Evaluation of the properties of osteogenic gene-activated matrices based on hydrogels impregnated with polyplexes with the BMP2 gene

Abstract

BACKGROUND: Currently, to effective bone regeneration, biocompatible matrices containing plasmid constructs with osteoinductor genes are developed.

AIM: To study in vitro the properties of gene-activated matrices based on collagen and platelet-rich plasma impregnated with polyplexes with the bone morphogenetic protein 2 gene.

METHODS: The methods of fluorescence microscopy, flow cytofluorimetry, ELISA, real-time PCR and MTT test were used in the study.

RESULTS: Using the MTT test and fluorescence microscopy to detect live and dead cells, it was shown that the obtained matrices have high cytocompatibility. Impregnation of plasmid constructs in hydrogel matrices based on collagen and fibrin ensures their prolonged release within 25 days. Using flow cytometry and fluorescence microscopy it was shown that polyplexes released from matrices are able to effectively transfect multipotent mesenchymal stromal cells derived from rat adipose tissue. 3 weeks after incubation of cells with matrices impregnated with polyplexes with BMP2 gene, it was shown a 25-fold increase in BMP-2 protein production by enzyme-linked immunosorbent assay. BMP-2 secreted by transfected cells induced osteogenic differentiation of multipotent mesenchymal stromal cells, as evidenced by increased expression of the osteopontin gene and extracellular matrix mineralization.

CONCLUSION: The developed matrices in the future can be used in gene therapy for damaged bone.

Keywords

gene-activated matrices / collagen / platelet-rich plasma / transfection / bone morphogenetic protein 2

References

[1] Shapiro G, Lieber R, Gazit D, Pelled G. Recent advances and future of gene therapy for bone regeneration. Curr Osteoporos Rep. 2018;16(4):504–511. doi: 10.1007/s11914-018-0459-3

[2] Shapiro G., Lieber R., Gazit D., Pelled G. Recent advances and future of gene therapy for bone regeneration // Curr Osteoporos Rep. 2018. Vol. 16, N 4. P. 504–511. doi: 10.1007/s11914-018-0459-3

[3] Balmayor ER, van Griensven M. Gene therapy for bone engineering. Front Bioeng Biotechnol. 2015;3:9. doi: 10.3389/fbioe.2015.00009

[4] Balmayor E.R., van Griensven M. Gene therapy for bone engineering // Front Bioeng Biotechnol. 2015. Vol. 3. P. 9. doi: 10.3389/fbioe.2015.00009

[5] Schmidt-Bleek K, Willie BM, Schwabe P, et al. BMPs in bone regeneration: less is more effective, a paradigm-shift. Cytokine Growth Factor Rev. 2016;27:141–148. doi: 10.1016/j.cytogfr.2015.11.006

[6] Schmidt-Bleek K., Willie B.M., Schwabe P., et al. BMPs in bone regeneration: less is more effective, a paradigm-shift // Cytokine Growth Factor Rev. 2016. Vol. 27. P. 141–148. doi: 10.1016/j.cytogfr.2015.11.006

[7] Nedorubova IA, Bukharova TB, Vasilyev AV, et al. Non-viral delivery of the BMP2 gene for bone regeneration. Genes & cells. 2020;15(4):33–39. (In Russ). doi: 10.23868/202012005

[8] Недорубова И.А., Бухарова Т.Б., Васильев А.В., и др. Невирусная доставка гена BMP2 для регенерации костной ткани // Гены и клетки. 2020. Т. 15, № 4. С. 33–39. doi: 10.23868/202012005

[9] Deev RV, Drobyshev AY, Bozo IY. Ordinary and activated osteoplastic materials. NN Priorov Journal of Traumatology and Orthopedics. 2015;22(1):51–69. (In Russ). doi: 10.32414/0869-8678-2015-1-51-69

[10] Деев Р.В., Дробышев А.Ю., Бозо И.Я. Ординарные и активированные остеопластические материалы // Вестник травматологии и ортопедии им. Н.Н. Приорова. 2015. № 1. С. 51–69. doi: 10.32414/0869-8678-2015-1-51-69

[11] Bozo IY, Presnyakov EV, Rochev ES, et al. Non-viral gene transfer in hydrogel matrices with octacalcium phosphate microgranules in optimization of reparative osteogenesis. Genes & cells. 2021;16(3):91–96. (In Russ). doi: 10.23868/202110013

[12] Бозо И.Я., Пресняков Е.В., Рочев Е.С., и др. Невирусный генный трансфер в гидрогелевых матриксах с микрогранулами октакальциевого фосфата в оптимизации репаративного остеогенеза // Гены и Клетки. 2021. Т. 16, № 3. С. 91–96. doi: 10.23868/202110013

[13] Bharadwaz A, Jayasuriya AC. Recent trends in the application of widely used natural and synthetic polymer nanocomposites in bone tissue regeneration. Mater Sci Eng C Mater Biol Appl. 2020;110:110698. doi: 10.1016/j.msec.2020.110698

[14] Bharadwaz A., Jayasuriya A.C. Recent trends in the application of widely used natural and synthetic polymer nanocomposites in bone tissue regeneration // Mate Sci Eng C Mater Biol Appl. 2020. Vol. 110. P. 110698. doi: 10.1016/j.msec.2020.110698

