唑来膦酸辅助治疗骨巨细胞瘤的基础研究与临床新方法的初步应用
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摘要
[背景]骨巨细胞瘤是常见的原发性骨肿瘤之一。多数研究者认为其为一种来源于骨髓间充质干细胞的良性骨肿瘤,以局部骨质破坏为主要特征,其中少数病例还可发生“良性”肺转移。目前研究显示,骨巨细胞瘤对传统的放疗及化疗不敏感,手术治疗仍然是骨巨细胞瘤最主要的治疗方式。术后高复发率是骨巨细胞瘤治疗最大的挑战。为手术彻底清除肿瘤病灶,临床医生曾经尝试在术中使用多种化学试剂或物理方法辅助治疗,进行病灶刮除术后囊壁的处理,清除残留的肿瘤细胞,从而减少骨巨细胞瘤的复发。病灶刮除囊壁高速磨除加骨水泥填充目前被认为是骨巨细胞瘤手术治疗的标准方式。随着对骨巨细胞瘤疾病的深入研究,双膦酸盐类及RANK单克隆抗体辅助治疗成为近年来骨巨细胞瘤治疗的新进展。双膦酸盐早期是作为一类抗代谢骨病药物进入市场,它通过与焦磷酸的类似结构,发挥抗骨吸收的作用。在临床应用于骨巨细胞瘤辅助治疗的研究中,取得了良好的效果。在体外实验研究中,人们发现双膦酸盐类不仅可抑制破骨细胞介导的骨吸收,延缓破骨细胞生成和成熟并诱导破骨细胞凋亡,而且能够直接作用于肿瘤细胞而发挥抑制肿瘤的作用。我们在对新一代双膦酸盐药物唑来膦酸对骨巨细胞瘤基质细胞的直接作用的研究中观察到,其不仅诱导肿瘤细胞凋亡,还可诱导肿瘤细胞的成骨分化,通过这两种方式而发挥显著的抗肿瘤作用。这一结论也解释了临床上报导的双膦酸盐辅助治疗骨巨细胞瘤后病灶周围明显骨化形成的现象,也为骨骼相关事件的降低提供理论依据。在证实双膦酸盐具有诱导骨巨细胞瘤肿瘤细胞分化这一重要的抗肿瘤方式后,进一步研究双膦酸盐的浓度及诱导作用时间与其成骨诱导效应的相关性可能具有重要的临床意义。在分子生物学水平深入探索双膦酸盐如何通过激活信号通路而诱发骨巨细胞瘤基质细胞向成骨细胞分化,也将为人们更进一步了解其生物学规律并促进其抗肿瘤效应具有重要意义。此外,目前临床医生尝试了双膦酸盐辅助治疗骨巨细胞瘤的不同途径和方式,我们也报道了常规术后双膦酸盐静脉辅助治疗方式的疗效。根据手术治疗骨巨细胞瘤的特点,寻找更加安全有效的辅助用药途径和方式,也将可能对提高肿瘤的疗效发挥重要的意义。
[目的]
1)探讨唑来膦酸诱导骨巨细胞瘤基质细胞向成骨细胞分化的最佳浓度及作用时间。
2)探讨唑来膦酸诱导骨巨细胞瘤基质细胞向成骨细胞分化机制。
3)探讨临床上应用双膦酸盐类辅助治疗骨巨细胞瘤的新方式及可行性。
[方法]
1)唑来膦酸诱导骨巨细胞瘤基质细胞向成骨细胞分化的浓度及时间相关性实验研究。
实验标本来自2011年11月至2013年1月广州军区广州总医院及南方医科大学附属南方医院收治的4例四肢骨巨细胞瘤患者。其中男性1例,女性3例,年龄13～31岁。肿瘤影像学分级按Campanacci分级标准,其中Ⅱ级3例,Ⅲ级1例。所有患者术前均未接受双膦酸盐药物辅助治疗,术后均经过病理诊断为骨巨细胞瘤。对术中切除的骨巨细胞瘤新鲜组织进行原代培养,待骨巨细胞瘤细胞经9次传代纯化后分别给予含有不同浓度唑来膦酸(0μM,0.01μM,0.1μM,1μM,5μM,30μM)培养基培养,处理72小时后,采用qRT-PCR方法检测各组骨巨细胞瘤基质细胞成骨相关基因cbfa-1、osterix、osteocalcin表达。应用SPSS19.0统计学软件将各组cbfa-1、osterix、osteocalcin的基因表达值进行单因素方差分析(one-way ANOVA)分析,组间多重比较采用LSD test。再采用1μM唑来膦酸与空白对照培养基分别行骨巨细胞瘤基质细胞培养,处理48小时后,给予细胞爬片ALP染色及BSP、Collagen Type I、Osteonectin、Osteocalcin细胞免疫染色检测。另于诱导后0小时、24小时、72小时及168小时行qRT-PCR方法检测两组骨巨细胞瘤基质细胞cbfa-1、osterix、osteocalcin的基因表达。统计结果,分析唑来膦酸诱导骨巨细胞瘤基质细胞向成骨细胞分化的最佳浓度及作用时间。
2)唑来膦酸诱导骨巨细胞瘤基质细胞向成骨细胞分化的信号途径实验研究。
取原代培养并已纯化的骨巨细胞瘤基质细胞,随机分组为对照组,诱导组和信号抑制组1,2,3。对照组采用常规培养液,诱导组加入1μM浓度的唑来膦酸,信号抑制组1和2在加入唑来膦酸前2h加入JNK信号抑制剂SP600125(1μM)和ERK信号抑制剂PD98059(20μM),抑制组3同时加入SP600125及PD98059。处理30分钟后,采用Western印迹杂交检测p-JNK,JNK,p-ERK,ERK蛋白质在各组骨巨细胞瘤基质细胞内的表达。
采用同样的实验分组的处理方法,72小时后,采用qRT-PCR方法检测各组细胞cbfa-1、osterix、osteocalcin的基因表达。统计学方法同前。分析双膦酸盐是否通过激活JNK和ERK通路(即信号通路的磷酸化)发挥成骨分化诱导作用。
3)双膦酸盐辅助治疗骨巨细胞瘤新方式的可行性探索。
数字化复合抗骨巨细胞瘤珊瑚羟基磷灰石／聚乳酸(CHA/PLA)人工骨的制备及性能探索：首先利用计算机辅助3D打印将按一定比例混合的CHA粉末与聚乳酸(PLA)制作成具有特定外形珊瑚羟基磷灰石/聚乳酸人工骨支架,通过浸泡后真空冻干燥等处理将唑来膦酸钠载入。将复合人工骨支架浸入细胞培养液,收集6h、12h,24h、48h及72h浸提液。采用四甲基偶氮唑盐比色法(MTT法)及Annexin-V FITC流式细胞定量分析法检测不同时期浸提液对骨巨细胞瘤基质细胞的增值影响及凋亡诱导作用。分析局部使用双膦酸盐的有效性。
采用局部灌洗法辅助治疗骨巨细胞瘤1例的观察：广州军区广州总医院1例左股骨下段骨巨细胞瘤复发患者,给予再次囊内病灶刮除骨水泥填充手术,术中采用唑来膦酸溶液(2mg/500ml)局部冲洗,术后48小时内给予病灶内唑来膦酸溶液(2mg/500ml)局部灌洗引流。观察患者一般情况,切口引流及愈合情况。术后定期病情复查。初步观察局部灌洗法可能存在的安全性。
通过双膦酸盐的临床新方法的初步应用观察,探索其不同应用方式的可行性,为进一步大样本临床应用和疗效观察提供指导。进一步促进骨巨细胞瘤的治疗效果。
[结果]
1)唑来膦酸诱导骨巨细胞瘤基质细胞向成骨细胞分化的浓度及时间相关性实验研究。
实验显示1μM唑来膦酸组中成骨分化相关基因表达最明显。48小时后1μM唑来膦酸组cbfa-1、osterix、osteocalcin的基因表达分别是对照组2.66倍、2.50倍及2.23倍,具有统计学显著性差异(P=-0.002,<0.001,<0.001)。此外,0.1μM唑来膦酸组cbfa-1基因表达是对照组1.6倍,具有显著性差异(P=0.003)。
在唑来膦酸诱导48小时后骨巨细胞瘤基质细胞ALP染色及BSP、Collagen Type I、Osteonectin、Osteocalcin免疫组化染色与对照组相比表现为轻度阳性或阳性。在诱导后24小时,诱导组cbfa-1的基因表达与对照组具有显著性差异(P<0.001)；在72小时监测点,诱导组与对照组osterix、osteocalcin的基因表达均具有显著性差异(P=0.001,0.003)；在168小时监测点,诱导组与对照组cbfa-1、osterix、osteocalcin的基因表达分别增高8.1,3.8及3.3倍,均具有显著性差异(P=0.002,0.002,0.002)。
2)唑来膦酸诱导骨巨细胞瘤基质细胞向成骨细胞分化的信号途径实验研究。
采用了Western印迹杂交检测ERK,JNK,p-ERK,p-JNK蛋白质在各组骨巨细胞瘤基质细胞内的表达。唑来膦酸组内可见ERK,JNK,p-ERK,p-JNK明显表达。同时加入两组阻断剂后JNK及ERK蛋白表达明显降低。
在处理72小时后,单纯1μM唑来膦酸诱导组组osteocalcin的基因与对照组及信号抑制组1,2,3均具有统计学显著性差异(P<0.001,=0.007,<0.001),cbfa-1基因与对照组及信号抑制组1,3具有统计学显著性差异(P).013,0.008)。唑来膦酸诱导组cbfa-1、osterix、osteocalcin的基因表达分别是SP600125+PD98059抑制组的5.01、3.54、3.39倍。Cbfa-1,osterix、osteocalcin的基因表达信号抑制组1,2,3与对照组无统计学显著性差异(P>0.05)。Osterix的基因表达中,1μM唑来膦酸诱导组与对照组无显著性差异(P=-0.213)；但与cbfa-1、osteocalcin存在显著性差异(P=0.048,0.005)。
3)双膦酸盐辅助治疗骨巨细胞瘤基质细胞新方式的可行性探索。
