| 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... |
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