Comparison and selective preference of preparation methods of platelet-rich gel

doi:10.3969/j.issn.2095-4344.2014.03.024

Abstract

Abstract:

BACKGROUND: Platelet-rich plasma gel contains some kinds of growth factors, which play important roles in repair of local trauma and bone defects. Platelet-rich plasma gel is now more and more studied as tissue engineering scaffolds, but there are no unified preparation methods.
OBJECTIVE: To summarize the studies about the preparation methods of platelet-rich plasma gel and the growth factors in platelet-rich plasma gel in recent years, and to compare different methods in order to find which one is better.
METHODS: A online search of PubMed database from January 1980 to January 2013 and China National Knowledge Infrastructure from January 1980 to January 2013 was performed with the key words of “platelet-rich plasma, platelet-rich plasma gel, tissue engineering” in English and Chinese, respectively. The studies about detection and preparation methods of platelet-rich plasma were included and those unrelated to platelet-rich plasma were excluded. Finally, 322 articles were gained and 67 articles were included in result analysis.
RESULTS AND CONCLUSION: All preparation methods have their advantages. We should select the suitable method according to the goal of study. With the further study of platelet-rich plasma, it will become more and more important for bone defect repair and tissue engineering research.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

全文链接：

Key words: biocompatible materials, blood platelets, gels, review

CLC Number:

R318

Kang Jian, Yuan Wen. Comparison and selective preference of preparation methods of platelet-rich gel[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(3): 476-481.