3D打印技术可制备个体化复合抗骨巨细胞瘤珊瑚羟基磷灰石／聚乳酸(CHA/PLA)人工骨。制备材料经过72小时、48小时、24小时、12小时及6小时后提取浸提液,在原代培养的骨巨细胞瘤基质细胞中予不同时期的复合唑来膦酸人工骨浸提液,48小时后,见细胞数量较阴性对照组减少,部分细胞形态发生变化,细胞外形发生皱缩,伪足减少,部分梭形基质细胞改变为圆形或椭圆形的形状,在早期的浸提液中最为明显。
采用四甲基偶氮唑盐比色法(MTT法)检测不同时期浸提液对骨巨细胞瘤基质细胞的增值影响,结果显示复合唑来膦酸人工骨浸提液对骨巨细胞瘤基质细胞抑制较阴性培养基对照组显著增强。48小时内早期高浓度唑来膦酸浸提液对骨巨细胞瘤基质细胞增殖具有明显抑制作用,与对照组浸提液有显著性差异(P<0.001,0.001,0.001,=0.002)。48小时后浸提液抑制作用减弱,并与前24小时有显著性差异(P=0.007,0.005,0.004)。复合唑来膦酸人工骨浸提液同样表现出很强的的诱导凋亡的能力,Annexin-V FITC凋亡染色及流式细胞仪定量分析结果显示对照组、72小时、48小时、24小时、12小时及6小时浸提液组原代培养的骨巨细胞瘤基质细胞凋亡占总数细胞的百分比均值依次为1.5%、2.05%、3.3%、5.23%、8.95%及12.79%。
局部唑来膦酸灌洗法预防骨巨细胞瘤复发安全性的病例观察：术中多功能引流管置于病灶周围。术后灌洗引流管引流通畅,总引流液约900m1。术后48小时拔出引流管。患者未发生流感样症状及肾功能异常。12天后手术切口拆线,愈合状况良好。唑来膦酸溶液局部灌洗治疗随访未发现明显相关并发症。
[结论]
1)双膦酸盐对骨巨细胞瘤基质细胞具有诱导凋亡及成骨分化的双重作用。其作用方式可能于药物浓度相关,低浓度唑来膦酸可能具有较强的骨巨细胞瘤基质细胞成骨诱导潜能,促进肿瘤细胞向正常细胞分化。在体外实验中,1μM浓度唑来膦酸具有最强的诱导分化能力。因此,维持低剂量唑来膦酸辅助治疗骨巨细胞瘤能够诱导残留肿瘤基质细胞分化,避免了骨巨细胞瘤手术中不必要的切除,促进病灶周围骨结构的强化或重建,减少术后骨折等并发症,还可能减少高浓度辅助用药的全身性副作用。
2)唑来膦酸诱导骨巨细胞瘤基质细胞成骨分化呈时间依赖性,诱导时间越久骨巨细胞瘤基质细胞成骨分化现象越明显。临床双膦酸盐类药物的使用较方便,可定期静脉或口服用药。在避免发生重大并发症的基础上,围手术期可维持低剂量唑来膦酸治疗,发挥持续抗肿瘤作用。辅助治疗时间越久,术后肿瘤复发率可能越低。
3) JNK和ERK信号通路在成骨细胞分化及生长增殖等多种生理过程发挥重要作用。实验证明唑来膦酸也可能激活JNK和ERK蛋白磷酸化,阻断JNK和ERK信号通路能够降低唑来膦酸诱导成骨相关基因的表达,因此唑来膦酸可能是通过JNK和ERK信号通路的激活来诱导骨巨细胞瘤基质细胞向成骨细胞分化。通过对骨巨细胞瘤基质细胞成骨分化机制的深入研究,为新型辅助用药的使用及提高成骨诱导效应提供新途径。
4)数字化复合抗骨巨细胞瘤珊瑚羟基磷灰石/聚乳酸(CHA/PLA)人工骨可为骨巨细胞瘤病灶清除术后的骨质缺损提供个性化支撑,其制作方便快捷。同时,唑来膦酸在洗脱支架周围局部释放,早期可提高局部药物浓度,对残留骨巨细胞瘤基质细胞发挥凋亡诱导作用。随着唑来膦酸药物降低,可持续发挥抗骨质吸收及促进肿瘤基质细胞成骨诱导作用,促进病灶内骨结构的强固或重建,减少骨折等并发症的发生率。局部使用数字化人工骨复合唑来膦酸为骨巨细胞瘤辅助治疗提供一种可能的有效治疗途径。
5)骨巨细胞瘤的复发给患者带来极大的痛苦。在1例骨巨细胞瘤复发后再次行病灶清除术,并且在囊内采用唑来膦酸溶液冲洗及留置唑来膦酸术后灌注方式,希望残留肿瘤细胞的清除更加可靠。这一局部辅助治疗方式既往未见报道。本病例观察见切口引流未见明显影响。患者未出现流感样症状及肾功能异常等系统性并发症。手术切口愈合良好。局部唑来膦酸灌注的使用方式,为骨巨细胞瘤辅助治疗提供一种可能的安全治疗手段,其有效性及安全性仍需要进一步的临床研究。
[Backgroud] Giant cell tumor of bone is one of the most common primary bone tumors. Most studies consider it as a kind of benign tumor derived from bone marrow mesenchymal cells. It is characterized as local bone destruction. Occasionally, rare cases of "benign" lung metastases also occur. Studies have shown that giant cell tumor is not sensitive to both traditional radiotherapy and chemotherapy. Surgical treatment is still the main treatment for giant cell tumor of bone. High recurrence rate is the biggest challenge in the treatment of giant cell tumor of bone. In order to completely remove the tumor lesions and reduce the recurrence rate, clinicians have tried to use a variety of chemical agents or physical methods as adjuvant therapies after the operation to remove the residual tumor cells. Intralegional curettage with high-speed blur and bone cement filling are now considered as the standard way of giant cell tumor of bone treatment. Later, bisphosphonates and RANK monoclonal antibody adjuvant therapy are thought to be new progresses in recent years in the treatment of giant cell tumor of bone. Diphosphonic acid was used as anti bone metabolic drugs. It has a similar structure as antiresorptive pyrophosphate in human body. In clinical studies, bisphosphonate application as adjuvant treatment for giant cell tumor of bone has achieved good results. In vitro study, researchers also found that bisphosphonates not only inhibits osteoclast-mediated bone resorption, delaying formation and maturation of osteoclasts, inducing osteoclast apoptosis, but also exerting a direct inhibition effect on tumor cells. We observed in previous study that zoledronic acid (a new generation of bisphosphonates) not only had direct effect on giant cell tumor stromal cells by inducing apoptosis, but also induced osteogenic differentiation of giant cell tumor stromal cells. This conclusion explains the clinical phenomenon that significant callus formation after bisphosphonates application as adjuvant