PRP对人皮肤成纤维细胞增殖、体外合成胶原与透明质酸的影响

目的研究不同浓度的富血小板血浆(PRP)对人皮肤成纤维细胞(HSFs)增殖、体外合成胶原与透明质酸的影响。方法原代培养人皮肤成纤维细胞,用含不同浓度PRP(1%、5%、10%、20%、30%、50%)的培养液进行细胞培养,无血清培养液为阴性对照,含10%新生小牛血清的培养液为阳性对照。培养第3、5、7天使用四甲基偶氮唑盐(MTT)法测定细胞增殖率,应用RT-PCR测定胶原蛋白表达情况,ELISA测定透明质酸功能变化。结果 MTT检测发现所有不同浓度的PRP均有明显促进HSFs增殖的作用,与阴性对照组均有明显差异(P<0.01),且随着时间递增,增殖效果也明显增强;RT-PCR测定50%PRP浓度组及阴性、阳性对照组中目的基因COL1及COL3均显著表达,但组间差异不明显;ELISA测定实验组透明质酸功能均增强,与阴性对照组有明显差异(P<0.01),PRP浓度在30%～50%时表达效果明显增强。结论PRP对HSFs的增殖、合成胶原与透明质酸的功能有明显促进效果。     

富血小板血浆对青山羊骨髓间充质干细胞增殖分化的影响

目的观察富血小板血浆（platelet-rich plasma,PRP）对体外培养的中国青山羊骨髓间充质干细胞（marrow mesenchymal stem cells,MSCs）增殖和成骨分化的影响,探讨PRP促进骨修复的细胞学机制。方法将体外培养第3代中国青山羊MSCs,分为对照组（单纯血清培养组）和PRP组（加入10%PRP复合培养）,通过相差显微镜观察细胞生长情况,于2、4、6、8d采用MTT法检测细胞增殖活性。将体外培养的第3代细胞分为对照组、地塞米松组（dexamethasone,DEX）组、PRP组,于培养第7天行碱性磷酸酶（alkaline phosphatase,ALP）、第18天行矿化结节染色检测细胞成骨分化活性。结果相差显微镜下见PRP组细胞最先达到汇合,MTT法显示PRP组2、4、6、8d的平均吸光度（A）值分别为0.252±0.026、0.747±0.042、1.173±0.067、1.242±0.056明显高于对照组（A值分别为0.137±0.019、0.436±0.052、0.939±0.036、1.105±0.070）（P〈0.01）。与对照组比较,PRP组ALP阳性细胞数增多,矿盐沉积减少;DEX组ALP阳性细胞数减少,矿化结节形成明显增多。结论PRP能明显促进中国青山羊MSCs增殖,抑制MSCs向成骨细胞分化。     

富血小板血浆诱导脂肪干细胞成骨作用的实验研究

目的探讨在体外培养中富血小板血浆（PRP）对脂肪干细胞的增殖及诱导成骨的影响。方法从Wistar大鼠自体动脉血中提取PRP，配制成条件培养液，并作用于培养状态的脂肪干细胞，MTT法检测细胞的增殖情况，Kaplow法染色检测细胞碱性磷酸酶表达情况，碱性磷酸酶活性检测，茜素红染色鉴定钙结节形成情况。结果脂肪干细胞经诱导培养后，PRP诱导组较对照组增殖明显（P〈0．01），碱性磷酸酶活性较对照组增高明显（P〈0．01），PRP诱导组碱性磷酸酶染色阳性，茜素红染色可见钙结节形成。结论体外培养时，PRP可促进脂肪干细胞的增殖并能诱导成骨分化，从而为骨组织工程提供一种新的方法。     

