The Development And Empirical Study Of The Composite Active Antitumor Coral Hydroxyapatite(CCHA) Artificial Bone

There are two most common issues in surgery treatments of bone tumors: repair ofbone defects and local recurrence of bone tumors after resections. To produce acomplexed material which can not only fill bone defects but also suppress tumorrecurrence is of great importance in bone tumor treatment. In recent years, majoradvances have been made in the development of novel matrials such as ceramics andpolymers to be used for the delivery of drugs to a local region of pathology. Theadvantages of such system are twofold: the ability to deliver the concentrations for arelatively long period of time, whilst minimizing the concentrations of the drug in thebloodstream and other organs and potential side effects produced by systemicadministration. In the last decade or so, crystals of calcium-phosphate have received agood deal of attentions as a delivery system because of their physical and chemicalproperties, high surface interaction properties, and their biocompatibility. In thisresearch, we use the coral hydroxyapatite(CHA) made by hydrothermal exchange inour laboratory.We used cis-diamminedichloroplatinum(Ⅱ) (CDDP) as the drug of choice in theseexperiments because it has been found to be an effective anti-cancer drug, widelyused despite the large number of analogs developed. The mechanism of its biologicalactivity has been investigated in detail as has its thermodynamic behavior in solutionin vitro.In this research, we used coral hydroxyapatite(CHA) as drug deliver system toproduce CDDP-compounded CHA(CCHA), and test its ability in tumor suppressionand effects on bone defects repair.The prepare and delayed released study of CHA-bound CDDP METHOD: CDDP was dissolved in phosphate buffer (PB) by rapid mixing in aVortex apparatus for 5 to 10 min just prior to their use in adsorption studies. Samplesof CHA crystals were dispersed in 0.6mg/ml CDDP-PB solution in 1.5ml capacityconical polyethylene tubes by rapid mixing in a Vortex apparatus for 30s, and thennegative pressure standing for 24 hours. The CHA samples were then cryodesiccatedunder subambient temperature. Because of the very limited solubility of CDDP inaqueous solutions, we dissolved CDDP in DMSO(0.5g/0.2g/0.1g in 1ml), andprepared the CDDP-bound CHA as mentioned above. The uptake of CDDP by CHAcrystals was determined by scanning electron microscopy and spectrum analysis.In-vitro test of sustained release of Pt(CDD) from the implant of CHA complexedwith 20% (w/w) CDDP. Each implant of CHA-CDDP complex and CHA was kept in50ml of 10mmol/L phosphate buffer (PH 7.4) in a chamber maintained at 37℃and30 rpm, and sustained releasee was tested. Ten ml of the phosphate buffer wascollected every two days for 12 weeks, and the platinum concentration in each samplewas determined, using a high performance liquid chromatography, in order tocalculate the releasee of CDDP from the implant. After each sampling, the phosphatebuffer was replenished (10ml). The cumulateive sustained releasee ratio and sustainedrelease rates were determined. In-vivo test of sustained release of Pt(CDDP) from theimplant of CHA complexed with 20% (w/w) CDDP. Sodium pentobarbital (3% w/w,30mg/kg) was given celiacly to the implant group. The SD rat was fixed on a surgicalplatform and the skin of back was shaved. The skin was sterilized with iodophors and75% alcohol. A longitudinal incision of about 2cm was made, and then thesarolemma was separated. The implants, containing 20% (w/w) CDDP wereimplanted manually in the muscle sack of back. 3 rats were killed each 2 weeks for 12weeks. In the implant group, Muscle tissue was collected from a site 1cm around theimplant and from a site 1cm to 2cm from the implant. The abdomens of animals werelongitudeinally incised after the muscle tissue had been collected, and small sliceswere removed from the kidneys, liver and myocardium. The Pt concentrateions inthese samples were determined using HPLC.RESULT: 1.CDDP were well-distributed in CHA pores.2.High performance liquidchromatography(HPLC) showed that the retention times of cisplatin and internal standard are 4.6min and 7.5min respectively. 