Tracing process of β-TCP ceramics in vivo with 45Ca

Vol. 14 No. 1Trans. Nonferrous Met. Soc. ChinaFeb. 2004Article ID: 1003 - 6326(2004)01 - 0020 - 05Tracing process of -TCP ceramics in vivo with $sCaLI Shi-pu(李世普)', DAI Hong-lian( 戴红莲)', YAN Yu-hua(闫玉华)'，WANG Xin-yu(王欣宇)，XIONG Xian-li( 熊先立)'，ZHENG Qi-xin( 郑启新)”，DU Jing-yuan(杜靖远)(1. Biomedical Materials and Engineering Research Center, W uhan University of Technology,W uhan 430070, China;2. Union Hospital, Tongji Medical College, Huazhong University of Science and Technology ,W uhan 430022, China)Abstract: The metabolic way of calcium ions which was released due to in vivo degradation of porous β TCP ceram-ics was studied by using the radioisotope “sCa as tracer. The result shows that some of the calcium ions enter bloodand take part in the circulation. These calcium ions distribute into organs and tissues (such as liver, kidney， brain,heart, lung， spleen and stomach) and participate in the metabolisms of body. There is neither the accumulation ofcalcium ions, nor the lesion or pathologic calcification of the organs and tissues. Some of the calcium ions that enterthe near end femur, ulna and skull are reused by bony tissue to take part in both local mineralization processes dur-ing bone healing, or are stored in calcium pool which can participate in the whole body circulating. In the cyclicalprocess, other calcium ions are excreted with urine and feces through kidney and liver. It is indicated that the degra-dation products of β TCP ceramics can take part in the physiological metabolic process of normal bone and tissue.Key words: B TCP ceramics; 45 Ca tracing; metabolism; mineralizationCLC number: R318. 08Document code: A1 INTRODUCTIONcalcium is more suitable than phosphate and oxy-gen[8]. The method of atom tracing was used inFor a long time, bone substance losing due to .this study. Calcium has various isotopes, in whichcongenital, tumorous or traumatic has been treatedthe half-life of 41Ca is 8X 104 a, the half-life of 45Cawith bone grafts. In order to avoid the drawbacksis 165d, and the half-life of 47Ca is 45 d. The re-of autografts ( blood loss， haematoma， pain， risksults of previous animal experiments showed thatof infection) or heterografts ( rejection，infection,β TCP ceramics began to degrade after being im-viral risk) ，various synthetic bone substitutes areplanted in vivo, and almost all of materials degrad-now being proposed, in which, calcium- phosphateed after 20 weeks. Hence, 45Ca is comparatively anceramics (CPC) appears to be suitable，since theirappropriate tracer. The β TCP ceramics labeled bychemical composition is very close to the mineral“5Ca was placed in the femoral condyle of rabbits.phase of natural bone. These materials have beenThe excreta， organ and tissue of animals and theproved to be well biocompatible, non- inflammatorysurplus materials were collected at intervals afterand non-variable1.2]. Being implanted in osseousimplantation，and then the radioactivity was test-sites，these materials can directly contact withed. Sequentially, the distribution，metabolism andbony tissue without fibrous interlayer-3]. The most .reuse process of the degraded products were esti-typical CPC is β tricalcium phosphate (β TCP) cemated.ramic that exhibits excellent biodegradable proper-ty. It can serve as bracket for new bone formation2 MATERIALS AND METHODSand play the role of osteoconduction'[4,5]， But it isstill not known whether calcium ions involved inthe mineralization process of new-formed extracel-2.1 Materials preparation and propertylular bone matrixes are provided by the blood flow .In order to obtain porous β TCP ceramics,or by released ions from the resorbing implant.high-purity β TCP micro-powder was mixed withThe metabolic way of the degradation products ispore- forming material and high-temperature binderalso not clarified6.7]. β TCP ceramic consists of the .rnnsisted, nf_ CaO and P2O3，thenelements of calcium，phosphate and oxygen, whichfoar中国煤化工ming method and sin-have multiple isotopes. As far as half-life， radioac-tereMHC N M H Gorous β TCP ceramicstivity intension and ray energy are concerned, thehas 50% porosity and exhibits interconnecting①Foundation item: Project(G1999064701) supported by the National Key Fundamental Research and Development Program of ChinaReceived _date: 2003 - 03 - 03; Accepted date: 2003 -06 -02Correspondeae.