Regeneration of critical-sized defects, in a goat model, using a dextrin-based hydrogel associated with granular synthetic bone substitute

Abstract

The development of injectable bone substitutes (IBS) have obtained great importance in the bone regeneration field, as a strategy to reach hardly accessible defects using minimally invasive techniques and able to fit to irregular topographies. In this scenario, the association of injectable hydrogels and bone graft granules is emerging as a well-established trend. Particularly, in situ forming hydrogels have arisen as a new IBS generation. An in situ forming and injectable dextrin-based hydrogel (HG) was developed, aiming to act as a carrier of granular bone substitutes and bioactive agents. In this work, the HG was associated to a granular bone substitute (Bonelike^®) and implanted in goat critical-sized calvarial defects (14 mm) for 3, 6 and 12 weeks. The results showed that HG improved the handling properties of the Bonelike^® granules and did not affect its osteoconductive features, neither impairing the bone regeneration process. Human multipotent mesenchymal stromal cells from the umbilical cord, extracellular matrix hydrolysates and the pro-angiogenic peptide LLKKK18 were also combined with the IBS. These bioactive agents did not enhance the new bone formation significantly under the conditions tested, according to micro-computed tomography and histological analysis.

Keywords: Bonelike®; bone regeneration; calvarial defect; granular ceramics; injectable hydrogel; polysaccharide.

Figure 1.

Calvarial critical-sized defects performed in the animals filled with the dextrin-based hydrogel formulations with BL granules. In each goat was performed a control defect (non-treated defect) marked with an asterisk (*)

Figure 2.

Human umbilical cord multipotent mesenchymal stromal cells (hMSCs) characterization: (a) hMSCs from umbilical cord at passage 5 in standard culture conditions. (b) Chondrogenic, adipogenic and osteogenic differentiation visualized through Alcian Blue, Oil Red O and Von Kossa histochemical staining.

Figure 3.

Representative micro-CT reconstruction images of calvarial critical-sized defects, after 3, 6 and 12 weeks, for the different dextrin-based hydrogel (HG) formulations.

Figure 4.

Results of micro-CT analysis for critical-sized calvarial defects, after 3, 6 and 12 weeks: (a) quantification of the new bone volume formed in the defects treated with the dextrin-based hydrogel (HG) alone and the non-treated defect (control). (b) Quantification of the total bone volume (new bone and granules volume), (c) the granules volume and (d) the new bone volume in the defects treated with BL granules alone or incorporated into HG matrix (HG + BL) and its combination with 4 mg/ml of SIS (HG + BL + SIS), 1 mg/ml of LLKKK18 (HG + BL + LLKKK18) or hMSCs (HG + BL + hMSCs). Data are presented as mean ± SD (n = 5 replicates per group) and were analysed by one-way ANOVA followed by Bonferroni’s post hoc test: *P < 0.05, **P < 0.01 and ***P < 0.001 vs. HG + BL treatment, ^#P < 0.05 and ^##P < 0.01 vs. 3 weeks (intra group).

Figure 5.

Haematoxylin- and eosin-stained histological sections from calvarial critical-sized defects of decalcified samples. BL, Bonelike^® granules; NB, new bone; NV, new vessels; V, vessels; OB, osteoblasts; OC, osteoclasts; UM, unmineralized bone.

Figure 6.

Haematoxylin- and eosin-stained histological sections of decalcified samples from calvarial critical-sized defects treated with 4 mg/ml of SIS (HG + BL + SIS), 1 mg/ml of LLKKK18 (HG + BL + LLKKK18) or hMSCs (HG + BL + hMSCs). BL, Bonelike^® granules; NB, new bone; NV, new vessels; OB, osteoblasts; OC, osteoclasts; UM, unmineralized bone.