3. In the first two weeks of in vitrodesorption study, CDDP release rapidly from CDDP-CHA complex, the concentrationof CDDP in leaching liquor is extremely high and still have 134.54±9.88μg/ml after12 weeks.4.After implantation into SD rats, CDDP-CHA complexes produced highCDDP concentration in local regions. With time passing and distance increasing, theconcentrations of CDDP decreased. 5. When implantated into SD rats, theconcentration of CDDP in blood stream remained at a low level, less than 17ug/ml,much lower than that in local tissue. 6. CDDP-CHA complex implantation has noharmful effects on all other organs, showed by histological sectionAnti-tumor study of CCHA in vitroObjective: To evaluate the inhibitory ability of self-made composite anti-tumor coralhydroxyapatite(CCHA) on human tumors in vitro. Methods The coralhydroxyapatite(CHA) was made by hydrothermal exchange. Cisplatin wasimpregnated into CHA by vacuum freeze-drying techniques. The leaching liquor ofCCHA was collected at different intervals during 8 weeks. Then the inhibitory effectsof the leaching liquor on primary culture cells of human breast cancer osseousmetastasis and human giant cell tumor of bone, human high-metastatic lung cancercells SPCA-1, human osseous metastasis prostatic carcinoma cells PC-3 and humanhigh-metastatic colorectal cancer cells LOVO were examined in vitro. ResultElectron microscope showed cisplatin were well-distributed in the pores of CHA.The inhibition ratios of the leaching liquor on all the tumors tested were more than50%, except for PC-3, which is 29.92% when treated with leaching liquor collected atthe eighth week, and primary cultured GCT cells, which are less sensitive to theleaching liquor than all other cell lines. Conclusions The CCHA had good sustainedreleasee function. It keeps excellent inhibitory ratio on human tumor cell for 8 weeksin vitro.Anti-tumor study of CCHA in vivoObjective: To evaluate anti-tumor ability of CCHA in vivo. METHOD: Primaryculture of human breast cancer osseous metastasis, primary culture of human giantcell tumor of bone, cell lines of SPCA-1, PC-3 and LOVO were all inoculated inathymic mouse. 10 days after the new tumors were formed, CCHAs and CHA(control) were put into the tumor nodus of the athymic mouse, and watched for another 10 days,Finally, the athymic mouse were killed and tumor tissues as well as hearts, livers,kidneys, lungs were made into sections and eosin-stained to evaluate the effects of theinoculated CCHA on tumors and organs. RESULT: The ability for each tumor celllines to form new tumors in athymic mouse is different. MDA-MB-231 failed to formany new tumors, GCT cells' ability to form new tumors is lower than other cell lines.For all the newly formed tumors in athymic mouse, inoculated CCHAs helped toinhibit the growth of tumor cells, induce necrosis of tumor tissues and made thetumor nodus become smaller compared to that inoculated with CHA.Osteogenesis study of CCHA in vivoBecause of the anti-tumor drugs complexed in CHA, when implanted into bodies,CCHA will kill normal tissue cells as well as tumor cells. This will definitely interferethe repair of bone defects because the growth of osteoblasts were severely inhibitedby CCHA. Objective: To investigate the degradation of self-made CCHA and itseffect on bone conduction and bone growth. Method: Implants ofCCHA(20%CDDP-CHA w/w)and CHA(control,0% CDDP w/w) were implanted intoholes of the metaphysic of the rabbit femur. X-rays and Decalcifyed histological sectionof rabbit femoral bone with hematoxylin and eosin stain were used regularly to investigatethe degradation of CCHA and CHA, and how bone tissues grow in the defect regionin 30 weeks. Result: After implantation, CHA crystals are faster than CCHA inconnecting with surrounding bone tissues and forming bone bridges. The borderlinesof implanted CHA became obscure in 4 weeks. Loose connective tissues were foundin pores of the CHA and osteoblasts were growing on the surface. Bone tissues ofthe surrounding area gradually grow into the CHA, finally repair the bone defects. Atthe beginning of implantation, CCHA mainly inhibited the growth of surroundingtissues until 6～12 weeks later, normal bone tissues gradually grew into pores ofCCHAs, and healed bone defects at the weeks of 26. At the 28th week, the x-raypictures of implanted CCHA looked the same as that of CHA.