PRP is an autologous blood products containing a variety of bioactive components with anti-inflammatory and tissue repair- and regeneration-promoting functions, which has been widely used in the treatment of chronic degenerative diseases9–11. In vitro experiments have confirmed the ability of PRP to enhance the proliferation of chondrocytes and MSCs12–15. However, the effect of PRP on cartilage differentiation of BMSCs in tissue engineering remains controversial. Although recent studies17, 18, 22 demonstrated the effects of different concentrations of PRP on chondrogenic differentiation of MSCs, they did not compare them with traditional commercial cartilage induction medium. The present study, therefore, aimed to compare the effects on cartilage differentiation of BMSCs between different PRP concentrations and commercial cartilage induction medium, and to evaluate the role of PRP in tissue-engineered cartilage formation.
In this study, concentrations of TGF-β3, IGF-1, PDGF, and VEGF were significantly increased in activated PRP compared with plasma. The addition of high concentrations of PRP to basic medium promoted the expansion of BMSCs in vitro compared with 10% FBS, revealing a concentration-time dependent effect. Higher growth factors and cytokines concentrations in PRP may induce this result. In addition, we also found that 10% PRP enhanced chondrogenic differentiation in cultured BMSC pellets, but still inferior to traditional commercial cartilage induction medium.
Commercial MSC chondrogenic differentiation media from different manufacturers have been widely used in chondrogenic differentiation experiments. These synthetic reagents contain not only basic cell culture medium, but also additional ingredients, such as TGF-β3, insulin, transferrin and selenium supplement, ascorbate, sodium pyruvate, proline, and dexamethasone. Among these, TGF-β3 is a vital regulatory factor promoting chondrogenic differentiation of MSCs, while the other components also improve cell metabolism, promote cell proliferation, and inhibit cell aging. The concentration of TGF-β3 in the cartilage differentiation medium used in this study was 10 ng/ml, which was higher than that in 10% PRP (3.05 ng/ml), while PRP itself may not contain any other additives. We therefore considered that basic cell culture medium with 10% PRP might have a limited effect on BMSC cartilage differentiation compared with commercial cartilage differentiation medium.
Whether the increased concentration of PRP in medium would enhance chondrogenic differentiation in BMSCs? Krüger et al. have reported that human PRP enhanced the migration and stimulated the chondrogenic differentiation of human BMSCs derived from spongious bone of the tibia or femur head16. Liou et al. investigated the effects of different concentrations of PRP on adipose-derived stem cells and found that increasing the PRP concentration did not enhance chondrogenic differentiation 18. Amaral et al. also observed cartilage differentiation medium containing different PRP concentrations on chondrogenesis of BMSC pellets and showed that increasing the PRP proportion from 1–10% reduced the expression levels of cartilage-specific genes and proteoglycans in pellets22. In addition, some previous studies reported that commercial induction medium containing 10% PRP did not enhance chondrogenic differentiation compared with induction medium alone17. The similar results were also observed in this study. Though certain PRP concentration induced chondrogenic differentiation in BMSCs pellets, this effect was not enhanced with the increasing of PRP contents.
In this study, we observed that 10% PRP promoted the chondrogenic differentiation of BMSCs pellets by analysis of gene expression levels, gross morphological observation, and pathological staining of cartilage-specific proteins. We supposed that the biological mechanism of PRP in chondrogenic differentiation of MSCs may be complicated and Intricate. Furthermore, whether MSC derived from different sources respond consistently to PRP in induction chondrogenesis, further studies are deserved.
Activated-PRP includes cytokines such as TGFβ, PDGF, VEGF, and epidermal growth factor (EGF), but the beneficial effects of these growth factors in chondrogenesis remain unclear. The TGFβ family is known to induce proliferation and chondrogenesis of BMSCs during cartilage formation, meanwhile, PDGF supports chondrocytes to maintain the hyaline-like chondrogenic phenotype and induces proteoglycan synthesis23. However, VEGF showed poor chondrogenic effects on muscle-derived stem cells in a rat model24, and an EGF receptor ligand promoted chondrocyte catabolic activity and inhibited anabolic activity in an osteoarthritis mouse model25. VEGF and/or EGF may therefore weaken the chondro-inductive effects of PRP. Further studies are needed to determine if specific depletion of such anti-chondrogenic factors and optimization of other stimulatory components in PRP might enhance BMSC chondrogenic differentiation.
In addition to optimizing the differentiation induction medium, it is also necessary to improve the seed cells to facilitate the construction of tissue-engineered cartilage. Some researchers have constructed a co-culture system composed of autologous chondrocytes and MSCs in order to observe the efficiency of cartilage differentiation and to inhibit chondrocyte hypertrophy and osteogenic differentiation by the paracrine effect of different cells26, 27. Although the results were not consistent, they provided a novel research direction for the construction of tissue-engineered cartilage. Recent studies has reported that human placental28, umbilical cord blood29, and amnion-derived MSCs30 all reveal high proliferative ability, multipotency, and low immunogenicity, and are thus becoming novel seed cells in tissue engineering. Whether PRP could enhance chondrogenic differentiation in these stem cells or not, further studies are deserved.
There are some limitations in this study. Firstly, we only observed the end-stage chondrogenic differentiation of BMSCs after 21 days of culture, and the continuous effect of PRP were not examined. Secondly, limited number of PRP-treated groups were designed, the details of concentration-dependent effect of PRP on chondrogenesis were limited. Additionally, due to commercial chondrogenic differentiation media from different manufacturers may contain various components and efficacy, the reagents used in this experiment are of limited representativeness.