摘Shi- pu, Professor; Tel: + 86-27-87651852; E mail: Lisp@ public. wh. hb. cnVol. 14 No. 1Tracing process of β-TCP ceramics in vivo with *5Ca●21.pores with a mean pore size of 240 - 510 μm. The β-2 d at 100 C，and evaporated to dryness in the-TCP ceramics, with a density of 1.05 - 2.00 g/balneum arenae at 280 C after being digested com-cm3 exhibits compression strength of 15 - 30 MPa.pletely. After slight cooling of the samples， 1 mLThe sintered β TCP structure was formed by neckglycerine (100 'C) and 6mL ethanol solution of N-connecting at grain boundaries. The grain size is a-N-dimethylformamide( volume ratio 1 : 3) werebout 2 - 3 μm. X-ray results show, and that thedissolved. The samples were displaced into themain crystal phase of the ceramic is βCa3 (PO)z，scintillation cup, in which 10 mL TPP3 scintilla-containing a few other calcium phosphate crystaltion liquor was added. The cycle time per minutephases and amorphous phase. Cylindrical implants(cpm) was measured using the scintillation counterof 8 mm in height and 5 mm in diameter were made(FJ21084，double canal and combination， China)and sterilized by high-pressure steam,after mixing uniformly. The efficiency curve wasplotted and revised by using the external standard2.2 Preparation of 45 Ca labeled B-TCPmethod, The decay time per minute(d) values wereSamples of porous β TCP ceramics were madecalculated referring to the formula of d=卡(E is effi-radioactive by thermal neutron bombardment ,which was produced by the nuclear reactor of theciency). The decay of radioactivity was adjusted refer-Atomic Energy Scientific Research Institute of Chi-ring to the formula of A。=-_AThe formulana. The radioactivity of per gram material was10.12 MBq before implantation.of1一( Being一implanted一material)X 100%2.3 Implantation experiments(Not- being- implanted- material)Ten New Zealand white rabbits in both sexeswas used to count the d value of implantation mate-( provided by Animal Experiment Center of Hubeirial. Consequently， the reduction rate of radioac-Province)，weighing 2- 2.5 kg，were divided intotivity that could reflect the degradation rate of ma-five groups according to different stages of implan-terial was worked out.tation. The animals were subjected to a standardThe blood, urine and feces of the rabbits wereoperative procedure in the areas of both condyle fe-respectively collected and the radioactivity wasmurs under general anesthesia. Bone defects of 5tested at 3，6，10, 16，30，77，107，126 and 158mm in diameter and 8 mm in height in the tibiaed. The decay time of nucleus per minute wascondyle for implanting ceramics samples were crea-shown as d.ted at low speed under an asepsis condition. Thecavity orientation including spongy and compact2.5 A utoradiograph of liver and kidney tissuebone was perpendicular to the longitudinal and sag-At 1 and 3 months the liver and kidney tissuettal axis of the tibia. After being washed andof animals were excised, the frozen sections werecleaned with normal saline， the cylindrical β- TCPprepared. The sections (6 μm) were placed on mi-ceramics were press fit inserted into the defects, .croscope slide，then were dried in a desiccator, inand each condyle received an implant. The cylinderwhich P2O3 was used for 48 h. The sections werewas lower than the surface of bony tissue. Afterfixed with anhydrous ethanol vapour， smeared byinstallation，each implant was