兩種血小板濃縮物修復(fù)兔硬腭軟組織缺損的形態(tài)學(xué)觀察

何婷婷,王 拓,陳紅亮,孫 勇

·論著·

兩種血小板濃縮物修復(fù)兔硬腭軟組織缺損的形態(tài)學(xué)觀察

何婷婷,王 拓,陳紅亮,孫 勇

目的對(duì)比觀察兩種血小板濃縮物修復(fù)兔硬腭軟組織缺損的形態(tài)學(xué)效果,評(píng)價(jià)其臨床應(yīng)用價(jià)值。方法將54只日本大耳白兔隨機(jī)分為A、B、C 3組,于兔硬腭前部中份制備軟組織缺損模型,缺損區(qū)分別植入自體改良型富血小板纖維蛋白(A-PRF)膜(A組)、自體富血小板纖維蛋白(PRF)膜(B組)或不做任何處理(C組),分別于術(shù)后 3、7、11、15、21、28 d行形態(tài)學(xué)觀察,計(jì)算創(chuàng)面愈合率。結(jié)果(1)形態(tài)學(xué)觀察：術(shù)后 7 d內(nèi),A組和B組創(chuàng)面炎性反應(yīng)輕微,A組更優(yōu),C組炎癥反應(yīng)明顯。術(shù)后15 d,A組和B組無顯著差異,創(chuàng)面黏膜上皮覆蓋完整,均無組織凹陷,無瘢痕形成;C組在愈合過程中出現(xiàn)了不同程度的組織凹陷,有瘢痕組織形成。(2)創(chuàng)面愈合率：術(shù)后 3 d,3組無明顯差異(P>0.05);術(shù)后 7、11 d,A組與B組之間無明顯差異(P>0.05),A組和B組均明顯高于C組(P<0.05)。結(jié)論APRF、PRF均能減輕創(chuàng)傷區(qū)域的炎癥反應(yīng),減少瘢痕形成,加速創(chuàng)面愈合。

血小板濃縮物;軟組織缺損;形態(tài)學(xué)觀察;創(chuàng)面愈合率

軟組織缺損的重建是口腔種植臨床上經(jīng)常面對(duì)的難題。外科手段、生物學(xué)和組織工程材料等常被應(yīng)用于口腔臨床軟組織重建中[1-3］。外科手段一般需開辟第二術(shù)區(qū),增加患者痛苦。有價(jià)值的生物細(xì)胞及生長(zhǎng)因子等則需要在嚴(yán)格的無菌培養(yǎng)環(huán)境中進(jìn)行較長(zhǎng)時(shí)間的培養(yǎng)才能獲得。人體血小板濃縮物(platelet concentration,PC)中含有大量能促進(jìn)骨和軟組織再生的生長(zhǎng)因子[4］,且可快速有效地獲得組織重建細(xì)胞,避免使用昂貴的外源性生長(zhǎng)因子,免去復(fù)雜手術(shù)的痛苦,亦更安全可靠。

Choukroun等[5］發(fā)現(xiàn)并制備的第二代血小板濃縮物-富血小板纖維蛋白(platelet-rich fibrin,PRF),臨床和動(dòng)物實(shí)驗(yàn)證實(shí)其具有促進(jìn)軟組織再生的作用[6-8］。Ghanaati等[9］于2014年提出了一種與PRF離心速度和時(shí)間不同的改良型富血小板纖維蛋白(advanced platelet-rich fibrin,A-PRF),具有比PRF總量更高、更豐富的生長(zhǎng)因子。但A-PRF較之PRF是否具有更好的促進(jìn)口腔軟組織再生的能力,尚待大量臨床和動(dòng)物實(shí)驗(yàn)證實(shí)。本研究在兔硬腭區(qū)制備軟組織缺損模型,對(duì)比觀察兩者促進(jìn)軟組織愈合的效果,為其臨床應(yīng)用提供實(shí)驗(yàn)依據(jù)。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物 54只雄性3月齡健康日本大耳白兔,2.0～2.5 kg,由四川省成都達(dá)碩實(shí)驗(yàn)動(dòng)物中心提供及飼養(yǎng)管理,生產(chǎn)許可證號(hào)：SYXK(川)2014～189。所有動(dòng)物均先在同一條件下適應(yīng)性分籠喂養(yǎng)1 w。實(shí)驗(yàn)過程均按照動(dòng)物倫理學(xué)要求進(jìn)行各項(xiàng)處置。

