富血小板血浆，浸渍的缝合材料的高，低血小板浓度相比，能够提高大鼠结肠吻合口愈合的伤口

Abstract

Objective。This study was designed to investigate the impact of using suture material impregnated with platelet-rich plasma (PRP) in different platelet concentrations on colonic anastomotic wound healing in rats.Methods。A total of 24 Sprague Dawley female rats were separated into 3 groups ( for each) including the control group (CON; standard vicryl suture repair), the low platelet concentrate PRP group (L-PRP; suture material impregnated with PRP containing average 2.7-fold (range, 2.0 to 3.1) higher amount of platelets vs. control), and the high platelet concentrate PRP group (H-PRP; suture material impregnated with PRP containing average 5.1-fold (range, 4.8 to 5.4) higher amount of platelets vs. control). Rats were sacrificed on the postoperative 7^thday for analysis of colonic anastomosis region including macroscopic observation, measurement of anastomotic bursting pressure (ABP), and the hydroxyproline levels and histopathological findings in colon tissue samples.Results。总损伤评分在L-PRP和H-PRP组比对照组中显著降低（中位数（范围）13.00（7.00）和11.50（6.00）15.50对比（4.00）， 和 ,分别)。ABP值（180.00（49.00）124.00对比（62.00）和121.00（57.00）mmHg时， for each) and tissue hydroxyproline levels (0.56 (0.37) vs. 0.25 (0.17) and 0.39 (0.10)μg/mg组织， 和 ,respectively) were significantly higher in the L-PRP group as compared with those in the control and H-PRP groups.结论。In conclusion, our findings revealed PRP application to colonic anastomosis sutures to promote the anastomotic healing process. The platelet concentration of PRP seems to have a significant impact on the outcome with superior efficacy of L-PRP over H-PRP in terms of bursting pressures and collagen concentration at the anastomotic site.

1. Introduction

Despite marked advances in preoperative management and suture techniques and materials, gastrointestinal anastomotic leakage or dehiscence remains a common complication in colorectal surgery being associated with an increased risk of perioperative morbidity and mortality [1–4]。This led to a continuing search for innovative methods or technical modifications to avoid anastomotic leakage [5,6]。

众所周知，富含血小板血浆（PRP）等血小板浓缩物作为一种天然的纤维蛋白凝块，通过输送大量生长因子来调节细胞增殖、基质重塑和修复过程，如血小板衍生生长因子（PDGF）、转化生长因子-b1（TGF-b1）和血管，从而促进伤口愈合内皮生长因子[7–11]。

In this regard, generation of a synergistic effect involving a multitude of growth factors, with specific impact on the cascade of wound healing, has become the main rationale for use of PRP to improve wound healing [6]。PRP应用胃肠anastomosis is considered a useful method to provide the surgical site with growth factors to facilitate wound healing [5]。然而，肠吻合的愈合局部使用PRP的影响已经在大鼠模型数量有限，其提供的结果有争议[解决5,6,12–14]。

采用导致不同的血小板浓度不同的PRP制备方法是建议在研究[之间的这场争论中的潜在作用6]。The opposite impact of low PRP (stimulatory effect) and high PRP (inhibitory effect) concentrates on intestinal wound healing has also been reported in a recent rat model study [6]。

Identification of anastomotic collagen deposition via tissue hydroxyproline levels and of anastomotic strength via anastomotic bursting pressure (ABP) measurement is considered to be the most reliable indicators of anastomotic wound healing and outcome of gastrointestinal anastomoses [5,15,16]。

This study was therefore designed to evaluate the impact of using suture repair augmented with different PRP concentrations on anastomotic wound healing in a rat model of colonic anastomosis based on bursting pressures, tissue hydroxyproline levels, and histopathological examination.