covered with itsusing maskant of 5% collodion before being shiftedperiosteal flap，and then the incision was stratifiedto darkroom. The emulsoid (Nucleus-4) was uni-closed. Each animal was maintained in a separateformly spread on the tissue slices. The slices werecage and allowed full weight- bearing.exposed, developed and fixed after natural drying,stained with HE and observed by optical micro-2.4 Radioactivity testingscope. .After 2, 4, 8, 12 and 20 weeks，the animalswere sacrificed with CO2 asphyxiating. The wholeRESLUTSimplanted material, together with organ and tissueof animals such as near-end femur， ulna， skull,3.1 Radioactivity of *Ca in organic tissueliver, kidney， brain， heart， lung and stomachRadioactivity could be tested in the organicwere taken out， and then the radioactivity wastisst中国煤化工brain， heart, spleen,tested respectively. 0. 25 g tissue of each organ andstonter implantation. Thisthe whole implant were respectively placed in theshovYHC N M H Gwhich were degradedground alimsntary test-tube with stopper. Eachfrom the implanted materials, were metabolized intube had 2 - 3 mL Perie's liquor， which was satu-various organs by force of blood circulation. Therated with magnesium nitrate(O. 5-0.6 g/L), andradioactivity variation in different organic tissuewas made of 60% nitric acid and one third of per-during 4，8 and 12 weeks was not significant. Alchloric ach万豹据samples were water -bathed for 1ter 20 weeks，the radioactivity in organic tissuesTrans. Nonferrous Met. Soc. ChinaFeb. 2004was lower than that of 2 weeks, which indicatedthat the calcium ions of degradation product were60 |not collected in all of organs (Figs. 1 and 2).1- UIna50.- Skull .3.0-一Femur●一Kidney. Brain▲一Liver302.0 t201.5101.0 F48216240.5Time/weekFig.3 RA in different bone tissues1263.0 [Fig.1 RA in tissue of kidney, brain and liver◆一Feces ..5 fUrine-Blood3.5三2.0 t3.0Stomach1.5 t2.5■一Splcen出1.0个2.01.5 I0.5 t40120160Time/d020 24Fig.4 RA in feces, urine and bloodsulted in the increasing of concentration of calciumFig.2 RA in tissue of lung. stomach,ions. Around 3 months, the radioactivity of "Caheart and spleenreached the ma ximal value in blood and feces, andthe radioactivity in urine was lower than that at 10The radioactivity of calcium in the tissue ofand 16d. But it was higher than that at 2 and 4near- end femur, ulna and skull was tested at 2months. This showed that the degradation rateweeks. But the radioactivity values in the near end，was the ma ximum in this period. The implants stillfemur and the skull were lower than those in all ofdegraded 3 months after implantation, but the deg-organs. The radioactivity gradually increased withradation rate was relatively reduced because sometime. The radioactivity in skull arrived to the max-of implants have been degraded.imum peak at 8 weeks. The radioactivity values inthe near-end femur and the skull were tenfold or3.3 Radioactivity of *Ca in implantstens-fold higher at 8 weeks than that at 2 weeks.The radioactivity in the implants was gradual-The value in bone tissue was far higher than thosely reduced with time. Compared to the material be-in other organs. Moreover, the radioactivity in thefore implantation, the reduction rate of radioactivi-near- end femur and skull was higher than the onesty was 5. 90% at 4 weeks, 32. 5% at 8 weeks,in ulna after 12 weeks(Fig. 3). It is shown that the45.67% at 12 weeks and 58. 16% at 20 weeks, re-caleium ions of degradation product were collectedspectively. This indicated that B- TCP ceramics de-in bony tissue.grad中国煤化工CNMHG3.2 Radioactivity of *Ca in blood and excreta3.4Aduraulograp UL lver and kidney tissue“Ca could be tested in blood, urine and fecesA few of silver particles were found to distrib-3 d after implantation. Their radioactivity general-ute in extracellular space and blood vessel of liverly increased. with time. This is indicated that theand kidney, ductuli hepaticus communis or urim-degradati5h7#据of implants increased, which re-ferous tubules. This reflected the distribution ofVol. 