不同浓度血清对人成纤维细胞生长的比较研究

目的探索和建立人真皮成纤维细胞体外培养所需最低浓度血清的方法。方法采用胶原酶消化法,在不同浓度血清(体积分数为2%、5%、10%)的培养基中进行条件培养。苏木素-伊红染色后,观察成纤维细胞的形态以及波形蛋白免疫细胞化学染色,MTT法进行细胞计数,用流式细胞仪对细胞生长周期进行检测。结果在3种条件培养基中,分离的成纤维细胞的生长状态有所不同,其含5%、10%血清培养液相对于含2%血清培养液的条件下,培养的成纤维细胞生长、增殖迅速,细胞活力强,MTT值明显增高(P<0.05),细胞周期G1/S期降低(P<0.01);5%血清与10%血清比较,差异无显著意义(P>0.05)。结论在5%自体血清的条件下培养的人真皮成纤维细胞,活力强、增殖迅速,可为临床应用提供参考依据。     

PRP促进光老化人皮肤成纤维细胞胶原与透明质酸合成的研究

目的体外研究人皮肤成纤维细胞（HSFs）发生光老化时,富血小板血浆（PRP）对其合成胶原与透明质酸的影响。方法使用紫外线（UVA）照射体外培养的原代人皮肤成纤维细胞,并检测光老化细胞的细胞增殖情况及细胞倍增时间。用含不同浓度PRP（10%、30%、50%）的培养液培养光老化细胞,正常培养的光老化细胞为阴性对照,原代人皮肤成纤维细胞为阳性对照。ELISA检测各组细胞外液中胶原蛋白与透明质酸含量。结果经紫外线照射后,细胞增殖减弱,加入PRP后,伴随PRP浓度的增加,细胞倍增时间缩短;ELISA检测发现,经过UVA照射的成纤维细胞COL1、COL3及透明质酸合成量,显著低于正常培养的成纤维细胞（P〈0.01）。在PRP作用后,成纤维细胞的COL1、COL3及透明质酸合成量均明显增加（P〈0.01）。结论PRP能显著促进光老化人皮肤成纤维细胞胶原与透明质酸合成,可应用于皮肤抗衰老的研究。     

重组人酸性成纤维细胞生长因子对人成骨细胞生长及增殖的影响

目的研究酸性成纤维细胞生长因子(acidfibroblastgrowthfactoraFGF)对成骨细胞生长及增殖的影响。方法取人胚胎颅盖骨骨内膜,利用组织块培养法进行成骨细胞原代培养,DMEM-F12培养液传代、扩增培养,以细胞形态学、细胞形成钙结节的能力及碱性磷酸酶活性鉴定成骨细胞。应用光镜、电镜、MTT法及流式细胞仪检测,分别从细胞形态、细胞生长及增殖特性分析不同浓度的aFGF对人成骨细胞生长和增殖的影响。结果10～100ng/mlaFGF可明显促进成骨细胞生长、增殖使S期细胞数增加,但高浓度aFGF(>1μg/ml)对成骨细胞生长具有抑制作用。结论aFGF对体外培养的人成骨细胞具有促生长和增殖作用,其有效浓度为10～100ng/ml。     

雷公藤提取物抑制增生性瘢痕成纤维细胞的实验研究

目的　为雷公藤提取物 (LLZ)治疗烧伤后增生性瘢痕提供实验依据。　方法　体外培养增生性瘢痕成纤维细胞 ,培养液中加入不同浓度的LLZ(5× 10 -3 、5× 10 -4、5× 10 -5、5× 10 -6g/L) ,2 4h后观察细胞形态、增殖活性及药物雷公藤提取物的细胞毒性。　结果　不同浓度的LLZ均能改变成纤维细胞形态 ,减少细胞数量 ,同时可明显降低细胞增殖活性。　结论　LLZ对烧伤后增生性瘢痕成纤维细胞形态和增殖均有明显的抑制作用 ,此作用并非毒性所至。     

