PRP促进半月板白—白区损伤愈合及对股骨髁软骨退变影响的实验研究
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摘要
目的：比较实验动物比格犬自体富血小板血浆(platelet-rich plasma, PRP)与静脉全血的血小板浓度差异；观察比格犬骨髓间质干细胞(marrow stroma cell, MSCs)在七种不同PRP浓度干预下体外培养时的生长特性；研究PRP和MSCs对半月板白-白区损伤愈合的影响,观察比格犬半月板白-白区损伤对股骨髁软骨退变及关节液中OPN含量变化的影响；探讨半月板白-白区损伤修复的重要性和促进其愈合的可行方法。
方法：实验动物24只健康成年雄性比格犬(体重：16.10±0.06Kg；年龄：25.87±0.34月)。1.采集比格犬静脉全血40ml利用二次离心法制备PRP,比较PRP与静脉全血之间的血小板浓度。采集比格犬2ml骨髓培养原代MSCs,利用贴壁生长特性传代纯化MSCs,用MTT实验绘制细胞生长曲线,并应用CD13-FITC、CD45-FITC、 CD11b-FITC、CD73-FITC抗体通过流式细胞仪鉴定纯化培养的MSCs。观察MSCs与不同浓度PRP(20%、40%、50%、60%、70%、80%、90%)共培养的生长特性。2.24只比格犬均建立双侧膝关节外侧半月板白-白区损伤模型。按照半月板损伤部位随机给予不同的干预因素分为4组,每组6只12膝,每只双侧干预因素相同,分别为(生理盐水)对照组、MSCs组、PRP组、PRP+MSCs组；成功造模3月后行膝关节MRI(3级信号评价标准)检查了解半月板愈合情况。造模3月后处死比格犬进行半月板大体标本观察及HE染色法组织学(Henning评价标准)比较各组比格犬半月板白-白区损伤愈合情况,通过免疫组织化学方法检测Ⅰ型胶原、Ⅱ型胶原在半月板白-白区损伤处周围的表达。3.采用ELISA检测造模前与造模后3个月时所有比格犬关节液中OPN含量。造模后3个月行X线和MRI检查关节退变情况。动物处死后通过大体标本观察和甲苯胺兰染色法组织学观察股骨外髁软骨退变情况。基于前部分实验结果根据半月板白-白区损伤愈合与否分为半月板损伤未愈合组和半月损伤愈合组,比较两组之间关节软骨退变情况和关节液中OPN含量差异。
结果：1.通过二次离心法制备的PRP血小板浓度(1368.67±52×109/L)高于全血血小板浓度(158.89±35×109/L)(P<0.05)；传代培养的MSC95%呈CD13和CD73阳性表达。浓度为20%、40%、50%、60%、70%和80%的PRP干预均对MSCs生长有促进作用,70%PRP浓度干预时达最高峰,与70%浓度组比较,50%、60%浓度组无显著性差异(p>0.05),20%、40%、80%组有显著性差异(p<0.05),浓度高于70%时其促进作用又逐渐减弱,至90%PRP浓度时与对照组无统计学差异(p>0.05)。2.造模后3月MRI评估半月板白-白区愈合率为：对照组愈合率0%；MSCs组愈合率为8.3%；PRP组愈合率为75%；PRP+MSCs组愈合率为83.3%。对照组与MSC组无统计学差异；PRP组与PRP+MSC组无统计学差异(P>0.05)。MSC组与PRP组,MSC组与PRP+MSC组存在显著性统计学差异(P<0.05,P<0.05),对照组与PRP组,对照组与PRP+MSC组有显著性统计学差异(P<0.05,P<0.05)。HE组织学评估：对照组完全愈合率为0,部分愈合率为8.3%；MSCs组完全愈合率为8.3%,部分愈合率为8.3%；PRP组完全愈合率为75%,部分愈合率为16.7%,未愈合率为8.3%; PRP+MSCs组完全愈合率为83.3%,部分愈合率为16.7%,未愈合率为0。对照组和MSCs愈合率无统计学差异(P>0.05); PRP组和PRP+MSCs组愈合率无统计学差异(P>0.05)；对照组与PRP组愈合率上有统计学差异(P<0.05); MSCs组与PRP组愈合率有统计学差异(P<0.05)；对照组与PRP+MSCs组愈合率有统计学差异(P<0.05); MSCs组与PRP+MSCs组愈合率有统计学差异(P<0.05).Ⅰ型胶原、Ⅱ型胶原检测显示对照组和MSC的棕色颗粒表达较PRP组和PRP+MSC组少,具有统计学差异(P<0.05)。3.X线、MRI检查和大体标本观察结果均显示未愈合组与愈合组之间的软骨退变率无统计学差异。组织学检查显示未愈合组与愈合组在股骨软骨退变率比较有统计学差异(P<0.05)(未愈合组轻度16/22,愈合组轻度6/26)。在造模前、后3月比格犬膝关节液中OPN含量变化：半月板白-白区未愈合组有明显统计学差异(P<0.05),半月板愈合组OPN含量有升高但无显著性差异(P>0.05)。
结论：1.PRP在一定浓度范围内(<70%)时促进MSCs生长的能力比较明显,PRP在50%-70%范围促进MSCs生长的能力强于其他浓度组,较高浓度时(>70%)促进MSCs生长的能力反而下降,即PRP所含有的血小板浓度为684-957.6×109/L时促进MSCs生长的能力最强。2.PRP在联合或者不联合MSCs使用的前提下能促进半月板白-白区损伤愈合,单独应用MSCs对于半月板白-白区并无促进作用。3.半月板白-白区损伤未愈合在早期会给股骨软骨带来一定程度的损伤,但不会表现出关节退变的影像学改变。膝关节内关节液OPN含量升高对于早期关节退变的检测有一定参考价值。
