Elsevier

Tissue and Cell

Volume 49, Issue 1, February 2017, Pages 122-130
Tissue and Cell

Expressions of pathologic markers in PRP based chondrogenic differentiation of human adipose derived stem cells

https://doi.org/10.1016/j.tice.2016.11.001Get rights and content

Highlights

  • Angiogenic/inflammatory potential of PRP-based chondrogenically-differentiated cell was examined by monitoring the secretion of the growth factor, activation of the relevant receptor and cell-transmigration.

  • PRP significantly decreases the angiogenic and inflammatory potentials of the chondrogenically differentiated cells, but did not inhibit it completely.

  • PRP enhanced the mineralization and hypertrophic differentiation of the cells in the matrix of the macro pellet neotissues.

  • PRP has no physiologic contribution to the integrity and functionality of the constructed tissue since significant portion of the synthesized GAG released out of the macro pellet.

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 TGFβ 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, TNFα 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 TGFβ for chondrogenic differentiation, yet further improvement of the PRP-based chondrogenic differentiation media is required to inhibit the production of angiogenic/inflammatory markers, calcification and the release of synthesized GAG out of the construct.

Section snippets

Background

In the recent decade, PRP has been widely used as a therapeutic for OA patients with pain killing effects, although its overall effects depend on the stage of the disease, or in the other words on the micro-environmental conditions. The application of activated Platelet-Rich Plasma (PRP), with its vast range of cytokines and growth factors has achieved a considerable attention for chondrogenic differentiation in tissue engineering fields.

PRP is rich in growth factors such as TGF-β, Insulin-like

Adipose stem cells isolation, expansion and characterization

Liposuctioned samples were collected from healthy patients after obtaining their written informed consent according to the ethics of Tehran University of Medical Sciences. For ADSCs isolation briefly, the liposuctioned sample was washed several times to remove the blood cells, 150 μg/ml collagenase I (Sigma) was added and the sample was incubated for 60 min at 37 °C while on shaker at 250 rpm. Collagenase was removed by centrifugation at 1200 rpm for 5 min. The pellet was then incubated with RBC

Chondrogenic marker expression in PRP-based differentiation

Cartilage specific markers such as SOX9, aggrecan and Collagen type II genes were expressed in all groups, and as presented PRP had significant stimulatory effects on transcription of the mentioned genes in comparison to that of TGFβ treated group (Fig. 1a–c). In all groups the data was obtained by normalizing the expression of the test gene to the house keeping gene (Table 1). Collagen type II was expressed in all studied groups and, qualitatively, T1 group showed higher ColII protein

Discussions

Improving the effectiveness of chondrogenic differentiation medium either by addition of cytokines or differentiation factors or by exploiting biophysico-chemical/mechanical cues has been of the focus of many researches in recent years. Given the cross talk between closely related signaling pathways, especially in tissues with overlapped differentiation pathways, it is rather likely that unwanted or side pathways occur or even overtake the desired fate, necessitating investigation of side-fates

Conclusion

It should be mentioned that although PRP significantly decreases the angiogenic and inflammatory potentials of chondrogenically differentiated cells, but it does not inhibit it completely. On the other hand, PRP enhances the mineralizatin in the matrix of the macro pellet neotissues. In addition, while the GAGs producrion increases, a sigificant portion is released. Furthermore, expression of hypertrophic marker ColX highlights that the constructed tissues under current protocols have inferior

Consent for publication

This manuscript in the present format has been approved by all authors for publication in the journal of Tissue and Cell.

Availability of data and material

Available.

Competing interest

The authors declare no conflicts of interest.

Ethics approval and consent to participate

Samples were collected from healthy patients after obtaining their written informed consent according to the ethics of Tehran University of Medical Sciences, Tehran, Iran.

Author’s contribution

H.HA and Sh. I designed the project. A.P conducted the experiments and analyzed the data. H. HA wrote the manuscript. All authors read and approved the final manuscript.

Funding

Not applicable.

Acknowledgment

This work was supported financially by Stem Cell Technology Research Center, Tehran, Iran.

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