Demineralized bone matrix combined with concentrated growth factors promotes intervertebral fusion in a novel rat extreme lateral interbody fusion model

Abstract

Background: Whether demineralized bone matrix (DBM) combined with concentrated growth factors (CGF) can accelerate intervertebral fusion remains uncertain. This study developed a novel rat model for extreme lateral interbody fusion (XLIF) and evaluated the fusion outcomes of DBM combined with CGF using imaging and histological analysis.

Methods: A total of 70 male SD rats (3 months old, average body weight 300 ± 50 g) were included in this study. Among them, 10 rats were used for the anatomical study of the lumbar spine. The remaining 48 rats were randomly divided into four groups (n = 12 per group): Group A (control), Group B (titanium plate fixation), Group C (DBM + titanium plate fixation), and Group D (DBM + CGF + titanium plate fixation). The remaining 12 rats were used as donors to prepare fresh CGF. Eight weeks after surgery, the rats were euthanized and lumbar spine specimens were collected, with interbody fusion evaluated by manual palpation. Subsequently, specimens from groups B, C, and D were analyzed by micro-CT and histological examinations to comprehensively assess the fusion outcome.

Results: The anatomical and surgical techniques for the rat XLIF model are described. Titanium plates (7 mm × 2.5 mm × 0.8 mm) and screws (3 mm × 1 mm) were designed based on the anatomical measurements. In Group A, spontaneous fusion occurred in 1 case; the remaining 11 cases showed intervertebral mobility. In Group B, 3 cases achieved fusion; in Group C, 8 cases; and in Group D, 11 cases. Micro-CT revealed fusion index scores (FIS) of 2.21 ± 0.51 for Group B, 3.62 ± 0.67 for Group C, and 4.57 ± 0.56 for Group D. Histological examination showed limited bone formation in Group B, with fibrous connective tissue filling the intervertebral space. Group C showed more bone formation, but some cartilage and fibrous tissue remained. Group D demonstrated abundant new bone formation and robust histological fusion, with substantial bridging between vertebrae.

Conclusion: The rat XLIF model for interbody fusion has been successfully established and validated. Using this model, it was preliminarily demonstrated that DBM combined with CGF can effectively promote intervertebral fusion in rats.

Keywords: Concentrated growth factor; Demineralized bone matrix; Intervertebral fusion; XLIF.

Fig. 1

Flow diagram of the study design indicating the group allocations and study procedures. A: control group; B:titanium plate fixation group; C:DBM + titanium plate fixation group; D:DBM + CGF + titanium plate fixation group. IHC:Immunohistochemistry

Fig. 2

Macroscopic examination and SEM analysis of CGF. A-C:The macroscopic appearance of CGF shows a three-layer structure in the collection tube: the top plasma layer, the middle yellow gel-like CGF layer, and the bottom red blood cell layer. D, E: SEM images at different magnifications reveal a dense fibrin network structure within the CGF

Fig. 3

Surgical procedure A: Surface landmarks were identified and marked. B: The surgical field was exposed. C: The iliac vessels were dissected and ligated. D: The intervertebral disc at the surgical segment was exposed. E: The intervertebral space was prepared. F: Lateral titanium plate fixation was applied

Fig. 4

Postoperative X-ray images of the rat lumbar spine in both anteroposterior (AP) and lateral views showed that the titanium plate and screws were properly positioned. The titanium plate was placed at the anterolateral aspect of the vertebral body, and the screws did not penetrate the spinal canal. A: Anteroposterior view B: Lateral view

Fig. 5

A small cube with a side length of 2 mm was selected as the region of interest (ROI) at the interbody fusion site, and analysis was performed using CTvox software. The red box indicates the ROI

Fig. 6

Measurement of Anatomical Specimens A: Sagittal and coronal maximum diameters of the inferior end of L5 vertebra. B: Angle between the vertebral body and transverse process at the inferior end of L5 vertebra. C: Maximum depth of the screw channel at the inferior end of L5 vertebra. D: Sagittal and coronal maximum diameters of the superior end of L6 vertebra. E: Angle between the vertebral body and transverse process at the superior end of L6 vertebra. F: Maximum depth of the screw channel at the superior end of L6 vertebra. G: Height of the intervertebral space between L4 and L6

Fig. 7

Gross Observation and Palpation of Rat Specimens After Collection.A,B:The anteroposterior and lateral views of the specimens show that the titanium plate is encapsulated by bone spurs.C,D: The specimens in hyperextension and flexion positions show no mobility at the surgical segment. E,F:X-ray images of the specimens in anteroposterior and lateral views confirm the proper placement of the internal fixation.G,H: After removal of the internal fixation, good bone bridging is visible between the vertebrae

Fig. 8

The Micro-CT images of Groups B, C, and D in the coronal, sagittal, and 3D reconstructions showed that in Group B (Titanium plate fixation), no obvious bony structures were present in the intervertebral space. In Group C (DBM + Titanium plate fixation group), bone ingrowth was observed, but fissures remained at the intervertebral connection. In Group D (DBM + CGF + Titanium plate fixation), significant bone ingrowth was seen across the intervertebral space, forming a stable intervertebral fusion

Fig. 9

The histological sections stained with HE and Safranin O-Fast Green from Groups B, C, and D showed bone growth in the fusion areas. Boxes labeled 1 to 6 on the right are magnified. Arrows indicate bone, triangles represent cartilage, and stars represent fibrous connective tissue. The scale bar is shown in the lower left corner of the image. Group B: Titanium plate fixation. Group C: DBM + Titanium plate fixation. Group D: DBM + CGF + Titanium plate fixation

Fig. 10

The figure displays immunohistochemical staining of the osteogenic proteins ALP, OPN, and Runx2 in Groups B, C, and D; the accompanying bar charts quantitatively compare their expression levels across the three groups. It can be observed that in the intervertebral bone implantation groups, C and D, the expression levels of MMP13, OPN, and Runx2 were significantly higher than in the titanium plate fixation group (Group B). Additionally, the expression levels of MMP13, OPN, and Runx2 were notably higher in Group D compared to Group C.Group B: Titanium plate fixation group; Group C: DBM + Titanium plate fixation; Group D: DBM + CGF + Titanium plate fixation