1.2 實(shí)驗(yàn)材料 PRF真空無菌采血管(5 ml),APRF真空無菌采血管(10 ml)(江蘇創(chuàng)英醫(yī)療器械有限公司);TDZ4-WS低速離心機(jī)(中國(guó)湘儀集團(tuán));纖維蛋白成型處理工具盒(法國(guó)Process,Nice);Surgic XT Plus種植機(jī)(日本NSK);Ti-SG20L種植手機(jī)(日本NSK);5.0 mm組織環(huán)切鉆(MCT,韓國(guó));普通透明薄膜等。

1.3 方法 54只日本大耳白兔隨機(jī)分為A、B、C 3組,每組18只。隨機(jī)選取實(shí)驗(yàn)動(dòng)物,稱重后固定,使用A-PRF/PRF專用離心管通過兔耳中央動(dòng)脈采集4 ml血液,迅速離心：A-PRF(2000 r/min,24 min)、PRF(3250 r/min,10 min)。離心后靜置3～5 min,棄去上清液,用無菌有齒鑷取出中間層凝膠,修剪并保留其下部少許紅細(xì)胞層后,置于專用工具盒中壓制成膜備用。

按1 ml/kg沿兔耳緣靜脈緩慢推注3%戊巴比妥鈉,麻醉起效后,將其以仰臥位固定于手術(shù)臺(tái),常規(guī)消毒鋪巾;于兔硬腭前部中份制備直徑5 mm軟組織缺損(環(huán)切鉆中心點(diǎn)位于距上頜后排門齒4.5 mm的硬腭中線處),剝離黏骨膜露出骨面,充分止血。分別于A、B兩組動(dòng)物軟組織缺損處填入按缺損大小修剪后的A-PRF膜或PRF膜,縫合固定。C組軟組織缺損經(jīng)充分止血后,不做任何處理。

術(shù)后連續(xù)3 d肌注青霉素鈉(10萬(wàn)U/kg,1次/d),食軟食1 w。口內(nèi)縫線任其自然脫落。

1.4 觀察指標(biāo) 分別于術(shù)后3、7、11、15、21、28 d行大體形態(tài)觀察。使用透明薄膜描記創(chuàng)面大小后,修剪成創(chuàng)面大小并稱重,以薄膜的重量來反映創(chuàng)面面積。創(chuàng)面愈合率=(原始創(chuàng)面面積-未愈合創(chuàng)面面積)/原始創(chuàng)面面積[10］。

1.5 統(tǒng)計(jì)學(xué)方法 應(yīng)用 SPSS 23.0統(tǒng)計(jì)軟件分析,計(jì)量資料以±s表示,采用單因素方差分析,P< 0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 形態(tài)學(xué)觀察 術(shù)后3 d,A、B兩組縫線均未脫落,A-PRF膜及PRF膜均穩(wěn)固附著于創(chuàng)面,且有少量吸收,創(chuàng)面輕度紅腫;C組創(chuàng)緣紅腫明顯,可見新鮮肉芽組織,觸之點(diǎn)狀出血。術(shù)后 7 d,A、B兩組肉眼見膜基本吸收,創(chuàng)面縫線大部分自行脫落,A組創(chuàng)面無明顯炎癥及紅腫反應(yīng),B組尚見輕微紅腫,兩組愈合面積均達(dá)原創(chuàng)面面積的1/2;C組創(chuàng)面紅腫仍明顯,呈凹陷缺損。術(shù)后11 d,A、B組愈合面積達(dá)原創(chuàng)面積的 4/5;C組創(chuàng)面基底也可見黏膜上皮覆蓋,仍可見明顯組織凹陷。術(shù)后15 d,A、B組創(chuàng)面上皮覆蓋基本完成,質(zhì)地較軟,色澤欠紅潤(rùn),未見明顯瘢痕組織;C組創(chuàng)面仍呈凹陷狀,創(chuàng)面邊緣有瘢痕形成。術(shù)后21 d,A、B兩組創(chuàng)面黏膜色澤、質(zhì)地均與正常黏膜無明顯差異,且A組更優(yōu);C組仍存少量組織凹陷,瘢痕組織明顯。術(shù)后28 d,C組見明顯瘢痕組織,未見組織凹陷。