Objective:The investigation first compared the platelet concentration of the platelet rich plasma with the venous whole blood, and then examined the growth ability of marrow stroma cells (MSCs) with different PRP concentration, and finally efficacy and mechanism of platelet-rich plasma combined with or without marrow stroma cell in the healing of white-white meniscal tears were further studied by building the meniscus white-white lesion model in Beagle dogs.Meanwhile the cartilage degeneration origination from white-white meniscal tears was investigated.
Methods:24mature Beagle dogs were used, which were about2years old (16.10±0.06Kg,25.87±0.34months). The platelet-rich plasma was prepared from40ml venous whole blood of Beagle dogs through the twice centrifugation method. MSCs were derived from bone marrow, and MSCs were naturally purified by the adherence characteristic during passage. The identification of MSCs was examined with the flow cytometer using antibodies CD13-FITC, CD45-FITC, CDllb-FITC, CD73-FITC. And meanwhile MSCs growth curve was observed by MTT assay. Platelet concentration was compared between the whole blood and the platelet rich plasma. The growth ability of MSCs at different concentration of platelet rich plasma (20%、40%、50%、60%、70%、80%、90%) was observed.24Beagle dogs were randomly divided into4groups:Physiological saline Group (controlled group), MSCs Group, PRP Group, and PRP plus MSCs Group. Bilateral knee menisci white-white tears models were set up. The menisci healing status was evaluated by the nuclear magnetic resonance imaging system at post-operation3months. The healing status of the menisci white-white tears were evaluated by gross pathological observation, hematoxylin-eosin staining histological method and Ⅰ, Ⅱ collagen immunohistochemistry assay was used to examine the expression of Ⅰ, Ⅱ collagen around white-white meniscal tears. Osteoarthritis was evaluated by X-radiation at post-operation3months.Osteopontin(OPN) concentration in knee synovial fluid was compared by enzyme-linked immunosorbent assay (ELSIA) at timing of lw pre-operaton,3months post-operation. Meanwhile the lateral femoral condyle cartilage lesion was observed by gross pathological observation, toluidine blue staining histological method.
