Lumbar spine intervertebral disc gene delivery of BMPs induces anterior spine fusion in lewis rats

Abstract

Minimally invasive techniques and biological autograft alternatives such as the bone morphogenetic proteins (BMPs) can reduce morbidity associated with spinal fusions. This study was a proof-of-concept for gene-therapy-mediated anterior spine fusion that could be adapted to percutaneous technique for clinical use. Isogeneic bone marrow stromal cells genetically programmed to express b-galactosidase (LACZ, a marker gene), BMP2, BMP7, a mixture of BMP2 and BMP7 infected cells (homodimers, HM), or BMP2/7 heterodimers (HT) were implanted into the discs between lumbar vertebrae 4 and 5 (L4/5) and L5/6 of male Lewis rats. Spine stiffening was monitored at 4, 8 and 12 weeks using noninvasive-induced angular displacement (NIAD) testing. At 12 weeks isolated spines were assessed for fusion and bone formation by palpation, biomechanical testing [four-point bending stiffness, moment to failure in extension, and in vitro angular displacement (IVAD)], faxitron x-rays, microCT, and histology. Progressive loss of NIAD occurred in only the HT group (p < 0.001), and biomechanical tests correlated with the NIAD results. Significant fusion occurred only in the HT group (94% of animals with one or both levels) as assessed by palpation (p < 0.001), which predicted HT bone production assessed by faxitron (p ≤ 0.001) or microCT (p < 0.023). Intervertebral bridging bone was consistently observed only in HT-treated specimens. Induced bone was located anterior and lateral to the disc space, with no bone formation noted within the disc. Percutaneous anterior spine fusions may be possible clinically, but induction of bone inside the disc space remains a challenge.

Figure 1

Coronal noninvasive in vivo angular displacement (NIAD) of rat lumbar motion segments had the greatest decline in mobility for the BMP2/7 heterodimer (HT)-treated spines at (A) lumbar 4/5 (L4/5), (B) L5/6, and (C) combined L4-6 segment at 4, 8 and 12 weeks following intradiscal administration of genetically-programmed bone marrow stromal cells. (D) Increased mobility was detected in all groups at the L6/S1 segment over the same time course, likely due to stiffening at the L4-6 levels. Time of assessment (horizontal axis) and treatment (white square is Naïve, blue square is LACZ, green square is BMP2, yellow square is BMP7, orange square is mixed BMP2 and BMP7 [homodimers, HM], red square is BMP2/7 [heterodimers, HT]) are indicated. Individual data points are shown, box plots represent treatment-group median, 25th and 75th percentiles and whiskers denote minimum and maximum data points. Significance: *** p ≤ 0.001, **p ≤ 0.01, and *p ≤ 0.05.

Figure 2

In vitro angular displacement (IVAD) (A) and four-point bending (B) assessments showed greatest stiffness induction in BMP2/7 heterodimer (HT) treated spines. Spines were assessed for IVAD and four-point bending over the combined lumbar segment 4–6 (L4-6) at 12 weeks after intradiscal delivery of genetically programmed bone marrow stromal cells. The bending moment at failure in extension (C) showed a treatment effect, but post-hoc testing did not reveal a unique treatment. In (A)–(C) treatments correspond to color (white square is Naïve, blue square is LACZ, green square is BMP2, yellow square is BMP7, orange square is mixed BMP2 and BMP7 [homodimers, HM], red square is BMP2/7 [heterodimers, HT]). Individual data points are shown, box plots represent treatment-group median, 25th and 75th percentiles and whiskers denote minimum and maximum data points. Significance: p ≤ 0.001 (***), p ≤ 0.01 (**), and p ≤ 0.05 (*). (D) scatter plot of paired IVAD:NIAD (black circle) and NIAD:four-point bending stiffness (orange circle is extension, green circle is flexion, red circle is right bend, and light blue circle is left bend). Note that the infrequently observed double-level fusions represent the low NIAD value specimens on the left side of the graph.

