| [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. |
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