2. Methods

2.1. Animals and Study Protocol

A total of 24 Sprague Dawley female rats (weighing 260-310 g) were kept in a light- and temperature-controlled room with a 12 hr light-dark cycle, temperature of 21°C, and relative humidity of 40-60%. The animals were fed standard rat pellets and provided with water ad libitum. This study was carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, while the study protocol was approved by the Institutional Animal Care and Use Committee (approval number: 2014/14).

将大鼠分成3组（ for each) including the control group (CON; standard vicryl suture repair), the low platelet concentrate PRP group (L-PRP; suture material impregnated with PRP containing average 2.7-fold (range, 2.0 to 3.1) higher amount of platelets vs. control blood), and the high platelet concentrate PRP group (H-PRP; suture material impregnated with PRP containing average 5.1-fold (range, 4.8 to 5.4) higher amount of platelets vs. control blood).

2.2. Preparation of PRP and Impregnated Sutures

八个供体老鼠用于获得PRP。在time of surgery, 8.5 mL of intracardiac homologous blood was drawn from each of the eight rats. The blood was aspirated into 10 mL GLO-PRP (Biotrend Medical, Istanbul, Turkey) tubes containing 1.5 mL of acid-citrate-dextrose (ACD) and transferred into a centrifugation chamber and centrifuged using a GT 416 centrifuge device (Glotech Inc., Glofinn, Korea) at 1200 × g, 20°C for 5 min. Of the 3 different layers obtained after centrifugation, erythrocytes at the bottom were removed through the RBC cap, while the remaining layers of buffy coat and acellular plasma on the top were mixed and placed into a second chamber for a second spin for 10 min at 1200 × g to create 0.5 mL H-PRP at the bottom, 1 mL L-PRP in the middle, and platelet poor plasma (PPP) at the top. Platelet concentrations were determined via a Sysmex T1800i (Sysmex Cor., Tokyo, Japan) hemogram device. When necessary, platelet concentrates were adjusted with the addition of minimum amounts of PPP. In this way, L-PRP and H-PRP were obtained containing average 2.7-fold (2.0-3.1) and 5.1-fold (4.8-5.1) higher platelet concentrates, respectively, as compared with blood samples used to prepare PRP. Average platelet concentration in blood sample was μL (range, 0.545 to μL) and white blood cell count (WBC) μL (range, 8.1 to μL), whereas platelet concentration was μL (range, 1.44 to μL) and WBC count μL (range, 0.9 to μL) in the L-PRP group and μL (range, 2.78 to μL) and WBC count μL（范围1.3至 μL) in the H-PRP group.

4/0 vicryl sutures were kept in sterile containers involving liquid forms of L-PRP or H-PRP for 3 minutes based on findings from a preliminary timeline analysis of weight increase per minute in PRP-impregnated sutures which revealed maximum saturation (from the baseline value of 0.0640 g to the maximum value of 0.1284 g) to be reached at the 3^rdminute (Figure1(a)).

Afterwards, for preanalysis to determine the platelet content absorbed by the sutures, the amount of PRP was measured inμ把薇乔缝合成PRP容器，并发现缝合吸收0.7之前大号 μL PRP within 3 minutes. The PRP-impregnated vicryl sutures were placed in an empty container and added with 2.8 μL distilled water which enabled the release of entire PRP content after a 3 min waiting period, while the fluid was analyzed in the same hemogram device to confirm the target platelet concentrations. Values obtained from the hemogram device were calculated by taking dilution rate into account. Average platelet concentration in blood sample was μL, whereas in platelet concentrations in the fluid obtained from L-PRP- and H-PRP-impregnated vicryl sutures were μL (2.3-fold) and μL (4.7-fold), respectively, indicating sufficient amount of platelet absorption in the vicryl sutures.