Figures/Tables 1

2.1 富血小板血浆中生长因子成分及作用富血小板血浆中含有丰富的血小板，其血小板浓度可达普通血浆血小板浓度的3-7倍[3]。在富血小板血浆与激活剂混合激活后，其中的血小板通过脱颗粒作用，大量释放血管内皮生长因子、血小板衍生生长因子、胰岛素样生长因子、转化生长因子β1和转化生长因子β2等多种生长因子[1]，具有促进多种组织损伤修复的作用。其中血小板衍生生长因子和转化生长因子β在骨缺损修复中的作用最为重要[4-5]。 2.1.1 血小板衍生生长因子血小板衍生生长因子是首先在血小板中发现的一种促细胞分裂因子，是一种耐热、耐酸、易被胰蛋白酶水解的阳离子多肽，由两条高度同源的A链及B链组成，A链和B链有56%的同源序列[6]。血小板衍生生长因子具有3种形式的二聚体结构，即血小板衍生生长因子AA，血小板衍生生长因子BB及血小板衍生生长因子AB。血小板衍生生长因子主要作用是促进有丝分裂，能使静止状态的G0、G1期细胞转变为具有分化潜能的细胞，加速间叶起源细胞的DNA合成，具有促进间充质细胞如成骨细胞、成纤维细胞等进行有丝分裂的潜能，使间充质细胞分化与生长，提高成骨细胞合成胶原蛋白的能力[7]，还可以刺激内皮细胞的生长促进损伤区毛细血管增生，增加巨噬细胞的活化，并通过增加破骨细胞数量来加速其骨吸收作用[8]，加快骨塑形重建，因此其对软组织及骨组织修复都具有重要作用[9-13]。 2.1.2 转化生长因子转化生长因子β属于一组调节细胞生长和分化的转化生长因子β超家族，由两个亚基组成，目前在哺乳动物体内发现的主要有转化生长因子β1、转化生长因子β2、转化生长因子β3、转化生长因子β1β2四个亚型。体内多个组织都可合成，但骨组织及血小板是其主要来源[14]。转化生长因子β对间充质起源的细胞起刺激作用，而对上皮或神经外胚层来源的细胞起抑制作用。其可促进成纤维细胞、成骨细胞和许旺细胞的生长，刺激成骨前体细胞增殖，直接促进骨胶原蛋白合成。同时还可诱导成骨细胞增殖分化及破骨细胞凋亡，减少骨吸收，促进骨折创伤缺损的修复[15-19]；并可促进细胞外基质如胶原蛋白、纤粘连蛋白的表达和抑制细胞外基质的降解，对细胞的形态发生、增殖和分化过程起着重要作用，有利于胚胎发育和细胞修复。 2.1.3 胰岛素样生长因子胰岛素样生长因子是生长激素产生生理作用过程中必须的一种含有两条链的活性蛋白多肽物质。现在已知的包括胰岛素样生长因子1和胰岛素样生长因子2两种。胰岛素样生长因子可明显地促进多种来源不同软骨细胞的分裂增殖并提高其合成软骨基质和Ⅱ型胶原的能力，提高糖胺聚酶及成骨细胞中碱性磷酸酶的活性[20]，从而促进骨的基质合成，抑制骨骼的分解代谢，防止骨骼中钙的流失，维持骨骼的正常结构和功能。同时胰岛素样生长因子还是人体内非常重要的细胞有丝分裂促进剂，并能调节成纤维细胞的增殖，促进软组织缺损修复[21]。 2.1.4 血管内皮生长因子血管内皮生长因子是由2个相同亚基以二硫键交联结合成的二聚体糖蛋白，血管内皮生长因子121、血管内皮生长因子165和血管内皮生长因子189三种亚型在人类表达；是一种强烈的促血管生长因子，在伤口愈合和血管化过程中起关键作用[22]，是血管形成早期的重要促进因子[23]。血管内皮生长因子可与血管内皮细胞表面相应受体结合，刺激血管内皮细胞增殖，并可诱导心血管形成并能增加血管尤其是微小血管的通透性，为损伤部位细胞修复及新生毛细血管网提供营养。此外，血管内皮生长因子可直接作用于成骨细胞，促进成骨细胞迁移、增殖和分化，并可增强碱性磷酸酶活性，使局部钙盐沉积，促进骨折创伤的修复[24]。 2.2 富血小板血浆的制备方法富血小板血浆的制备方法较多，通常分为手工制备法和全自动制备法。手工制备法是通过手工操作，利用离心机等仪器设备获取富血小板血浆的一种方法，容易开展，但操作繁琐，样本污染风险高；全自动制备法是利用血小板分离机通过离心将血细胞和血浆分离，再将红细胞、白细胞和血浆回输体内，得到血小板的浓缩物[25]。 2.2.1 手工制备法离心法：手工制备富血小板血浆时，根据离心次数、离心时间和离心力的不同，主要有以下5种常用方法：①Anitua法[26]：以160 ×g的力离心6 min，试管里的全血分为3层，上层是上清液，下层是红细胞，两层交界处可见一薄浅黄层，即为富血小板血浆层。