Osteoinductivity and biomechanical assessment of a 3D printed demineralized bone matrix-ceramic composite in a rat spine fusion model

Abstract

We recently developed a recombinant growth factor-free bone regenerative scaffold composed of stoichiometric hydroxyapatite (HA) ceramic particles and human demineralized bone matrix (DBM) particles (HA-DBM). Here, we performed the first pre-clinical comparative evaluation of HA-DBM relative to the industry standard and established positive control, recombinant human bone morphogenetic protein-2 (rhBMP-2), using a rat posterolateral spinal fusion model (PLF). Female Sprague-Dawley rats underwent bilateral L4-L5 PLF with implantation of the HA-DBM scaffold or rhBMP-2. Fusion was evaluated using radiography and blinded manual palpation, while biomechanical testing quantified the segmental flexion-extension range-of-motion (ROM) and stiffness of the fused segments at 8-weeks postoperatively. For mechanistic studies, pro-osteogenic gene and protein expression at 2-days and 1-, 2-, and 8-weeks postoperatively was assessed with another cohort. Unilateral fusion rates did not differ between the HA-DBM (93%) and rhBMP-2 (100%) groups; however, fusion scores were higher with rhBMP-2 (p = 0.008). Both treatments resulted in significantly reduced segmental ROM (p < 0.001) and greater stiffness (p = 0.009) when compared with non-operated controls; however, the degree of stabilization was significantly higher with rhBMP-2 treatment relative to the HA-DBM scaffold. In the mechanistic studies, PLGA and HA scaffolds were used as negative controls. Both rhBMP-2 and HA-DBM treatments resulted in significant elevations of several osteogenesis-associated genes, including Runx2, Osx, and Alp. The rhBMP-2 treatment led to significantly greater early, mid, and late osteogenic markers, which may be the mechanism in which early clinical complications are seen. The HA-DBM scaffold also induced osteogenic gene expression, but primarily at the 2-week postoperative timepoint. Overall, our findings show promise for this 3D-printed composite as a recombinant growth factor-free bone graft substitute for spinal fusion. STATEMENT OF SIGNIFICANCE: Despite current developments in bone graft technology, there remains a significant void in adequate materials for bone regeneration in clinical applications. Two of the most efficacious bone graft options are the gold-standard iliac crest bone graft and recombinant human-derived bone morphogenetic protein-2 (rhBMP-2), available commercially as Infuse™. Although efficacious, autologous graft is associated with donor-site morbidity, and Infuse™ has known side effects related to its substantial host inflammatory response, possibly associated with a immediate, robust osteoinductive response. Hence, there is a need for a bone graft substitute that provides adequate osteogenesis without associated adverse events. This study represents a significant step in the design of off-the-shelf growth factor-free devices for spine fusion.

Keywords: 3D printing; Bone regeneration; Demineralized bone matrix; Hydroxyapatite; Spine fusion.

Figure 1.

Materials and experimental model overview. The hydroxyapatite-demineralized bone matrix (HA-DBM) composite is 3D-printed and trimmed to size prior to implantation. The HA-DBM group was compared to recombinant human-derived bone morphogenetic protein-2 (rhBMP-2) delivered on a collagen sponge, known to be highly efficacious in yielding successful fusion. An established L4-L5 posterolateral lumbar fusion (PLF) model in rats was used to compare the efficacy of the HA-DBM to rhBMP-2 in achieving spinal fusion and bone regeneration.

Figure 2.

Biomechanics Overview. (A) Schematic of the biomechanics testing apparatus from both a posterior and a left lateral view. Samples of explanted L4-L5 vertebrae were loaded with 5 cycles of flexion-extension using a hybrid control algorithm, namely rotation control with a torque limit of 0.01 Nm. (B) Representative load-displacement curve of moment (Nm) versus rotation angle (°). The range-of-motion (ROM) was measured as the difference in rotation angle at the torque limits (−0.01 Nm and 0.01 Nm) of the flexion and extension cycles. The stiffness (K₁) was measured as a linear fit to the load-displacement curve from 0 Nm to the first third of the flexion range-of-motion. The red arrows indicate the bounds of the curve to which the linear fit is made. The slope of the red line yields the stiffness (K₁). SP = spinous process, TP = transverse process, PMMA = polymethylmethacrylate

Figure 3.

Comparison of the fusion capacity of rhBMP-2 and HA-DBM groups. (A) Representative radiographs of the lumbar spine at 8 weeks postoperatively for the rhBMP-2 and HA-DBM groups. The fusion area is indicated bilaterally with white arrows. (B) Mean fusion score was significantly greater in the rhBMP-2 group relative to the HA-DBM group. (C) The fusion rate was not significantly different between the rhBMP-2 and HA-DBM groups. * Indicates statistical significance (p<0.05)

Figure 4.

Histological evaluation of representative spines for each experimental group tested, 2 and 8 weeks post-operatively. Sections are stained with Gill’s hematoxylin and eosin and alcian blue. Acronym legend: transverse process (TP), fusion mass (FM), scaffold struts (ST). Scale bars of large images: 1 mm. Yellow arrows in the 8 weeks rhBMP-2 magnified images indicate trabeculae-like structures. Yellow arrows in the 2 and 8 weeks HA-DBM magnified images indicate DBM particles. The yellow arrow in the 8 weeks HA magnified image indicates remaining HA particles (black). Scale bars of magnified images: 500 µm.

Figure 5.

Biomechanics assessment of explanted specimens. (A) Representative load-displacement curves of a fused and unfused specimen. The stiffness (K₁) – determined by the linear fit of the first third of the flexion cycle for each specimen (indicated by red lines on this plot) – was significantly greater in the fused specimen relative to the unfused specimen. Comparison of the (B) range-of-motion (ROM, °) and (C) stiffness between the non-operated control, rhBMP-2, and HA-DBM groups. The rhBMP-2 group had a significantly lower ROM and greater stiffness than both the HA-DBM and control groups, respectively. Additionally, the HA-DBM had a significantly lower ROM and greater stiffness than the non-operated control group. The first stiffness plot demonstrates the data with an unadjusted y-axis. The second stiffness plot utilizes a y-axis break to illustrate representative differences in stiffness between the three groups. * indicates statistical significance (p<0.05).

Figure 6.

Gene Expression Studies. A. Expression of select early osteogenic markers in fusion bed tissue 2 days after implantation of 3DP PLGA, 3DP HA, 3DP HA-DBM, or rhBMP-2/ACS scaffolds. B. Heat map representation of the relative expression levels of genes important in osteogenic differentiation in explanted fusion bed tissue taken at 1 and 2 weeks post-operative. Light green signifies significantly elevated expression between 2–5-fold higher than PLGA, and dark green signifies >5 fold-higher expression than the PLGA-treated control group. C. Line graphs depicting the relative expression levels of several key early (Runx2, Osx), early/mid (Alp, Col1a), and late osteogenic markers (Opn, Ocn). Statistical significance (p<0.05) is indicated for HA (#), rhBMP-2 (^) and HA-DBM (*), relative to PLGA control explants.

Figure 7.

Western Blot Analysis. A. Western blot visualization and B. Quantified protein expression of markers of interest– DLX5, RUNX2, OSX, OPN, and PHEX – for PLGA, HA, HA-DBM, and rhBMP-2 treatment groups at 2-weeks postoperatively. * indicates statistical significance relative to PLGA.