混合接种法构建组织工程皮肤

目的 探讨混合接种法体外构建复合皮的可行性.方法 在异种猪脱细胞真皮的真皮面接种人成纤维细胞7 d后,将其分两组进行实验.实验组:将表皮细胞按5×105/cm2与成纤维细胞0.2 ×105/cm2混合后接种于表皮面,培养液用K-SFM与成纤维细胞上清的1:1混合液.对照组:仅接种表皮细胞5×105/cm2,培养液用K-SFM.培养1、3周后取材观察其形态变化,并行免疫组化鉴定.结果 培养3周后,实验组可见表皮层连续,细胞层数3～4层,与真皮连接紧密,有表皮突形成;对照组表皮细胞层仅1～2层,且与真皮分离.实验组Laminin强阳性提示基底膜形成充分,并经透射电镜也可观察到完整的基底膜.结论 将表皮细胞与少量成纤维细胞混合接种,可促进表皮细胞在脱细胞真皮上黏附增殖,并有助于基底膜充分形成.     

不同浓度血清对人成纤维细胞生长的比较研究

目的 探索和建立人真皮成纤维细胞体外培养所需最低浓度血清的方法。方法 采用胶原酶消化法，在不同浓度血清（体积分数为2%、5%、10%）的培养基中进行条件培养。苏木素-伊红染色后，观察成纤维细胞的形态以及波形蛋白免疫细胞化学染色，MTT法进行细胞计数，用流式细胞仪对细胞生长周期进行检测。结果 在3种条件培养基中，分离的成纤维细胞的生长状态有所不同，其含5%、10%血清培养液相对于含2%血清培养液的条件下，培养的成纤维细胞生长、增殖迅速，细胞活力强，MTT值明显增高（P〈0.05），细胞周期G1/S期降低（P〈0.01）；5%血清与10%血清比较，差异无显著意义（P〉0.05）。结论 在5%自体血清的条件下培养的人真皮成纤维细胞，活力强、增殖迅速，可为临床应用提供参考依据。     

不同培养基对皮肤来源的成纤维细胞增殖、衰老及胶原蛋白分泌的影响

目的:探讨不同培养基组成对皮肤成纤维细胞的增殖、衰老及胶原蛋白分泌的影响。方法:采用两步酶消化法从健康人耳后皮肤中获得成纤维细胞,依据培养基种类不同分为A组(FM无血清培养基)、B组(DMEM+10%FBS)、C组(RPMI-1640+10%FBS)、D组(DMEM+F12(1∶1)+10%FBS),采用MTT法、细胞划痕实验、酶联免疫吸附实验、β-半乳糖苷酶试验、软琼脂克隆形成试验比较不同培养基对细胞增殖、迁移、胶原分泌、老化、变异的影响。结果:采用酶消化法在2d~4d内即可从组织块中获得大量成纤维细胞;其中B、D组较A、C组细胞增殖较快,且趋势与MTT细胞增殖相同;划痕实验显示B、D组较A、C组向缺损部位迁移更为明显;此外,D组胶原蛋白分泌量高于其余三组(P0.05),且连续传代30次后,细胞无明显衰老及瘤变迹象。结论:两步酶消化法可快速高效的从人组织块中获得成纤维细胞,且采用DMEM+F12(1∶1)完全培养基有利于促进细胞增殖、分泌胶原蛋白及延缓细胞衰老。     

The effect of platelet-rich plasma on the cellular response of rat bone marrow cells in vitro.

The aim of this study was to investigate the effect of platelet-rich plasma (PRP) on the proliferation and the differentiation of rat bone marrow cells (RBMCs). PRP, platelet-poor plasma (PPP), and bone marrow cells were derived from the rats (hearts and tibia) and the cells were cultured with or without PRP or PPP (0 [control]), 0.2 approximately 10 microL/mL). The proliferation of RBMCs was measured on days 2 and 4, and alkaline phosphatase (ALP) staining and activity measurement were evaluated to determine the effect of PRP on the differentiation on days 4 and 8. PRP enhanced the proliferation significantly compared to the control group (P < .05). These enhancements were greater than ones induced by the addition of PPP. ALP staining appeared to show that PRP decreased the number of ALP positive cells and ALP activity significantly (P < .05). Our results demonstrate that PRP stimulates the proliferation but suppresses the differentiation of RBMCs.     

