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. 2021 Jan 7;26(2):261.
doi: 10.3390/molecules26020261.

Influence of the Degree of Deacetylation of Chitosan and BMP-2 Concentration on Biocompatibility and Osteogenic Properties of BMP-2/PLA Granule-Loaded Chitosan/β-Glycerophosphate Hydrogels

Andrey Vyacheslavovich Vasilyev  1   2   3 , Valeriya Sergeevna Kuznetsova  1   2 , Tatyana Borisovna Bukharova  1   2 , Timofei Evgenevich Grigoriev  4   5 , Yuriy Dmitrievich Zagoskin  4 , Irina Alekseevna Nedorubova  1   2 , Igor Ivanovich Babichenko  2   3 , Sergey Nicolaevich Chvalun  4 , Dmitry Vadimovich Goldstein  1   2   3 , Anatoliy Alekseevich Kulakov  1
Affiliations

Influence of the Degree of Deacetylation of Chitosan and BMP-2 Concentration on Biocompatibility and Osteogenic Properties of BMP-2/PLA Granule-Loaded Chitosan/β-Glycerophosphate Hydrogels

Andrey Vyacheslavovich Vasilyev et al. Molecules. .

Abstract

Compositions based on chitosan/β-glycerophosphate hydrogels with highly porous polylactide granules can be used to obtain moldable bone graft materials that have osteoinductive and osteoconductive properties. To eliminate the influence of such characteristics as chain length, degree of purification, and molecular weight on a designed material, the one-stock chitosan sample was reacetylated to degrees of deacetylation (DD%) of 19.5, 39, 49, 55, and 56. A study of the chitosan/β-glycerophosphate hydrogel with chitosan of a reduced DD% showed that a low degree of deacetylation increased the MSCs (multipotent stromal cells) viability rate in vitro and reduced the leukocyte infiltration in subcutaneous implantation to Wistar rats in vivo. The addition of 12 wt% polylactide granules resulted in optimal composite mechanical and moldable properties, and increased the modulus of elasticity of the hydrogel-based material by approximately 100 times. Excessive filling of the material with PLA (polylactide) granules (more than 20%) led to material destruction at a ~10% strain. Osteoinductive and osteoconductive properties of the chitosan hydrogel-based material with reacetylated chitosan (39 DD%) and highly porous polylactide granules impregnated with BMP-2 (bone morphogenetic protein-2) have been demonstrated in models of orthotopic and ectopic bone formation. When implanted into a critical-size calvarial defect in rats, the optimal concentration of BMP-2 was 10 μg/mL: bone tissue areas filled the entire material's thickness. Implantation of the material with 50 μg/mL BMP-2 was accompanied with excessive growth of bone tissue and material displacement beyond the defect. Significant osteoinductive and osteoconductive properties of the material with 10 μg/mL of BMP-2 were also shown in subcutaneous implantation.

Keywords: BMP-2; chitosan; deacetylation degree; osteoinduction; osteoplastic material; porous polylactide granules.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
The effect of chitosan reacetylation on the biocompatibility of the thermosetting chitosan–β-glycerophosphate hydrogel. (A) MSC viability in the presence of chitosan hydrogels, seven days, MTT test. (B) Cell adhesion of the chitosan-based hydrogels at the lowest and highest degrees of deacetylation (65 and 39 DD%), PKH26 staining. (C) Level of leukocyte infiltration 14 days after subcutaneous implantation of chitosan hydrogels. (D) Tissue sections of the implantation area of the chitosan-based hydrogels with high and low degrees of deacetylation (65 and 39 DD%).
Figure 2
Figure 2
Mechanical properties of composition based on the chitosan and β-glycerophosphate hydrogel and the PLA granules. (Left) The deformation curves of the composite materials during compression test. (Right) The elastic modulus of the compositions containing different PLA granule mass fractions.
Figure 3
Figure 3
Volume of newly formed bone in the critical-size calvarial bone defect after implantation of the composition based on the reacetylated chitosan/β-glycerophosphate hydrogel and 12 wt% PLA granules containing different BMP-2 doses in rats after 28 days. The significance of differences was indicated in accordance with the requirements of the American Psychological Association (APA): ns—p > 0.05; ***—p ≤ 0.001.
Figure 4
Figure 4
Bone regeneration capacity in the critical-size calvarial bone defect area of rats after implantation of the material based on the reacetylated chitosan/β-glycerophosphate hydrogel and 12 wt% PLA granules containing 0, 10, and 50 µg/mL of BMP-2. H&E and Masson’s trichrome staining after 28 days. PLA—highly porous polylactide granules, NB—newly formed bone, BV—blood vessel, GC—foreign-body giant cell, CT—connective tissue, IM—implanted material, and BM—bone marrow.
Figure 5
Figure 5
Bone formation after subcutaneous implantation of the composition containing 10 µg/mL of BMP-2. H&E and Masson’s trichrome staining after 28 days. PLA—highly porous polylactide granules, NB—newly formed bone, BV—blood vessel, GC—foreign-body giant cell, LYM—lymphocytes, and plasma cells.
Figure 6
Figure 6
Scheme of a two-step chitosan acetylation reaction.
Figure 7
Figure 7
Infrared spectroscopy for the initial and reacylated chitosan.
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