Science and Innovations in MedicineScience and Innovations in MedicineНаука и инновации в медицине2500-13882618-754XFSBEI of Higher Education SamSMU of Ministry of Health of the Russian Federation63582210.35693/SIM635822BiotechnologyБиотехнологияResearch ArticleEvaluation of biocompatibility and osteoconductivity of a hybrid cell-tissue graft for bone regenerative medicineОценка биосовместимости и остеокондуктивности гибридного клеточно-тканевого трансплантата для регенеративной медицины костной тканиhttps://orcid.org/0000-0002-1245-0426DanilkovichNataliya N.ДанилковичН. Н.
Belarus

Scientific officer of the laboratory of Stem Cell Biology and Genetics

научный сотрудник лаборатории биологии и генетики стволовых клеток

nndanilkovich@gmail.comhttps://orcid.org/0000-0002-1617-8845KosmachevaSvetlana M.КосмачеваСветлана. М.
Belarus

PhD, Associate professor, Head of the laboratory of Stem Cells Biology and Genetics

заведующая лабораторией биологии и генетики стволовых клеток

4kosmacheva@mail.ruhttps://orcid.org/0009-0000-3884-9112IonovaAleksandra G.ИоноваА. Г.
Belarus

Junior researcher of the laboratory of Stem Cells Biology and Genetics

младший научный сотрудник лаборатории биологии и генетики стволовых клеток

al_ionova96@mail.ruhttps://orcid.org/0000-0003-0456-2839KrivorotKirill A.КриворотК. А.
Belarus

PhD, Associate professor, Deputy Director for Organizational and Methodological work, neurosurgeon of the highest qualification category

канд. мед. наук, доцент, заместитель директора по организационно-методической работе, врач-нейрохирург высшей квалификационной категории

kirill.doc@mail.ruhttps://orcid.org/0000-0001-7092-2615MazurenkoAndrei N.МазуренкоА. Н.
Belarus

Head of Neurosurgical Department №2, PhD, Associate professor

канд. мед. наук, заведующий нейрохирургическим отделением №2

mazurenko@mail.ruhttps://orcid.org/0000-0003-4185-0709AlekseevDenis G.АлексеевД. Г.
Russian Federation

PhD, Associate professor, Leading researcher at the BioTech Research Institute

канд. мед. наук, доцент, ведущий научный сотрудник НИИ «БиоТех»

D.G.Alekseev@samsmu.ruRepublican Scientific and Practical Center of Transfusiology and Medical BiotechnologyРеспубликанский научно-практический центр трансфузиологии и медицинских биотехнологийRepublican Scientific and Practical Center of Traumatology and OrthopedicsРеспубликанский научно-практический центр травматологии и ортопедииSamara State Medical UniversityФГБОУ ВО «Самарский государственный медицинский университет» Минздрава России2910202415122024942562670809202418092024Copyright ©; 2024, Danilkovich N.N., Kosmacheva S.M., Ionova A.G., Krivorot K.A., Mazurenko A.N., Alekseev D.G.Copyright ©; 2024, Данилкович Н.Н., Космачева С.М., Ионова А.Г., Криворот К.А., Мазуренко А.Н., Алексеев Д.Г.2024Danilkovich N.N., Kosmacheva S.M., Ionova A.G., Krivorot K.A., Mazurenko A.N., Alekseev D.G.Данилкович Н.Н., Космачева С.М., Ионова А.Г., Криворот К.А., Мазуренко А.Н., Алексеев Д.Г.https://creativecommons.org/licenses/by/4.0https://innoscience.ru/2500-1388/article/view/635822

Aim – to evaluate in vitro the biocompatibility and osteoconductivity of a hybrid graft based on a bioorganic matrix, human bone marrow mesenchymal stromal cells (BM-MSC) and osteogenic growth factors.

Material and methods. Bioorganic matrices were studied for biocompatibility with human BM-MSC culture used in traumatology and orthopedics. For promoted osteogenic differentiation of BM-MSCs, allogeneic plasma enriched with soluble platelet factors was used. The osteogenic potential of BM-MSCs by the synthesis of mRNAs of early (transcription factor 2 (Run X2), alkaline phosphatase (ALP)) and late genes (osteopontin (OSP)) of osteogenesis was analyzed. The properties of cell adhesion and proliferation of MSCs in the conditions of a three-dimensional hybrid graft by the MTT test and fluorescence microscopy were assessed.