Proliferation and differentiation of human tenocytes in response to platelet rich plasma: an in vitro and in vivo study

Platelet rich plasma (PRP) is the autologous plasma fraction with a platelet-rich cellular component which is enriched with a number of growth factors. Due to its availability and low cost, PRP has become an increasingly popular clinical tool as an alternative source of growth factors for various applications, for example, tendon regeneration but with limited success in clinical trials. The main objective of the current study was to determine whether activated PRP [i.e., platelet rich plasma-clot release (PRCR)] could be used to induce the proliferation and collagen synthesis in human tenocyte in vitro. The advantage of using PRCR is that the platelet-derived bioactive factors are more concentrated and could initiate a more rapid and accelerated healing response than PRP. Our results demonstrated that 10% PRCR treatment accelerated the extent of cell proliferation and collagen production by human tenocytes in vitro. The expression of specific tenocyte markers were similar to conventional fetal bovine serum (FBS)-treated tenocytes implanted in mice within 14 days of implantation in diffusion chambers. Moreover, relatively more collagen fibrils were evident in PRCR-treated tenocytes in vivo as compared to 10% FBS-treated cells. Overall, our feasibility study has indicated that PRCR can induce human tenocyte proliferation and collagen synthesis which could be implemented for future tendon regeneration in reconstructive surgeries.     

可注射组织工程材料壳聚糖-β-磷酸三钙/富血小板血浆对体外培养骨髓基质干细胞增殖的影响

目的研究富血小板血浆(PRP)与可注射性材料壳聚糖-β-磷酸三钙(β-TCP)复合后对体外培养骨髓基质干细胞(BM-SCs)的增殖作用,探讨PRP作为壳聚糖-β-TCP生长因子来源的可行性。方法将体外培养并扩增的中国青山羊BMSCs与可注射的壳聚糖-β-TCP支架体外复合培养,分为空白对照组、单纯材料组和PRP/材料组,通过倒置显微镜观察细胞生长情况,MTT法检测材料中PRP对细胞增殖的影响。结果PRP/材料组中体外培养的BMSCs增殖优于单纯材料组和空白对照组(P<0·05),单纯材料组和空白对照组细胞增殖无明显差异。结论复合材料中的PRP能促进体外培养的BMSCs增殖,PRP作为可注射性材料壳聚糖-β-TCP中的生长因子是可行的。     

Can thrombin‐activated platelet releasate compensate the age‐induced decrease in cell proliferation of MSC?

Mesenchymal progenitor cells (MSCs) are promising for cell‐based regeneration therapies. In elderly patients a reduced proliferation of MSCs has been described. Platelet‐rich plasma (PRP) contains important factors necessary for osteogenic regeneration. The aim of this study was to find out whether the age‐induced decrease in cell proliferation can be compensated by the use of supernatant of centrifuged, activated PRP (tPR). MSCs of donors of three age groups (A: young, 14–16 years, B: middle age, 36–46 years, C: older, 74–83 years) were expanded with 20% FCS alone or supplemented with thrombin‐activated platelet releasate (tPR) (1%, 2.5%, and 5%) or platelet‐poor plasma (PPP 5%). Cell proliferation and differentiation was measured on days 0, 3, and 7. Proliferation increased significantly in groups A and B with tPR, and non‐significantly in group C. The generation times of MSCs of elderly patients were significantly increased in group C compared to groups A and B. Addition of 1% or 2.5% tPR significantly reduced population doubling times of all age groups. Adding tPR stimulates the proliferation rate of MSCs independent of donor age. For juvenile and middle‐aged patients this influence was significant. Cells differentiation into osteoblasts was not influenced by addition of tPR. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1786–1795, 2013     

The contribution of platelet-derived growth factor, transforming growth factor-beta1, and insulin-like growth factor-I in platelet-rich plasma to the proliferation of osteoblast-like cells.