2.2 創(chuàng)面愈合率 在術(shù)后3 d,各組創(chuàng)面愈合率無統(tǒng)計(jì)學(xué)差異(P>0.05);術(shù)后 7、11 d,A組與B組的創(chuàng)面愈合率無顯著差異,但均較C組更高(P< 0.05)。在術(shù)后15、21、28 d,A組及B組創(chuàng)面愈合率為100%。在各時(shí)間節(jié)點(diǎn),A組創(chuàng)面愈合率均最高,B組次之,C最低。見表1。

表1 各組不同時(shí)間點(diǎn)創(chuàng)面愈合率的比較(n=18)

3 討論

創(chuàng)傷后的組織愈合大致經(jīng)歷止血期、炎性反應(yīng)期、肉芽形成期、組織重塑期4個(gè)階段[10］,主要包括機(jī)體抵御炎性反應(yīng)、血管新生、結(jié)締纖維組織形成幾個(gè)方面。當(dāng)損傷發(fā)生后,血小板迅速在創(chuàng)面局部聚集形成血凝塊,同時(shí),血小板被活化后脫顆粒釋放各種生長(zhǎng)因子,這些生長(zhǎng)因子能夠促進(jìn)大量中性粒細(xì)胞、單核/巨噬細(xì)胞在創(chuàng)傷區(qū)聚集浸潤(rùn),釋放大量炎性介質(zhì), 如白細(xì)胞介素(1L-1、1L-4、1L-6)、TNF-α等,吞噬殺滅細(xì)菌、清除創(chuàng)面的壞死組織及碎屑,發(fā)揮著抗炎、抗感染的免疫作用[12］。各種生長(zhǎng)因子與纖維蛋白結(jié)合可促進(jìn)與軟組織愈合相關(guān)的環(huán)節(jié),成纖維細(xì)胞不斷成熟,并繼續(xù)增殖分化,在相關(guān)生子因子作用下不斷合成膠原纖維,損傷區(qū)被結(jié)締纖維組織、新生血管及上皮組織等組織覆蓋,從而具有一定強(qiáng)度,最終形成穩(wěn)定的瘢痕[13-14］?？傊?血小板及其釋放的各種生長(zhǎng)因子在創(chuàng)傷愈合各階段都很重要[15］。

PRF凝膠中富含全血中約97%的血小板及＞50%的白細(xì)胞,具有與血凝塊結(jié)構(gòu)類似的疏松三維立體纖維蛋白網(wǎng)狀結(jié)構(gòu),能夠?yàn)榧?xì)胞的遷移、附著及增殖分化提供支架,血小板及白細(xì)胞如黏結(jié)劑一樣嵌入其中,各種細(xì)胞因子被滯留于其中緩慢節(jié)律性釋放[12,16］。A-PRF與PRF制備的離心速度及時(shí)間不同,由＞97%的血小板及少量血細(xì)胞組成,呈疏松三維立體網(wǎng)狀結(jié)構(gòu),具有比PRF總量更高的血小板和白細(xì)胞[9］。Eizaburo等將PRP、PRF和A-PRF行體外溫育培養(yǎng),評(píng)估15 min、60 min、8 h、1 d、3 d、10 d時(shí)生長(zhǎng)因子的釋放情況,采用ELISA定量分析PDGF-AA、PDGF-AA、PDGF-AB、PDGF-BB、TGFB1、VEGF、EGF、IGF等生長(zhǎng)因子的釋放量,結(jié)果3種血小板濃縮物中釋放最多的生長(zhǎng)因子依次是PDGF-AA、PDGF-BB、TGFB1、VEGF和PDGF-AB,溫育15～60 min,PRP釋放的生長(zhǎng)因子顯著高于PRF和A-PRF,60 min后至第10 d,A-PRF釋放的總生長(zhǎng)因子及血清總蛋白量最高,可長(zhǎng)期穩(wěn)定高效地釋放生長(zhǎng)因子[4］。