吸取上清液至交界面上3 mm弃掉，再吸取全部上清直至交界面下约3 mm，轻摇即得到富血小板血浆，其血小板的平均浓度约为433 000/mm3。②Sonnleitner法[27]：离心两次，第1次以160×g离心20 min后，吸取全部上清弃掉，再吸取薄层血小板层和交界面6 mm以上成分转入另一支离心管，再以400×g离心15 min，移除交界面上成分，剩余成分振荡混匀，既得富血小板血浆。其血小板浓度甚至超过2 000 000/mm3，达正常血小板浓度的近10倍。③Petrungaro法[28]：采用2次离心，第1次1 500×g离心6 min后，吸取全部上清液至交界面下3 mm，移至另一离心管，平衡后再次1 000×g离心6 min。离心管中液体分为两层，上层上清液为贫血小板血浆，下层为血小板浓缩物。吸取约3/4上清液弃掉，剩余成分振荡摇匀，即为富血小板血浆。④Landersberg法[29]：先以200×g离心10 min，吸取全部上清液至交界面下3 mm，移至另一离心管，平衡后以200×g再次离心10 min；离心管中液体分为2层，上层上清液为贫血小板血浆，下层为血小板浓缩物，吸取约3/4上清液弃掉，剩余振荡混匀即为富血小板血浆。Landersberg认为离心力超过250×g易导致过多血小板破坏，此法得到的富血小板血浆血小板浓度平均可达到 687 000/mm3。⑤Aghaloo法[30]：同样两次离心且成分转移方式同Landersberg法，第1次以215×g的力离心10 min，第2次以863×g的力离心10 min得到富血小板血浆，其血小板浓度为1 050 000/mm3。白细胞层法：白细胞层法又称白膜层法，首先将新鲜全血在6 h内离心后分离出白膜层(富血小板血浆层)，再将白膜层轻摇混匀后再次低离心力离心，既可得富血小板血浆[31]。与传统离心法相比，白细胞层法得到的富血小板血浆中白细胞含量低，血小板损伤降低，反应血小板激活程度的血小板膜表面CD62p表达率明显降低[32]。同时可降低血小板糖分解率，提高氧代谢率，维持二氧化碳及pH值得稳定[3-34]。 2.2.2 全自动制备法全自动制备法是使用血细胞分离仪器等专业设备离心分离，从而获取富血小板血浆的一种方法，同时红细胞、白细胞和血浆还可输回供血者体内。目前主要有SmatPReP、PCCS、PRGF Kit、Trissee、Clinaseal Sealed Technology Centrifuge、ACE Surgical、Curasan、Platelet concerntrate collection等[35]，其中SmatPReP和PCCS已经获得了FDA的认可。对于各系统来说，其获得的富血小板血浆中血小板浓度不尽相同。Marx[36]报道PCCS获得的血小板浓度平均值为939 000/mm3，Curasan系统为 344 000/mm3，SmartPReP系统为1 086 000/mm3；Weibrich等[37]报道PCCS获得血小板浓度最高可达 2 232 500/mm3，Curasan系统最高可达到 1 140 500/mm3。通常认为以PCCS和SmartPReP系统得到的富血小板血浆血小板和生长因子浓度最高，并且血小板破坏最少。但此类方法操作技术要求较高，设备昂贵，多为血液制品相关机构使用，且制备的血小板不易保存，总体成本较高，因此目前并未在临床广泛应用。 2.3 富血小板凝胶的制备方法将富血小板血浆激活凝固，形成凝胶样物，即为富血小板凝胶。获取富血小板血浆凝胶的主要方法有两种：一种是向富血小板血浆中加入一定量的激活剂，富血小板血浆可在30 min内凝聚成凝胶状，从而获得富血小板血浆凝胶。另一种方法是利用不同的离心次数和离心力将未加入抗凝剂的新鲜全血进行离心，以此改变富血小板血浆的体外凝固时间和凝缩率，从而获得富血小板血浆凝胶。富血小板血浆中生长因子浓度及比例与全血中相似[38]，而加入激活剂后富血小板血浆凝胶中生长因子浓度可达到全血中的3-7倍[3]。 2.3.1 加入激活剂法获得富血小板血浆凝胶将富血小板血浆与不同比例的激活剂混匀，静置30 min，即可获得富血小板凝胶。目前大多数学者所用的激活剂主要为牛凝血酶和氯化钙的混合物(1 000 U牛凝血酶： 1 mL 10%氯化钙溶液)[39]。