Effect of platelet-rich fibrin matrix in complex with artificial material Nubiplant on expression of chondrogenic marker genes and morphogenesis of the nucleus pulposus cells of intervertebral discs in rats
Keywords:nucleus pulposus, platelet-rich fibrin matrix, gene expression
The purpose was to study the barrier and biological properties of platelet-rich fibrin matrix (PRFM), an artificial biopolymer Nubiplant, and a mixture of PRFM / Nubiplant by assessing the viability and morphological characteristics of nucleus pulposus (NP) cells in rats, as well as the expression level of chondrogenic marker genes during cell cultivation in the presence of these matrices.
Materials and methods. PRFM was obtained from platelet-rich plasma using a SiO2 coagulation activator. A suspension of nucleus pulposus cells was obtained from the caudal spine of rats. Cultivation was carried out in the presence of one of three matrices — PRFM, Nubiplant, or their mixture for 3, 7, and 14 days under standard culture conditions in an EC-160 incubator (Nüve, Turkey). Observation of the living culture was carried out in the area bordering with the matrix within one field of view using an inverted microscope (Nicon TS100, Japan). The expression of chondrogenic marker genes in the cell culture of the NP was determined by the method of PCR with reverse transcription.
Results. The study of the viability and morphological characteristics of NP cells during their cultivation for 3, 7, and 14 days in the presence of PRFM, PRFM / Nubiplant, or Nubiplant showed a decrease in the content of living cells in control samples; in cultures with PRFM and PRFM / Nubiplant, the number of living cells significantly exceeded the control values, aggregation of cells was observed in the area bordering with the matrices from the side of the application. None of the experimental samples showed the outflow of cells to the opposite side of the matrix after 14 days of cultivation; thus, PRFM, Nubiplant, and their mixture can perform barrier functions to keep the cell population in a certain location. Expression of the COL II, ACAN, GPC3, ANXA3, PTN, MGP, and VIM genes by the NP cells during cultivation for 3 and 7 days in the presence of PRFM and PRFM / Nubiplant increased as compared to the control samples.
Conclusions. The use of PRFM, Nubiplant, or a mixture of PRFM / Nubiplant during the cultivation of NP cells demonstrated the absence of cell outflow to the opposite side of the studied matrices during the study period (14 days). The use of PRFM, Nubiplant, or a mixture of PRFM / Nubiplant promoted the formation of cell colonies with chondrocyte-like morphology in the zone bordering with the matrices and maintained cell viability throughout the study period. PRFM and PRFM / Nubiplant contributed to the maintenance of the expression of chondrogenic genes in the NP cells in the zone bordering the matrices. The results obtained indicate the positive effect of the matrix based on platelet-rich fibrin on the NP cells and its barrier functions, which is promising for the use of PRMF for preventing the formation of cicatricial adhesion.
1. Khyzhnyak MV, Bodnarchuk YuA. [The instable thoracolumbar spine fractures, modern methods of management]. Ukrainian Neurosurgical Journal. 2012;(4):6-10. Ukrainian. [CrossRef]
2. Osterman H, Seitsalo S, Karppinen J, Malmivaara A. Effectiveness of microdiscectomy for lumbar disc herniation: a randomized controlled trial with 2 years of follow-up. Spine (Phila Pa 1976). 2006 Oct 1;31(21):2409-14. [CrossRef] [PubMed]
4. Coskun E, Süzer T, Topuz O, Zencir M, Pakdemirli E, Tahta K. Relationships between epidural fibrosis, pain, disability, and psychological factors after lumbar disc surgery. Eur Spine J. 2000 Jun;9(3):218-23. [CrossRef] [PubMed] [PubMed Central]
5. Sae-Jung S, Jirarattanaphochai K, Sumananont C, Wittayapairoj K, Sukhonthamarn K. Interrater Reliability of the Postoperative Epidural Fibrosis Classification: A Histopathologic Study in the Rat Model. Asian Spine J. 2015 Aug;9(4):587-94. [CrossRef] [PubMed] [PubMed Central]
6. Tural Emon S, Somay H, Orakdogen M, Uslu S, Somay A. Effects of hemostatic polysaccharide agent on epidural fibrosis formation after lumbar laminectomy in rats. Spine J. 2016 Mar;16(3):414-9. [CrossRef] [PubMed]
7. Gürer B, Kahveci R, Gökçe EC, Ozevren H, Turkoglu E, Gökçe A. Evaluation of topical application and systemic administration of rosuvastatin in preventing epidural fibrosis in rats. Spine J. 2015 Mar 1;15(3):522-9. [CrossRef] [PubMed]
8. Karanci T, Kelten B, Karaoglan A, Cinar N, Midi A, Antar V, Akdemir H, Kara Z. Effects of 4% Icodextrin on Experimental Spinal Epidural Fibrosis. Turk Neurosurg. 2017;27(2):265-271. [CrossRef] [PubMed]
10. Zhang C, Kong X, Ning G, Liang Z, Qu T, Chen F, Cao D, Wang T, Sharma HS, Feng S. All-trans retinoic acid prevents epidural fibrosis through NF-κB signaling pathway in post-laminectomy rats. Neuropharmacology. 2014 Apr;79:275-81. [CrossRef] Erratum in: Neuropharmacology. 2020 Aug 1;172:107927. [PubMed]
11. Xu H, Liu C, Sun Z, Guo X, Zhang Y, Liu M, Li P. CCN5 attenuates profibrotic phenotypes of fibroblasts through the Smad6-CCN2 pathway: Potential role in epidural ﬁbrosis. Int J Mol Med. 2015 Jul;36(1):123-9. [CrossRef] [PubMed] [PubMed Central]
12. Chen F, Wang C, Sun J, Wang J, Wang L, Li J. Salvianolic acid B reduced the formation of epidural fibrosis in an experimental rat model. J Orthop Surg Res. 2016 Nov 16;11(1):141. [CrossRef] [PubMed] [PubMed Central]
13. Theys T, Van Hoylandt A, Broeckx CE, Van Gerven L, Jonkergouw J, Quirynen M, van Loon J. Plasma-rich fibrin in neurosurgery: a feasibility study. Acta Neurochir (Wien). 2018 Aug;160(8):1497-1503. [CrossRef] [PubMed]
14. García M.C. Drug delivery systems based on nonimmunogenic biopolymers. In: Parambath A., editor. Engineering of Biomaterials for Drug Delivery Systems. Elsevier; 2018. p.317-344.