2.3. Surgery

After overnight fasting, the rats were anesthetized by intraperitoneal injection of ketamine 35 mg/kg (Ketalar; Parke Davis, Eczacibasi, Istanbul, Turkey) and xylazine 5 mg/kg (Rompun; Bayer AG, Leverkusen, Germany). The same surgeon performed all operations. The abdominal skin of the rat was shaved, and a 3 cm midline incision was made under aseptic conditions. The left colon was cut into two 3-4 cm over the peritoneal reflection. A colocolonic single-layer end-to-end anastomosis was performed with standard 4/0 and 45 mm length vicryl suture (polyglactin 910) (Ethicon Inc., NJ, USA) in the control group, whereas with L-PRP-impregnated and H-PRP-impregnated 4/0 vicryl suture in the L-PRP and H-PRP groups, respectively (Figure1(b)).

Standard length sutures were used for each anastomosis. Midline closure was performed using interrupted 3/0 silk sutures (Ethicon Inc.). No analgesic or antibiotic was administered in the postoperative period, and oral intake was started on the 1^stpostoperative day in each group of rats. After operation, the animals were fed standard rat pellets and provided with water ad libitum.

2.4。ABP测量

Rats were sacrificed on the postoperative 7^th经腹腔注射2 mL大剂量戊巴比妥钠（200 mg/mL，KU life，哥本哈根，丹麦）。安乐死和腹部切口重新开放后，评估腹腔是否存在吻合口漏或裂开、腹膜炎、脓肿、吻合口部位或其他内脏粘连。结肠被仔细地外部化，并确定了吻合口的位置。仔细切除中间有吻合口的节段，用等渗盐水冲洗，去除粪便。一根18号硅胶导管穿过两端，通过3/0丝线连接。使用输液泵（Argus Medical AG，Heimberg，Switzerland）进行腔内亚甲基蓝等渗溶液灌注（5 m/min），同时通过附在另一端导管上的传感器（Beneview T5，中国深圳）监测和记录腔内压力（图1(c)和1(d)). The pressure recorded just before the leak was considered to be the ABP. After measurement of ABP, half of the colon segment containing the anastomosis line was used for histopathological analysis and the other half for hydroxyproline analysis.

2.5. Macroscopic Examination and Histopathological Analysis

Tissue samples involving the colon anastomosis line were fixed in 10% buffered formalin for 48-72 hours and then trimmed and processed for routine histopathological examination. Tissue samples perpendicular to the direction of anastomosis line were embedded in paraffin for serial sectioning. 4 μm sections were stained with hematoxylin and eosin (HE) and examined under a light microscope by the same pathologist who was unaware of the experimental groups. Semiquantitative scoring of histopathological parameters (necrosis, PMN cells, MN cells, edema, mucosal epithelium, submucosal/mucosal layer, and granulation tissue; each scored from 0 to 3) was performed using the Verhofstad wound healing scale [17]。Lower and higher scores were considered to indicate good and worse healing, respectively, based on the Verhofstad injury scoring system (Table1).

2.6条。羟脯氨酸测定

The hydroxyproline level in the tissue was measured colorimetrically with the Hydroxyproline Test Kit (Elabscience, E-BC-K061, Houston, Texas, USA). The principle of measurement was based on the purplish red color occurring upon the reaction of dimethylaminobenzaldehyde with the oxidation product under the effect of oxidizer. The content of hydroxyproline was calculated by measuring the OD value at 550 nm.

2.7。统计分析

统计分析了使用IBM SPSS Statistics for Windows, version 25.0 software (IBM Corp., Armonk, NY, USA). The Kruskal-Wallis test with post hoc Tamhane’s test was used to analyze differences in platelet and wound healing parameters between the study groups. Data were expressed as median (range). was considered statistically significant. Power of the study was calculated to be 0.99 (alpha 0.05), considering a mean (SD) 4 (0.5) unit difference in mean score ( , )between more than two groups ( ).

3. Results

3.1条。一般特征

除了对照组中一只大鼠，其死在术后3^rd一天，所有的老鼠都活了下来。对结肠吻合口的宏观评估显示，大鼠没有腹腔内脓肿或渗漏。研究组之间的基线体重和术后体重没有显著差异。各组从基线检查到术后体重略有下降（表1).