Results:The platelet concentration of platelet rich plasmas(1368.67±52×109/L) was higher than that of the whole blood (158.89±35×109/L) after platelet rich plasma was made by the twice centrifugation method(p<0.05). CD13and CD73positive expression of95%MSCs was determinated by flow cytometer. MSCs was cultured at low concentration of platelet rich plasma, the growth capacity was enhanced, while MSCs was cultured at high concentration of platelet rich plasma when the concentration of platelet rich plasma was higher than70%, the growth ability was inhibited. Healing ability of Beagle dogs lateral white-white menisci tears was enhanced by PRP combined with or without MSCs,compared with the controlled group. And there was significant difference in statistics when PRP group or PRP plus MSCs group was compared with controlled group(p<0.05).There was no significant difference in statistics between the PRP group and PRP plus MSCs group(p>0.05,p=1.00). While when the MSCs alone were used, healing ability of Beagle dogs lateral white-white menisci tears was not enhanced, compared with the control group. There was no significant difference in statistics between MSCs and controlled group(p>0.05). And there was significant difference in statistics between PRP group and MSCs group or between PRP plus MSCs group and MSCs group(p<0.05,p=0.000). Post-operation OPN concentration in knee synovial fluid in meniscus unhealing group (control group and MSCs group) was obviously higher than pre-operation OPN concentration; there was a significant difference in statistics(p<0.05,p=0.000). There was no significant difference in meniscus healing group (PRP group and PRP+MSCs group) when OPN concentration was observed at respectively pre-operation and post-operation(p>0.05,p=0.98). Femur cartilage lesion was observed in meniscus unhealing group (MSCs group and control group), femur cartilage lesion was not observed in meniscus healing group (PRP group and PRP+MSCs group).
Conclusion:PRP can stimulate the MSCs growth in vitro under70%PRP concentration, while up to70%, the growth ability was inhibited. And PRP can enhance the healing ability of menisci white-white tears combined with or without MSCs. The unhealing of white-white meniscal tears results in femur cartilage lesion,and at early stage it does not result in any radiological change of osteoarthritis.OPN can be used as a biological marker to detect early osteoarthritis
