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. 2017 Dec 12;53(1):45-52.
doi: 10.1016/j.rboe.2017.12.004. eCollection 2018 Jan-Feb.

Proliferation and differentiation of stem cells in contact with eluate from fibrin-rich plasma membrane

Fernanda Gimenez de Souza  1 , Beatriz Luci Fernandes  1 , Carmen Lucia Kuniyoshi Rebelatto  1 , Alessandra Melo de Aguiar  2 , Letícia Fracaro  1 , Paulo Roberto Slud Brofman  1
Affiliations

Proliferation and differentiation of stem cells in contact with eluate from fibrin-rich plasma membrane

Fernanda Gimenez de Souza et al. Rev Bras Ortop. .

Abstract
in English, Portuguese

Objective: To evaluate the ability of the eluate from fibrin-rich plasma (FRP) membrane to induce proliferation and differentiation of isolated human adipose-derived stem cells (ASCs) into chondrocytes.

Method: FRP membranes were obtained by centrifugation of peripheral blood from two healthy donors, cut, and maintained in culture plate wells for 48 h to prepare the fibrin eluate. The SCATh were isolated from adipose tissue by collagenase digestion solution, and expanded in vitro. Cells were expanded and treated with DMEM-F12 culture, a commercial media for chondrogenic differentiation, and eluate from FRP membrane for three days, and labeled with BrdU for quantitative assessment of cell proliferation using the High-Content Operetta® imaging system. For the chondrogenic differentiation assay, the SCATh were grown in micromass for 21 days and stained with toluidine blue and aggrecan for qualitative evaluation by light microscopy. The statistical analysis was performed using ANOVA and Tukey's test.

Results: There was a greater proliferation of cells treated with the eluate from FRP membrane compared to the other two treatments, where the ANOVA test showed significance (p < 0.001). The differentiation into chondrocytes was visualized by the presence of mucopolysaccharide in the matrix of the cells marked in blue toluidine and aggrecan.

Conclusions: Treatment with eluate from FRP membrane stimulated cell proliferation and induced differentiation of the stem cells into chondrocytes, suggesting a potential application of FRP membranes in hyaline cartilage regeneration therapies.

Objetivo: Avaliar a capacidade do eluato proveniente da membrana de plasma rico em fibrina (PRF) de induzir proliferação e diferenciação das células-tronco humanas isoladas de tecido adiposo (CTDAh) em condrócitos.

Método: As membranas de PRF foram obtidas por centrifugação de sangue periférico de dois indivíduos saudáveis, cortadas, colocadas em poços de placa de cultivo por 48 h para obtenção do eluato de fibrina. As CTDAh foram isoladas do tecido adiposo por digestão com solução de colagenase e expandidas in vitro. As células foram expandidas e tratadas com meio de cultivo DMEM-F12, meio comercial para diferenciação condrocítica, e eluato de fibrina durante três dias e marcadas com BrdU para avaliação quantitativa da proliferação celular com o uso do sistema de imagens High-Content Operetta®. Para o ensaio de diferenciação condrogênica, as CTDAh foram cultivadas em micromassa por 21 dias e coradas com azul de toluidina e agrecana para avaliação qualitativa em microscópio óptico. As avaliações estatísticas foram feitas por meio dos testes Anova e Tukey.

Resultados: Houve uma maior proliferação das células tratadas com o eluato de fibrina comparativamente com os outros dois tratamentos, nos quais o teste Anova apontou significância (p < 0,001). A diferenciação em condrócitos foi visualizada pela presença de mucopolissacarídeos na matriz das células tratadas com meio de diferenciação ou eluato e marcação positiva para agrecana.

Conclusões: O tratamento com o eluato da membrana de fibrina estimulou a proliferação celular e induziu a diferenciação das células-tronco em condrócitos, o que sugere uma potencial aplicação da membrana de PRF nas terapias de regeneração de cartilagem hialina.

Keywords: Cartilage; Membranes; Platelet-rich plasma; Regeneration.

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Figures

Fig. 1
Fig. 1
Microscopic analysis of the fibrin membrane. (A) Platelet accumulation in the lower region (arrows) in transition with the mixed region, which shows whitish streaks (stars); (B) Superior area of the clot with the extensive fibrin network. Staining: Hematoxylin-Eosin. Magnified 10×.
Fig. 2
Fig. 2
Statistical results showing: (A) Total cells stained by condition of culture; (B) Percentage of 24 h proliferation cells (BrDU-stained).
Fig. 3
Fig. 3
Microscopic evaluation of a chondrogenic differentiation assay: (A, C, and E) Extracellular matrix evidenced by toluidine blue staining, which shows higher intensity in the differentiation and eluate; (B, D, and F) Immunocytochemical staining for aggrecan, extracellular matrix protein, expressed by cells in chondrocyte differentiation (arrows). Magnified 40×.
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References

    1. Fuller R. Osteoartrose. In: Greve J.M.D.A., editor. Tratado de medicina de reabilitação. Roca; São Paulo: 2007. pp. 889–904.
    1. Myers K.R., Sgaglione N.A., Grande D.A. Trends in biological joint resurfacing. Bone Joint Res. 2013;2(9):193–199. - PMC - PubMed
    1. Betsch M., Schneppendahl J., Thuns S., Herten M., Sager M., Jungbluth P. Bone marrow aspiration concentrate and platelet rich plasma for osteochondral repair in a porcine osteochondral defect model. PLOS ONE. 2013;8(8):e71602. - PMC - PubMed
    1. Pei M., He F., Li J., Tidwell J.E., Jones A.C., McDonough E.B. Repair of large animal partial-thickness cartilage defects through intraarticular injection of matrix-rejuvenated synovium-derived stem cells. Tissue Eng Part A. 2013;19(9–10):1144–1154. - PubMed
    1. Gobbi A., Karnatzikos G., Scotti C., Mahajan V., Mazzucco L., Grigolo B. One-step cartilage repair with bone marrow aspirate concentrated cells and collagen matrix in full-thickness knee cartilage lesions: results at 2-year follow-up. Cartilage. 2011;2(3):286–299. - PMC - PubMed

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