3.2. Histopathological Findings on Wound Healing

Total injury scores were significantly lower in the L-PRP and H-PRP groups than those in the control group (13.00 (7.00) and 11.50 (6.00) vs. 15.50 (4.00), 和 ,分别)。Specifically, edema score was significantly lower in the L-PRP and H-PRP groups than that in the control group (1.00 (0.00) and 0.00 (0.00) vs. 2.00 (1.00), 和 ,分别)。Mucosal epithelium scores were significantly lower in the H-PRP group than those in the L-PRP and control groups (1.00 (2.00) vs. 2.50 (1.00) and 3.00 (1.00), for each). Granulation tissue scores were significantly lower in the L-PRP and H-PRP groups as compared with those in the control group (2.00 (1.00) and 2.00 (1.00) vs. 3.00 (1.00), for each) (Figure2).

3.3. ABP Values

中位数（范围）ABP values were significantly higher in the L-PRP group as compared with those in the control and H-PRP groups (180.00 (49.00) vs. 124.00 (62.00) and 121.00 (57.00) mmHg, for each), and although ABP values were slightly higher in the H-PRP group compared to the control group, the difference was not statistically significant (124.00 (62.00) vs 121.00 (57.00) mmHg) (Table2).

3.4. Tissue Hydroxyproline Levels

中位数（范围）tissue hydroxyproline levels were significantly higher in the L-PRP group as compared with those in the control and H-PRP groups (0.56 (0.37) vs. 0.25 (0.17) and 0.39 (0.10)μg/mg组织， 和 ,分别)。意味着(SD)组织羟脯氨酸列弗els in the H-PRP group were also significantly higher than levels in the control group ( )（表2).

4. Discussion

我们在大鼠结肠端对端吻合模型中的发现支持PRP的吻合口愈合作用，同时表明PRP的疗效可能因所用血小板浓度的不同而发生变化。经L-PRP浸渍的缝合线和经H-PRP浸渍的缝合线在组织病理学评估上的总损伤分数较低，尽管与使用标准缝合线相比，经L-PRP和H-PRP处理的缝合线在与伤口愈合相关的总损伤分数方面没有显著差异。然而，与H-PRP组和对照组相比，L-PRP组的ABP值和组织羟脯氨酸水平显著升高。H-PRP组ABP值略高于对照组，但差异无统计学意义。另一方面，H-PRP组组织羟脯氨酸水平显著高于对照组。因此，在本研究中，L-PRP而不是H-PRP似乎与改善吻合口愈合相关，评估的总体标准包括吻合口强度和完整性（ABP）、组织胶原（羟脯氨酸）和组织再生（损伤评分）。

同样地，在对三种不同浓度的PRP的影响进行研究过去包括L-PRP（ /mm³), H-PRP (platelet count /mm³), and platelet-poor plasma (PPP) on the intestinal anastomotic healing process in rats, a significant increase, decrease, and no change from control values were noted in ABP and hydroxyproline levels in the L-PRP, H-PRP, and PPP groups, respectively [6]。作者表示,likelihood of L-PRP to promote anastomotic wound healing, whereas the association of H-PRP with adverse effects leading to inhibition of the healing process [6]。

同样，在过去一项关于不同PRP浓度对成骨细胞和成纤维细胞（FBs）细胞增殖的影响的研究中，据报道，血小板浓度为2.5x（约为最大浓度的一半）可达到最大效果，而较高浓度可导致细胞增殖减少[18]。在另一项研究调查上的FB不同血小板浓度的效果，8.8％，17.5％，和35％的最终的血小板浓度中，作者报告与8.8％和17.5％的制剂，得到的是优异的增殖与35相比％ 浓度 [19]。The authors also emphasized the association of fibroblast proliferation with maintenance of acid environment and thus improved wound healing [19]。In a study by Vahabi et al., the effects of PRP at concentrations of 10, 25, 50, and 75% activated or not activated with calcium gluconate on human gingival fibroblasts (HGFs), and it was reported that the rate of proliferation decreased in both groups as the concentrations increased. In the same study, although the proliferation rate was higher in the activated PRP group, the difference was not statistically significant [20]。PRP used in our study was not activated.

