In vitro and in vivo evaluation of heparin mediated growth factor release from tissue‐engineered constructs for anterior cruciate ligament reconstruction

Anterior cruciate ligament (ACL) rupture is a common injury often necessitating surgical treatment with graft reconstruction. Due to limitations associated with current graft options, there is interest in a tissue‐engineered substitute for use in ACL regeneration. While they represent an important step in translation to clinical practice, relatively few in vivo studies have been performed to evaluate tissue‐engineered ACL grafts. In the present study, we immobilized heparin onto electrospun polycaprolactone scaffolds as a means of incorporating basic fibroblast growth factor (bFGF) onto the scaffold. In vitro, we demonstrated that human foreskin fibroblasts (HFFs) cultured on bFGF‐coated scaffolds had significantly greater cell proliferation. In vivo, we implanted electrospun polycaprolactone grafts with and without bFGF into athymic rat knees. We analyzed the regenerated ACL using histological methods up to 16 weeks post‐implantation. Hematoxylin and eosin staining demonstrated infiltration of the grafts with cells, and picrosirius red staining demonstrated aligned collagen fibers. At 16 weeks postop, mechanical testing of the grafts demonstrated that the grafts had approximately 30% the maximum load to failure of the native ACL. However, there were no significant differences observed between the graft groups with or without heparin‐immobilized bFGF. While this study demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture, it also demonstrates that in vitro results do not always predict what will occur in vivo. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:229–236, 2015.     

碱性成纤维细胞生长因子对体外大鼠牙胚分化的影响

目的：观察外源性ｂＦＧＦ对体外培养牙胚发育和分化的作用。方法：采用体外培养１７ｄＳＤ胎鼠第一磨牙牙胚，培养液中加入１０ｎｇ／ｍｌ人重组ｂＦＧＦ（ｈｂＦＧＦ），分别培养３、６、９ｄ，组织学观察。结果：外源性ｈｂＦＧＦ可使大鼠牙胚的细胞分化和牙本质基质分泌加快。结论：外源性ｂＦＧＦ可以促进大鼠牙胚的发育，加速牙齿发育。     

贝复济(bFGF)促进拔牙创早期愈合的临床疗效观察

目的：探讨贝复济（bFGF）对拔牙创愈合的临床疗效。方法：采用随机双盲对照临床研究，对59例患者拔牙后创口愈合情况进行临床疗效观察，拔牙后1，3，6，8周摄X线牙片观察牙槽骨骨密度改变情况。结果：局部应用bFGF的实验组拔牙创上皮愈合较对照组早，X线牙片观察拔牙窝内新骨生长量较对照组多，速度较快，拔牙后8周骨密度即趋于正常。结论：拔牙后局部应用贝复济（bFGF）具有良好的促进拔牙创骨愈合的作用，使用简便，易于推广。     

碱性成纤维细胞生长因子和表皮生长因子对Balb/c胎鼠下颌突外胚间充质细胞增殖的影响

目的：探讨碱性成纤维细胞生长因子(bFGF)和表皮生长因子（EGF）对Balb/c胎鼠下颌突外胚间充质细胞增殖的剂量效应和时间效应。方法：用体外细胞培养技术和噻唑蓝（MTT）比色法，观察不同浓度和不同时间bFGF和EGF对外胚间充质细胞增殖的影响。结果：0.1-100ng/mL bFGF,0.1-10ng/mL EGF对细胞有促增殖作用，并呈浓度依赖性,两种生长因子均在10ng/mL浓度时作用最强，并于培养第5d达到促增殖高峰。结论：bFGF和EGF能促进外胚间充质细胞的增殖。     

PTTG和bFGF在舌癌细胞株Tca8113中表达相关性研究

目的：探讨垂体肿瘤转化基因（Pituitary Tumor Transforming Gene,PTrG）蛋白在舌癌细胞株Tca8113中的表达及其与碱性成纤维细胞生长因子（basic Fibroblast Growth Factor,bFGF）间的关系.方法：应用免疫细胞化学方法,检测加入bFGF抗体后孵育的癌细胞中PTTG蛋白表达的变化.结果：Tca8113中PTTG蛋白呈阳性表达,并受bFGF影响,加入bFGF抗体的培养液中的癌细胞PTTG蛋白荧光明显减弱,随着时间延长和bFGF抗体滴度增加,减弱趋势明显.结论：舌癌细胞株中PTTG蛋白的表达受bFGF制约,对二者的联合治疗可能是一种治疗舌癌的有效方法.     