14 No. 1Tracing process of β-TCP ceramics in vivo with *5Ca●23●calcium in tissues after degradation of β-TCP. Themineral of bone matrix， are CaO and P2O3. Asnumber and distribution of silver particles at 12shown in Figs. 1 and 2, the radioactivity in organicweeks were the same as that at 4 weeks. I hetissue firstly reduced, and then increased gradual-structures of liver and kidney tissue were normal,ly. Moreover, 45Ca was found in blood, urine andwithout damage and calcification (Figs. 5 and 6).. feces in a short time. T his proved that calcium ionshave already released from the material due to deg-radation. The initial degradation took place on thesurface and in the pore of material. It began withthe dissolution of the binder. The radioactivity inimplants gradually reduced with the implantationtime. The reduction rate was 58. 16% at 20weeks. As an important index of degradation ob-servation，the reduction rate of radioactivity reflec-ted that β- TCP ceramic evidently degraded， athough it incompletely corresponded with the deg-radation rate of material[9].In our research, we found that βTCP ceram-ics degraded in vivo in two ways: the dissolutionprocess in body fluid and the degradation processmediated by cells. The degradation products wereFig. 5Tissue structure of liver and distributioncalcium and phosphate ionsh0.' The above resultsof silver grain 12 weeks after implantationshowed that the calcium ions released from the materials due to degradation entered blood and tookpart in the circulation immediately. They were dis-tributed into organs and tissues to participate inthe metabolism. Some of the calcium ions were ex-creted with urine and feces through kidney and liv-er. The radioactivity of "Ca was continuously test-ed in urine and feces after implantation. This indi-cated that the excretion was going on in successionas the material continually degraded.Aftermonths, the radioactivity of“Ca in blood, urineand feces did not increase continuously but reducedgradually. This showed that the metabolism of4Ca was not an amount of accumulation, but onlya procedure. Therefore， the degradation productsof β TCP ceramics took part in circulation and me-Fig.6‘Tissue structure of kidney and distributiontabolism by force of blood, and were excreted withurine and feces. There was no forming of accumu-lation.4 DISCUSSIONThe distribution of calcium ions that producedby degradation was homogeneous in organs and tis-The isotopic tracing is extensively used in bio-sue. More calcium ions entered organs such as liver .logic research because it has high sensitivity，sim-and kidney due to the great amount of blood flow，ple operation and is almost close to normal physio-so the calcium concentration was higher in theselogic biochemistry condition of living beings. Irorgans. The results of autoradiograph showed thatthis study, β-TCP ceramics that were made radio-calcium ions were generally in the blood vessel,active by nuclear activation were placed in the fem-extracellular enzyme and tissue space. The calcifi-oral condyle of rabbits and monitored for 20 .cation and structural damage of organs were notweeks. By force of the technique of isotopic trac-ob中国煤化radioactivity in organsing，the degradation process of β TCP ceramics andredutestified that the deg-the metabolism process of the degradation productsradaMYHC N M H G accumulated. On thein vivo were investigated scientifically and dynami-contrary, the radioactivity of 4Ca in bony tissue,cally by testing the radioactivity of 45Ca. The por-especially in skull， increased rapidly with time.ous β TCP ceramics used in this experiment consis-The ability of new bone formation was most vigor-ted of βCa3(PQ2)z and calcium phosphate solubleous in bony tissue, which is far higher than that inglasses. 