Results. The biocompatibility of the studied bioorganic matrices with human BM-MSCs was established. The collagen matrix promoted rapid cell adhesion and proliferation between the scaffold fibrils. It has also been established that allogeneic platelet-rich plasma affects the osteogenic differentiation of human BM-MSCs in vitro, increasing the expression of marker genes RunX2, ALP, OSP. When modeling a hybrid graft in vitro, the formation of a tight contact between the alloimplant and collagen biopolymer using MSCs was shown.

Conclusion. The biological properties of the developed hybrid cell-tissue graft characterize its biocompatibility and osteoconductivity of its constituent components, which makes it promising for use in regenerative medicine, especially in reconstructive surgery of bone defects.

Цель – оценить биосовместимость и остеокондуктивность in vitro гибридного клеточно-тканевого трансплантата для регенеративной медицины костной ткани на основе биоорганического матрикса, мезенхимальных стромальных клеток костного мозга (КМ-МСК) человека и остеогенных факторов роста.

Материал и методы. Исследованы на биосовместимость с культурой КМ-МСК человека биоорганические матриксы, используемые в травматологии и ортопедии. Для направленной остеогенной дифференцировки КМ-МСК использовали аллогенную плазму, обогащенную растворимыми факторами тромбоцитов. Остеогенный потенциал КМ-МСК анализировали по синтезу последними мРНК ранних (фактора транскрипции 2 / Run X2, щелочная фосфатаза / ALP) и поздних генов (остеопонтин / OSP) остеогенеза. Свойства клеточной адгезии и пролиферации КМ-МСК в условиях трехмерного гибридного трансплантата оценивали с помощью МТТ-теста и флуоресцентной микроскопии.

Результаты. Установлена биосовместимость исследуемых биоорганических матриксов с КМ-МСК человека. Отмечена быстрая адгезия и пролиферация клеток между волокнами используемых матриксов. Также установлено, что аллогенная плазма, обогащенная растворимыми факторами тромбоцитов, достоверно влияет на остеогенную дифференцировку КМ-МСК человека in vitro, усиливая экспрессию маркерных генов RunX2, ALP, OSP. При имитации трехмерного гибридного клеточно-тканевого трансплантата in vitro показано формирование плотного контакта между аллогенной спонгиозой (костной тканью) и биоорганическим матриксом с помощью остеогенно предифференцированных КМ-МСК.

Выводы. Биологические свойства разработанного гибридного клеточно-тканевого трансплантата характеризуются биосовместимостью и остеокондуктивностью, что делает его перспективным для применения в регенеративной медицине, особенно в реконструктивной хирургии костных дефектов.

