自体富血小板血浆对膝关节退行性变和难愈性创面治疗作用的实验研究

发布时间：2018-03-01 15:40

本文关键词： 富血小板血浆关节软骨难愈性创面修复　出处：《山东大学》2016年博士论文　论文类型：学位论文

【摘要】：目的：本研究应用两步离心法制备PRP,通过分析PRP中血小板、白细胞和生长因子的浓度,计算回收率和富集系数,并做相关性分析,探讨PRP制备方法的实用性和稳定性。关节退变临床产生关节疼痛,活动受限和关节畸形等症状,是导致患者长期痛苦和身体残疾的常见原因,不仅极大地影响了人们的生活质量,导致人力资源的丧失,也给家庭和社会带来沉重负担。传统方法存在诸多弊端和生物学技术不断取得新进展的今天,PRP技术为我们治疗骨性关节炎提供了一种崭新的治疗思路和方法。通过关节内注射的方法,将PRP与玻璃酸钠作比较,探讨关节内注射PRP治疗关节软骨退行性变的安全性和疗效。慢性难愈性创面的治疗一直是医学研究的热点,传统的伤口换药、清创手术、负压真空吸引和局部应用各种药物等治疗,能清除局部感染灶或坏死组织,减少溃疡分泌物,但仍有许多创面难以愈合,我们采用局部喷洒PRP的方法治疗慢性难愈性创面,探索PRP治疗慢性难愈性创面的安全性和疗效,同时通过体外实验评价其抗感染作用。方法：(取30例成年健康自愿者捐献的外周血,男女各15例；年龄24-76岁,平均47岁。用预先装有5 mL枸橼酸钠注射液的50 mL一次性注射器以18 G针头于每例志愿者肘前静脉取血40 mL。摇匀后,取1 mL作血细胞分析,4 mL作生长因子浓度测定；剩余40 mL注入PRP制备套装离心管中,于严格无菌状态下制备PRP。根据血液中各组分的沉降系数不同,以离心半径15 cm、2000 r/min离心10 min后,全血分为3层,上层为上清液,下层为红细胞,两层交界处一浅黄色层即PRP层。分别将1 mL抗凝静脉血和制备的PRP采用全自动血液分析仪进行血常规分析,测定血小板及白细胞浓度,并计算PRP中血小板和白细胞浓度变异系数。计算PRP中血小板或白细胞回收率和富集系数,回收率=PRP中血小板或白细胞总数／全血中血小板或白细胞总数×100%,富集系数=PRP中血小板或白细胞浓度／全血中血小板或白细胞浓度。分别测定男、女自愿者PRP血小板及白细胞浓度。生长因子浓度测定于4 mL抗凝静脉血中加入0.4 mL凝血酶和氯化钙混合物(2000 U凝血酶中加入5 mL 5%氯化钙溶液),3 min后血液凝聚成冻胶状,常温下静置1 h后,同上法离,010 min,吸取上清液转移到冻存管中。将制备的PRP采用相同方法制备得到富含生长因子的上清液。取制备的全血及PRP上清液,按照ELISA试剂盒说明测定PDGF、TGF-β及VEGF浓度。将30例慢性关节疼痛或肿胀(不得少于4月),X或MRI有退行性变表现的膝关节软骨退行性变患者纳入研究。根据患者的年龄、性别、体重指数以及Kellgren Lawrence骨性关节炎分级情况将30例病人分配到PRP组(试验组)和透明质酸钠(Sodium hyaluronate, SH)。试验组15例患者(15膝),每次关节内注射3.5 mLPRP,对照组15例患者(15膝),每次关节内注射透明质酸钠(Sodium hyaluronate, SH) 2 mL。所有患者每3周注射一次,连续3次为一个疗程。通过记录治疗过程中相关不良事件的数量、发生时间、严重程度、持续时间和处理结果来评估该治疗方法的安全性。通过比较注射前和3、4、6月随访时的国际膝部文件委员会(IKDC)主观膝部评估表、WOMAC评价表、Lequesne指数评估表来评估其临床疗效。将符合选择标准的21例慢性难愈性创面患者纳入本项研究。男8例,女13例；年龄48-78岁,平均58.5岁。开放原始创面,彻底清创,按照1：10的比例将制备的2-8mL自体PRP与促凝剂(2000U凝血酶中加入5mL 5%氯化钙溶液)同时缓慢喷洒入创面并尽量覆盖所有创面。3min后创面表面形成一层PRP凝胶,取大小合适的无菌透明薄膜覆盖于创口,四周密封确保PRP不流失,术后1周内建议患者尽量减少创面临近关节的活动量。术后可视情况更换无菌敷料,一般1周左右查看创面。嘱患者避免用手碰触患处,根据创面情况决定PRP使用的次数。采用滴注无菌生理盐水的方法测定伤口体积和计算创面覆盖率,我们将3个月内的创面愈合情况分为如下五个等级：Ⅰ级完全愈合,覆盖率100%；Ⅱ级明显愈合,覆盖率在75%-100%之间；Ⅲ级有效愈合,覆盖率在50%-75%之间；Ⅳ级部分有效愈合,覆盖率在25%-50%之间；V级无效愈合,覆盖率低于25%。对于3个月后没有痊愈的患者,在征求他们同意的情况下给予再次PRP治疗或继续观察治疗。于治疗前及3月随访结束时分别行创面内分泌物细菌培养,对比观察。选取创面感染最常见的金黄色葡萄球菌作为实验菌株,分别在PRP凝胶及PRP等培养基中孵育,分别于2、4、6、8、12、24h取样计数菌落数,绘制时间-抑菌曲线图,计算细菌减少率。结果：研究发现全血和PRP中血小板浓度分别为(131.40±29.44)×109/L和(819.47±136.32)×109/L,比较差异有统计学意义(t=-27.020,P=0.000)；PRP中血小板回收率为60.85%±8.97%,富集系数为6.40±1.06。全血和PRP中白细胞浓度分别为(5.57±1.91)×1012/L和(32.20±10.42)×1012/L,比较差异有统计学意义(t=-13.780,P=0.000)；PRP中白细胞回收率为58.30%±19.24%,富集系数为6.10±1.93。PRP中血小板浓度和白细胞浓度分别与全血中血小板浓度(r=0.652,P=0.000)和白细胞浓度(r=0.460,P=0.011)成正相关。男性组和女性组PRP中血小板浓度和白细胞浓度比较差异均无统计学意义(P0.05)。PRP中PDGF、TGF-β、VEGF浓度分别为(698.15±64.48)、(681.36±65.90)(1071.55±106.04)ng/mL,是全血的(5.67±1.18)、(6.99±0.61)、(5.74±0.83)倍。PRP中PDGF浓度(r=0.832,P=0.020)、TGF-∞浓度(r=0.835,P=0.019)、VEGF浓度(r=0.824,P=0.023)均与PRP中血小板浓度成正相关。在关节软骨退变部分的临床研究中未发生关节内感染、肌肉萎缩、深静脉血栓、发热、血肿和异常组织增生等不良反应,与对照组相比试验组未表现出有更长的不良反应持续时间(P=0.862),所有出现不良反应的患者在得到合理治疗后都在4天内恢复到治疗前状态。试验组患者的评分在6个月内保持了稳定,而对照组只在前4个月内保持了稳定,在第6个月随访时就表现出明显的减低(PIDC=0.003, PWOMAC=0.048, PLequesne=0.002)。在前4个月内试验组患者的状态改善并不优于对照组(P0.05),但在随访到6月时IKDC评分和Lequesne指数的改变提示此时试验组患者膝关节状态优于对照组(P,KDC=0.000, PLequesne=0.013)。短期结果证实关节内注射PRP治疗关节软骨退行性病变是安全的,其长期疗效可能会优于透明质酸钠。在难愈性创面的研究中未发生创面感染加重、菌血症、局部异常组织增生等不良反应。治疗后第1、2、4周随访时创面覆盖率分别为(55.1±22.6)%、(72.0±16.2)%,(84.3±14.0)%,周间差异有统计学意义(P0.05),说明在治疗的4周内,创面处于持续修复状态。3个月随访时,创面体积为(1.5±1.8)mL,创面平均覆盖率为85.6%,治愈率为57.1%,治疗的有效率和患者主观满意率均为95.23%。体外抑菌结果显示PRP凝胶抗菌作用明显,细菌数量于孵育4 h时降至最低。结论：结合二次离心法使用PRP制备套装可以稳定地制备出富含高浓度血小板、白细胞和生长因子的PRP。关节内注射PRP为临床治疗膝关节退行性变提供了一种可能的选择,但是需要大样本的随机对照研究的长期随访结果以进一步证实其安全性和疗效。PRP不但能有效安全的促进慢性难愈性创面的愈合,并有潜在的抗感染作用。意义：通过对PRP细胞成分的检测、抗菌活性的测定等多个层面的研究,确定了制备PRP的最佳离心时间及离心力,为PRP在骨科的临床应用提供理论依据和现实指导。利用自体PRP来解决关节软骨损伤和难愈性创面修复的难题,无免疫排斥反应,制备简便,费用低廉,能有效地缩短治疗周期,避免了传统治疗方法效果差及花费高等缺点,具有良好的社会和经济效益,具有广阔的应用前景。