treatment for giant cell tumor of bone. Ii may provide a posibility for reducing skeletal-related events. In the process of osteogenic induction of giant cell tumor stromal cells, the application concentration and time of bisphosphonate are important factors which may has important clinical significance. Through revealing the activation signaling pathways of bisphosphonate on giant cell tumor stromal cells, it may also promote its anti-tumor effect. In addition, clinicians tried different ways and means of adjuvant bisphosphonate therapy for giant cell tumor of bone. It is also important to find a safe and effective ways of adjvant administration.
[Object]
1) To investigate the optimum concentration and reaction time that zoledronic acid induces osteogenic differentiaon on giant cell tumor stromal cells.
2) To investigate the mechanism of giant cell tumor stromal cell osteogenic differentiaon induced by zoledronic acid.
3) To explore the feasibility of clinical noval ways that bisphosphonates applied as adjuvant therapy for giant cell tumor.
[Method]
1) Experimental study of the correlation of zoledronic acid concentration and reaction time with osteogeinc inducing effect of giant cell tumor stromal cells.
Experimental samples were taken four giant cell tumor patients from General Hospital of Guangzhou Military Command and Nanfang Hospital, Southern Medical University in November2011to January2013. One patient was male and the others were females. They aged from13to31years. The tumors were graded by Campanacci grading standards. There were three cases of grade II and one case of grade III. All patients had not received bisphosphonate adjuvant therapy before the operation. Postoperative pathological diagnosis was confirmed as giant cell tumor of bone. Primary cell cultures were got from resected fresh tissue of giant cell tumor. Purified giant cell tumor stromal cells were got after nine passages. The cultured giant cell tumor stromal cells were treated with different concentrations of zoledronic acid (0μM,0.01μM,0.1μM,1μM,5μM,30μM) in the culture medium. After72hours, qRT-PCR was used to detect cbfa-1, osterix, osteocalcin (osteogeinc related gene) expression of giant cell tumor of bone stromal cells. SPSS19.0statistical software was used. The values of cbfa-1, osterix, osteocalcin gene expression of each group were analysis with single-factor analysis of variance method(one-way ANOVA). Multiple comparisons between groups using LSD test. Then1μM zoledronic acid medium stromal cell culture was performed with the black control group. After48hours, ALP staining and BSP, Collagen Type I, Osteonectin, Osteocalcin immunostaining were given. Also at0hours,24hours,72hours and168hours after culture, qRT-PCR method was used to detect cbfa-1, osterix, osteocalcin gene expression in both groups. Then, analyse of the optimal concentration of zoledronic acid inducing osteogenic differentiation of giant cell tumor stromal cells.
2) Experimental study of signaling pathway the zoledronic acid inducing osteogenic differentiation of giant cell tumor stromal cells.
Primary cultured and purified giant cell tumor stromal cells were randomized to different groups. The control group, induction group and signal suppression group1,2,3. Control group with conventional medium, induction group with1μM concentration of zoledronic acid, signal suppression groups1and2with zoledronic acid and JNK inhibitor SP600125(1μM) or ERK signaling inhibitor PD98059 (20μM), suppression group3with both SP600125and PD98059. After30minutes treatment, Western blot was used to detecte the exsistance of p-JNK, expression of JNK, p-ERK, ERK proteins within each group of giant cell tumor stromal cells.
