Introduction

Researchers have investigated the use of platelet-rich plasma (PRP) therapy. However, the mechanisms through which PRP affects tissue repair remain unclear. We hypothesize that PRP promotes tissue repair through not only via direct manner on the local cells but also via indirect manner that encourage the recruitment of reparative cells such as macrophages (MPs), and it depends on the quality of PRP including the concentration of leukocytes. The aim of this study is to elucidate the actions of the MPs in the mechanisms of PRP on tissue repair processes.

Methods

Leukocyte-rich (LR) PRP and leukocyte-poor (LP) PRP were prepared from 12-week-old C57BL6 mice. Full-thickness defects were created in central third of patellar tendons of 12-week-old C57BL/6 mice for histologic analysis (n = 36) and 12-week-old B6.129P-Cx3cr1tm1Litt/J mice for flow cytometry analysis (n = 108). B6.129P-Cx3cr1tm1Litt/J mouse is GFP-positive only in the MP-linage cells thus MPs recruited to the repair tissue can be distinguished whether it had originated from administrated PRP or recruited from host mouse. Mice were treated either with LR-PRP, LP-PRP, or without PRP (control group). Histological analyses were performed to evaluate the tendon healing using Bonar score as semi-quantitative histological scoring system. Flow cytometric analyses were performed to count the number of GFP-positive cells around repaired patellar tendon. In addition, the ratio of pro-inflammatory MPs (M1)/anti-inflammatory MPs (M2) were analyzed in those GFP-positive cells. The statistical analysis was performed using GraphPad Prism ver6. P values < 0.05 were considered statistically significant.

Results

In LR-PRP and LP-PRP groups, all variables in Bonar score such as cell morphology, cellularity, vascularity, and collagen arrangement were significantly improved in comparison with control group, indicating that both PRPs promote tendon hearing. LP-PRP promoted the tendon healing significantly faster than that of LR-PRP on postoperative day 28 (P < 0.001). LR-PRP enhanced angiogenesis (vascularity: P < 0.001), while LP-PRP improved the collagen arrangement on postoperative day 28 (collagen arrangement: P < 0.01). In other variables such as cell morphology and cellularity score, there were no significant differences between LR-PRP and LP-PRP groups in any time points. Flow cytometric findings showed that recruitment of GFP-positive MPs in the LR and LP-PRP groups were significantly increased from postoperative day 4 compared with control group without PRP treatment (P < 0.001). The majority of GFP-positive MPs were M1 at the initiation of tendon healing phase, and M2 were gradually increased from postoperative day 4. The number of M1 was significantly high both in the LP- and LR-PRP groups (day 4 and 7, p < 0.001), but the number of M2 was high only in the LP-PRP group (day 7 and 14, P < 0.05) when it compared with control group. The M1/M2 ratio on postoperative day 7 was significantly lower in the LP-PRP group than those in the control group (P < 0.05).

Conclusions

This study demonstrated that PRP enhanced the tendon healing and promoted the recruitment of MPs to the injured tissue. The subtypes of MPs were different depends on the types of PRPs, suggesting that leukocytes in PRP influence the effect of PRP therapy.

中文翻译：

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富含血小板的血浆促进肌腱愈合过程中巨噬细胞的募集。

 介绍

研究人员研究了富血小板血浆（PRP）疗法的使用。然而，PRP 影响组织修复的机制仍不清楚。我们假设PRP不仅通过直接作用于局部细胞的方式促进组织修复，还通过间接方式鼓励巨噬细胞（MP）等修复细胞的募集，并且这取决于PRP的质量，包括白细胞的浓度。本研究的目的是阐明 MP 在 PRP 组织修复过程机制中的作用。

 方法

富含白细胞（LR）的 PRP 和缺乏白细胞（LP）的 PRP 是从 12 周龄的 C57BL6 小鼠中制备的。在 12 周龄 C57BL/6 小鼠的髌腱中央三分之一处创建全层缺损用于组织学分析 (n = 36) 和 12 周龄 B6.129P-Cx3cr1tm1Litt/J 小鼠用于流式细胞术分析 (n = 36) = 108）。 B6.129P-Cx3cr1tm1Litt/J 小鼠仅在 MP 谱系细胞中呈 GFP 阳性，因此招募到修复组织的 MP 可以区分其是否源自施用的 PRP 或从宿主小鼠招募。小鼠接受 LR-PRP、LP-PRP 或不接受 PRP 治疗（对照组）。使用Bonar评分作为半定量组织学评分系统进行组织学分析以评估肌腱愈合。进行流式细胞术分析以计算修复后的髌腱周围 GFP 阳性细胞的数量。此外，还分析了 GFP 阳性细胞中促炎 MP（M1）/抗炎 MP（M2）的比率。使用 GraphPad Prism ver6 进行统计分析。 P值<0.05被认为具有统计学意义。

 结果

在LR-PRP和LP-PRP组中，与对照组相比，Bonar评分的所有变量，如细胞形态、细胞结构、血管分布和胶原排列均显着改善，表明两种PRP均促进肌腱听力。术后第 28 天，LP-PRP 促进肌腱愈合的速度明显快于 LR-PRP（P < 0.001）。 LR-PRP 增强血管生成（血管分布：P < 0.001），而 LP-PRP 改善术后第 28 天的胶原排列（胶原排列：P < 0.01）。在细胞形态和细胞结构评分等其他变量中，LR-PRP 和 LP-PRP 组在任何时间点都没有显着差异。流式细胞术结果显示，与未进行 PRP 治疗的对照组相比，术后第 4 天起 LR 组和 LP-PRP 组中 GFP 阳性 MP 的募集显着增加（P < 0.001）。大多数 GFP 阳性 MP 在肌腱愈合阶段开始时为 M1，M2 从术后第 4 天开始逐渐增加。LP- 和 LR-PRP 组（第 4 天和第 7 天）M1 的数量显着较高。 p < 0.001），但与对照组相比，仅 LP-PRP 组的 M2 数量较高（第 7 天和 14 天，P < 0.05）。 LP-PRP组术后第7天M1/M2比值显着低于对照组（P < 0.05）。

 结论

这项研究表明，PRP 可以增强肌腱愈合并促进 MPs 募集到受伤组织。根据PRP的类型，MP的亚型也不同，提示PRP中的白细胞影响PRP治疗的效果。