human bone marrow mesenchymal stromal cellsscaffoldsbiopolymersbioorganic matrixbiocompatibilityplatelet-rich plasmahybrid cell-tissue graftмезенхимальные стромальные клеткикостный мозгбиополимерыбиоорганический матриксбиосовместимостьобогащенная тромбоцитами аллогенная плазмагибридный клеточно-тканевый трансплантатKosmacheva SM, Danilkovich NN, Shchepen AV, et al. Effect of platelet release on osteogenic differentiation of human bone marrow mesenchymal stem cells. Bull Exp Biol Med. 2014;156(4):560-5. DOI: https://doi.org/10.1007/s10517-014-2396-1Deev RV, Isaev AA, Kochish AYu, Tikhilov RM. Pathways for the development of cell technologies in bone surgery. Traumatology and orthopedics in Russia. 2008;47(1):65-75. (In Russ.). [Деев Р.В., Исаев А.А., Кочиш А.Ю., Тихилов P.M. Пути развития клеточных технологий в костной хирургии. Травматология и ортопедия в России. 2008;47(1):65-75]. URL: file:///C:/Users/Persona/Downloads/puti-razvitiya-kletochnyh-tehnologiy-v-kostnoy-hirurgii.pdfSzpalski C, Sagebin F, Barbaro M, Warren SM. The influence of environmental factors on bone tissue engineering. Tissue Engineering Part B. 2012;18(4):258-269. DOI: https://doi.org/10.1002/jbm.b.32849Wang W, Yeung KWK. Bone graft substitutes for bone defect repair: a review. Bioactive Materials. 2017;2:224-247. DOI: https://doi.org/10.1016/j.bioactmat.2017.05.007Polo-Corrales L, Latorre-Esteves M, Ramirez-Vick J. Scaffold design for bone regeneration. Journal of Nanoscience and Nanotechnology. 2014;14(1):15-56. DOI: https://doi.org/10.1166/jnn.2014.9127Makarevich S, Mazurenko A, Krivorot K, et al. The use of autologous mesenchymal stem cells for the purpose of spinal fusion. Science and innovation. 2019;11:79-84. (In Russ.). [Макаревич С., Мазуренко А., Криворот К. Применение аутологичных мезенхимальных стволовых клеток с целью спондилодеза. Наука и инновации. 2019;11:79-84]. DOI: https://doi.org/10.29235/1818-9857-2019-11-79-84Lagenbach F, Handschel J. Effects of dexamethasone, ascorbic acid and ß-glycerophosphate on the osteogenic differentiation of stem cells in vitro. Stem Cell Res Therapy. 2013;4(5):117. DOI: https://doi.org/10.1186/scrt328Hutchings G, Moncrieff L, Dompe C, et al. Bone regeneration, reconstruction and use of osteogenic cells; from basic knowledge, animal models to clinical trials. J Clin Med. 2020;9(1):139. DOI: https://doi.org/10.3390/jcm9010139Steinert AF, Rackwitz L, Gilbert F, et al. The clinical application of mesenchymal stem cells for muskuloskeletal regeneration: current status and perspectives. Stem Cells Trans Med. 2012;1(3):237-247. DOI: https://doi.org/10.5966/sctm.2011-0036Correia C, Grayson WL, Park M, et al. In vitro model of vascularized bone: synergizing vascular development and osteogenesis. PLOS One. 2011;6(12):e28352. DOI: https://doi.org/10.1371/journal.pone.0028352Filippi M, Born G, Chaaban M, Scherberich A. Natural polymeric scaffolds in bone regeneration. Frontiers in Bioengineering and Biotechnology. 2020;8:1-28. DOI: https://doi.org/10.3389/fbioe.2020.00474Ripamonti, U. Induction of bone formation by recombinant human osteogenic protein-1 and sintered porous hydroxyapatite in adult primate. Plastic &Reconstructive Surgery. 2001;107:977-988. DOI: https://doi.org/10.1097/00006534-200104010-00012Zhukova Y, Hiepen Ch, Knaus P, et al. The role of titanium surface nanostructuring on preosteoblast morphology, adhesion, and migration. Advanced Healthcare Materials. 2017;6(15):1-13. DOI: https://doi.org/10.1002/adhm.201601244Garg T, Singh O, Arora S, Murthy R. Scaffold: a novel carrier for cell and drug delivery. Critical Review in Therapeutic Drug Carrier Systems. 2012;29(1):1-63. DOI: https://doi.org/10.1615/CritRevTherDrugCarrierSyst.v29.i1.10Seeherman H, Wozney J, Lee R. Bone morphogenetic protein delivery systems. Spine. 2002;27:16-23. DOI: https://doi.org/10.1097/00007632-200208151-00005Polo-Corrales L, Latorre-Esteves M, Ramirez-Vick J. Scaffold design for bone regeneration. Journal of Nanoscience and Nanotechnology. 