Use the same grouping and experiment method above. After72hours, qRT-PCR method was used to detect cbfa-1, osterix, osteocalcin gene expression. With the same statistical methods, analyse the possibility that bisphosphonates induced osteogenic differentiation through activation of JNK and ERK pathway.
3) Explore the feasibility of clinical noval ways that bisphosphonates applied as adjuvant therapy for giant cell tumor.
Preparation and properties of exploring of digitized composite anti GCT coralline hydroxyapatite/polylactic acid (CHA/PLA):Use computer-aided3D printing to architech a specific shape coralline hydroxyapatite/polylactic acid bone frame with CHA powder mixed with a certain proportion of polylactic acid (PLA). Then, zoledronic sodium was load on the bone scaffold by immersion and vacuum freeze drying. The composite artificial scaffold immersed in cell culture medium. The leaching solution was collected after6h,12h,24h,48h and72h. Using tetrazolium salt assay (MTT method) and Annexin-V FITC flow cytometry assay to analyse the impact of the leaching solution on proliferation and apoptosis of giant cell tumor stromal cells. Analyse the effectiveness of local use of bisphosphonates.
A case report of a giant cell tumor patient with local lavage of bisphosphonate:A patient of right femur giant cell tumor from Guangzhou General Hospital of Guangzhou Military Command was given intralegional curettage and cement filled. Intraoperative irrigation of zoledronic acid solution (2mg/500ml) was taken. The wound irrigation was given within48hours after the surgery with zoledronic acid solution (2mg/500ml). The patients was obscured with normal systemic condition, incision healing and drainage. Follow up was taken after surgery.
[Result]
1) Experimental study of the correlation of zoledronic acid concentration and reaction time with osteogeinc inducing effect of giant cell tumor stromal cells.
The experiment showed that1μM zoledronic acid group induced osteogenic differentiation most obviously. After72hours treatment, cbfa-1, osterix, osteocalcin gene expression in1μM zoledronic acid groups were2.66times,2.50times and2.23times more than the control group, with statistically significant difference (P=0.002,<0.001,<0.001). In addition, cbfa-1gene expression in0.1μM zoledronic acid group is1.6times more than the control group, with significant difference (P=0.003).
After48hours, ALP staining and BSP, Collagen Type I, Osteonectin, Osteocalcin immunohistochemical staining of the giant cell tumor stromal cells in the zoledronic acid group showed mild positive or positive compared with the control group. At24hours after treatment, cbfa-1gene expression of induction group are more than the control group with significant difference (P<0.001). At72hours, induction group and the control group osterix, osteocalcin in gene expression were significant differenct (P=0.001,0.003). At168hours, cbfa-1, osterix, osteocalcin gene expression in induction group has increased8.1,3.8and3.3times, respectively, compared with the control group, all with significant difference (P=0.002,0.002,0.002)
2) Experimental study of signaling pathway the zoledronic acid inducing osteogenic differentiation of giant cell tumor stromal cells.
Western blot was used to detecte ERK, JNK, p-ERK, p-JNK protein expression within each group of giant cell tumor stromal cells. In zoledronic acid group, ERK, JNK, p-ERK, p-JNK were likely to expressed significantly. After adding two blockers, JNK and ERK protein expression were significantly decreased.
After72hours, the1μM zoledronic acid group induced more quantity of osteocalcin, osterix, osteocalcin genes expression than the control and signal suppression group1,2,3, with statistically significant differences (P<0.001,=0.007,<0.001).1μM zoledronic acid group expressed5.01,3.54,3.39times more than SP600125+PD98059inhibiting group in cbfa-1, osterix, osteocalcin gene expression respectively. Signal suppression1,2,3group and the control group had no statistically significant difference in osterix, osteocalcin gene expression (P>0.05).1μM zoledronic acid group and the control group had no significant difference in cbfa-1gene expression (P=0.213), but existed significant difference in cbfa-1、 osteocalcin gene expression (P=0.048,0.005)
3) The feasibility of clinical noval ways that bisphosphonates applied as adjuvant therapy for giant cell tumor..
3D printing technology can produce individualized composite anti coralline hydroxyapatite/poly lactic acid (CHA/PLA) bone frame for giant cell tumor of bone. Leaching solution was prepared after72hours,48hours,24hours,12hours and6hours after the extraction. Primary cultured giant cell tumor stromal cells were incubated in different stages of the composite bone zoledronic acid leaching solution for48hours. We can see fewer cells than the negative control group, some changes in cell morphology, cell shape shrinkage occurs pseudopods was partly changed to spindle stromal cells round or oval shape in the early leaching most obvious solution.
Tetrazolium salt assay (MTT assay) was used to evaluate the impact of cell proliferation of the leaching solution of different stages on giant cell tumor stromal cells. The leaching solution of composite bone had more signicficant impact on cell proliferation of giant cell tumor stromal cells, compared to the control group. Early stage high concentrations zoledronic acid extracts inhibited cell proliferation of giant cell tumor of bone stromal significantly. Zoledronic acid composite artificial bone extract also showed a strong ability to induce apoptosis. Annexin-V FITC apoptosis staining and flow cytometry analysis showed that the mean percentage of apoptotic of tumor stromal cells were1.5%,2.05%,3.3%,5.23%,8.95%and12.79%in the control group,72hours,48hours,24hours12hours and6hours groups.
Local administration of zoledronic acid to prevent recurrence of giant cell tumor of bone:Intraoperative multifunctional drainage tube was placed around the lesion. Postoperative irrigation drainage tube patency, total drainage of about900ml. Pull out the drainage tube after48hours. Special discomfort did not occur in patients like influenza-like symptoms. The surgical incision healed well.
[Conclusion]
1) Low concentrations of zoledronic acid may have a strong giant cell tumor of bone stromal cells osteogenic differentiation potential. In vitro,1μM concentration of zoledronic acid expressed the strongest ability to induce differentiation. Therefore, maintaining a low dose adjuvant zoledronic acid therapy in giant cell tumor of bone can not only induce osteogenic differentiation of residual tumor stromal cells avoided unnecessary surgical resection, promote strengthening, reconstruct the bone structure around the lesion, reduce postoperative fractures but also reduce other complications and side effects.
2) Zoledronic acid-induced giant cell tumor of bone stromal cells into osteoblasts showed a time-dependent manner. The longer of the induction time, more obvious of giant cell tumor of bone stromal cells osteogeinc differentiation. Clinical use of bisphosphonate drugs is convenient. Without major complications, perioperative use of sustainable low-dose zoledronic acid as adjuvant therapy may play consistent anti-tumor effect. The longer the time of adjuvant therapy, the lower of tumor recurrence rate.
