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

Abstract

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.