Expression of receptors for basic fibroblast growth factor on human periodontal ligament cells

Basic fibroblast growth factor (FGF-2; bFGF) is a major mitogen for connective tissue cells, and participates in the healing process. It has already been reported that FGF-2 could be applicable to enhance periodontal regeneration. In the present study, we examined FGF receptor (FGFR) expression on human periodontal ligament (PDL) cells. The binding of [¹²⁵I]-labeled FGF-2 to human PDL cells was studied by radioreceptor assay. The binding of [¹²⁵I]-FGF-2 to PDL cells reached a plateau after 2.5 h incubation at 4°C and was inhibited by the addition of unlabeled FGF-2 and acidic FGF (FGF-1; aFGF), but not insulin-like growth factor-I, platelet-derived growth factor and transforming growth factor-β₁. Scatchard analysis revealed the presence of approximately 1.0 × 10⁵ FGF-2 binding sites per cell with an apparent Kd of 1.2 × 10-¹⁰ M. Interestingly, the binding of [¹²⁵I]-FGF-2 on PDL cells reached its maximum at d 6 of the culture and then gradually decreased. Scatchard analysis also demonstrated that the number of FGFRs on a PDL cell was altered during the course of the culture, while the affinity between FGF-2 and its receptor was not. The responsiveness of PDL cells to FGF-2, which was monitored by the inhibitory effect on alkaline phosphatase activity, was reduced in proportion to the decrease in the number of FGFRs on the PDL cells. The present study suggests that PDL cells alter the responsiveness to FGF-2 during the course of the culture by changing the density of its receptor, and that the density of FGFR expression might be a marker of the cytodiflerentiation of PDL cells into mineralized tissue forming cells.     

Efficient differentiation of vascular endothelial cells from dermal-derived mesenchymal stem cells induced by endothelial cell lines conditioned medium

Objective

To directionally-differentiate dermis-derived mesenchymal stem cells (DMSCs) into vascular endothelial cells (VECs) in vitro, providing an experimental basis for studies on the pathogenesis and treatment of vascular diseases.

碱性成纤维细胞生长因子对兔骨髓基质细胞生物行为的影响

目的:观察兔骨髓基质细胞(MSC)经不同剂量碱性成纤维细胞生长因子(bFGF)刺激后,促血管内皮细胞增生的重要递质血管内皮细胞生长因子(VEGF)在MSC中的表达及分布情况,并与相应成骨情况做一对比,借以预测MSC经bFGF刺激后成骨效能与成血管效能的变化,探讨其对剂效关系。方法:取兔双侧股骨MSC,采用MSC体外培养技术,分别以不同剂量bFGF刺激细胞,并设立空白对照组。培养5d后,进行细胞形态(HE染色)、增殖情况(细胞记数法与MTT法)、碱性磷酸酶(改良钙钴法染色)、钙化结节(图像分析)、VEGF阳性细胞率(免疫组化染色)等项目的检测。结果:不同剂量组间在细胞记数法、MTT法检测、碱性磷酸酶活性、矿化面积百分率、VEGF阳性细胞率5项观测指标上F值分别为65.50,22.47,11.12,8.33和224.37,P<0.01,差别具有显著意义,结合各组均值来看,bFGF剂量为1200U/mL左右时效果最佳。结论:应用bFGF在促进MSC增殖的同时,还可促进促血管内皮细胞增生的重要递质-VEGF的表达,其应用剂量与效果之间存在明显相关关系。bFGF应用剂量为1200U/mL左右时,其促进MSC表达VEGF及促进细胞增殖作用同时达到最佳效果,超过此应用剂量,两者则有下降趋势。     

碱性成纤维细胞生长因子对大鼠真皮下血管网皮片愈合的影响

目的:探讨碱性成纤维细胞因子(bFGF)对大鼠真皮下血管网皮片愈合的影响。方法:Wistar大白鼠30只,每只背部切取2个真皮下血管网皮片并原位移植缝合,上部皮片下注入bFGF为实验组。下部皮片为对照组,术后大体肉眼观察皮片成后情况,并于2,4,6,8d取材镜下组织学观察CD34阳性细胞数、血管内皮细胞生长情况,微血管密度及肉芽组织生长情况。结果:实验组与对照组皮片成活率分别为93%及64%,经配对t检验差异显著(t=3.701,P<0.01)。其镜下观察CD34阳性细胞数、微血管数及肉芽组织厚度两组均有明显差异(P<0.01)。结论:bFGF具有广泛的细胞增殖效应,可通过多个环节促进小血管形成及肉芽组织增长,加速移植皮片与周围皮肤的愈合过程。