## 4 Discussion  
Even though some studies have already examined the effects of ultra-weak electromagnetic fields on mature human chondrocytes, their interaction with chondrogenic lineage commitment of MSCs sources such as ADSCs, has not been completely elucidated. This study aimed to investigate the biological effects of ultra-low complex electromagnetic  
Figure 3: Western Blot analysis of Collagen I and Collagen II expression in different cultures and treatment conditions. Results are expressed as arbitrary units (a.u.) and normalized on Tubulin input. Basal—Basal medium; Diff—differentiation medium; Limfa—Limfa® Therapy treatment. Experiments are means of three different repeats. Error standard is reported. * = \(p\) < 0.05; ** = \(p\) < 0.025 (One-Way ANOVA). fields delivered by an innovative medical device, Limfa(r) Therapy, on an in vitro cartilage regeneration model. Our results indicate that ADSCs treatment with Limfa Therapy is able to induce the modulation of some of the main genetic chondrogenesis markers. Evidence has emerged that SOX9 did not show significant differences when compared to untreated and Basal medium conditions. This is in line with the early-stage nature of this chondrogenesis marker. Such a transcription factor is described as an early gene driving the initial switch to the chondrogenic commitment of undifferentiated progenitor cells [17]. The genetic markers related to extracellular matrix composition showed modifications induced by Limfa Therapy which are highly representative of ADSCs chondrogenic lineage commitment. Cartilage-specific collagen type-II expression showed the most significant increase in cells treated with Limfa Therapy and maintained in a Differentiation medium (Diff + Limfa) when compared to both its untreated relative control (Diff) and Basal conditions. Limfa Therapy was also able to significantly decrease collagen type 10 expression, both in Basal and conditioned medium cultures. As previously described [18], COL10A1 is known to be a specific marker for late chondrocyte hypertrophy, found in network-like rather than in fibril-like collagen structures, suggesting that Limfa Therapy preferentially promotes hyaline cartilage formation instead of bone tissue. This represents an innovative finding considering that many studies show how cartilage derived in vitro from MSCs commonly shows hypertrophic rather than hyaline features, making it unsuitable for functional cartilage tissue regeneration purposes [19]. Finally, collagen type-II protein expression evaluated by Western blot confirms the hypothesis that Limfa Therapy is able to potentiate the induction of chondrogenesis, exploiting the soluble factors contained in pro-chondrogenic medium and therefore enhancing the deposition of cartilage-specific ECM. Moreover, we have envisaged a significant downregulation of collagen type-I protein expression after performing Limfa Therapy treatment on cells maintained with differentiation medium. Such findings strengthen the data obtained so far by our group, as collagen type I represents one of the main osteogenic markers, which is repressed during chondrogenic lineage commitment [20]. Overall, our results suggest that Limfa Therapy efficacy might be ascribable to its ability to promote adipose mesenchymal stem cells' chondrogenic lineage commitment and tissue repair [10,11]. Moreover, concerning extracellular matrix deposition, Limfa Therapy application effectively promotes ADSC differentiation when coupled with biochemical stimuli contained in a pro-chondrogenic medium. Such results open a new path as it is known that the literature lacks studies regarding the use of ADSCs in humans for orthopedic pathologies [13]. Nevertheless, preliminary outcomes are very encouraging, with a low rate of complications. Different delivery systems for these stem cells have been tested so far. ADSCs can be administered either with a simple injection or during a surgical procedure.