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. 2022 Nov 25;23(23):14720.
doi: 10.3390/ijms232314720.

Comparative Efficiency of Gene-Activated Matrices Based on Chitosan Hydrogel and PRP Impregnated with BMP2 Polyplexes for Bone Regeneration

Irina Alekseevna Nedorubova  1 , Tatiana Borisovna Bukharova  1 , Viktoria Olegovna Mokrousova  1   2 , Maria Aleksandrovna Khvorostina  1   3 , Andrey Vyacheslavovich Vasilyev  1   2 , Andrey Anatolevich Nedorubov  4 , Timofei Evgenevich Grigoriev  5 , Yuriy Dmitrievich Zagoskin  5 , Sergei Nicolaevich Chvalun  5 , Sergey Ivanovich Kutsev  1 , Dmitry Vadimovich Goldshtein  1
Affiliations

Comparative Efficiency of Gene-Activated Matrices Based on Chitosan Hydrogel and PRP Impregnated with BMP2 Polyplexes for Bone Regeneration

Irina Alekseevna Nedorubova et al. Int J Mol Sci. .

Abstract

Gene therapy is one of the most promising approaches in regenerative medicine. Gene-activated matrices provide stable gene expression and the production of osteogenic proteins in situ to stimulate osteogenesis and bone repair. In this study, we developed new gene-activated matrices based on polylactide granules (PLA) impregnated with BMP2 polyplexes and included in chitosan hydrogel or PRP-based fibrin hydrogel. The matrices showed high biocompatibility both in vitro with mesenchymal stem cells and in vivo when implanted intramuscularly in rats. The use of porous PLA granules allowed the inclusion of a high concentration of polyplexes, and the introduction of the granules into hydrogel provided the gradual release of the plasmid constructs. All gene-activated matrices showed transfecting ability and ensured long-term gene expression and the production of target proteins in vitro. At the same time, the achieved concentration of BMP-2 was sufficient to induce osteogenic differentiation of MSCs. When implanted into critical-size calvarial defects in rats, all matrices with BMP2 polyplexes led to new bone formation. The most significant effect on osteoinduction was observed for the PLA/PRP matrices. Thus, the developed gene-activated matrices were shown to be safe and effective osteoplastic materials. PLA granules and PRP-based fibrin hydrogel containing BMP2 polyplexes were shown to be the most promising for future applications in bone regeneration.

Keywords: bone regeneration; chitosan; gene-activated matrices; plasmid DNA; platelet rich plasma; 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
Matrices based on PLA granules, PLA/Chit hydrogel and PLA/PRP hydrogel in saline solution.
Figure 2
Figure 2
In vitro cytocompatibility of matrices: (a) adhesion of MSCs to PLA granules, SEM; (b) assessment of cytotoxicity of matrices using fluorescent microscopy of live MSCs stained with calcein AM (green) and dead cells stained with DAPI (blue); and (c) viability assessment of MSCS at 1 and 7 days after incubation with the matrices, MTT-test. * p < 0.05 (relative to control).
Figure 2
Figure 2
In vitro cytocompatibility of matrices: (a) adhesion of MSCs to PLA granules, SEM; (b) assessment of cytotoxicity of matrices using fluorescent microscopy of live MSCs stained with calcein AM (green) and dead cells stained with DAPI (blue); and (c) viability assessment of MSCS at 1 and 7 days after incubation with the matrices, MTT-test. * p < 0.05 (relative to control).
Figure 3
Figure 3
In vivo biocompatibility of matrices 28 days after intramuscular implantation: (a) tissue sections of the implantation area, H&E staining. Light microscopy. BV—blood vessel, GC—giant cells; (b) quantitative assessment of the resorption degree of the matrices; and (c) quantitative assessment of the degree of inflammation. * p < 0.05 (comparison between the groups).
Figure 4
Figure 4
Release of TF/pGFP polyplexes from matrices, spectrophotometry.
Figure 5
Figure 5
Transfecting ability of gene-activated matrices: (a) the ability of TF/pGFP-Matrices to transfect MSCs (green) prestained with PKH-26 (red), fluorescent microscopy; (b) relative BMP2 gene expression 14 days after incubation of MSCs with TF/pBMP2-Matrices, RT-PCR; and (c) BMP-2 protein production 14 days after incubation of MSCs with TF/pBMP2-Matrices, ELISA. * p < 0.05 (relative to control without matrices).
Figure 6
Figure 6
Osteoinductive properties of TF/pBMP2-Matrices 14 days after incubation with MSCs: (a) relative Alpl gene expression, RT-PCR. * p < 0.05 (relative to negative control); (b) Alpl protein production (green), nuclei (blue) ICH; and (c) ECM mineralization of MSCs, alizarin red staining.
Figure 7
Figure 7
Critical-size calvarial bone defect regeneration 56 days after implantation of the matrices: (a) nonactivated matrices; (b) TF/BMP2-Matrices. H&E and Masson’s trichrome staining. Light microscopy. CT—connective tissue, NB—newly formed bone.
Figure 7
Figure 7
Critical-size calvarial bone defect regeneration 56 days after implantation of the matrices: (a) nonactivated matrices; (b) TF/BMP2-Matrices. H&E and Masson’s trichrome staining. Light microscopy. CT—connective tissue, NB—newly formed bone.
Figure 8
Figure 8
Critical-size bone defect regeneration in rats: (a) micro-CT data of critical-size calvarial defect; (b) the volume of newly formed bone (Nb.V.) measured using micro-CT; and (с) the area of newly formed bone (Nb.Ar.) inside the defect measured using histomorphometrical analysis. * p < 0.05 (relative to nonactivated matrices).
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