方法：实验动物24只健康成年雄性比格犬(体重：16.10±0.06Kg；年龄：25.87±0.34月)。1.采集比格犬静脉全血40ml利用二次离心法制备PRP,比较PRP与静脉全血之间的血小板浓度。采集比格犬2ml骨髓培养原代MSCs,利用贴壁生长特性传代纯化MSCs,用MTT实验绘制细胞生长曲线,并应用CD13-FITC、CD45-FITC、 CD11b-FITC、CD73-FITC抗体通过流式细胞仪鉴定纯化培养的MSCs。观察MSCs与不同浓度PRP(20%、40%、50%、60%、70%、80%、90%)共培养的生长特性。2.24只比格犬均建立双侧膝关节外侧半月板白-白区损伤模型。按照半月板损伤部位随机给予不同的干预因素分为4组,每组6只12膝,每只双侧干预因素相同,分别为(生理盐水)对照组、MSCs组、PRP组、PRP+MSCs组；成功造模3月后行膝关节MRI(3级信号评价标准)检查了解半月板愈合情况。造模3月后处死比格犬进行半月板大体标本观察及HE染色法组织学(Henning评价标准)比较各组比格犬半月板白-白区损伤愈合情况,通过免疫组织化学方法检测Ⅰ型胶原、Ⅱ型胶原在半月板白-白区损伤处周围的表达。3.采用ELISA检测造模前与造模后3个月时所有比格犬关节液中OPN含量。造模后3个月行X线和MRI检查关节退变情况。动物处死后通过大体标本观察和甲苯胺兰染色法组织学观察股骨外髁软骨退变情况。基于前部分实验结果根据半月板白-白区损伤愈合与否分为半月板损伤未愈合组和半月损伤愈合组,比较两组之间关节软骨退变情况和关节液中OPN含量差异。
结果：1.通过二次离心法制备的PRP血小板浓度(1368.67±52×109/L)高于全血血小板浓度(158.89±35×109/L)(P<0.05)；传代培养的MSC95%呈CD13和CD73阳性表达。浓度为20%、40%、50%、60%、70%和80%的PRP干预均对MSCs生长有促进作用,70%PRP浓度干预时达最高峰,与70%浓度组比较,50%、60%浓度组无显著性差异(p>0.05),20%、40%、80%组有显著性差异(p<0.05),浓度高于70%时其促进作用又逐渐减弱,至90%PRP浓度时与对照组无统计学差异(p>0.05)。2.造模后3月MRI评估半月板白-白区愈合率为：对照组愈合率0%；MSCs组愈合率为8.3%；PRP组愈合率为75%；PRP+MSCs组愈合率为83.3%。对照组与MSC组无统计学差异；PRP组与PRP+MSC组无统计学差异(P>0.05)。MSC组与PRP组,MSC组与PRP+MSC组存在显著性统计学差异(P<0.05,P<0.05),对照组与PRP组,对照组与PRP+MSC组有显著性统计学差异(P<0.05,P<0.05)。HE组织学评估：对照组完全愈合率为0,部分愈合率为8.3%；MSCs组完全愈合率为8.3%,部分愈合率为8.3%；PRP组完全愈合率为75%,部分愈合率为16.7%,未愈合率为8.3%; PRP+MSCs组完全愈合率为83.3%,部分愈合率为16.7%,未愈合率为0。对照组和MSCs愈合率无统计学差异(P>0.05); PRP组和PRP+MSCs组愈合率无统计学差异(P>0.05)；对照组与PRP组愈合率上有统计学差异(P<0.05); MSCs组与PRP组愈合率有统计学差异(P<0.05)；对照组与PRP+MSCs组愈合率有统计学差异(P<0.05); MSCs组与PRP+MSCs组愈合率有统计学差异(P<0.05).Ⅰ型胶原、Ⅱ型胶原检测显示对照组和MSC的棕色颗粒表达较PRP组和PRP+MSC组少,具有统计学差异(P<0.05)。3.X线、MRI检查和大体标本观察结果均显示未愈合组与愈合组之间的软骨退变率无统计学差异。组织学检查显示未愈合组与愈合组在股骨软骨退变率比较有统计学差异(P<0.05)(未愈合组轻度16/22,愈合组轻度6/26)。在造模前、后3月比格犬膝关节液中OPN含量变化：半月板白-白区未愈合组有明显统计学差异(P<0.05),半月板愈合组OPN含量有升高但无显著性差异(P>0.05)。
结论：1.PRP在一定浓度范围内(<70%)时促进MSCs生长的能力比较明显,PRP在50%-70%范围促进MSCs生长的能力强于其他浓度组,较高浓度时(>70%)促进MSCs生长的能力反而下降,即PRP所含有的血小板浓度为684-957.6×109/L时促进MSCs生长的能力最强。2.PRP在联合或者不联合MSCs使用的前提下能促进半月板白-白区损伤愈合,单独应用MSCs对于半月板白-白区并无促进作用。3.半月板白-白区损伤未愈合在早期会给股骨软骨带来一定程度的损伤,但不会表现出关节退变的影像学改变。膝关节内关节液OPN含量升高对于早期关节退变的检测有一定参考价值。
Objective:The investigation first compared the platelet concentration of the platelet rich plasma with the venous whole blood, and then examined the growth ability of marrow stroma cells (MSCs) with different PRP concentration, and finally efficacy and mechanism of platelet-rich plasma combined with or without marrow stroma cell in the healing of white-white meniscal tears were further studied by building the meniscus white-white lesion model in Beagle dogs.Meanwhile the cartilage degeneration origination from white-white meniscal tears was investigated.