Figure 3

Microscopic morphology of fusion bone and intervertebral disc space tissues 12 weeks after intradiscal implantation of genetically programmed bone marrow stromal cells. Spines were explanted, decalcified, paraffin embedded, sagittal sectioned, and assessed for: general architecture (H&E stain—A, B, C and D), proteoglycan/cartilage (Alcian Blue stain—E, F, G and H), or collagen (Picrosirus Red stain and circular polarized light—I, J, K and L). Representative images from specimen 29 from a non-operated normal L3/4 level (A, E and I), specimen 26 LACZ (marker gene)-treated L4/5 level (B, F and J), specimen 29 BMP2 and BMP7 homodimer (HD)-treated L4/5 level (C, G and K), and specimen 24 BMP2/7 heterodimer (HT)-treated L5/6 level (D, H and J) are shown. H&E stained images are marked to show: anterior longitudinal ligament (ALL) (white triangle) (partially discontinuous distally as an artifact of preparation in A), annulus fibrosus (AF) (white asterisks), and epiphyseal bony endplates (black triangle). Alcian Blue stained images are marked to show: endplate growth plates (white triangle), anterior and posterior AF lamellae (white asterisks), and nucleus pulposus (NP) (black triangle). Picrosirus Red polarized images are marked to show: bright yellow/red and green coloring of high collagen content structures (ALL [as white triangle], AF [as yellow triangle], and vertebral body bone network) and black coloring of low collagen content (growth plates [as white asterisks], NP space, and bone marrow compartments in the vertebral bodies). (A)–(H) shows annulus fibrosus (AF) and nucleus pulposus (NP) are well stained and generally preserved. (A) and (E) shows the non-operated NP has a central volume that stains lighter with Alcian Blue and contains small eosinophilic (physaliphorous) cells, which are not seen in the more homogeneously dark blue staining NP compartments of the operated levels (B–D and F–H). Note scar formation and disorganization of the bony and cartilaginous growth plate epiphyses (red asterisks in B, C and D) at what were interpreted to be endplate puncture sites. Comparing (E) with (F) and (G) shows additional operative changes including: new cartilage-appearing cells between the ALL and anterior fibers of the AF (red asterisks) staining similarly and in apparent continuity with the cartilaginous growth plate, and new vertebral body bone extending anterior to the anterior AF that is minor in Panel F but more noticeable in G (yellow asterisks). D & H shows impressive bone production anterior to the disc space in an HT treated specimen, contiguous with the marrow space, and fatty appearing marrow. Segments are shown with top left of sections being ventral and proximal, magnification at 20 × , and scale bar representing 1 mm.

Figure 4

Fusion status assigned by palpation, radiography (faxitron or micro-CT) and histology don’t always directly translate at 12 weeks after disc-implantation of BMP-expressing cells. (A) Lateral faxitron and midsagittal micro-CT images (when available) for several specimens where palpation (“Palpation” – fused (F) / not fused (NF)) was inconsistent or non-correlative to dichotomous radiographic [fused (+)/not fused (−)] or the 0–6 sum score (sum shown, 0–4 is not fused and 5–6 is fused). BMP treatment and specimen number are indicated to allow cross-comparison with Figs. 3, 4B–D, and Supplementary Fig. S1. Note that segmental kyphosis or disc height loss of an L4/5 or L5/6 segment tends to correlate with anterior fusion mass status (compare levels fused/not for specimens 24, 31, 44, and 63). (B)–(D) Alcian blue stained histology sections demonstrating cartilage clefts through the anterior induced bone fusion masses, noted in some of the other heterodimer-treated specimens not shown in Fig. 3. Images are marked to show: endplate growth plates (white triangle), anterior and posterior AF lamellae (white asterisks), nucleus pulposus (NP) (black triangle), and presence of cartilage clefts (red asterisks). Images are of the L4/5 level of each specimen, (B) shows specimen 24, (C) shows specimen 31, and (D) shows specimen 63. All images are oriented with top left being ventral and proximal, magnification is 20 × , and scale bar represents 1 mm.