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Platelet rich concentrate promotes early cellular proliferation and multiple lineage differentiation of human mesenchymal stromal cells in vitro.

ABSTRACT: Platelet rich concentrate (PRC) is a natural adjuvant that aids in human mesenchymal stromal cell (hMSC) proliferation in vitro; however, its role requires further exploration. This study was conducted to determine the optimal concentration of PRC required for achieving the maximal proliferation, and the need for activating the platelets to achieve this effect, and if PRC could independently induce early differentiation of hMSC. The gene expression of markers for osteocytes (ALP, RUNX2), chondrocytes (SOX9, COL2A1), and adipocytes (PPAR-γ) was determined at each time point in hMSC treated with 15% activated and nonactivated PRC since maximal proliferative effect was achieved at this concentration. The isolated PRC had approximately fourfold higher platelet count than whole blood. There was no significant difference in hMSC proliferation between the activated and nonactivated PRC. Only RUNX2 and SOX9 genes were upregulated throughout the 8 days. However, protein expression study showed formation of oil globules from day 4, significant increase in ALP at days 6 and 8 (P ≤ 0.05), and increased glycosaminoglycan levels at all time points (P < 0.05), suggesting the early differentiation of hMSC into osteogenic and adipogenic lineages. This study demonstrates that the use of PRC increased hMSC proliferation and induced early differentiation of hMSC into multiple mesenchymal lineages, without preactivation or addition of differentiation medium.

SUBMITTER: Shani S

PROVIDER: S-EPMC4243129 | biostudies-other | 2014

REPOSITORIES: biostudies-other

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Project description:Previous studies have shown that platelet concentrates used in conjunction with appropriate growth media enhance osteogenic differentiation of human mesenchymal stromal cells (hMSCs). However, their potential in inducing osteogenesis of hMSCs when cultured in serum free medium has not been explored. Furthermore, the resulting osteogenic molecular signatures of the hMSCs have not been compared to standard osteogenic medium. We studied the effect of infrequent supplementation (8-day interval) of 15% non-activated platelet-rich concentrate (PRC) in serum free medium on hMSCs proliferation and differentiation throughout a course of 24 days, and compared the effect with those cultured in a standard osteogenic medium (OM). Cell proliferation was analyzed by alamar blue assay. Gene expression of osteogenic markers (Runx2, Collagen1, Alkaline Phosphatase, Bone morphogenetic protein 2, Osteopontin, Osteocalcin, Osteonectin) were analyzed using Q-PCR. Immunocytochemical staining for osteocalcin, osteopontin and transcription factor Runx2 were done at 8, 16 and 24 days. Biochemical assays for the expression of ALP and osteocalcin were also performed at these time-points. Osteogenic differentiation was further confirmed qualitatively by Alizarin Red S staining that was quantified using cetylpyridinium chloride. Results showed that PRC supplemented in serum free medium enhanced hMSC proliferation, which peaked at day 16. The temporal pattern of gene expression of hMSCs under the influence of PRC was comparable to that of the osteogenic media, but at a greater extent at specific time points. Immunocytochemical staining revealed stronger staining for Runx2 in the PRC-treated group compared to OM, while the staining for Osteocalcin and Osteopontin were comparable in both groups. ALP activity and Osteocalcin/DNA level were higher in the PRC group. Cells in the PRC group had similar level of bone mineralization as those cultured in OM, as reflected by the intensity of Alizarin red stain. Collectively, these results demonstrate a great potential of PRC alone in inducing proliferation of hMSCs without any influence from other lineage-specific growth media. PRC alone has similar capacity to enhance hMSC osteogenic differentiation as a standard OM, without changing the temporal profile of the differentiation process. Thus, PRC could be used as a substitute medium to provide sufficient pool of pre-differentiated hMSCs for potential clinical application in bone regeneration.

Project description:BackgroundBiologics containing growth factors are frequently used to enhance healing after musculoskeletal injuries. One mechanism of action is thought to be though the ability of biologics to induce homing and migration of endogenous mesenchymal stromal cells (MSCs) to a target tissue. However, the ability of biologics to stimulate chemotaxis (directed migration of cells) and chemokinesis (increase rate of cell migration) of MSCs is unknown.Hypothesis/purposeThe aim of this study was to directly compare the ability of biologics including platelet rich plasma (PRP) and bone marrow concentrate (BMC) to induce MSC migration. The hypothesis was that leukocyte-low platelet rich plasma (Llo PRP) would induce migration to a greater extent than leukocyte-high platelet rich plasma (Lhi PRP) or BMC.MethodsBone marrow-derived MSCs were isolated from 8 horses. Migration of MSCs toward a biologic (BMC, Llo PRP, and Lhi PRP) or the positive control platelet derived growth factor (PDGF) was continuously traced and measured for 24hrs using time-lapse microscopy and a microfluidics device. Cell migration, chemotaxis and chemokinesis were determined by measurements of displacement, number of cells migrated, and cell flux.ResultsAll biologics resulted in a significantly greater percentage of MSCs migrated compared to the positive control (PDGF). MSCs migrated further toward BMC compared to Llo PRP. Cell migration, measured as cell flux, was greater toward BMC and Lhi PRP than Llo PRP.ConclusionThe biologics BMC and Lhi PRP elicit greater chemotaxis and chemokinesis of MSCs than Llo PRP. However, all biologics recruited the same number of MSCs suggesting that differences in other regenerative effects, such as growth factor concentration, between biologics should be strongly considered when choosing a biologic for treatment of musculoskeletal injuries. The results of this study have the potential to reduce the need, risks, and costs associated with MSC culture and delivery.

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Platelet rich concentrate promotes early cellular proliferation and multiple lineage differentiation of human mesenchymal stromal cells in vitro.

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