BMP-4, TGF-β and Smad3 as Modulators of Synovial Fluid Cells Viability

Abstract

Objective Our goal was to evaluate the modulation of the synovial fluid cells (SFC) from patients with and without osteoarthritis (OA) by bone morphogenetic protein 4 (BMP-4), Smad-3 and transforming growth factor beta (TGF-β). Methods Synovial fluid was collected from patients submitted to knee arthroscopy or replacement and were centrifuged to isolate cells from the fluid. Cells were cultured for 21 days and characterized as mesenchymal stem cells (MSCs) according to the criteria of the International Society of Cell Therapy. Then, we performed an [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (MTT) assay after exposing cells with and without OA to TGF-β, Smad3 and BMP-4 pathway inhibitors and to different concentrations of BMP4. Results Exposure to the TGF-β, Smad3 and BMP-4 inhibitors modifies the mitochondrial activity of the SFCs. The activity of the SFCs is modified by influences of increasing concentrations of BMP4, but there is no difference in cellular activity between patients with and without OA. Conclusion TGF-β, Smad3 and BMP-4 modulate the activity of SFCs from patients with and without knee OA.

Keywords: TGF-β pathway; mesenchymal stem cells; osteoarthritis; synovial fluid cells.

Fig. 1

Evaluation of mitochondrial activity of SFCs exposed to different concentrations of BMP-4. Comparison of the cellular viability of SFCs from patients with (W/OA) and without OA (W/O OA) exposed to different concentrations of BMP-4 did not show statistically significant differences between the groups ( p  > 0.05), although mitochondrial activity has increased up to 2nM ( p  < 0.001) and decreased at 10 nM ( p  < 0.001) in both groups. Control/Cells: Well containing only cells without BMP-4; Control/Factor: Wells containing medium with BMP-4 without cells; * p  < 0.001.

Fig. 2

Mitochondrial activity of SFCs after TGF-β inhibitor treatment. A reduction of mitochondrial activity was observed in SFCs of W/O OA subjects after TGF-β inhibitor treatment for 24 hours ( p  = 0.016) and an increase after 48 hours ( p  < 0.0001), while no differences were found after 72 hours ( p  > 0.05). On the other hand, the SFCs of W/OA subjects showed an increase in activity only after 48 hours in the presence of the inhibitor ( p  = 0.0046), but no differences were observed after 24 hours and 72 hours ( p  > 0.05).

Fig. 3

Mitochondrial activity of SFCs after Smad3 inhibitor treatment. The activity of the SFCs of W/O OA subjects reduced after 24 hours of treatment using Smad3 inhibitor ( p  = 0.046) but increased after 48 hours ( p  = 0.0002) and 72 hours ( p  = 0.006). On the other hand, the SFCs of W/OA subjects showed a reduction in activity only after 72 hours in the presence of the Smad3 inhibitor ( p  = 0.0102), but no differences were found after 24 and 48 hours ( p  > 0.05).

Fig. 4

Mitochondrial activity of SFCs after BMP4 inhibitor treatment. The activity of the SFCs of W/O OA subjects increased after 48 hours ( p  = 0.0064) and 72 hours ( p  < 0.0001) of treatment with the BMP-4 inhibitor. The activity of the SFCs of W/OA subjects also increased after 48 hours ( p  < 0.0001) and 72 hours in the presence of the BMP-4 inhibitor ( p  < 0.0001). There were no differences in activity neither between the SFCs of W/O OA nor W/OA subjects after treatment with BMP-4 inhibitor after 24 hours ( p  > 0.05).

Fig. 1

Avaliação da atividade mitocondrial das SFCs expostas a diferentes concentrações de BMP4. A comparação da viabilidade celular de SFCs de pacientes com (W/OA) e sem OA (W/O OA) exposta a diferentes concentrações de BMP-4 não apresentou diferenças estatisticamente significativas entre os grupos ( p  > 0,05), embora a atividade mitocondrial tenha aumentado até 2nM ( p  < 0,001) e diminuído em 10 nM ( p  < 0,001) em ambos os grupos. Controle/Células: Poço contendo apenas células sem BMP-4; Controle/Fator: Poços contendo meio com BMP-4 sem células; *p < 0,001.

Fig. 2

Atividade mitocondrial de SFC após tratamento inibidor com TGF-β. Observou-se redução da atividade mitocondrial nas SFCs do grupo W/O OA após tratamento inibidor TGF-β por 24 horas ( p  = 0,016) e aumento após 48 horas ( p  < 0,0001), enquanto não foram encontradas diferenças após 72h ( p  > 0,05). Por outro lado, as SFCs do grupo W/OA apresentaram aumento da atividade somente após 48 horas na presença do inibidor ( p  = 0,0046), mas não foram observadas diferenças após 24 horas e 72 horas ( p  > 0,05).

Fig. 3

Atividade mitocondrial das SFCs após tratamento inibidor de Smad3. A atividade de SFC W/O OA reduziu após 24 horas de tratamento usando inibidor Smad3 ( p  = 0,046), mas aumentou após 48 horas ( p  = 0,0002) e 72 horas ( p  = 0,006). Por outro lado, as SFCs do grupo W/OA apresentaram redução da atividade somente após 72 horas na presença do inibidor Smad3 ( p  = 0,0102), mas não foram encontradas diferenças após 24 e 48 horas ( p  > 0,05).

Fig. 4

Atividade mitocondrial das SFCs após tratamento inibidor BMP4. A atividade dass SFCs do grupo W/O OA aumentou após 48 horas ( p  = 0,0064) e 72 horas ( p  < 0,0001) de tratamento com inibidor de BMP4. A atividade das SFCs do grupo W/OA também aumentou após 48 horas ( p  < 0,0001) e 72 horas na presença do inibidor BMP4 ( p  < 0,0001). Não houve diferenças na atividade nem entre as SFCs do grupo W/O OA, nem do grupo W/OA após tratamento com inibidor BMP4 após 24 horas ( p  > 0,05).