[Abstract]:Objective: To study the two step prepared by centrifugal PRP, through the analysis of PRP in platelet concentration, white blood cells and growth factors, the recovery rate was calculated and the enrichment coefficient, and correlation analysis, to explore the practicability and stability of the preparation methods of PRP. Clinical joint degeneration joint pain, limited mobility and joint deformities and other symptoms that is the result of patients with long-term pain and physical disability common cause, not only greatly affect people's quality of life, resulting in loss of human resources, but also brings heavy burden to family and society. The traditional method has many disadvantages and biological technology have made new progress today, PRP Technology provides a new idea and treatment our method for treatment of osteoarthritis by intra-articular injection. Methods, compare the PRP with sodium hyaluronate intra-articular injection of PRP, to explore the treatment of articular cartilage degeneration safety And the curative effect. For the treatment of chronic refractory wounds is a hotspot of medical research, traditional wound dressing, debridement, negative pressure vacuum and topical application of various drugs and other treatment, can remove local infection or necrosis, reduce ulcer secretions, but there are still many difficult to heal the wound, we adopt the method of local spraying of PRP the treatment of chronic refractory wounds, to explore the safety and efficacy of PRP in treating chronic wounds, and in vitro experiments to evaluate the anti infection effect. Methods: 30 cases of healthy adult volunteers (donor peripheral blood, 15 cases of male and female; the average age is 24-76 years old, 47 years old. The pre loaded with 5 mL citrate acid sodium injection 50 mL disposable syringe with 18 G needle in each volunteers antecubital vein blood 40 mL. after shaking, 1 mL blood cell analysis, 4 mL growth factor concentration determination; the remaining 40 mL injection of PRP preparation Set centrifugal tube, in strict aseptic conditions for preparation of PRP. according to the blood sedimentation coefficient of the components in the different centrifugal radius of 15 cm, 2000 r/min, 10 min after centrifugation, the blood is divided into 3 layers, the upper layer is lower for the supernatant, red blood cell, the two layer at the junction of a light yellow layer is PRP layer respectively. 1 mL anticoagulant venous blood and preparation of PRP using automatic blood analyzer for blood analysis, determination of platelet and leukocyte concentration, PRP in platelet and leukocyte concentration variation coefficient were calculated. The calculation of platelets or white blood cell recovery rate PRP and enrichment coefficient, the recovery rate of platelets or leukocytes in whole blood or / the total number of white blood cells in =PRP * 100%, platelets or leukocytes in whole blood concentration / or leukocyte concentration enrichment coefficient in =PRP were measured. The male, female volunteers PRP platelet and WBC concentration. The concentrations of growth factors measured on 4 mL Add 0.4 mL of thrombin and calcium chloride mixture of anticoagulant venous blood (adding 5 calcium chloride solution for 5% mL 2000 U in 3 min after thrombin), blood condenses into jelly, standing at room temperature for 1 h, 010 min, from the above method, supernatant was transferred to cryotubes. The preparation of PRP use the same method to prepare the supernatant rich in growth factors. Whole blood and PRP supernatant preparation, in accordance with the ELISA kit for determination of PDGF, TGF- and beta VEGF concentration. 