0s h节components, which resemble theother organs. This indicated that most of the calci-●24●Trans. Nonferrous Met. Soc. ChinaFeb. 2004um ions degraded from β TCP ceramics were storedscopic evaluationbone implant contact betweenin the calcium-pool of body and reused by bony tis-hydroxyapatite, bioactive glass and tricalcium phos-phate implanted in sheep diaphyseal dects[J]. Bio-sue to form new bone. Metsger et al11]and Irriga-materials, 1995，16: 1175- 1179.ray et al42J found that the calcium ions that degrad-[2]BegleyC T, Doherty M J, MollanR A B, et al. Com-ed from the calcium phosphate ceramics implantedparative study of the bioceramic, coral and processedin bone were precipitated in the implantation regionbone graft substitutes [J ]. Biomaterials， 1995， 16:and reused by new bone，but were not used by the1181 - 1185.mature host bone tissue.[3] Basle M F, Rebel A，Grizon F, et al. Cellular respinseTherefore it could be seen that not all of theto calcium phosphate ceramics implanted in rabbit bonecalcium ions released from materials took part in[J]. J Mater Sci, 1993, 2: 273 - 280.the new bone formation. Some calcium ions par-[4] Koerten H K, Van der Meulen J. Degradation of calci-um phosphate ceramics [J ]. J Biomed Mater Res,took in local mineralization process， some ones1999, 47: 78- 86.went into the circulation and participated in the cir-[5] Malard O, Bouler J M, GuicheuxJ, et al. Influence ofculation of calcium pool, and other ones participa-biphasic calcium phosphate granulometry on bone in-ted in the metabolism of body and discharged withgrowth, ceramic resorption， and inflammatory reac-the excretion. The degradable calcium phosphatetions: Preliminary in vitro and in vivo study[J]. J Bi-omed Mater Res, 1999, 46:103- 111.materials that were used as bone substitutes repre-[6]Schwartz Z，Boyan B D. Underlying mechanisms atsent a store, which was probably involved in both .the bone- biomaterial interface[J]. J Cell Biochem,local mineralization process during bone healing1994，56: 340 - 347.and circulation of calcium pool，just like physiolog-[7] LU Jian-xi , Descamps M, Dejou J, et al. The biode-ical bone mineral. This showed that the lifeless de-gration mechanism of calcium phosphate biomaterialsgradable calcium phosphate ceramics could be incor-in bone[J]. J Biomed Mater Res，2002， 63: 408412.porated in the organic tissue activity by force of degra-[8] REN Shi-ren. Radioactive Nucleus Technology in Biol-dationthe normal physiologic metabolic process.ogy[M]. Beijing: Beiing University Press, 2002. 23 -45. (in Chinese)5 CONCLUSIONS[9]Den Hollander W，Patke P. Macroporous calciumphosphate ceramics for bone substitution: A tracerThe calcium ions that released from βTCP ce-study on biodegradation with “Ca tracer[J]. Biomate-rials, 1991. 12: 569 - 573.ramic enter the blood immediately after implanta-tion. They distribute in all organs and take part in[10] Chen F, LiSP, Yan Y H, et al. Effect of TCP material on pH value inside and outside phagocytes by u-the metabolism of body. Some ones are excretedsing nanometric microelectrode [ . J]. Biocermics,with urine and feces. There are no damages of or-1996，9: 209 -212.ganic tissue and pathologic calcification. Other[11] Metsger DS, DephilipR M, Hayes T G. An autro-ones are stored in the calcium pool of body，andradiographic study of calcium phosphate ceramic boneare used to take part in the local mineralization ofimplants in turkeys[J]. Clin Orthop, 1993， 291: 283- 294.new bone in implanted area or the metabolism of[12] Irrigaray J L, Oudadesse J P, Terver S, et al. Com-host bony tissue, then constitute a part of body.parison of the ossification kinetics after implantationof a radioactivated coral and a natural coral[J]. J Ma-REFERENCESter Sci, 1995， 6: 230 - 234.(Edited by PENG Chao-qun)[1] Gao T J, Lindholm T S, Kommonen B, et al. Micro-中国煤化工MHCNMHG.