Tough magnesium phosphate-based 3D-printed implants induce bone regeneration in an equine defect model

Abstract

One of the important challenges in bone tissue engineering is the development of biodegradable bone substitutes with appropriate mechanical and biological properties for the treatment of larger defects and those with complex shapes. Recently, magnesium phosphate (MgP) doped with biologically active ions like strontium (Sr²⁺) have shown to significantly enhance bone formation when compared with the standard calcium phosphate-based ceramics. However, such materials can hardly be shaped into large and complex geometries and more importantly lack the adequate mechanical properties for the treatment of load-bearing bone defects. In this study, we have fabricated bone implants through extrusion assisted three-dimensional (3D) printing of MgP ceramics modified with Sr²⁺ ions (MgPSr) and a medical-grade polycaprolactone (PCL) polymer phase. MgPSr with 30 wt% PCL (MgPSr-PCL30) allowed the printability of relevant size structures (>780 mm³) at room temperature with an interconnected macroporosity of approximately 40%. The printing resulted in implants with a compressive strength of 4.3 MPa, which were able to support up to 50 cycles of loading without plastic deformation. Notably, MgPSr-PCL30 scaffolds were able to promote in vitro bone formation in medium without the supplementation with osteo-inducing components. In addition, long-term in vivo performance of the 3D printed scaffolds was investigated in an equine tuber coxae model over 6 months. The micro-CT and histological analysis showed that implantation of MgPSr-PCL30 induced bone regeneration, while no bone formation was observed in the empty defects. Overall, the novel polymer-modified MgP ceramic material and extrusion-based 3D printing process presented here greatly improved the shape ability and load-bearing properties of MgP-based ceramics with simultaneous induction of new bone formation.

Keywords: 3D printing; Bone tissue engineering; Composites; In vivo; Magnesium phosphates; Osteoinduction; Strontium.

Fig. 1

Preparation and printability characterization of the bioactive materials. A) Schematic illustration of the low-temperature printing process and the composition of the ink. B) Printability evaluation. Filament test: different compositions extruded over pillar support with different spacings. Effect of ceramics concentration on the angle of deflection θ, in radians, as a function of half the gap distance L, in mm (n = 3 for each group). Fusion test: Pictures from stereo microscopy after deposition on the glass slides (n = 3). The exponential fitting of the fused filament length is normalized by filament thickness as a function of the filament distance for the tested compositions. C) Designed and printed scaffolds with various shapes of MgPSr-PCL30.

Fig. 2

Evaluation of the static and dynamic mechanical properties. A) Longitudinal compression profile of 3D printed scaffolds with various rations of PCL. B) Corresponding photographs showed the plastic deformation of the scaffolds begins at 10% strain and proceeds to buckle and barrel. Interpreted data (scale bar 10 mm) C) Elastic modulus, D) Yield stress, and E) Toughness from compressive loading profile for various concentrations of PCL. The compression and recovery profile of 3D printed scaffolds (50 cycles) in F) strain domain and G) time domain for MgPSr-PCL30 scaffolds. Additional information on mechanical properties can be found in the Supplementary Information.

Fig. 3

In-vitro assessment of bioactivity of the printed scaffolds. A) confocal images from the live-dead staining assay during 14 days culturing of eMSCs in basal media. B) Quantified results of the distribution of live and dead cells per scaffold. C and G) alkaline phosphatase (ALP) images of the printed samples. ALP activity levels were normalized to DNA content. D and H) Formation of the calcified matrix by eMSCs investigated using Alizarin Red S staining after 30 days of culture. Quantified amount of calcified matrix evaluated by Alizarin Red S content on the 3D printed scaffolds using a colorimetric assay. Scale bars represent 1 mm. E and I) XRD and F and J) FTIR analysis of the scaffolds after culturing of eMSCs. Peaks corresponding to hydroxyapatite are marked with red circles. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

In vivo study preparation and implantation. A) Schematic representation of the implantation of cylindrical constructs in the equine tuber coxae. B) Drawing and photograph of the large-size printed implants (10 mm × 10 mm). C) Intraoperative views of the surgical procedure, showing empty defect and defect filled with the construct. D) μ-CT analysis of new bone formation after 6 months. Representative reconstructed images of longitudinal and transverse cross-sections of defects implanted with the MgPSr-PCL30 and empty defect after 6 months in vivo (scale bar = 10 mm).

Fig. 5

Histology assessment after 6-months in vivo study for the equine model. A) Histological assessment of new bone (*) within the MgPSr-PCL30 scaffolds after 6 months. Representative hematoxylin and eosin, Basic fuchsin/methylene blue-stained MMA samples, immunohistochemical staining for collagen type I (brown region), and picrosirius red–stained tissue sections of defects filled by MgPSr-PCL30 scaffolds (S) and of empty defects. The scale bar is 50 μm. B) EDX analysis of newly formed bone. Representative BSE image of newly formed bone adjacent to the scaffold strut. C) Calcium and Phosphorous analysis for newly formed bone and native one. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)