Effects of platelet rich plasma and chondrocyte co-culture on MSC chondrogenesis, hypertrophy and pathological responses.
Effects of platelet rich plasma and chondrocyte co-culture on MSC chondrogenesis, hypertrophy and pathological responses.
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DOI：
10.17179/excli2017-453
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发表时间：
2017
期刊：
影响因子：
4.6
通讯作者：
Hanaee Ahvaz H
中科院分区：
文献类型：
--
作者：
Ramezanifard R;Kabiri M;Hanaee Ahvaz H
关于软骨组织的愈合能力不足，基于细胞的治疗正在创造软骨修复和再生的未来。间充质干细胞（MSC）在软骨再生中显示出巨大的前景。然而，一个尚未解决的问题是在体外MSC软骨形成过程中肥大和病理标志物的出现。关节软骨细胞（AC）可以抑制不希望的肥大时，与MSC共培养。另一方面，富血小板血浆（PRP）被认为对软骨修复和体外软骨形成潜在有效。因此，我们的目的是利用PRP的软骨促进作用和AC：MSC共培养的肥大抑制作用，以获得功能更强的软骨新组织。我们在MSC单培养物和AC：MSC共培养物中使用PRP或常规分化软骨形成培养基（ConvDiff）。我们使用实时RT-PCR和免疫染色评估软骨形成和肥大标记物的基因表达。对颗粒的碱性磷酸酶活性（ALP）和钙含量进行定量。我们还通过ELISA测定了VEGF和TNF-α的分泌。结果显示PRP在mRNA和蛋白水平上具有更高的成软骨潜能，并具有比Conv-Diff（mRNA水平）更强的肥大抑制作用。共培养降低ALP，而PRP增加钙沉积。在所有四组中，与MSC对照相比，TNF-α下调，接受ConvDiff培养基的共培养物分泌最少。同时，VEGF分泌增加的唯一组是PRP单培养物。我们观察到PRP和AC：MSC共培养在增强软骨形成方面的协同效应。在PRP组中，AC的纳入降低了肥大标志物和血管生成潜力。因此，我们提出PRP和共培养的组合将有利于软骨形成，同时减轻但不能完全根除不希望的肥大和病理反应。
Regarding the inadequate healing capability of cartilage tissue, cell-based therapy is making the future of cartilage repair and regeneration. Mesenchymal stem cells (MSC) have shown great promise in cartilage regeneration. However, a yet-unresolved issue is the emergence of hypertrophic and pathologic markers during in vitro MSC chondrogenesis. Articular chondrocytes (AC) can suppress the undesired hypertrophy when co-cultured with MSC. On the other hand, platelet rich plasma (PRP), is considered potentially effective for cartilage repair and in-vitro chondrogenesis. We thus aimed to harness chondro-promotive effects of PRP and hypertrophic-suppressive effects of AC:MSC co-culture to achieve a more functional cartilage neo-tissue. We used PRP or conventional-differentiation chondrogenic media (ConvDiff) in MSC mono-cultures and AC:MSC co-cultures. We assessed gene expression of chondrogenic and hypertrophic markers using real-time RT-PCR and immunostaining. Alkaline-phosphatase activity (ALP) and calcium content of the pellets were quantified. We also measured VEGF and TNF-α secretion via ELISA. We showed PRP had higher chondrogenic potential (in mRNA and protein level) and hypertrophic-suppressive effects than Conv-Diff (mRNA level). Co-culturing reduced ALP while PRP increased calcium deposition. In all four groups, TNF-α was down-regulated compared to MSC controls, with co-cultures receiving ConvDiff media secreting the least. Meanwhile, the only group with increased VEGF secretion was PRP-mono-cultures. We observed synergistic effects for PRP and AC:MSC co-culture in enhancing chondrogenesis. Inclusion of AC reduced hypertrophic markers and angiogenic potential in PRP groups. We thus propose that combination of PRP and co-culture would favor chondrogenesis while alleviate but not totally eradicate undesired hypertrophic and pathologic responses.