Unlike these studies, Arpornmaeklong et al. found that proliferation was increased as the concentration increased when rat osteoblastic bone marrow cells were cultured with PRP at different concentrations [21]。

同样，Kawasumi等人。报道了大鼠骨髓细胞与含1.2倍、3.5倍和10.6倍血小板浓度的PRP培养时，含10.6倍血小板浓度的PRP对大鼠骨髓细胞增殖的影响^ND, 4^th, and 6^thdays [22]。

In a study by Yoshida et al., the effects of PRPs containing platelets at 1x concentration same as the blood sample and 3x and 5x folds higher concentrations on in vitro anterior crural ligament cells in terms of proliferation, metabolism, and production of type 1 and type 3 procollagen were examined, and it was found that PRP at 1x concentration provided higher cellular metabolism, lower cellular apoptosis, and increased gene expression for collagen that are among the important factors in wound healing. The authors argued that difference results between their study and those reporting increased proliferation as concentrations increase might be resulted from cell types [23]. 成骨细胞和成纤维细胞在氧气、营养和外周血管系统方面生活在不同的环境中。骨损伤通常发生在血管化良好的床内，ACL损伤通常发生在没有血管化的滑膜介质中。因此，细胞可以对不同的血小板浓度产生反应。

Association of topical PRP application to anastomosis line with better wound healing has also been reported in other studies of rat colon anastomosis models, based on increased ABP values and higher tissue hydroxyproline levels accompanied with histopathological findings of decreased inflammatory cell infiltration, marked fibroblast development, and rich collagen production identified in the PRP vs. the control group of rats [12,14]。Similarly, Ocak et al. reported that PRP administration to intestinal anastomosis in rats that underwent hyperthermic intraperitoneal chemotherapy (HIPEC) decreased inflammatory response, increased anastomotic bursting pressure, and increased hydroxyproline levels [24]。

Notably, in contrast to topical gel application reported previously, liquid form of PRP was used in our study to impregnate vicryl sutures for the first time in the literature, which is a 3 min process versus a 45 min waiting period needed for topical gel application. In a study by Daradka et al. including anastomosis applied in rabbit bowel with a suture material similar to that we used, the suture was first treated with 70% ethanol, kept in PRP containing /microL platelets gelled with sodium acetate for 30 minutes to provide covering of the suture; the suture was then dried in the room air and used in the anastomosis. In that study, when the suture covered with PRP gel was compared with uncovered suture or the suture covered only with sodium citrate, a significant increase was found in tissue hydroxyproline levels and anastomotic bursting pressure [25]。Although the results of that study were consistent with our results, in our technique, much shorter time is needed to cover the suture and the effectiveness of PRP at different concentrations was compared.

The advantageous biological effects of PRP on bone regeneration was also reported with a platelet concentration of approximately 1,000,000/μL, whereas suboptimal efficacy with lower concentrations and paradoxically inhibitory effect with higher concentrations [26]。因此，我们的研究结果支持PRP对大鼠吻合愈合的增加ABP和组织羟脯氨酸水平方面的血小板浓度依赖性的影响，用L-PRP过H-PRP的卓越的功效，而强调较温和的，而不是一个在愈合过程中H-PRP的抑制作用。