部分学者认为由于牛凝血酶的使用过程中通常会形成凝血因子V 、XI抗体，因此存在发生致命性凝血紊乱的风险[40-41]，但Marx[36]认为制备富血小板血浆凝胶过程中所用牛凝血酶剂量小，且未进入循环系统，与组织接触之前就已固化在富血小板血浆凝胶内，且可被巨噬细胞吞噬清除，因此不会引起免疫系统紊乱。由于在牛凝血酶激活剂使用中存在理论上的不确定性，因此寻找一种新型的激活剂成为当前富血小板血浆凝胶激活剂研究领域的重要方向，获取对机体安全性更高的激活剂。Shen等[42]研究认为壳聚糖刺激血小板并使血小板激活后同样可以释放生长因子，可作为牛凝血酶的替代激活剂。Landersberg等[43]研究显示固定化单宁酸酶胶凝剂可替代牛凝血酶作为富血小板血浆的激活剂，且凝胶释放的血小板衍生生长因子和转化生长因子β量与牛凝血酶相似。Landersberg等[44]使用凝血酶受体激动剂替代牛凝血酶，结果显示凝血酶受体激动剂组凝胶聚合最慢，牛凝血酶组聚合最快。凝血酶受体激动剂组血块凝缩率为27%，牛凝血酶组则高达59%。而凝胶释放的血小板衍生生长因子和转化生长因子β量与牛凝血酶相似。因此与牛凝血酶相比，凝血酶受体激动剂在激活富血小板血浆时比较安全，可以提供充足的工作时间，血块凝缩比例较小。但加入牛凝血酶激活剂时，由于凝血酶浓度和凝血酶与氯化钙溶液配比的不同，激活后所得的富血小板血浆凝胶中各种生长因子的浓度也各不相同，同时富血小板血浆凝胶形成后，其中的各种生长因子浓度随时间变化规律也与激活剂密切相关，因此针对不同的实验和临床用途，需要对激活剂的浓度和配比，以及富血小板血浆凝胶凝固后的使用时间进行一定的控制，这些因素也使其在临床中尚未得到广泛应用，需要进一步的试验及临床研究。 2.3.2 离心法获得富血小板血浆凝胶离心法是通过改变对新鲜全血的离心次数和离心力大小，从而改变富血小板血浆凝胶的体外凝固时间和血块凝缩率，从而获得所需富血小板血浆凝胶的一种新的制备方法。在Dohan等[45]的实验中使用不抗凝的试管取代抗凝试管，采取静脉血后立即以400×g低速离心10 min，由于没有抗凝剂，离心过程中血小板即被活化、纤维蛋白聚集，离心后静置3-5 min后，离心管内成分分为3层：上层为血浆层，下层为红细胞层，中间层即为富血小板纤维凝块，将中间层取出压在2块纱布之间即可形成凝胶膜状的富血小板血浆凝胶。Dohan等[45]认为此法获得的富血小板血浆凝胶内部纤维蛋白可将血小板和生长因子以化学键的方式结合起来并缓慢释放，延长生长因子作用时间。此种方法不需要利用复杂的步骤制备富血小板血浆后再进行激活，制备过程操作简便，成本消耗低廉，且制备过程中并无抗凝剂及激活剂等化学试剂的加入，避免了因加入化学试剂后对后续实验所带来的不确定因素及对人体安全的潜在风险，因此被越来越多的实验及临床工作人员采用，在一些国家更是作为富血小板血浆凝胶制备的主要方法被广泛采用。但此方法制备的富血小板血浆凝胶中各种生长因子浓度的变化及峰值时间尚无具体的实验研究形成统一意见，在此方面仍需科学严密的实验研究提供可靠数据。 2.4 富血小板凝胶在临床中的应用传统的自体骨、异体骨及生物工程材料修复骨缺损存在供骨区缺损、术中神经损伤、异体骨的免疫排斥反应等多种缺点，生物工程材料则存在价格昂贵、异物排斥反应等问题，因此寻找一种廉价、高效、不良反应小的骨缺损修补材料越来越受到重视。根据部分学者的早期动物试验显示[46-49]，富血小板血浆凝胶应用在骨及软组织缺损修复中可获得的良好效果。目前富血小板血浆凝胶临床应用主要集中在口腔颌面外科及整形外科中的骨缺损修复[50-57]。此外，由于富血小板血浆凝胶中含有多种高浓度的生长因子，能够有效促进软组织的修复及创伤愈合，因此也被一些学者用于软组织缺损的修复[58-61]。但是也有一些学者认为富血小板血浆凝胶对于骨缺损修复并无明显促进作用。Pryor等[62]试验显示，富血小板血浆凝胶对骨缺损修复并无明显促进作用。Aghaloo等[63]在研究中认为富血小板血浆凝胶对比自体骨移植在成骨率上并无统计学差异。Choi[64]和Butterfield等[65]的研究表明，富血小板血浆凝胶复合材料在修复骨缺损方面并不优于自体骨移植。Arpornmaeklong[66]和Ranly等[67]的研究结果显示，富血小板血浆凝胶对骨缺损修复过程存在抑制作用。因此，富血小板血浆凝胶在正式临床应用之前，还需大量的试验及循证医学研究来确定其安全性及可行性。"