2014;14(1):15-56. DOI: https://doi.org/10.1166/jnn.2014.9127Oliveira JF, Aguiar PF, Rossi AM, Soares GA. Effect of process parameters on the characteristics of porous calcium phosphate ceramics for bone tissue scaffolds. Artificial Organs 2003;27:406-411. DOI: https://doi.org/10.1046/j.1525-1594.2003.07247.xKruyt MC, Gaalen SM, Oner FC, et al. Bone tissue engineering and spinal fusion: the potential of hybrid constructs by combining osteoprogenitor cells and scaffolds. Biomaterials. 2004;25:1463-1473. DOI: https://doi.org/10.1016/s0142-9612(03)00490-3Neman J, Hambrecht A, Cadry C, Jandal R. Stem cell-mediated osteogenesis: therapeutic potential for bone tissue engineering. Biologics: Targets and Therapy. 2012;6:47-57. DOI: https://doi.org/10.2147/BTT.S22407Kitoh H, Kitakoji T, Tsuchiya H, et al. Transplantation of culture expanded bone marrow cells and platelet rich plasma in distraction osteogenesis of the long bones. Bone. 2007;40(2):522-528. DOI: https://doi.org/10.1016/j.bone.2006.09.019Tsiklin LI, Pugachev EI, Kolsanov AV, et al. Biopolymer material from human spongiosa for regenerative medicine application. Polymers. 2022;14(5):941-953. DOI: https://doi.org/10.3390/polym14050941Kim HN, Jiao A, Hwang AS, et al. Nanotopography-guided tissue engineering and regenerative medicine. Advanced Drug Delivery Reviews 2013;65;536-558. DOI: https://doi.org/10.1016/j.addr.2012.07.014Thurairajah K, Broadhead LM, Balogh ZJ. Trauma and stem cells: biology and potential therapeutic Implications. International Journal of Molecular Science. 2017;18:577-595. DOI: https://doi.org/10.3390/ijms18030577Armiento AR, Hatt PhL, Rosenberg GS, et al. Functional biomaterials for bone regeneration: a lesson in complex biology. Advanced Functional Materials. 2020;30(44):1-41. DOI: https://doi.org/10.1002/adfm.201909874Paladini F, Pollini M. Novel approaches and biomaterials for bone tissue engineering: a focus on silk fibroin. Materials. 2022;15(19):1-22. DOI: https://doi.org/10.3390/ma15196952Potapnev MP, Arabey AA, Kondratenko GG, et al. A soluble platelet-derived growth factors and regenerative medicine. Healthcare. 2014;9:32-40. (In Russ.). [Потапнев М.П., Арабей A.A., Кондратенко Г.Г., и др. Растворимые факторы тромбоцитов и регенеративная медицина. Здравоохранение. 2014;9:32-40].Marx RE, Carlson ER, Eichstaedt RM, et al. Platelet-rich plasma: Growth factor enhancement for bone grafts. Oral Surg Oral Med Oral Pathol Oral Radiol Oral Endod. 1998;85 (6):538-46. DOI: https://doi.org/10.1016/S1079-2104(98)90029-4Laquinta MR, Mazzoni E, Manfrini M, et al. Innovative biomaterials for bone regrowth. Int J Mol Sci. 2019;20(3):618. DOI: https://doi.org/10.3390/ijms20030618Kotelnikov GP, Kolsanov AV, Volova LT, et al. Technology of manufacturing of personalized reconstructive allogenic bone graft. Pirogov Russian Journal of Surgery. 2019;3:65-72. [Котельников Г.П., Колсанов А.В., Волова Л.Т., и др. Технология производства персонифицированного реконструктивного аллогенного костного имплантата. Хирургия. Журнал им. Н.И. Пирогова. 2019;(3):65 72]. DOI: https://doi.org/10.17116/hirurgia201903165Tsiklin IL, Shabunin AV, Kolsanov AV, Volova LT. In Vivo Bone Tissue Engineering Strategies: Advances and Prospects. Polymers. 2022;14(15):3222. DOI: https://doi.org/10.3390/polym14153222World Health Organization. Blood Transfusion Safety Team. (2001). The Clinical use of blood: handbook.Available online: https://apps.who.int/iris/handle/10665/42396 (accessed on 25 August 2023).Pierce J, Benedetti E, Preslar A, et al. Comparative analyses of industrial-scale human platelet lysate preparations. Transfusion. 2017;57(12):2858-2869. DOI: https://doi.org/10.1111/trf.14324Shakhbazov AV, Goncharova NV, Kosmacheva SM, et al. Plasticity of human mesenchymal stem cell phenotype and expression profile under neurogenic conditions. Bulletin of Experimental Biology and Medicine (Cell Technologies in Biology and Medicine). 2009;147(4):513-516. DOI: https://doi.org/10.1007/s10517-009-0547-6Chen X, Huang J, Wu J. Human mesenchymal stem cells. Cell Proliferation. 