Demineralized Bone Matrix Injection in Consolidation Phase Enhances Bone Regeneration in Distraction Osteogenesis via Endochondral Bone Formation

Abstract

Background: Distraction osteogenesis (DO) is a promising tool for bone and tissue regeneration. However, prolonged healing time remains a major problem. Various materials including cells, cytokines, and growth factors have been used in an attempt to enhance bone formation. We examined the effect of percutaneous injection of demineralized bone matrix (DBM) during the consolidation phase on bone regeneration after distraction.

Methods: The immature rabbit tibial DO model (20 mm length-gain) was used. Twenty-eight animals received DBM 100 mg percutaneously at the end of distraction. Another 22 animals were left without further procedure (control). Plain radiographs were taken every week. Postmortem bone dual-energy X-ray absorptiometry and micro-computed tomography (micro-CT) studies were performed at the third and sixth weeks of the consolidation period and histological analysis was performed.

Results: The regenerate bone mineral density was higher in the DBM group when compared with that in the saline injection control group at the third week postdistraction. Quantitative analysis using micro-CT revealed larger trabecular bone volume, higher trabecular number, and less trabecular separation in the DBM group than in the saline injection control group. Cross-sectional area and cortical thickness at the sixth week postdistraction, assessed using micro-CT, were greater in the regenerates of the DBM group compared with the control group. Histological evaluation revealed higher trabecular bone volume and trabecular number in the regenerate of the DBM group. New bone formation was apparently enhanced, via endochondral ossification, at the site and in the vicinity of the injected DBM. DBM was absorbed slowly, but it remained until the sixth postoperative week after injection.

Conclusions: DBM administration into the distraction gap at the end of the distraction period resulted in a significantly greater regenerate bone area, trabecular number, and cortical thickness in the rabbit tibial DO model. These data suggest that percutaneous DBM administration at the end of the distraction period or in the early consolidation period may stimulate regenerate bone formation and consolidation in a clinical situation with delayed bone healing during DO.

Keywords: Bone development; Bone substitute; Distraction; Osteogenesis.

Fig. 1. Demineralized bone matrix was injected under the guidance of C-arm fluoroscopy.

Fig. 2. At week 0, distraction gap in demineralized bone matrix (DBM) group looked more radiopaque due to injected DBM. At week 3, more exuberant bone formation was observed in DBM group. At week 6, cortical remodeling was observed in DBM group while it was scarce even at week 8 in control group.

Fig. 3. The three-dimensional (3D) reconstruction image of one voxel of interest showed that demineralized bone matrix (DBM) group (A) had denser trabecular bones than control group (B) at third week of consolidation period. The 3D reconstruction image of whole regenerate in distraction gap showed that DBM group (C) had well remodeled cortices when compared with control group (D) at the sixth week of consolidation period.

Fig. 4. (A) Demineralized bone matrix (DBM) group showed higher pixel values than control group during early 3 weeks of consolidation period (^*p < 0.05). However, during late 4 weeks of consolidation period, pixel value of DBM group was similar to that of control group. (B) There was significant difference of bone mineral density (BMD) between DBM group and control group at the 3rd week of consolidation period (^*p < 0.05). BMD of DBM group was similar to that of control group at the 6th week of consolidation period.

Fig. 5. (A, B) Fig. 5B is a 4 times magnified picture of the black square in Fig. 5A. On the second day of consolidation period, injected demineralized bone matrix (DBM) distorted bone regenerate in distraction gap and inflammatory cells accumulated around the injected DBM (H&E). (C, D) At the second week of consolidation period, endochondral ossification around injected DBM (safranin O and fast green staining) and resorption of DBM by multi-nucleated giant cell (arrows, H&E) were observed. (E, F) At the third week of consolidation period, DBM group (E) had denser bone trabeculae than control group (F) (H&E). (G, H) At the sixth week of consolidation period, DBM group (G) showed thicker cortical bone and wider total bone diameter than control group (H) (H&E). The remnants of injected DBM were still observed at the sixth week of consolidation period (G).