30 cases of chronic joint pain or swelling (not less than April), X or MRI showed degeneration of articular cartilage of knee degenerative variable were included in the study. According to the patient's age, gender, body mass index and Kellgren Lawrence osteoarthritis grading of 30 patients were assigned to group PRP (experimental group) and sodium hyaluronate (Sodium hyaluronate, SH). 15 patients in experimental group (15 knees), each intra-articular injection of 3.5 m LPRP, a control group of 15 patients (15 knees), each intra-articular injection of sodium hyaluronate (Sodium hyaluronate SH) 2 mL. all patients were injected once every 3 weeks, 3 times for a course of treatment. The number of records related adverse events during the treatment time of occurrence, severity, duration time and processing results to assess the safety of the treatment. Compared before injection and 3,4,6 month follow-up international documents Committee (IKDC) subjective knee knee scoring scale, WOMAC evaluation, Lequesne index evaluation table to evaluate the clinical curative effect. The study included 21 cases of chronic wound patients will meet the selection criteria. 8 cases were male, 13 were female; the average age is 48-78 years old, 58.5 years old. The open wound debridement, in accordance with the 1:10 proportion will be prepared 2-8mL autologous PRP with coagulant (adding calcium chloride solution for 5% 5mL 2000U in thrombin) at the same time slowly into a spray And try to cover all the wounds after.3min wound surface to form a layer of PRP gel, taking the sterile transparent film of suitable size to cover the wound, to ensure no loss of seal around PRP, suggesting that patients should reduce the wound near the joint activity within 1 weeks after surgery. After surgery can replace a sterile dressing, generally about 1 weeks to see the wound avoid touching the affected area. The patients with hand wound, number according to the decision PRP use. Determination of wound volume and calculate the wound coverage by infusion of sterile saline, we will be within 3 months of the wound healing is divided into the following five grades: Grade I completely healed. The coverage rate of 100%; grade II significantly healing coverage between 75%-100%; III effective healing, coverage of 50%-75%; Part IV effective healing, coverage of 25%-50%; V invalid healing, coverage is lower than 25%. for 3 months After not healed patients treated observation PRP treatment again or continue to seek their consent. At the end of observation before treatment and at follow-up in March were cultured within the wound. The wound secretions of bacteria selected the most common infection of Staphylococcus aureus as the experimental strains were incubated in medium PRP gel PRP and 2,4,6,8,12,24h, respectively in the sample count the number of colonies, rendering time antibacterial curve, calculation bacteria reduction rate. Results: the study found that PRP in whole blood and platelet concentrations were (131.40 + 29.44) * 109/L and (819.47 + 136.32) * 109/L, the difference was statistically significant (t=-27.020, P=0.000); for 60.85% + 8.97% recovery rate of platelet PRP, white blood cell concentration enrichment factor was 6.40 + 1.06. blood and PRP were (5.57 + 1.91) * 1012/L and (32.20 + 10.42) * 1012/L, the difference was statistically significant (t=-13 .780, P=0.000); 58.30% + 19.24% white blood cell recovery rate PRP, enrichment coefficient was 6.10 + 1.93.PRP in platelet and leukocyte concentration and concentration respectively in whole blood platelet concentration (r=0.652, P=0.000) and leukocyte concentration (r=0.460, P=0.011) were positively correlated. Platelet concentration