References

[1]Amy DB,May JR,Helene S.SPARC-null mice exhibit accelerated cutaneous wound closure.J Histochem Cytochem. 2002;50(1):1-10．

[2]Whitman DH,Berry RL,Green DM.Platelet gel: an autologous alternative to fibrin glue with applications in oral and maxillofacial surgery.J Oral Maxillofac Surg.1997;55(11): 1294-129．

[3]Doucet C,Ernou I,Zhang Y,et al.Platelet lysates promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-based therapy applications.J Cell Physiol. 2005;205:228-236.

[4]Celotti F,Colciago A,Negri-Cesi P,et al.Effect of platelet-rich plasma on migration and proliferation of SaOS-2 osteoblasts: role of platelet-derived growth factor and transforming growth factor-beta.Wound Repair Regen.2006; 14(2):195-202．

[5]Tonelli P,Mannelli D,Brancato L,et al.Counting of platelet derived growth factor and transforming growth factor-beta in platelet-rich-plasma used in jaw bone regeneration.Minerva Stomatol.2005;54:23-34.

[6]Betsholtz C,Johnsson A,Heldin CH,et al.cDNA sequence and chromosomal localization of the human plateletderived growth factor A-chain and it expression in tumor cell lines.Nature. 1986;320:695-699.

[7]Bouletreau PJ,Warren SM,Spector JA,et al.Factors in the fracture microenvironment induceprimary osteoblast angiogenic cytokine production. Plast Reconstr Surg. 2002; 110(1):139-148.

[8]Horner A,Bord S,Kemp P,et al.Distribution of platelet-derived growth factor(PDGF)A chain mRNA，protein,and PDGF-alpha receptor inrapidly forming human bone.Bone.1996;19: 353-362.

[9]Antoniades HN. Human platelet-derived growth factor (PDGF): purification of PDGF-I and PDGF-II and separation of their reduced subunits.Proc Natl Acad Sci USA.1981;78 (12):7314-7317.

[10]Marx RE,Carlson ER,Eichstaedt RM,et al.Platelet-rich plasma: Growthfactor enhancement for bone grafts.Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 1998;85:638-646.

[11]Howes R,Bowness JM,Grotendorst GR,et al.Platelet-derived growth factor enhances demineralized bone matrix-induced cartilage and bone formation. Calcif Tissue Int.1988;42:34-38.

[12]Nash TJ,Howlett CR,Martin C,et al.Effect of platelet-derived growth factor on tibial osteotomies in rabbits.Bone.1994;15(2): 203-208.

[13]Vikjaer D,Blom S,Horting-Hansen E,et al.Effect of PDGF on bone formation in calvarial defects. An experimental study in rabbits.Eur J Oral Sci.1997; 105:59-66.

[14]Assoian RK,Komoriya A,Meyers CA,et al.Transforming growth factor-beta in human platelets. Identification of a major storage site, purification and characterization.J Biol Chem. 1983;258:7155-7160.

[15]Centrella M,McCarthy T,Canalis E.Transforming growth factors beta is a bifunctional regulator of replication and collagen synthesis in osteoblast enriched cell cultures from fetal rat bone.J Biol Chem.1987;262:2869-2874.

[16]Carlson ER.Bone grafting the jaws in the 21st century：the use ofplatelet-richplasma and bone morphogenetic protein. Alpha Omegan.2000;93(3):26-30．

[17]Roberts AB,Sporn MB.Physiological actions and clinical applications of transforming growth factor-B (TGF-B).Growth Factors.1993;8:1-9.

[18]Carano RA,Filvaroff EH. Angiogenesis and bone repair. Drug Discov Today. 2003;8(21):980-989.

[19]Kanno T,Takahashi T,Tsujisawa T,et al.Platelet-rich plasma enhances human osteoblast-like cellproliferation and differentiation.J OralMaxillofac Surg. 2005;63(3): 362-369.

[20]Trippel SB.Potential role of insulin-like growth factors in fracture healing. Clin OrthopRelat Res.1998;355(Suppl): 301-313.

[21]Galiano RD,Zhao LL,Clemmons DR,et al.Interaction between the insulin-like growth factor family and the integrin receptor family in tissue repair processes. Evidence in arabbit ear dermal ulcer model.J Clin Invest.1996;98:2462-2468.

[22]Freymiller EG,Agbaloo TL.Platelet-rich plasma:ready or not?J Oral Maxillofac Surg.2004;62(4):484-488.

[23]段小军,杨柳,李起鸿.骨再生与血管生成协同作用的分子机制[J].临床骨科杂志,2005,8(1):88-91.

[24]MayrWohlfart U,Waltenberger J,Hausser H,et al.Vascular endothelial growthfactor stimulates chemotacticmigration of primary human osteoblasts. Bone. 2002;30(3):472-477.

[25]David M,Dohan E,Lars R,et al.Classification of platelet concentrates from pure platelet-rich plasma(P-PRP)to leucocyte and platelet-rich fibrin(L-PRF). Trends Biotechnol. 2009;27(3):158-167.

[26]Anitua E.Plasma rich in growth factors: preliminary results of use in the preparation of future sites for implants.Int J Oral Maxillofac Implants.1999;14: 529-535.

[27]Sonnleitner D,Huemer P,Sullivan DY.A simplified technique for producing platelet-rich plasma and platelet concentrate for intraoral bone grafting techniques: a technical note. Int J Oral Maxillofac Implants.2000;15: 879-882.

