In-Vivo Degradation Behavior and Osseointegration of 3D Powder-Printed Calcium Magnesium Phosphate Cement Scaffolds

Abstract

Calcium magnesium phosphate cements (CMPCs) are promising bone substitutes and experience great interest in research. Therefore, in-vivo degradation behavior, osseointegration and biocompatibility of three-dimensional (3D) powder-printed CMPC scaffolds were investigated in the present study. The materials Mg225 (Ca_0.75Mg_2.25(PO₄)₂) and Mg225d (Mg225 treated with diammonium hydrogen phosphate (DAHP)) were implanted as cylindrical scaffolds (h = 5 mm, Ø = 3.8 mm) in both lateral femoral condyles in rabbits and compared with tricalcium phosphate (TCP). Treatment with DAHP results in the precipitation of struvite, thus reducing pore size and overall porosity and increasing pressure stability. Over 6 weeks, the scaffolds were evaluated clinically, radiologically, with Micro-Computed Tomography (µCT) and histological examinations. All scaffolds showed excellent biocompatibility. X-ray and in-vivo µCT examinations showed a volume decrease and increasing osseointegration over time. Structure loss and volume decrease were most evident in Mg225. Histologically, all scaffolds degraded centripetally and were completely traversed by new bone, in which the remaining scaffold material was embedded. While after 6 weeks, Mg225d and TCP were still visible as a network, only individual particles of Mg225 were present. Based on these results, Mg225 and Mg225d appear to be promising bone substitutes for various loading situations that should be investigated further.

Keywords: 3D powder printing; biocompatibility; degradable bone substitutes; farringtonite; in-vivo Micro-Computed Tomography; osseointegration; scaffold; stanfieldite.

Figure 1

Three-dimensional (3D) reconstruction of the distal rabbit femur with implanted scaffold in the lateral condyle: (a) in craniolateral view and (b) in caudal view.

Figure 2

Cylindrical region of interest (ROI) in the scaffold center for measurement of scaffold volume (SV): (a) in the original in-vivo µCT scan (Xtreme CT II) (h = 60 slices ≙ 1.82 mm), (b) cylindrical ROI in the reoriented in-vivo µCT scan (∅ = 126 voxels (≙ 3.82 mm)), (c) second ROI for measurement of bone volume (BV), trabecular number (Tb. N) and trabecular thickness (Tb. Th) in the scaffold environment (inner ring: ∅ = 128 voxels (≙ 3.88 mm), outer ring: ∅ = 160 voxels (≙ 4.85 mm), h = 60 slices ≙ 1.82 mm).

Figure 3

X-Ray diffraction pattern of Mg225, Mg225d and TCP powders. The most relevant peaks are marked for ▼: α-TCP, *: β-TCP, ‘: stanfieldite, f: farringtonite, s: struvite and o: periclase.

Figure 4

Porosity, pore size and relative pore volume of: (a) Mg225, (b) Mg225d and (c) TCP.

Figure 5

(a) Ventrodorsal (VD) X-ray view of the hind limbs directly after surgery with TCP implanted in the right and Mg225 in the left femoral condyle. (b) Magnification of the left knee joint with implanted Mg225 scaffold.

Figure 6

In-vivo µCT images (Xtreme CT II) of the scaffolds and the surrounding cancellous bone in the reoriented scans in cross-section over time (immediately after surgery up to 6 weeks); scale bar = 1 mm.

Figure 7

Volume degradation of the scaffolds in in-vivo µCT (Xtreme CT II) over time (immediately after surgery up to 6 weeks) and scaffold volume in µCT 100 (6 weeks after surgery).

Figure 8

Cancellous bone volume (BV) in the scaffold environment in in-vivo µCT (Xtreme CT II) over time (immediately after surgery up to 6 weeks) and in µCT 100 (6 weeks after surgery) compared to intact cancellous bone of the lateral femoral condyle.

Figure 9

(a) Trabecular number and (b) trabecular thickness in the scaffold environment in in-vivo µCT (Xtreme CT II) over time (immediately after surgery up to 6 weeks) and in µCT 100 (6 weeks after surgery) compared to intact cancellous bone of the lateral femoral condyle.

Figure 10

Scaffolds and surrounding cancellous bone (a,c,e) in the original scan in longitudinal-section and (b,d,f) in the reoriented scan in cross-section in µCT 100 6 weeks after surgery.

Figure 11

Histological thick sections (toluidine blue staining) from the center of the CMPC (calcium magnesium phosphate cement) scaffolds in cross-section (a,b) after 1 week and (c,d) after 6 weeks (magnification 2.5× /0.085). After 1 week, bone trabeculae from the surrounding had grown onto the scaffold and already slightly into the scaffold margin ((*) old bone in lighter blue, (↑) newly formed bone in dark blue, (°) bone marrow). After 6 weeks, new bone trabeculae and (∧) connective tissue traversed the scaffold completely and in the case of (c) Mg225, bone marrow had already grown into the scaffold.

Figure 12

Histological thick sections (toluidine blue staining) of the femoral condyle with TCP scaffold after 6 weeks: (a) as overview and (b) with magnification 2.5× /0.085. Numerous bone trabeculae from the surrounding had grown into the scaffold and traversed it completely ((*) old bone in lighter blue, (↑) newly formed bone in dark blue), as well as (∧) connective tissue. (°) Bone marrow was also already visible within the scaffold.

Figure 13

Histological thick sections (toluidine blue staining) from the scaffold center of (a) Mg225, (b) Mg225d and (c) TCP after 6 weeks with magnification 20× /0.5. (•) Remaining scaffold material was surrounded by numerous (*) new bone trabeculae, on the margins of which several (↑) osteoblasts were deposited. In (a) Mg225 and (c) TCP, (◊) cell-rich connective tissue with numerous fibrocytes and (∆) bone marrow (adipocytes with fat vacuoles) were visible between the trabeculae.