Methods:24mature Beagle dogs were used, which were about2years old (16.10±0.06Kg,25.87±0.34months). The platelet-rich plasma was prepared from40ml venous whole blood of Beagle dogs through the twice centrifugation method. MSCs were derived from bone marrow, and MSCs were naturally purified by the adherence characteristic during passage. The identification of MSCs was examined with the flow cytometer using antibodies CD13-FITC, CD45-FITC, CDllb-FITC, CD73-FITC. And meanwhile MSCs growth curve was observed by MTT assay. Platelet concentration was compared between the whole blood and the platelet rich plasma. The growth ability of MSCs at different concentration of platelet rich plasma (20%、40%、50%、60%、70%、80%、90%) was observed.24Beagle dogs were randomly divided into4groups:Physiological saline Group (controlled group), MSCs Group, PRP Group, and PRP plus MSCs Group. Bilateral knee menisci white-white tears models were set up. The menisci healing status was evaluated by the nuclear magnetic resonance imaging system at post-operation3months. The healing status of the menisci white-white tears were evaluated by gross pathological observation, hematoxylin-eosin staining histological method and Ⅰ, Ⅱ collagen immunohistochemistry assay was used to examine the expression of Ⅰ, Ⅱ collagen around white-white meniscal tears. Osteoarthritis was evaluated by X-radiation at post-operation3months.Osteopontin(OPN) concentration in knee synovial fluid was compared by enzyme-linked immunosorbent assay (ELSIA) at timing of lw pre-operaton,3months post-operation. Meanwhile the lateral femoral condyle cartilage lesion was observed by gross pathological observation, toluidine blue staining histological method.
Results:The platelet concentration of platelet rich plasmas(1368.67±52×109/L) was higher than that of the whole blood (158.89±35×109/L) after platelet rich plasma was made by the twice centrifugation method(p<0.05). CD13and CD73positive expression of95%MSCs was determinated by flow cytometer. MSCs was cultured at low concentration of platelet rich plasma, the growth capacity was enhanced, while MSCs was cultured at high concentration of platelet rich plasma when the concentration of platelet rich plasma was higher than70%, the growth ability was inhibited. Healing ability of Beagle dogs lateral white-white menisci tears was enhanced by PRP combined with or without MSCs,compared with the controlled group. And there was significant difference in statistics when PRP group or PRP plus MSCs group was compared with controlled group(p<0.05).There was no significant difference in statistics between the PRP group and PRP plus MSCs group(p>0.05,p=1.00). While when the MSCs alone were used, healing ability of Beagle dogs lateral white-white menisci tears was not enhanced, compared with the control group. There was no significant difference in statistics between MSCs and controlled group(p>0.05). And there was significant difference in statistics between PRP group and MSCs group or between PRP plus MSCs group and MSCs group(p<0.05,p=0.000). Post-operation OPN concentration in knee synovial fluid in meniscus unhealing group (control group and MSCs group) was obviously higher than pre-operation OPN concentration; there was a significant difference in statistics(p<0.05,p=0.000). There was no significant difference in meniscus healing group (PRP group and PRP+MSCs group) when OPN concentration was observed at respectively pre-operation and post-operation(p>0.05,p=0.98). Femur cartilage lesion was observed in meniscus unhealing group (MSCs group and control group), femur cartilage lesion was not observed in meniscus healing group (PRP group and PRP+MSCs group).
Conclusion:PRP can stimulate the MSCs growth in vitro under70%PRP concentration, while up to70%, the growth ability was inhibited. And PRP can enhance the healing ability of menisci white-white tears combined with or without MSCs. The unhealing of white-white meniscal tears results in femur cartilage lesion,and at early stage it does not result in any radiological change of osteoarthritis.OPN can be used as a biological marker to detect early osteoarthritis
引文
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