3) JNK and ERK signaling pathway is an important way of osteoblast differentiation. It is also possible that zoledronic acid caused protein phosphorylation further induced giant cell tumor of bone stromal cells to differentiate into osteoblasts through JNK and ERK activation. Its comprehensive mechanism still needs further study, and it is likely to promote the anti-tumor effect.
4) Digitized composite anti giant cell tumor of bone coralline hydroxyapatite/polylactic acid (CHA/PLA) bone can provide personalized support for the giant cell tumor of bone defect after operation. It can also be produced quickly and easily. Meanwhile, zoledronic acid elution stents locally release around. In early stage, it may improve the local drug concentration, play a role in apoptosis induction of residual bone giant cell tumor stromal cells. With the zoledronic acid concentration decreases, it may still reduce bone resorption, promote osteogenic role, rebuild the structure, reduce the incidence of fractures and other complications. Localized application of digital composite bone zoledronic acid may be an effective way for giant cell tumor adjuvant therapy.
5) Intralesional zoledronic acid solution rinse and postoperative zoledronic acid irrigation of giant cell tumor of bone. The surgical incision and drainage was likely to be normal. Patients had no systemic complication, such as flu-like symptoms. Surgical incision healed well. Local irrigation of zoledronic acid may be a safe adjuvant therapy method to reduce the recurrence of giant cell tumor of bone.
[目的]
1)探讨唑来膦酸诱导骨巨细胞瘤基质细胞向成骨细胞分化的最佳浓度及作用时间。
2)探讨唑来膦酸诱导骨巨细胞瘤基质细胞向成骨细胞分化机制。
3)探讨临床上应用双膦酸盐类辅助治疗骨巨细胞瘤的新方式及可行性。
[方法]
1)唑来膦酸诱导骨巨细胞瘤基质细胞向成骨细胞分化的浓度及时间相关性实验研究。
实验标本来自2011年11月至2013年1月广州军区广州总医院及南方医科大学附属南方医院收治的4例四肢骨巨细胞瘤患者。其中男性1例,女性3例,年龄13～31岁。肿瘤影像学分级按Campanacci分级标准,其中Ⅱ级3例,Ⅲ级1例。所有患者术前均未接受双膦酸盐药物辅助治疗,术后均经过病理诊断为骨巨细胞瘤。对术中切除的骨巨细胞瘤新鲜组织进行原代培养,待骨巨细胞瘤细胞经9次传代纯化后分别给予含有不同浓度唑来膦酸(0μM,0.01μM,0.1μM,1μM,5μM,30μM)培养基培养,处理72小时后,采用qRT-PCR方法检测各组骨巨细胞瘤基质细胞成骨相关基因cbfa-1、osterix、osteocalcin表达。应用SPSS19.0统计学软件将各组cbfa-1、osterix、osteocalcin的基因表达值进行单因素方差分析(one-way ANOVA)分析,组间多重比较采用LSD test。再采用1μM唑来膦酸与空白对照培养基分别行骨巨细胞瘤基质细胞培养,处理48小时后,给予细胞爬片ALP染色及BSP、Collagen Type I、Osteonectin、Osteocalcin细胞免疫染色检测。另于诱导后0小时、24小时、72小时及168小时行qRT-PCR方法检测两组骨巨细胞瘤基质细胞cbfa-1、osterix、osteocalcin的基因表达。统计结果,分析唑来膦酸诱导骨巨细胞瘤基质细胞向成骨细胞分化的最佳浓度及作用时间。
2)唑来膦酸诱导骨巨细胞瘤基质细胞向成骨细胞分化的信号途径实验研究。
取原代培养并已纯化的骨巨细胞瘤基质细胞,随机分组为对照组,诱导组和信号抑制组1,2,3。对照组采用常规培养液,诱导组加入1μM浓度的唑来膦酸,信号抑制组1和2在加入唑来膦酸前2h加入JNK信号抑制剂SP600125(1μM)和ERK信号抑制剂PD98059(20μM),抑制组3同时加入SP600125及PD98059。处理30分钟后,采用Western印迹杂交检测p-JNK,JNK,p-ERK,ERK蛋白质在各组骨巨细胞瘤基质细胞内的表达。
采用同样的实验分组的处理方法,72小时后,采用qRT-PCR方法检测各组细胞cbfa-1、osterix、osteocalcin的基因表达。统计学方法同前。分析双膦酸盐是否通过激活JNK和ERK通路(即信号通路的磷酸化)发挥成骨分化诱导作用。
3)双膦酸盐辅助治疗骨巨细胞瘤新方式的可行性探索。
数字化复合抗骨巨细胞瘤珊瑚羟基磷灰石／聚乳酸(CHA/PLA)人工骨的制备及性能探索：首先利用计算机辅助3D打印将按一定比例混合的CHA粉末与聚乳酸(PLA)制作成具有特定外形珊瑚羟基磷灰石/聚乳酸人工骨支架,通过浸泡后真空冻干燥等处理将唑来膦酸钠载入。将复合人工骨支架浸入细胞培养液,收集6h、12h,24h、48h及72h浸提液。采用四甲基偶氮唑盐比色法(MTT法)及Annexin-V FITC流式细胞定量分析法检测不同时期浸提液对骨巨细胞瘤基质细胞的增值影响及凋亡诱导作用。分析局部使用双膦酸盐的有效性。
采用局部灌洗法辅助治疗骨巨细胞瘤1例的观察：广州军区广州总医院1例左股骨下段骨巨细胞瘤复发患者,给予再次囊内病灶刮除骨水泥填充手术,术中采用唑来膦酸溶液(2mg/500ml)局部冲洗,术后48小时内给予病灶内唑来膦酸溶液(2mg/500ml)局部灌洗引流。观察患者一般情况,切口引流及愈合情况。术后定期病情复查。初步观察局部灌洗法可能存在的安全性。
通过双膦酸盐的临床新方法的初步应用观察,探索其不同应用方式的可行性,为进一步大样本临床应用和疗效观察提供指导。进一步促进骨巨细胞瘤的治疗效果。
[结果]
1)唑来膦酸诱导骨巨细胞瘤基质细胞向成骨细胞分化的浓度及时间相关性实验研究。
实验显示1μM唑来膦酸组中成骨分化相关基因表达最明显。48小时后1μM唑来膦酸组cbfa-1、osterix、osteocalcin的基因表达分别是对照组2.66倍、2.50倍及2.23倍,具有统计学显著性差异(P=-0.002,<0.001,<0.001)。此外,0.1μM唑来膦酸组cbfa-1基因表达是对照组1.6倍,具有显著性差异(P=0.003)。
在唑来膦酸诱导48小时后骨巨细胞瘤基质细胞ALP染色及BSP、Collagen Type I、Osteonectin、Osteocalcin免疫组化染色与对照组相比表现为轻度阳性或阳性。在诱导后24小时,诱导组cbfa-1的基因表达与对照组具有显著性差异(P<0.001)；在72小时监测点,诱导组与对照组osterix、osteocalcin的基因表达均具有显著性差异(P=0.001,0.003)；在168小时监测点,诱导组与对照组cbfa-1、osterix、osteocalcin的基因表达分别增高8.1,3.8及3.3倍,均具有显著性差异(P=0.002,0.002,0.002)。
2)唑来膦酸诱导骨巨细胞瘤基质细胞向成骨细胞分化的信号途径实验研究。
采用了Western印迹杂交检测ERK,JNK,p-ERK,p-JNK蛋白质在各组骨巨细胞瘤基质细胞内的表达。唑来膦酸组内可见ERK,JNK,p-ERK,p-JNK明显表达。同时加入两组阻断剂后JNK及ERK蛋白表达明显降低。
在处理72小时后,单纯1μM唑来膦酸诱导组组osteocalcin的基因与对照组及信号抑制组1,2,3均具有统计学显著性差异(P<0.001,=0.007,<0.001),cbfa-1基因与对照组及信号抑制组1,3具有统计学显著性差异(P).013,0.008)。唑来膦酸诱导组cbfa-1、osterix、osteocalcin的基因表达分别是SP600125+PD98059抑制组的5.01、3.54、3.39倍。Cbfa-1,osterix、osteocalcin的基因表达信号抑制组1,2,3与对照组无统计学显著性差异(P>0.05)。Osterix的基因表达中,1μM唑来膦酸诱导组与对照组无显著性差异(P=-0.213)；但与cbfa-1、osteocalcin存在显著性差异(P=0.048,0.005)。
3)双膦酸盐辅助治疗骨巨细胞瘤基质细胞新方式的可行性探索。
