg scaffold group, a fibrotic walling-off phenomenon based on the formation of regular dense fibrous tissues (green arrows) with rare inflammatory cells was observed, whereas less fibrous tissue accompanied with more lymphocytes, macrophages and foreign body giant cells (FBGCs)were present around the 400-PMg scaffold. Compared with the 250-PMg scaffold, more new bone formed around 400-PMg scaffold, and more abundant cuboid osteoblasts (blue arrows) were observed between the fibrous tissue and new bone. Furthermore, in the 400-PMg scaffold group, more newly blood vessels (yellow arrows) formed than in the 250-PMg scaffold group. (b) Relatively higher expressions of collagen type 1 and OPN (red arrows) in the 400-PMg scaffold group than that in the 250-PMg scaffold group. (c) Representative histological analysis (Van Gieson’s picrofuchsin staining) of bone formation in the 250-PMg and 400-PMg scaffold group. Significantly more new bone formed in the 400-PMg scaffold group was observed.

Figure 8. Histological and sequential polychrome labels analysis of bone formation at 16 weeks after the implantation.

(a) Representative histological analysis (H&E staining and Van Gieson’s picrofuchsin staining) of bone formation in the 250-PMg and 400-PMg scaffold group. The inflammatory responses to both 250-PMg and 400-PMg scaffolds recede with progressive time after implantation and there are more bone formed in the 400-PMg scaffold group; (b) Sequential polychrome labels observed for 16 weeks in rabbit models: alizarin red S (red) and calcein (green).