15^th Asia-Pacific Biotechnology Congress

Biography

Biography: Arezou Pakfar

Abstract

Background: Optimization of the differentiation medium through using autologous factors such as PRP is of great consideration, but due to the complex, variable and undefined composition of PRP on one hand and lack of control over the absolute regulatory mechanisms in in vitro conditions or disrupted and different mechanisms in diseased tissue microenvironments in in vivo conditions on the other hand, it is complicated and rather unpredictable to get the desired effects of PRP making it inevitable to monitor the possible pathologic or undesired differentiation pathways and therapeutic effects of PRP. Therefore, in this study the probable potential of PRP on inducing calcification, inflammation and angiogenesis in chondrogenically differentiated cells was investigated.
Methods: The expressions of chondrogenic, inflammatory, osteogenic and angiogenic markers from TGF or PRP-treated cells during chondrogenic differentiation of human adipose-derived stem cells (ADSCs) was evaluated. Expressions of Collagen II (Col II), Aggrecan, Sox9 and Runx2 were quantified using q-RT PCR. Expression of Col II and X was investigated by immunocytochemistry as well. Glycosaminoglycans (GAGs) production was also determined by GAG assay. Possible angiogenic/inflammatory potential was determined by quantitatively measuring the secreted VEGF, TNF and phosphorylated VEGFR2 via ELISA. In addition, the calcification of the construct was monitored by measuring ALP activity and calcium
deposition.
Results: Our data showed that PRP positively induced chondrogenesis; meanwhile the secretion of
angiogenic and inflammatory markers was decreased. VEGFR2 phosphorylation and ALP activity had
a decreasing trend, but tissue mineralization was enhanced upon treating with PRP.
Conclusions: Although reduction in inflammatory/angiogenic potential of the chondrogenically differentiated constructs highlights the superior effectiveness of PRP in comparison to TGF for chondrogenic differentiation, yetfurther improvement ofthe PRP-based chondrogenic differentiation media is required to inhibitthe production of angiogenic/inflammatory markers, calcification and the release of synthesized GAG out of the construct.

e
Fig. 1. Gene expression in chondrogenically differentiated cells; cartilage specific genes were expressed in all groups (a–c). TGF: Dif, T1:5%PRP, and T2:15% PRP. Data werenormalized to expresion of house-keeping gene. “*” indicates significant difference.

Fig. 2. Collage Type II, X and glycosaminoglycans expression in chondrogenically differentiated cells; Collagen type II expression and the DAPI staining of TGF: Dif (A1,2),T1:5%PRP (B1,2), and T2:15% PRP (C1,2) groups and Collagen type X expression and the DAPI staining of TGF: Dif (A3,4), T1 (B3,4), T2 (C3,4) groups were visualized byPE-immunostaining; nuclei were counterstained with DAPI. Glycosaminoglycan expression was confirmed by Alician blue staining Dif (D), T1 5% PRP (E), T2 15%PRP (F). (Forinterpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


Fig. 3. Monitoring the angiogenic potential of the differentiated cells. VEGF was secreted by all differntiated cell (a), tyrosine 1175 residue of VEGFR2 was phosphorylatedunder all studied conditions (b), chemotactic potential of conditioned media of the TGF: Dif (c), T1:5% PRP (d), T2 15% PRP(e) at day 21. “*” indicates significant difference.

Fig. 4. Monitoring the inflammatory potential of the differentiated cells. TNF wassecreted by all differntiated cell (a), all conditioned media had stimulatory effects onU937 migration(b); pos: treated with 10% FBS. “*” indicates significant difference.TGF: Dif, T1:5%PRP, and T2:15% PRP.

Fig. 5. Expression of osteogenic markers in chondrogically differentiated cells. PRPlowered the ALP activity (a), Calcium content increased in PRP treated groups (b),RUNX2 was down regulated in all studied groups. “*” indicates significant difference.TGF: Dif, T1:5%PRP, T2:15% PRP and pos:10% FBS.

Fig. 6. GAG production. All groups represented decreasing trend towards day 21 (a),considerable portion of syntjesized GAG was released out in PRP-based differenti-ated cells (b). “*” indicates significant difference. TGF: Dif, T1:5%PRP, and T2:15%PRP.