3D打印技术可制备个体化复合抗骨巨细胞瘤珊瑚羟基磷灰石／聚乳酸(CHA/PLA)人工骨。制备材料经过72小时、48小时、24小时、12小时及6小时后提取浸提液,在原代培养的骨巨细胞瘤基质细胞中予不同时期的复合唑来膦酸人工骨浸提液,48小时后,见细胞数量较阴性对照组减少,部分细胞形态发生变化,细胞外形发生皱缩,伪足减少,部分梭形基质细胞改变为圆形或椭圆形的形状,在早期的浸提液中最为明显。
采用四甲基偶氮唑盐比色法(MTT法)检测不同时期浸提液对骨巨细胞瘤基质细胞的增值影响,结果显示复合唑来膦酸人工骨浸提液对骨巨细胞瘤基质细胞抑制较阴性培养基对照组显著增强。48小时内早期高浓度唑来膦酸浸提液对骨巨细胞瘤基质细胞增殖具有明显抑制作用,与对照组浸提液有显著性差异(P<0.001,0.001,0.001,=0.002)。48小时后浸提液抑制作用减弱,并与前24小时有显著性差异(P=0.007,0.005,0.004)。复合唑来膦酸人工骨浸提液同样表现出很强的的诱导凋亡的能力,Annexin-V FITC凋亡染色及流式细胞仪定量分析结果显示对照组、72小时、48小时、24小时、12小时及6小时浸提液组原代培养的骨巨细胞瘤基质细胞凋亡占总数细胞的百分比均值依次为1.5%、2.05%、3.3%、5.23%、8.95%及12.79%。
局部唑来膦酸灌洗法预防骨巨细胞瘤复发安全性的病例观察：术中多功能引流管置于病灶周围。术后灌洗引流管引流通畅,总引流液约900m1。术后48小时拔出引流管。患者未发生流感样症状及肾功能异常。12天后手术切口拆线,愈合状况良好。唑来膦酸溶液局部灌洗治疗随访未发现明显相关并发症。
[结论]
1)双膦酸盐对骨巨细胞瘤基质细胞具有诱导凋亡及成骨分化的双重作用。其作用方式可能于药物浓度相关,低浓度唑来膦酸可能具有较强的骨巨细胞瘤基质细胞成骨诱导潜能,促进肿瘤细胞向正常细胞分化。在体外实验中,1μM浓度唑来膦酸具有最强的诱导分化能力。因此,维持低剂量唑来膦酸辅助治疗骨巨细胞瘤能够诱导残留肿瘤基质细胞分化,避免了骨巨细胞瘤手术中不必要的切除,促进病灶周围骨结构的强化或重建,减少术后骨折等并发症,还可能减少高浓度辅助用药的全身性副作用。
2)唑来膦酸诱导骨巨细胞瘤基质细胞成骨分化呈时间依赖性,诱导时间越久骨巨细胞瘤基质细胞成骨分化现象越明显。临床双膦酸盐类药物的使用较方便,可定期静脉或口服用药。在避免发生重大并发症的基础上,围手术期可维持低剂量唑来膦酸治疗,发挥持续抗肿瘤作用。辅助治疗时间越久,术后肿瘤复发率可能越低。
3) JNK和ERK信号通路在成骨细胞分化及生长增殖等多种生理过程发挥重要作用。实验证明唑来膦酸也可能激活JNK和ERK蛋白磷酸化,阻断JNK和ERK信号通路能够降低唑来膦酸诱导成骨相关基因的表达,因此唑来膦酸可能是通过JNK和ERK信号通路的激活来诱导骨巨细胞瘤基质细胞向成骨细胞分化。通过对骨巨细胞瘤基质细胞成骨分化机制的深入研究,为新型辅助用药的使用及提高成骨诱导效应提供新途径。
4)数字化复合抗骨巨细胞瘤珊瑚羟基磷灰石/聚乳酸(CHA/PLA)人工骨可为骨巨细胞瘤病灶清除术后的骨质缺损提供个性化支撑,其制作方便快捷。同时,唑来膦酸在洗脱支架周围局部释放,早期可提高局部药物浓度,对残留骨巨细胞瘤基质细胞发挥凋亡诱导作用。随着唑来膦酸药物降低,可持续发挥抗骨质吸收及促进肿瘤基质细胞成骨诱导作用,促进病灶内骨结构的强固或重建,减少骨折等并发症的发生率。局部使用数字化人工骨复合唑来膦酸为骨巨细胞瘤辅助治疗提供一种可能的有效治疗途径。
5)骨巨细胞瘤的复发给患者带来极大的痛苦。在1例骨巨细胞瘤复发后再次行病灶清除术,并且在囊内采用唑来膦酸溶液冲洗及留置唑来膦酸术后灌注方式,希望残留肿瘤细胞的清除更加可靠。这一局部辅助治疗方式既往未见报道。本病例观察见切口引流未见明显影响。患者未出现流感样症状及肾功能异常等系统性并发症。手术切口愈合良好。局部唑来膦酸灌注的使用方式,为骨巨细胞瘤辅助治疗提供一种可能的安全治疗手段,其有效性及安全性仍需要进一步的临床研究。
[Backgroud] Giant cell tumor of bone is one of the most common primary bone tumors. Most studies consider it as a kind of benign tumor derived from bone marrow mesenchymal cells. It is characterized as local bone destruction. Occasionally, rare cases of "benign" lung metastases also occur. Studies have shown that giant cell tumor is not sensitive to both traditional radiotherapy and chemotherapy. Surgical treatment is still the main treatment for giant cell tumor of bone. High recurrence rate is the biggest challenge in the treatment of giant cell tumor of bone. In order to completely remove the tumor lesions and reduce the recurrence rate, clinicians have tried to use a variety of chemical agents or physical methods as adjuvant therapies after the operation to remove the residual tumor cells. Intralegional curettage with high-speed blur and bone cement filling are now considered as the standard way of giant cell tumor of bone treatment. Later, bisphosphonates and RANK monoclonal antibody adjuvant therapy are thought to be new progresses in recent years in the treatment of giant cell tumor of bone. Diphosphonic acid was used as anti bone metabolic drugs. It has a similar structure as antiresorptive pyrophosphate in human body. In clinical studies, bisphosphonate application as adjuvant treatment for giant cell tumor of bone has achieved good results. In vitro study, researchers also found that bisphosphonates not only inhibits osteoclast-mediated bone resorption, delaying formation and maturation of osteoclasts, inducing osteoclast apoptosis, but also exerting a direct inhibition effect on tumor cells. We observed in previous study that zoledronic acid (a new generation of bisphosphonates) not only had direct effect on giant cell tumor stromal cells by inducing apoptosis, but also induced osteogenic differentiation of giant cell tumor stromal cells. This conclusion explains the clinical phenomenon that significant callus formation after bisphosphonates application as adjuvant treatment for giant cell tumor of bone. Ii may provide a posibility for reducing skeletal-related events. In the process of osteogenic induction of giant cell tumor stromal cells, the application concentration and time of bisphosphonate are important factors which may has important clinical significance. Through revealing the activation signaling pathways of bisphosphonate on giant cell tumor stromal cells, it may also promote its anti-tumor effect. In addition, clinicians tried different ways and means of adjuvant bisphosphonate therapy for giant cell tumor of bone. It is also important to find a safe and effective ways of adjvant administration.