[28]Petrungaro PS.Using platelet-rich plasma to accelerate soft tissue maturation in esthetic periodonatal surgery.Compend Contin Educ Dent.2001; 22(9):729-32,734,736passim ; quiz 746.

[29]Landesberg R,Roy M,Glickman RS,et al.Quantification of Growth Factor Levels Using a Simplifed Method of Platelet-Rich Plasma Gel Preparation.J Oral Maxillofac Suig.2000;58:297-300.

[30]Aghaloo TL,Moy PK,Freymiller EG.Investigation of platelet-rich plasma in rabbit cranial defects: A pilot study.J Oral Maxillofac Surg.2002;60:1176-1181.

[31]陈龙菊,程亚媛,刘义明,等.汇集滤白细胞血小板的研制及临床应用[J].广东医学院学报,2009,27(3):242-244.

[32]Bertolini F,Rebulla P,Porretti L,et al.Platelet qualityafter 152 day storage of Platelet concent rates prepared from pooled buffy coats and stored in a glucose free crystalloid medium.Transfusion.1992;32(1):9-16.

[33]Fijnheer R,Veldman HA,Van den Eertwegh AJ,et al.In vitro evaluation of buffy coat derived platelet concent rates stored in a synthetic medium.Vox Sang. 1991;60(1):16-22.

[34]Eriksson L,Hogman CF.Platelet concent rates stored in an additive solution prepared from pooled buffy coats.Vox Sang. 1990;59(3):140-145.

[35]赵耀,瞿文亮.富血小板血浆促进骨再生与修复的影响因素研究进展[J].中国修复重建外科杂志,2010,24(8):1004-1008.

[36]Marx RE. Platelet-Rich Plasma: Evidence to Support Its Use. J Oral Maxillofac Surg.2004;62(4):489-496.

[37]Weibrich G,Kleis WK,Hafner G.Growth factor levels in the platelet-rich plasma produced by 2 different methods: curasan-type PRP kit versus PCCS PRP system.Int J Oral Maxillofac Implants.2002;17:184-190.

[38]Okuda K,Kawase T,Momose M,et al.Platelet-rich Plasma contains high levels ofPlateletderivedgrowthfactor and transforming growth factor-betaand modulates theProliferation ofperiodontallyrelated cells in vitro.J Periodontol.2003; 74(6):849-857.

[39]Jakse N,Tangl S,Chili R,et al.Influence of PRP on autogenous sinus graft. An experimental study in sheep.Clin Oral Implant Res.2003;14(5):578-583.

[40]Landesberg R,Moses M,Karpatkin M.Risks of using platelet-rich plasma gel. J Oral Maxillofac Surg.1998;56(9): 1116-1117.

[41]Carano RA,Filvaroff EH.Angiogenesis and bone repair.Drug Discov Today.2003;8(21):980-989.

[42]Shen EC,Chou TC,Gau CH,et al.Releasing growth factors from activated human platelets after chitosan stimulation: a possible bio-material for platelet-rich plasma preparation.Clin Oral Implants Res.2006;17(5):572-578.

[43]Landesberg R,Roy M,Glickman RS.Quantification of growth factor levels using a simplified method of platelet-rich plasma gel preparation.J Oral Maxillofac Surg.2000;58(3):297-300.

[44]Landesberg R,Burke A,Pinsky D,et al.Activation of platelet-rich plasma using thrombin receptor agonist peptide.J Oral Maxillofac Surg.2005;63(4):529-535.

[45]Dohan DM,Choukroun J,Diss A,et al.Platelet-rich fibrin (PRF): a second-generation platelet concentrate.Part I: technological concepts and evolution . Oral Surg Oral Med Oral Pathol Oral Radiol Endod.2006;101(3):37-44.

[46]Fennis JP,Stoelinga PJ,Jansen JA.Mandibular reconstruction: a histologicaland histomorphometric studyon the use of autogenous scaffolds, particulate cortico-cancellous bonegrafts andplateletrich plasma in goats.Int J Oral Maxillofac Surg.2004;33:48-55.

[47]Suba Z,Takacs D,Gyulai-Gaal S,et al.Facilitation of beta-tricalcium phosphate-induced alveolar boneregeneration by platelet-rich plasma inbeagle dogs: a histologic and histomorphometric study.Int J Oral Maxillofac Implants. 2004; 19:832-838.