本實(shí)驗(yàn)形態(tài)學(xué)觀察顯示,在創(chuàng)傷愈合早期(術(shù)后7 d內(nèi)),C組的炎性反應(yīng)明顯較A組及B組嚴(yán)重,而A組與B組相比,前者紅腫炎癥程度相對(duì)較輕,說明APRF與PRF均有一定的抗炎能力,但在創(chuàng)傷愈合早期(術(shù)后7 d內(nèi)),A-PRF抗炎效果更好。而在創(chuàng)傷愈合中后期,APRF與PRF抗炎效果相當(dāng),這與二者的生長(zhǎng)因子釋放特點(diǎn)基本吻合。

本實(shí)驗(yàn)僅通過形態(tài)學(xué)肉眼觀察創(chuàng)面愈合情況,分析各組的創(chuàng)面愈合率,初步評(píng)估創(chuàng)面愈合質(zhì)量,尚待后續(xù)通過HE染色、Masson染色、CD105免疫組化染色等對(duì)各組的炎性反應(yīng)、新生血管化、膠原的合成與降解進(jìn)行量化比較,進(jìn)一步評(píng)估創(chuàng)面愈合質(zhì)量。

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Morphological observation on two kinds of PC in repair of rabbit hard palate soft tissue defects

He Tingting1,2,Wang Tuo3,Chen Hongliang2,Sun Yong1,21.Medical College of Stomatology,Southwest Medical University,Luzhou, Sichuan,646000,China;2.Department of Stomatology,the Institutional Hospital of Chengdu Military Command,Chengdu,Sichuan, 610011,China;3.Department of Stomatology,Affiliated Hospital of North Sichuan Medical College,Nanchong,Sichuan,637000, China

ObjectiveTo compare the morphological observation of two kinds of platelet concentration (PC)in repair of rabbit hard palate soft tissue defects and evaluate the clinical application value.MethodsA total of 54 Japanese big ear white rabbits were selected and randomly divided into groups A,B and C.Soft tissue defect models were made in rabbit hard palate.A-PRF membrane and PRF membrane were implanted into the defects in groups A and B respectively.The defects in group C were not treated.The morphology was observed three,seven,11,15,21,28 days after the treatment to calculate the wound healing rate.Results(1) Morphological observation:seven days after the operation,there was minor wound inflammatory response in groups A and B.Group A was better.Inflammatory response in group C was obvious.15 days after the operation,there was no significant difference between groups A and B.The mucosal epithelial cover was complete without tissue depression or cicatrization,and there was tissue depression at varying degrees and cicatrization in the process of healing in group C.(2)Wound healing rate:three days after the operation,there was no significant difference among the three groups(P>0.05).Seven and 11 days after the operation,there was no significant difference between groups A and B(P>0.05),and the wound healing rate in groups A and B was much higher than that in group C (P<0.05).ConclusionBoth APRF and PRF can relieve the inflammatory response in wound area,reduce scar formation,and accelerate the wound healing.

PC;soft tissue defect;morphological observation;wound healing rate

R 782.22

A

1004-0188(2016)11-1225-03doi:10.3969/j.issn.1004-0188.2016.11.001

2016-06-30)

成都軍區(qū)“十二五”科研面上項(xiàng)目(C14050)

646000四川瀘州,西南醫(yī)科大學(xué)口腔醫(yī)學(xué)院(何婷婷,孫 勇);成都軍區(qū)機(jī)關(guān)醫(yī)院口腔科(何婷婷,陳紅亮,孫 勇);川北醫(yī)學(xué)院附屬醫(yī)院口腔科(王 拓)

孫 勇,電話：028-86687041