[Object]
1) To investigate the optimum concentration and reaction time that zoledronic acid induces osteogenic differentiaon on giant cell tumor stromal cells.
2) To investigate the mechanism of giant cell tumor stromal cell osteogenic differentiaon induced by zoledronic acid.
3) To explore the feasibility of clinical noval ways that bisphosphonates applied as adjuvant therapy for giant cell tumor.
[Method]
1) Experimental study of the correlation of zoledronic acid concentration and reaction time with osteogeinc inducing effect of giant cell tumor stromal cells.
Experimental samples were taken four giant cell tumor patients from General Hospital of Guangzhou Military Command and Nanfang Hospital, Southern Medical University in November2011to January2013. One patient was male and the others were females. They aged from13to31years. The tumors were graded by Campanacci grading standards. There were three cases of grade II and one case of grade III. All patients had not received bisphosphonate adjuvant therapy before the operation. Postoperative pathological diagnosis was confirmed as giant cell tumor of bone. Primary cell cultures were got from resected fresh tissue of giant cell tumor. Purified giant cell tumor stromal cells were got after nine passages. The cultured giant cell tumor stromal cells were treated with different concentrations of zoledronic acid (0μM,0.01μM,0.1μM,1μM,5μM,30μM) in the culture medium. After72hours, qRT-PCR was used to detect cbfa-1, osterix, osteocalcin (osteogeinc related gene) expression of giant cell tumor of bone stromal cells. SPSS19.0statistical software was used. The values of cbfa-1, osterix, osteocalcin gene expression of each group were analysis with single-factor analysis of variance method(one-way ANOVA). Multiple comparisons between groups using LSD test. Then1μM zoledronic acid medium stromal cell culture was performed with the black control group. After48hours, ALP staining and BSP, Collagen Type I, Osteonectin, Osteocalcin immunostaining were given. Also at0hours,24hours,72hours and168hours after culture, qRT-PCR method was used to detect cbfa-1, osterix, osteocalcin gene expression in both groups. Then, analyse of the optimal concentration of zoledronic acid inducing osteogenic differentiation of giant cell tumor stromal cells.
2) Experimental study of signaling pathway the zoledronic acid inducing osteogenic differentiation of giant cell tumor stromal cells.
Primary cultured and purified giant cell tumor stromal cells were randomized to different groups. The control group, induction group and signal suppression group1,2,3. Control group with conventional medium, induction group with1μM concentration of zoledronic acid, signal suppression groups1and2with zoledronic acid and JNK inhibitor SP600125(1μM) or ERK signaling inhibitor PD98059 (20μM), suppression group3with both SP600125and PD98059. After30minutes treatment, Western blot was used to detecte the exsistance of p-JNK, expression of JNK, p-ERK, ERK proteins within each group of giant cell tumor stromal cells.
Use the same grouping and experiment method above. After72hours, qRT-PCR method was used to detect cbfa-1, osterix, osteocalcin gene expression. With the same statistical methods, analyse the possibility that bisphosphonates induced osteogenic differentiation through activation of JNK and ERK pathway.
3) Explore the feasibility of clinical noval ways that bisphosphonates applied as adjuvant therapy for giant cell tumor.
Preparation and properties of exploring of digitized composite anti GCT coralline hydroxyapatite/polylactic acid (CHA/PLA):Use computer-aided3D printing to architech a specific shape coralline hydroxyapatite/polylactic acid bone frame with CHA powder mixed with a certain proportion of polylactic acid (PLA). Then, zoledronic sodium was load on the bone scaffold by immersion and vacuum freeze drying. The composite artificial scaffold immersed in cell culture medium. The leaching solution was collected after6h,12h,24h,48h and72h. Using tetrazolium salt assay (MTT method) and Annexin-V FITC flow cytometry assay to analyse the impact of the leaching solution on proliferation and apoptosis of giant cell tumor stromal cells. Analyse the effectiveness of local use of bisphosphonates.
