## 3 Results
### Effects of Linffa(r) Therapy on Chondrogenic Molecular Marker Expression  
RT-qPCR analysis was then applied to determine the effects of chondrogenic differentiation, as well as Limfa(r) Therapy treatment, on the following markers of chondrogenesis: SOX9, RUNN2, COL2A1, COL10A1 and ACAN. COL2A1 showed a significant upregulation in its expression rate following the treatment schedule with Limfa(r) Therapy under defined medium cultures compared to untreated controls.  
The RUNN2 marker exhibited a significant increase in Limfa(r) Therapy-treated cultures, both under Basal and differentiation medium conditions.  
Figure 1: **Morphological examination of ADSCs 2D cultures after 10 and 21 days of culture.** Cells have been examined under different conditions. Basal, which corresponds to cells cultured in basal adipose stem cells medium; Basal + Limfa, which corresponds to cells cultured in basal adipose stem cells medium and treated with Limfa® Therapy; Diff, which corresponds to cells cultured in chondrogenic differentiation medium; Diff + Limfa, which corresponds to cells cultured in chondrogenic differentiation medium and treatment with Limfa® Therapy. Scoring system: +, weak differentiation; ++, moderate differentiation; ++, strong differentiation. (**A**) cell images after 21 days of Limfa treatment (**B**) cell images after 10 days of Limfa treatment (**C**) Cytotoxicity assay performed after 10 and 21 days of treatment. The scale bar corresponds to 100 \(\upmu\)m.  
ACAN showed significant expression differences between untreated cells maintained in Basal compared to conditioned medium, but no difference was observed due to the Limfa(r) Therapy treatment per se.  
Interestingly, the COL10A1 marker displayed a significant decrease in cells under both Basal and differentiation conditions when treated with Limfa(r) Therapy, compared to the untreated group. SOX9 also significantly decreased when comparing Basal vs. Diff + Limfa groups.  
SOX9 expression only showed a significant reduction in cells treated with Limfa(r) Therapy cultured in conditioned media (Diff + Limfa) compared to untreated Basal conditions (Figure 2).  
Figure 2: **Expression analysis of the main chondrogenic differentiation markers in different culture and treatment conditions.** Cells were examined under different conditions. Basal, which corresponds to cells cultured in basal adipose stem cells medium; Basal + Limfa, which corresponds to cells cultured in basal adipose stem cells medium and treated with Limfa(r) Therapy; Diff, which corresponds to cells cultured in chondrogenic differentiation medium; Diff + Limfa, which corresponds to cells cultured in chondrogenic differentiation medium and treatment with Limfa(r)therapy. SOX9 (**A**), RUNX2 (**B**), COL2A1 (**C**), COL10A1 (**D**) and ACAN (**E**) expression levels were assessed by RT-qPCR. Experiments are means of three different repeats. Results are expressed as relative gene expression (2-ACt) normalized on Basal culture conditions values. Error standard is reported. * = \(p<0.05\); ** = \(p<0.025\); *** = \(p<0.01\) (One-Way ANOVA).  
Overall, RT-qPCR expression analysis of the main chondrogenesis markers revealed that, for most of the transcripts taken into account, the treatment with Limfa(r) Therapy (Diff + Limfa) induced an increase in the relative gene expression levels when compared to untreated ones.