The PRP preparation technique of the current study revealed PRP concentrates that approximate the appropriate increase over the blood baseline [13,27], including an increase by 3.7-fold in L-PRP and by 10-fold in H-PRP groups over the average platelet concentrations in the control group. Therefore, higher efficacy of L-PRP vs. H-PRP in improved colonic anastomotic healing in our study seems in accordance with the association of PRP concentrations of a 2.5-fold increase over the original platelet concentration with optimal efficacy with a decrease in efficacy for PRP concentrations of 4.2- to 5.5-fold increases over the original platelet concentration [18]。

ABP is considered to be a reliable marker of early postoperative anastomotic mechanical strength, particularly within the first postoperative week [16,28]。It is considered to reflect not only the intestinal physiologic strain but also the indirect collagen formation related to collagen deposition and lysis [5,6,29]。Therefore, an association of using L-PRP-impregnated sutures for colonic anastomosis with increased ABP values in our study seems important given that ABP is considered not only a composite measure of anastomotic wound healing but also a potential indicator of growing anastomotic strength and thus the outcome of gastrointestinal anastomoses [6,12,16]。

Moreover, as a surrogate of collagen deposition at the anastomosis site with low levels considered to negatively affect the wound healing [12,16,30]在本研究中，L-PRP组的组织羟脯氨酸水平也显著高于H-PRP组和对照组。

In addition, lack of significant difference between study groups in terms of body weight reduction during the postoperative period in our study also seems notable given the association of body weight reduction with impaired wound healing [6,14]。

4.1. Study Limitations

这项研究有一定的局限性。首先，使用同源PRP。我们不得不使用动物供体，因为血液产生PRP量不小动物足够的如老鼠。从同源血液产生的PRP是可能产生一个免疫反应，并给faşse结果。然而，H-PRP组中，特别是在L-PRP组获得的阳性结果可以排除了这种可能性。尽管如此，为了避免这种情况，我们推荐使用由能在今后的研究中获得的自体PRP较大的动物。

Second, given that postoperative days 3 or 4 of gastrointestinal anastomosis have been associated with the lowest value of anastomotic mechanical strength and thus the highest risk of anastomotic leakage [31], L-PRP seems to prevent the risk of anastomotic leak by enabling an increased anastomotic strength starting from the earliest period of inflammatory process, possibly with acceleration of the stimulation of fibroblasts and collagen formation via platelet-derived growth factors [6]。Nonetheless, it should be noted that in the clinical practice, anastomotic leak is a multifactorial phenomenon that is quite difficult to ascribe to a single factor or intervention and most leaks in actual practice occur in the 3- to 5-day period after surgery, while in the current study the rats were assessed rather late (postoperative day 7) for the healing process. Hence, our findings should be interpreted to the extent of the differences observed, within the limitations of an experimental animal study.

Third, in our study, we focused on describing an easier and different method of PRP containing platelets at different concentrations for intestinal anastomosis, which can be performed in a much shorter time in clinical practice. Further studies are needed to investigate effects of platelet-derived growth factors on anastomotic healing.

Finally, we preferred to use the sutures in the control group without subjecting it to any treatment and this caused us to have knowledge about the control group despite the use of blind manner in ABP measurement, histopathologic evaluation, and hydroxyproline level measurement. It may be possible to impregnate the suture with PPR during surgical process in a blinded manner also in control groups.

5个。结论

总之，我们的研究结果表明使用PRP-浸渍结肠吻合缝合材料，以促进吻合口愈合的过程，同时用在吻合部位破裂压力和胶原浓度方面L-PRP过H-PRP的优良功效。在对照，随机和前瞻性临床研究是合理的，这强调了在预防吻合口瘘手术后的高危期L-PRP的潜在效用，从而提高伤口愈合的胃肠吻合的更好的结果的实现。

Data Availability

Data used in the study are included in the manuscript.

Conflicts of Interest

The authors declare no conflict of interest regarding this study.

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版权© 2020 Mehmet Akif Aydin et al. This is an open access article distributed under theCreative Commons Attribution License，它允许在任何媒体中不受限制地使用、分发和复制，前提是正确引用了原始作品。