A case report of a giant cell tumor patient with local lavage of bisphosphonate:A patient of right femur giant cell tumor from Guangzhou General Hospital of Guangzhou Military Command was given intralegional curettage and cement filled. Intraoperative irrigation of zoledronic acid solution (2mg/500ml) was taken. The wound irrigation was given within48hours after the surgery with zoledronic acid solution (2mg/500ml). The patients was obscured with normal systemic condition, incision healing and drainage. Follow up was taken after surgery.
[Result]
1) Experimental study of the correlation of zoledronic acid concentration and reaction time with osteogeinc inducing effect of giant cell tumor stromal cells.
The experiment showed that1μM zoledronic acid group induced osteogenic differentiation most obviously. After72hours treatment, cbfa-1, osterix, osteocalcin gene expression in1μM zoledronic acid groups were2.66times,2.50times and2.23times more than the control group, with statistically significant difference (P=0.002,<0.001,<0.001). In addition, cbfa-1gene expression in0.1μM zoledronic acid group is1.6times more than the control group, with significant difference (P=0.003).
After48hours, ALP staining and BSP, Collagen Type I, Osteonectin, Osteocalcin immunohistochemical staining of the giant cell tumor stromal cells in the zoledronic acid group showed mild positive or positive compared with the control group. At24hours after treatment, cbfa-1gene expression of induction group are more than the control group with significant difference (P<0.001). At72hours, induction group and the control group osterix, osteocalcin in gene expression were significant differenct (P=0.001,0.003). At168hours, cbfa-1, osterix, osteocalcin gene expression in induction group has increased8.1,3.8and3.3times, respectively, compared with the control group, all with significant difference (P=0.002,0.002,0.002)
2) Experimental study of signaling pathway the zoledronic acid inducing osteogenic differentiation of giant cell tumor stromal cells.
Western blot was used to detecte ERK, JNK, p-ERK, p-JNK protein expression within each group of giant cell tumor stromal cells. In zoledronic acid group, ERK, JNK, p-ERK, p-JNK were likely to expressed significantly. After adding two blockers, JNK and ERK protein expression were significantly decreased.
After72hours, the1μM zoledronic acid group induced more quantity of osteocalcin, osterix, osteocalcin genes expression than the control and signal suppression group1,2,3, with statistically significant differences (P<0.001,=0.007,<0.001).1μM zoledronic acid group expressed5.01,3.54,3.39times more than SP600125+PD98059inhibiting group in cbfa-1, osterix, osteocalcin gene expression respectively. Signal suppression1,2,3group and the control group had no statistically significant difference in osterix, osteocalcin gene expression (P>0.05).1μM zoledronic acid group and the control group had no significant difference in cbfa-1gene expression (P=0.213), but existed significant difference in cbfa-1、 osteocalcin gene expression (P=0.048,0.005)
3) The feasibility of clinical noval ways that bisphosphonates applied as adjuvant therapy for giant cell tumor..
3D printing technology can produce individualized composite anti coralline hydroxyapatite/poly lactic acid (CHA/PLA) bone frame for giant cell tumor of bone. Leaching solution was prepared after72hours,48hours,24hours,12hours and6hours after the extraction. Primary cultured giant cell tumor stromal cells were incubated in different stages of the composite bone zoledronic acid leaching solution for48hours. We can see fewer cells than the negative control group, some changes in cell morphology, cell shape shrinkage occurs pseudopods was partly changed to spindle stromal cells round or oval shape in the early leaching most obvious solution.
Tetrazolium salt assay (MTT assay) was used to evaluate the impact of cell proliferation of the leaching solution of different stages on giant cell tumor stromal cells. The leaching solution of composite bone had more signicficant impact on cell proliferation of giant cell tumor stromal cells, compared to the control group. Early stage high concentrations zoledronic acid extracts inhibited cell proliferation of giant cell tumor of bone stromal significantly. Zoledronic acid composite artificial bone extract also showed a strong ability to induce apoptosis. Annexin-V FITC apoptosis staining and flow cytometry analysis showed that the mean percentage of apoptotic of tumor stromal cells were1.5%,2.05%,3.3%,5.23%,8.95%and12.79%in the control group,72hours,48hours,24hours12hours and6hours groups.
Local administration of zoledronic acid to prevent recurrence of giant cell tumor of bone:Intraoperative multifunctional drainage tube was placed around the lesion. Postoperative irrigation drainage tube patency, total drainage of about900ml. Pull out the drainage tube after48hours. Special discomfort did not occur in patients like influenza-like symptoms. The surgical incision healed well.
[Conclusion]
1) Low concentrations of zoledronic acid may have a strong giant cell tumor of bone stromal cells osteogenic differentiation potential. In vitro,1μM concentration of zoledronic acid expressed the strongest ability to induce differentiation. Therefore, maintaining a low dose adjuvant zoledronic acid therapy in giant cell tumor of bone can not only induce osteogenic differentiation of residual tumor stromal cells avoided unnecessary surgical resection, promote strengthening, reconstruct the bone structure around the lesion, reduce postoperative fractures but also reduce other complications and side effects.
2) Zoledronic acid-induced giant cell tumor of bone stromal cells into osteoblasts showed a time-dependent manner. The longer of the induction time, more obvious of giant cell tumor of bone stromal cells osteogeinc differentiation. Clinical use of bisphosphonate drugs is convenient. Without major complications, perioperative use of sustainable low-dose zoledronic acid as adjuvant therapy may play consistent anti-tumor effect. The longer the time of adjuvant therapy, the lower of tumor recurrence rate.
3) JNK and ERK signaling pathway is an important way of osteoblast differentiation. It is also possible that zoledronic acid caused protein phosphorylation further induced giant cell tumor of bone stromal cells to differentiate into osteoblasts through JNK and ERK activation. Its comprehensive mechanism still needs further study, and it is likely to promote the anti-tumor effect.
4) Digitized composite anti giant cell tumor of bone coralline hydroxyapatite/polylactic acid (CHA/PLA) bone can provide personalized support for the giant cell tumor of bone defect after operation. It can also be produced quickly and easily. Meanwhile, zoledronic acid elution stents locally release around. In early stage, it may improve the local drug concentration, play a role in apoptosis induction of residual bone giant cell tumor stromal cells. With the zoledronic acid concentration decreases, it may still reduce bone resorption, promote osteogenic role, rebuild the structure, reduce the incidence of fractures and other complications. Localized application of digital composite bone zoledronic acid may be an effective way for giant cell tumor adjuvant therapy.
5) Intralesional zoledronic acid solution rinse and postoperative zoledronic acid irrigation of giant cell tumor of bone. The surgical incision and drainage was likely to be normal. Patients had no systemic complication, such as flu-like symptoms. Surgical incision healed well. Local irrigation of zoledronic acid may be a safe adjuvant therapy method to reduce the recurrence of giant cell tumor of bone.
引文
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