The aim of this study was to investigate the effect of platelet-rich plasma (PRP) on the proliferation of osteoblast-like cells in vitro. PRP was prepared using a centrifuge; the number of platelets (n = 32) and the levels of platelet-derived growth factor-AB (PDGF-AB), transforming growth factor-beta1 (TGF-beta1), and insulin-like growth factor-I (IGF-I) were measured (n = 16). For the proliferation assay, SaOS-2 was cultured in the presence of platelet-poor plasma (PPP), whole blood, or PRP. The cell number was counted after 36 and 72 hours. To investigate the effect of each growth factor, the cells were cultured with PRP in the absence or presence of neutralizing antibodies, and counted as described. The mean platelet count of PRP was 1546.36 +/- 382.25 x 10(3)/microL, and the mean levels of PDGF-AB, TGF-beta1 and IGF-I were 0.271 +/- 0.043, 0.190 +/- 0.039, and 0.110 +/- 0.039 ng/1500 x 10(3) platelets, respectively. Cell proliferation was enhanced in all PRP groups in a dose-dependent manner, and all neutralizing antibodies significantly suppressed proliferation compared with the PRP group, lacking antibody, at 36 hours. However, at 72 hours, the neutralizing antibodies of PDGF and TGF-beta1, but not IGF-I, significantly suppressed proliferation. These results show the beneficial abilities of PRP in the proliferation of osteoblast-like cells from the standpoint of growth factors, including the contribution of each factor.     

Platelets and plasma stimulate sheep rotator cuff tendon tenocytes when cultured in an extracellular matrix scaffold

The addition of platelet‐rich plasma (PRP) to rotator cuff repair has not translated into improved outcomes after surgery. However, recent work stimulating ligament healing has demonstrated improved outcomes when PRP or whole blood is combined with an extracellular matrix carrier. The objective of this study was to evaluate the effect of three components of blood (plasma, platelets, and macrophages) on the in vitro activity of ovine rotator cuff cells cultured in an extracellular matrix environment. Tenocytes were obtained from six ovine infraspinatus tendons and cultured over 14 days in an extracellular matrix scaffold with the following additives: (1) plasma (PPP), (2) plasma and platelets (PAP), (3) plasma and macrophages (PPPM), (4) plasma, platelets and macrophages (PAPM), (5) phosphate buffered saline (PBS), and (6) PBS with macrophages (PBSM). Assays measuring cellular metabolism (AlamarBlue), proliferation (Quantitative DNA assay), synthesis of collagen and cytokines (SIRCOL, TNF‐α and IL‐10 ELISA, and MMP assay), and collagen gene expression (qPCR) were performed over the duration of the experiment, as well as histology at the conclusion. Plasma was found to stimulate cell attachment and spreading on the scaffold, as well as cellular proliferation. Platelets also stimulated cell proliferation, cellular metabolism, transition of cells to a myofibroblast phenotype, and contraction of the scaffolds. The addition of macrophages did not have any significant effect on the sheep rotator cuff cells in vitro. In vivo studies are needed to determine whether these changes in cellular function will translate into improved tendon healing. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:623–629, 2016.     

不同比例激活富血小板血浆对其生长因子浓度及人骨髓基质干细胞增殖的影响

目的 确定激活剂凝血酶与富血小板血浆(PRP)混合的最适比例,并探讨凝血酶与PRP中血小板衍生生长因子.AB(PDGF.AB)和转化生长因子-β(TGF-β)对人骨髓基质干细胞(hBMSCs)增殖的联合作用.方法 按1000 U凝血酶溶于1 mL质量百分比为10%的CaCl2配制激活剂.按PRP与激活剂混合比例为1:1、5:1、10:1、20:1、40:1分为A、B、C、D、E 5组.ELISA法测定各组0、1、8、24、120 h后PRP凝胶中PDGF-AB、TGF-β1的浓度;MTT法评价各组PRP凝胶上清对hBMSCs增殖的影响.结果 PRP与凝血酶混合后,PDGF-AB、TGF-β1浓度立即升高,在激活1 h后达到高峰.B、C、D组PRP凝胶中PDGF-AB、TGF-β1的浓度最高,并明显促进hBMSCs的增殖;而A组则抑制hBMSCs的增殖.结论 PRP对hBMSCs增殖的作用与其所含有PDGF-AB、TGF-β1存在浓度依赖性,而凝血酶可能在一定浓度范尉内主要通过对PRP中生长因子浓度的调控来影响hBMSCs的增殖,但是当凝血酶浓度过高时则抑制hBMSCs的增殖.     