[15] Gibbs DM, Black CR, Dawson JI, Oreffo RO. A review of hydrogel use in fracture healing and bone regeneration. J Tissue Eng Regen Med. 2016;10(3):187–198. doi: 10.1002/term.1968

[16] Gibbs D.M., Black C.R., Dawson J.I., Oreffo R.O. A review of hydrogel use in fracture healing and bone regeneration // J Tissue Eng Regen Med. 2016. Vol. 10, N 3. P. 187–198. doi: 10.1002/term.1968

[17] Sorushanova A, Delgado LM, Wu Z, et al. The collagen suprafamily: from biosynthesis to advanced biomaterial development. Adv Mater. 2019;31(1):1801651. doi: 10.1002/adma.201801651

[18] Sorushanova A., Delgado L.M., Wu Z., et al. The collagen suprafamily: from biosynthesis to advanced biomaterial development // Adv Mater. 2019. Vol. 31, N 1. P. 1801651. doi: 10.1002/adma.201801651

[19] Vasilyev AV, Kuznetsova VS, Bukharova TB, et al. Osteoinductive moldable and curable bone substitutes based on collagen, BMP-2 and highly porous polylactide granules, or a mix of HAP/β-TCP. Polymers (Basel). 2021;13(22):3974. doi: 10.3390/polym13223974

[20] Vasilyev A.V., Kuznetsova V.S., Bukharova T.B., et al. Osteoinductive moldable and curable bone substitutes based on collagen, BMP-2 and highly porous polylactide granules, or a mix of HAP/β-TCP // Polymers (Basel). 2021. Vol. 13, N 22. P. 3974. doi: 10.3390/polym13223974

[21] Etulain J. Platelets in wound healing and regenerative medicine. Platelets. 2018;29(6):556–568. doi: 10.1080/09537104.2018.1430357

[22] Etulain J. Platelets in wound healing and regenerative medicine // Platelets. 2018. Vol. 29, N 6. P. 556–568. doi: 10.1080/09537104.2018.1430357

[23] Desai AP, Sahoo N, Pal AK, Roy Chowdhury SK. Efficacy of platelet-rich plasma in enhancing the osteogenic potential of bone graft in oral and maxillofacial region. J Maxillofac Oral Surg. 2021;20(2):282–295. doi: 10.1007/s12663-020-01378-z

[24] Desai A.P., Sahoo N., Pal A.K., Roy Chowdhury S.K. Efficacy of platelet-rich plasma in enhancing the osteogenic potential of bone graft in oral and maxillofacial region // J Maxillofac Oral Surg. 2021. Vol. 20, N 2. P. 282–295. doi: 10.1007/s12663-020-01378-z

[25] Buharova TB, Volkov AV, Antonov EN, et al. Tissue-engineered construction made of adipose derived multipotent mesenchymal stromal cells, polylactide scaffolds and platelet gel. Cellular Transplantation and Tissue Engineering. 2013;8(4):61–68. (In Russ).

[26] Бухарова Т.Б., Волков А.В., Антонов Е.Н., и др. Тканеинженерная конструкция на основе мультипотентных мезенхимальных стромальных клеток жировой ткани, полилактидных носителей и тромбоцитарного геля // Клеточная трансплантология и тканевая инженерия. 2013. Т. 8, № 4. С. 61–68.

[27] Aggarwal R, Targhotra M, Kumar B, et al. Polyplex: a promising gene delivery system. International Journal of Pharmaceutical Sciences and Nanotechnology (IJPSN). 2019;12(6):4681–4686. doi: 10.37285//ijpsn.2019.12.6.1

[28] Aggarwal R., Targhotra M., Kumar B., et al. Polyplex: a promising gene delivery system // International Journal of Pharmaceutical Sciences and Nanotechnology (IJPSN). 2019. Vol. 12, N 6. P. 4681–4686. doi: 10.37285//ijpsn.2019.12.6.1

[29] Nedorubova IA, Bukharova TB, Vasilyev AV, et al. Comparative study of BMP-2 gene delivery to human adipose tissue-derived mesenchymal stem cells with turbofect and polyethylenimine. IOP Conf Ser Earth Environ Sci. 2021;677(4):042024. doi: 10.1088/1755-1315/677/4/042024

[30] Nedorubova I.A., Bukharova T.B., Vasilyev A.V., Khvorostina M. Comparative study of BMP-2 gene delivery to human adipose tissue-derived mesenchymal stem cells with turbofect and polyethylenimine // IOP Conf Ser Earth Environ Sci. 2021. Vol. 677, N 4. P. 042024. doi: 10.1088/1755-1315/677/4/042024

[31] Nedorubova IA, Bukharova TB, Vasilyev AV, et al. Development of osteoplastic material impregnated with plasmid encoding bone morphogenetic protein-2. Appl Biochem Microbiol. 2021;57(7):818–22. doi: 10.1134/S000368382107005X

[32] Nedorubova I.A., Bukharova T.B., Vasilyev A.V., et al. Development of osteoplastic material impregnated with plasmid encoding bone morphogenetic protein-2 // Appl Biochem Microbiol. 2021. Vol. 57, N 7. P. 818–822. doi: 10.1134/S000368382107005X

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