15. Kardos D, Hornyák I, Simon M, Hinsenkamp A, Marschall B, Várdai R, Kállay-Menyhárd A, Pinke B, Mészáros L, Kuten O, Nehrer S, Lacza Z. Biological and Mechanical Properties of Platelet-Rich Fibrin Membranes after Thermal Manipulation and Preparation in a Single-Syringe Closed System. Int J Mol Sci. 2018 Nov 1;19(11):3433. [CrossRef] [PubMed] [PubMed Central]
16. Wu I, Elisseeff J. Biomaterials and Tissue Engineering for Soft Tissue Reconstruction. In: Laurencin C, Deng M, editors. Natural and synthetic biomedical polymers. Newnes; 2014 Jan 21. Elsevier; 2014;235–41. [CrossRef]
18. Wang ZS, Feng ZH, Wu GF, Bai SZ, Dong Y, Chen FM, Zhao YM. The use of platelet-rich fibrin combined with periodontal ligament and jaw bone mesenchymal stem cell sheets for periodontal tissue engineering. Sci Rep. 2016 Jun 21;6:28126. [CrossRef] [PubMed] [PubMed Central]
21. Peng L, Jia Z, Yin X, Zhang X, Liu Y, Chen P, Ma K, Zhou C. Comparative analysis of mesenchymal stem cells from bone marrow, cartilage, and adipose tissue. Stem Cells Dev. 2008 Aug;17(4):761-73. [CrossRef] [PubMed]
22. Lee CR, Sakai D, Nakai T, Toyama K, Mochida J, Alini M, Grad S. A phenotypic comparison of intervertebral disc and articular cartilage cells in the rat. Eur Spine J. 2007 Dec;16(12):2174-85. [CrossRef] [PubMed] [PubMed Central]
23. Bertrand RL, Senadheera S, Tanoto A, Tan KL, Howitt L, Chen H, Murphy TV, Sandow SL, Liu L, Bertrand PP. Serotonin availability in rat colon is reduced during a Western diet model of obesity. Am J Physiol Gastrointest Liver Physiol. 2012 Aug 1;303(3):G424-34. [CrossRef] [PubMed]
24. Chen S, Hu ZJ, Zhou ZJ, Lin XF, Zhao FD, Ma JJ, Zhang JF, Wang JY, Qin A, Fan SW. Evaluation of 12 Novel Molecular Markers for Degenerated Nucleus Pulposus in a Chinese Population. Spine (Phila Pa 1976). 2015 Aug 15;40(16):1252-60. [CrossRef] [PubMed]
25. Massey CJ, van Donkelaar CC, Vresilovic E, Zavaliangos A, Marcolongo M. Effects of aging and degeneration on the human intervertebral disc during the diurnal cycle: a finite element study. J Orthop Res. 2012 Jan;30(1):122-8. [CrossRef] [PubMed]
26. Vo NV, Hartman RA, Patil PR, Risbud MV, Kletsas D, Iatridis JC, Hoyland JA, Le Maitre CL, Sowa GA, Kang JD. Molecular mechanisms of biological aging in intervertebral discs. J Orthop Res. 2016 Aug;34(8):1289-306. [CrossRef] [PubMed] [PubMed Central]
30. Vo NV, Hartman RA, Patil PR, Risbud MV, Kletsas D, Iatridis JC, Hoyland JA, Le Maitre CL, Sowa GA, Kang JD. Molecular mechanisms of biological aging in intervertebral discs. J Orthop Res. 2016 Aug;34(8):1289-306. [CrossRef] [PubMed] [PubMed Central]
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Copyright (c) 2020 Eugene G. Pedachenko, Iryna G. Vasylieva, Mykhaylo V. Khyzhnyak, Natalia G. Chopyk, Natalia P. Oleksenko, Iryna M. Shuba, Olga I. Tsjubko, Olena S. Galanta, Anzhela B. Dmytrenko, Tetiana A. Makarova
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