Healing in transplanted teeth with periodontal ligament cultured in vitro

Regeneration of connective tissue attachment is the ultimate goal of periodontal therapy. It has been suggested that periodontal ligament cells possess the potential to create new connective tissue attachment. However, as cells from gingiva and alveolar bone occupy the root surface during initial wound healing, population by periodontal ligament cells is limited in vivo. We have been developing a new periodontal regeneration technique using in vitro tissue culture of periodontal ligament remaining on a periodontally involved root. The purpose of this study was to examine the periodontal healing after transplantation of teeth with reduced periodontal ligament that had been cultured in vitro. Twenty-five incisors from four beagles were used. After the teeth were extracted, the periodontal ligament and cementum were removed from coronal part of the roots and the roots were planed. The periodontal ligament of the apical part was retained. Fourteen teeth of the experimental group were transplanted following culture for 6 weeks. Eleven teeth of the control group were similarly prepared and immediately transplanted without tissue culture. Four weeks after transplantation, the specimens were prepared for histological analysis. Downgrowth of junctional epithelium on the root of experimental group was significantly less than control. Most of the root planed surfaces of experimental group were covered with periodontal ligament fibers oriented parallel or inclined to the root surfaces and limited new cementum formation was observed near the apical end of the planed root. There was no significant difference between groups in observations on the root surface with remaining periodontal ligament. From the above results, it was concluded that periodontal tissue culture of teeth with root planed surface and remaining periodontal ligament could reduce the extent of epithelium downgrowth and increase connective tissue adhesion on the root planed surface, as well as minimize damage to remaining periodontal ligament, after transplantation of teeth.     

Does the application of GaAlAs laser and platelet-rich plasma induce cell proliferation and increase alkaline phosphatase activity in human dental pulp stem cells?

Blood extracts containing platelet products are gaining popularity in promoting healing and pulp regeneration. This study was designed to evaluate the effect of platelet-rich plasma (PRP) and gallium–aluminum–arsenide (GaAlAs) laser on proliferation and differentiation of human dental pulp stem cells (hDPSCs). In this ex vivo study, hDPSCs isolated from impacted mandibular third molars were cultured in Dulbecco’s Modified Eagle’s medium )DMEM(with 10% fetal bovine serum (FBS). After reaching the desired confluence, the cells were distributed into 4 groups, namely, control, PRP, laser, and PRP+laser for MTT assay and alkaline phosphatase (ALP) test. In the PRP and PRP+laser groups, 10% PRP was added to each well on the plate. In the laser and PRP+laser groups, as for the proliferation test, laser irradiation was carried out for 45 s, while 135 s was designated for ALP test. After 1, 3, and 5 days, cell proliferation and ALP activity were assessed using MTT and ALP colorimetric assay, respectively. Two-way ANOVA was utilized to analyze data. In PRP and PRP+laser groups, cell proliferation and viability increased until day 3 but began to decline afterwards until the 5th day. In the laser group, the increase in proliferation and viability was observed till day 5 which was less than the control group. Laser and control groups exhibited significantly higher cell viability and proliferation than both PRP and PRP+laser groups. ALP activity was more pronounced in PRP+laser, PRP, and laser in descending order; however, all were less than that of the control group. Only in the control group did the ALP activity augment during the 5-day period. Laser irradiation could induce pulp cell proliferation and demonstrated a better performance than PRP in this regard.