and leukocyte concentration difference between male group and women in the PRP group had no statistical significance (P0.05) in.PRP PDGF, TGF- beta, VEGF concentrations were (698.15 + 64.48), (681.36 + 65.90) (1071.55 + 106.04) ng/mL, the blood is (5.67 + 1.18), (6.99 + 0.61), (5.74 + 0.83) PDGF concentration times.PRP (r=0.832, P=0.020), TGF- (r=0.835, P=0.019) - concentration, the concentration of VEGF (r=0.824, P=0.023) were positively correlated with PRP concentration in platelet. No joint infection, muscle atrophy and degeneration of articular cartilage in the clinical research part, deep vein thrombosis, fever, adverse reactions and abnormal tissue hematoma hyperplasia, Compared with the control group, experimental group showed no adverse reaction with longer duration (P=0.862), all patients with adverse reactions to recover before treatment in a reasonable state after treatment in 4 days. In the test group the score remained stable in 6 months, while the control group only in the first 4 months remained stable, in sixth months follow-up showed significantly decreased (PIDC=0.003, PWOMAC=0.048, PLequesne=0.002). In the first 4 months of test group improvement was better than the control group (P0.05), but in the follow-up to June when the IKDC score and Lequesne index change it in the test group the knee joint state is better than the control group (P, KDC=0.000, PLequesne=0.013). The short-term results confirmed that intra-articular injection of PRP for the treatment of articular cartilage degeneration is safe, may the long-term efficacy is superior to sodium hyaluronate. In the study of more difficult wounds No wound infection occurred aggravating, bacteremia, adverse reactions of local abnormal tissue hyperplasia. Treatment after 1,2,4 weeks wound coverage rates were (55.1 + 22.6)% and (72 + 16.2)% and (84.3 + 14)%, there was statistical significance difference between the week (P0.05), in the treatment of within 4 weeks, continue to repair wounds in a state of.3 months of follow-up, the wound volume (1.5 + 1.8) mL, the average wound coverage rate was 85.6%, the cure rate was 57.1%, the treatment efficiency and subjective satisfaction rate of patients was 95.23%. in vitro showed significant antibacterial effect of PRP gel, the number of bacteria in the at 4 h of incubation to a minimum. Conclusion: the combination of two centrifugal method using PRP preparation kit can be stably prepared with high concentration of platelets, white blood cells and growth factor PRP. intra-articular injection of PRP provides a possible choice for the clinical treatment of knee joint degeneration, but requires a large sample Long term follow-up results of randomized studies to further confirm the safety and efficacy of.PRP security can not only effectively promote the healing of chronic wounds, and anti infection effect. The potential significance: through the detection of PRP cell components, research activity determination and many other aspects of antibacterial, the optimum centrifugal time the centrifugal force and the preparation of PRP PRP, to provide a theoretical basis and practical guidance in the clinical application of Department of orthopedics. Using autologous PRP to solve the problem of repairing articular cartilage injury and wound healing, no immune rejection, simple preparation, low cost, can effectively shorten the treatment cycle, to avoid the effect of traditional treatment method and the cost higher shortcomings, it has good social and economic benefits, and has broad application prospects.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R684

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