2022;55:1-11. DOI: https://doi.org/10.1111/cpr.13141Schendzielorz P, Froelich K, Rak K, et al. Labeling adipose-derived stem cells with Hoechst 33342: usability and effects on differentiation potential and DNA damage. Stem Cells Int. 2016;2016:6549347. DOI: https://doi.org/10.1155/2016/6549347Danilkovich NN, Derkachev VS, Kosmacheva SM, Potapnev MP. Application of bone-marrow mesenchymal stem cells and platelet-derived growth factors for human osteogenic graft engineering, 5th International conference on Tissue engineering and Regenerative Medicine, Berlin, Germany, 12-14 September, 2016:155. DOI: https://doi.org/10.4172/2157-7552.C1.025Santos VH, Hubbe Pfeifer JP, Souza BJ, et al. Culture of mesenchymal stem cells derived from equine synovial membrane in alginate hydrogel microcapsules. BMS Veterinary Research. 2018;14(1):1-10. DOI: https://doi.org/10.1186/s12917-018-1425-0Chevallier N, Anagnostou F, Zilber S, et al. Osteoblastic differentiation of human mesenchymal stem cells with platelet lysate. Biomaterials. 2010;31:270-278. DOI: https://doi.org/10.1016/j.biomaterials.2009.09.043Kuvyrkov EV, Severin IN, Belyasova NA. Osteogenic differentiation of human bone marrow mesenchymal stem cells. News of the National Academy of Sciences of Belarus. 2015;2:89-91. [Кувырков Е.В., Северин И.Н., Белясова Н.А. Остеогенная дифференцировка мезенхимальных стволовых клеток костного мозга человека. Весці Нацыянальнай акадэміі навук Беларусі. 2015;2:89-91]. URL: file:///C:/Users/Persona/Downloads/255-253-1-PB.pdfSzustak M, Gendaszewska-Darmach E. Extracellular nucleotides selectively induce migration of chondrocytes and expression of type II Collagen. Int J Mol Sci. 2020;21(15):5227. DOI: https://doi.org/10.3390/ijms21155227Zhang D, Yi C, Qi S, Yao X. Yang. M. Effects of Carbon Nanotubes on the Proliferation and Differentiation of Primary Osteoblasts. Methods in Molecular Biology. 2010;625:41-53. DOI: https://doi.org/10.1007/978-1-60761-579-8_5Meesuk L, Suwanprateeb J, Thammarakcharoen F, et al. Osteogenic differentiation and proliferation potentials of human bone marrow and umbilical cord-derived mesenchymal stem cells on the 3D-printed hydroxyapatite scaffolds. Scientific Reports. 2022;12;1-19. DOI: https://doi.org/10.1038/s41598-022-24160-2Janicki P, Boeuf S, Steck E, et al. Prediction of in vivo bone forming potency of bone marrow-derived human mesenchymal stem cells. European Cells and Materials. 2011;21:488-507. DOI: https://doi.org/10.22203/ecm.v021a37Yoon Y, Khan IU, Choi KU, et al. Different bone healing effects of undifferentiated and osteogenic differentiated mesenchymal stromal cell sheets in canine radial fracture model. Tissue Engineering and Regenerative Medicine. 2018;15(1):115-124. DOI: https://doi.org/10.1007/s13770-017-0092-8Kuk M, Kim Y, Lee SH, et al. Osteogenic ability of canine adipose-derived mesenchymal stromal cell sheets in relation to culture time. Cell Transplantation. 2015;25(7):1415-1422.DOI: https://doi.org/10.3727/096368915X689532Szpalski C, Barbaro M, Sagebin F, Warren SM. Bone Tissue Engineering: Current Strategies and Techniques – Part II: Cell Types. Tissue Engineering Part B: Reviews. 2012;14(4):258-269. DOI: https://doi.org/10.1089/ten.teb.2011.0440Zheng J, Xie Y, Yoshitomi T, et al. Stepwise Proliferation and Chondrogenic Differentiation of Mesenchymal Stem Cells in Collagen Sponges under different Microenvironments. International Journal of Molecular Science. 2022;23(12):6406. DOI: https://doi.org/10.3390/ijms23126406Kasten P, Vogel J, Beyen I, et al. Effect of plate-let-rich plasma on the in vitro proliferation and osteogenic differentiation of human mesenchymal stem cells on distinct calcium phosphate scaffolds: the specific surface area makes a difference. Journal of Biomaterials Applications. 2008;23(2):169-188. DOI: https://doi.org/10.1177/0885328207088269Kocaoemer A, Kern S, Kluter H, Bieback K. Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue. Stem Cells. 2007;25:1270-1278. DOI: https://doi.org/101634/stemcells.2006-0627Borzini P, Mazzucco L. Tissue regeneration and in loco administration of platelet derivatives: clinical outcome, heterogeneous products, and heterogeneity of the effector mechanisms. Transfusion. 2005;45:1759-1767. DOI: https://doi.org/10.1111/j.1537-2995.2005.00600.x