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Platelet-rich concentrate in serum free medium enhances osteogenic differentiation of bone marrow-derived human mesenchymal stromal cells.

ABSTRACT: 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.

SUBMITTER: Samuel S

PROVIDER: S-EPMC5018671 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Platelet-rich concentrate in serum free medium enhances osteogenic differentiation of bone marrow-derived human mesenchymal stromal cells.

Samuel Shani S Ahmad Raja Elina RE Ramasamy Thamil Selvee TS Karunanithi Puvanan P Naveen Sangeetha Vasudevaraj SV Murali Malliga Raman MR Abbas Azlina A AA Kamarul Tunku T

PeerJ 20160907

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 1 ...[more]

PMID: 27651984

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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.

Project description:Background: In the last decade, regenerative therapies have become one of the leading disease modifying options for treatment of knee osteoarthritis (OA). Still, there is a lack of trials with a direct comparison of different biological treatments. Our aim was to directly compare clinical outcomes of knee injections of Bone Marrow Aspirate Concentrate (BMAC), Platelet-rich Plasma (PRP), or Hyaluronic acid (HA) in the OA treatment. Methods: Patients with knee pain and osteoarthritis KL grade II to IV were randomized to receive a BMAC, PRP, and HA injection in the knee. VAS, WOMAC, KOOS, and IKDC scores were used to establish baseline values at 1, 3, 6, 9, and 12 months. All side effects were reported. Results: A total of 175 patients with a knee osteoarthritis KL grade II-IV were randomized; 111 were treated with BMAC injection, 30 with HA injection, and 34 patients with PRP injection. There were no differences between these groups when considering KL grade, BMI, age, or gender. There were no serious side effects. The mean VAS scores after 3, 7, 14, and 21 days showed significant differences between groups with a drop of VAS in all groups but with a difference in the BMAC group in comparison to other groups (p < 0.001). There were high statistically significant differences between baseline scores and those after 12 months (p < 0.001) in WOMAC, KOOS, KOOS pain, and IKDC scores, and in addition, there were differences between these scores in the BMAC group in comparison with other groups, except for the PRP group in WOMAC and the partial IKDC score. There were no differences between the HA and PRP groups, although PRP showed a higher level of clinical improvement. Conclusions: Bone marrow aspirate concentrate, Leukocyte rich Platelet Rich Plasma, and Hyaluronic acid injections are safe therapeutic options for knee OA and provide positive clinical outcomes after 12 months in comparison with findings preceding the intervention. BMAC could be better in terms of clinical improvements in the treatment of knee OA than PRP and HA up to 12 months. PRP provides better outcomes than HA during the observation period, but these results are not statistically significant. More randomized controlled trials and high quality comparative studies are needed for direct correlative conclusions.

Project description:Aging tissues present a progressive decline in homeostasis and regenerative capacities, which has been associated with degenerative changes in tissue-specific stem cells and stem cell niches. We hypothesized that amino acids could regulate the stem cell phenotype and differentiation ability of human bone marrow-derived mesenchymal stromal cells (hBMSCs). Thus, we performed a screening of 22 standard amino acids and found that D-tryptophan (10 μM) increased the number of cells positive for the early stem cell marker SSEA-4, and the gene expression levels of OCT-4, NANOG, and SOX-2 in hBMSCs. Comparison between D- and L-tryptophan isomers showed that the latter presents a stronger effect in inducing the mRNA levels of Oct-4 and Nanog, and in increasing the osteogenic differentiation of hBMSCs. On the other hand, L-tryptophan suppressed adipogenesis. The migration and colony-forming ability of hBMSCs were also enhanced by L-tryptophan treatment. In vivo experiments delivering L-tryptophan (50 mg/kg/day) by intraperitoneal injections for three weeks confirmed that L-tryptophan significantly increased the percentage of cells positive for SSEA-4, mRNA levels of Nanog and Oct-4, and the migration and colony-forming ability of mouse BMSCs. L-kynurenine, a major metabolite of L-tryptophan, also induced similar effects of L-tryptophan in enhancing stemness and osteogenic differentiation of BMSCs in vitro and in vivo, possibly indicating the involvement of the kynurenine pathway as the downstream signaling of L-tryptophan. Finally, since BMSCs migrate to the wound healing site to promote bone healing, surgical defects of 1 mm in diameter were created in mouse femur to evaluate bone formation after two weeks of L-tryptophan or L-kynurenine injection. Both L-tryptophan and L-kynurenine accelerated bone healing compared to the PBS-injected control group. In summary, L-tryptophan enhanced the stemness and osteoblastic differentiation of BMSCs and may be used as an essential factor to maintain the stem cell properties and accelerate bone healing and/or prevent bone loss.

Project description:BackgroundThere is a need to identify and quantify mesenchymal stromal cells (MSCs) in human bone marrow aspirate concentrate (BMAC) source tissues, but current methods to do so were established in cultured cell populations. Given that surface marker and gene expression change in cultured cells, it is doubtful that these strategies are valid to quantify MSCs in fresh BMAC.PurposeTo establish the presence, quantity, and heterogeneity of BMAC-derived MSCs in minimally manipulated BMAC using currently available strategies.Study designDescriptive laboratory study.MethodsFive published strategies to identify MSCs were compared for suitability and efficiency to quantify clinical-grade BMAC-MSCs and cultured MSCs at the single cell transcriptome level on BMAC samples being used clinically from 15 orthopaedic patients and on 1 cultured MSC sample. Strategies included (1) the guidelines by the International Society for Cellular Therapy (ISCT), (2) CD271 expression, (3) the Ghazanfari et al transcriptional profile, (4) the Jia et al transcriptional profile, and (5) the Silva et al transcriptional profile.ResultsISCT guidelines did not identify any MSCs in BMAC at the transcriptional level and only 1 in 9 million cells at the protein level. Of 12,850 BMAC cells, 9 expressed the CD271 gene. Only 116 of 396 Ghazanfari genes were detected in BMAC, whereas no cells expressed all of them. No cells expressed all Jia genes, but 25 cells expressed at least 13 of 22. No cells expressed all Silva genes, but 19 cells expressed at least 8 of 23. Most importantly, the liberalized strategies tended to identify different cells and most of them clustered with immune cells.ConclusionCurrently available methods need to be liberalized to identify any MSCs in fresh human BMAC and lack consensus at the single cell transcriptome and protein expression levels. These different cells should be isolated and challenged to establish phenotypic differences.Clinical relevanceThis study demonstrated that improved strategies to quantify MSC concentrations in BMAC for clinical applications are urgently needed. Until then, injected minimally manipulated MSC doses should be reported as rough estimates or as unknown.

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Platelet-rich concentrate in serum free medium enhances osteogenic differentiation of bone marrow-derived human mesenchymal stromal cells.

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Platelet-rich concentrate in serum free medium enhances osteogenic differentiation of bone marrow-derived human mesenchymal stromal cells.

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