[48]Thorwarth M,Rupprecht S,Falk S,et al.Expression of bone matrix proteins duringde novo bone formationusinga bovinecollagen andplatelet-rich plasma (prp)-an immunohistochemical analysis. Biomaterials.2005; 26:2575-2584.

[49]Hokugo A,Ozeki M,Kawakami O,et al.Augmented bone regenerationactivityof platelet-rich plasma by biodegradable gelatin hydrogel.Tissue Eng.2005; 11:1224-1233.

[50]Papli R,Chen S.Surgical treatment of infrabony defects with autologous platelet concentrate or bioabsorbable barrier membrane: A prospective case series.J Periodontol. 2007; 78:185-193.

[51]Dori F,Huszar T,Nikolidakis D,et al.Effect of platelet-rich plasma on the healing of intra-bony defects treated with a natural bone mineral and a collagen membrane. J Clin Periodontol.2007;34:254-261.

[52]Yassibag-Berkman Z,Tuncer O,Subasioglu T,et al.Combined use of platelet-rich plasma and bone grafting with or without guided tissue regeneration in the treatment of anterior interproximal defects.J Periodontol.2007;78: 801-809.

[53]Ilgenli T,Dundar N,Kal BI.Demineralized freeze-dried bone allograft and platelet-rich plasma vs platelet-rich plasma alone in infrabony defects: a clinical andradiographic evaluation.Clin Oral Investig.2007;11:51-59.

[54]Demir B,Sengun D,Berberoglu A.Clinical evaluation of platelet-rich plasma and bioactive glass in the treatment of intra-bony defects.J Clin Periodontol. 2007;34:709-715.

[55]Keles GC,Cetinkaya BO,Albayrak D,et al.Comparison of platelet pellet and bioactive glass in periodontal regenerative therapy. Acta Odontol Scand.2006;64: 327-333.

[56]Christgau M,Moder D,Wagner J,et al.Influence of autologous platelet concentrate on healing in intra-bony defects following guided tissue regeneration therapy: A randomized prospective clinical split-mouth study.J Clin Periodontol. 2006; 33:908-921.

[57]Czuryszkiewicz-Cyrana J, Banach J. Autogenous bone and platelet-rich plasma (PRP) in the treatment of intrabony defects.Adv Med Sci.2006; 51(suppl 1): 26-30.

[58]Mannai C. Early implant loading in severely resorbed maxilla using xenograft, autograft, and platelet-rich plasma in 97 patients. J Oral Maxillofac Surg.2006; 64:1420-1426.

[59]Hartwig D,Harloff S,Liu L,et al.Epitheliotrophic capacity of a growth factor preparation produced from platelet concentrates on corneal epithelial cells: a potential agent for the treatment of ocular surface defects? Transfusion.2004; 44:1724-1731.

[60]Crovetti G,Martinelli G,Issi M,et al.Platelet gel for healing cutaneous chronic wounds.Transfus Apher Sci.2004;30(2): 145-151.

[61]Caloprisco G,Borean A.Chronic skin ulcers: A regenerative simulation by topical hemotherapy.Int J Artif Organs. 2004;27(9):816-817.

[62]Pryor ME,Yang J,Polimeni G,et al.Analysis of rat calvaria defects implanted with a platelet-rich plasma preparation: radiographic observations.J Periodontol.2005;76(8): 1287-1292.

[63]Aghaloo TL,Moy PK,Freymiller EG.Investigation of platelet-rich plasmain rabbit cranial defects : A pilot study.J Oral Maxillofac Surg.2002; 60(10):1176-1181.

[64]Choi BH,Im CJ,Huh JY,et al.Effect ofplatelet-rieh plasma on boneregeneration in autogenous bone graft.J Oral Maxillofac Surg.2004;33(1):56-59.

[65]Butterfield KJ, Bennett J,Oronowicz G,et al.Effect of platelet-rich plasma with autogenous bone graft for maxillarysinus augmentation in a rabbit model. J Oral Maxillofac Surg.2005;63(3):370-376.

[66]Arpommaeklong P,Kochel M,Depprich K,et al.Influence of platelet-rich plasma (PRP) on osteogenic differentiation of rat bone marrow stromal cells. An in vitro study.Int J Oral Maxillofac Surg.2004;33:60-70.

[67]Ranly DM,Lohmann CH,Andreacchio D,et al.Platelet-rich plasma inhibits demineralized bone matrix-induced bone formation in nude mice.J Bone Joint Surg Am.2007;89(1): 139-147.