Osteogenesis process of tricalcium phosphate ceramics in vivo

Vol. 13 No. 1Trans. Nonferrous Met. Soc. ChinaFeb. 2003Article ID: 1003 - 6326<2003>01 - 0065 - 04Osteogenesis process of tricalcium phosphate ceramics in vivoDAI Hong-lian(戴红莲), LI Shi pu(李世普), YAN Yu-hua(闫玉华)，LI Xiao-xi(李小溪), JIA Li(贾莉)(Biomaterials and Engineering Research Center，W uhan University of Technology, w uhan 430070，China)Abstract: To investigate the osteogenesis o[ calcium phosphate ceramics, β -TCP ceramics were implented into thecondyle femur of rabbits, and tetracycline was injected termly. Specimens were host at 1, 2, 3, 4, 5, 6 months af-ter implanted. The new bone formation and osteogenesis process were observed by the histomorphology, fluorescentmicroscope, SEM and EPMA. The results demonstrate that, osteogenesis is active, there are abundant osteoblastson the surface of osteoid, mesenchymal cell hyperplasia and incursion is found in materials after 1 month. After 2months, there is blood vessel formation and macrophage soakage within materials. Bone island appears and connectsby bone-bridge after 3 months. β - TCP ceramics degrade and are dispersed by new formation bone. Woven boneturns into bone lamella by rebuilding and calcification. The materials entirely change theit original shape and com-bines with bone tissue as a whole after 6 months. The typical structure of spongy bone forms. It is confirmed that β-TCP is a degradable biocompatible artificial boneimaterial which can incorporating in life.Key words: β - tricalcium phosphate; osteogenesis; fluorescence; biodegradationCLC number: R318. 08Document code: A1 INTRODUCTIONpatibility and biodegradable capability. In thiswork the process of bone tissue formation in por-Losses of bone substance for congenital, tuous β -TCP in vivo was studied in order to investi-moural or traumatic reasons need a long time treat-gate its osteogenesis.ment with bone graftsl. To avoid the drawbacksof autografts(blood loss, haematoma, pain, risk of2 EXPERIMENTALinfection) or heterografts ( rejection, infection, viral risk), various synthetic bone substitutes are2.1 Implant materialsnow proposed. Among them, calcium-phosphateIn order to obtain porous β - TCP ceramics,ceramics ( CPC) appear suitable ，since theirthe β -TCP powder(average size of 1 μm) preparedchemical composition is very close to the mineralby wet chemical precipitation method, was mixedphase of natural bone. Such materials have beenwith high-temperature binder and pore-formingapplied clinically as a bone substitute and shown tomaterial, foamed by the resin foaming method andbe biocompatible and good bioaffinity as well as os-sintered in air at 900C for 2h. The porosity wasteointegrativeld.4. When implanted in osseous40%- 50%，and the mean pore size ranged fromsites, these materials can bind directly with bonewithout an intervening fibrous layerf's6]. CPC,100 to 800 μm, the density was 1. 05 -2.00g●which are usually used for this purpose, are madecm~d and the average compression strength rangedbeta-tricalcium phosphate ( β- TCP ),from 15 to 30 MPa. The structure was found thathydroxyapatite or their mixture!1]. Though therethe erystalline grains are connected necked, and allis significant experience in application of CPC, thethe pores are interconnected. Cylindrical blocknature and mechanism of osteogenesis, namely thewith d 5 mmX 8 mm was implanted in bone defects.process of bone tissue formation on CPC in the bio-logical environment resulting in physicochemical2.2 Surgical procedureintegration of ceramics with living bone, are notTwenty-four New Zealand white rabbits, .still clear[0. Thus the problem of osteogenesisweighing 2.0- 2. 5 kg were divided into six groupsmust be studied in close connection with the ce-according to different stages of implantation. Ani-ramic state at different stages of implantation. It is mals under general anesthesia were subjected to awell known that日-TCP ceramics have better bio-standard operative procedure in the area of bothactivity than other CPC. It had excellent biocom-中国煤化工5mmX8mm) were .a Foundation iten: Pee1990470) sppred by the Vational Key FTYHCNMHGProgramofChineReelved date: 2001 - 12 - 06; Accepted dale; 2002- 01 -28Correspodence;Ll Shi-pu, Professor.Trans. Nonferrous Met. Soc. ChinaFeb. 2003created with a dental drill perpendicular to the longpeared in the center of masses(Fig. 1). Osteoidsaxis of the femoral shaft. Following press fit of theand thickened trabeculae of bone almost completelyceramic blocks were inserted into the defects. Eachflled the outside of ceramie pores after two .animal was implanted with two cylinders. In eachmonths. More deep pores were flled by wovengroup, one rabbit was not be implanted acted as abone and partially mineralized matrix. There wascontrol. One rabbit from each experiment group .blood vessel formation and macrophage soakageand control group were injected twice with 15%within materials, and the osteoid turned into la-tetracycline(30 mg" kg ) liquor before sacrificed.mella bone through calcification. After threeAfter1,2,3,4,5or6months,aswellasthemonths, thickened trabeculae and dense plates ofcontrol group， the implants and the surroundinglamellar bone were deterred in all pores of the ce-tissue were excised and placed into 10% bufferedramic block. The bone ingrowth patterns resem-formalin solution or 2. 5% buffered glutaraldehydebled Haversian canal structures instead of wovensolution.bone structures by rebuilding. Bone-island ap-peared and connected by bone -bridge. A great deal2.3 Histologic studyof medullary tissue has grown into the lacunae ofFor un-decalcified sections, six fixed speeci-the materials after four months. After six months,mens from each group were dehydrated in an etha-the residual materials are surrounded by bony tis-nol scries, and then embedded in methylmethacry-sue(Fig. 2). Prior to and during bone formation,late. Three specimens were cut into 5 μm sectionsthe number of osteoclast-like multinucleated giantin thickness perpendicular to the long axis of thecells decreased with the increase of already miner-specimen, and stained with toluidine blue. Thealized areas of the pore inner surface.others tetracycline labeled were cut into sections of10 μm to observe under fluorescent microscope.The rest specimens refrigerated in glutaraldehydesolution, was washed in an ultrasonic cleaner, de-hydrated in 95% alcohol and examined using SEMand electron probe X-ray microanalyzer.WB3 RESULTSAccording to the roentgenogram， the spaceIstechiast。2:50μbetween ceramics and bony tissue became vague af-ter one month. After five months, ceramics and"g1 New hone(NR) frmation a: periphery ofbony tissue were integrated and the external formceraria(TCP) and iregular wovenof the material became incomplete. After sixbon:(WB) Dalteru a. ler one monta imp antat onmonths, some part of the materials were disap-peared and the residual became to be fragments orparticles. After implantation, the implant insertedinto the medial cortex of rabbit's femur was tightlyunited with the host bone in all cases, without anysign of moverment at the interface. The portion ofthe porous β- TCP exposed outside of the cortexwas partially covered with new bone after onmonth.Light microscopy showed that, β - TCP has noearly adverse effects， inflammation and foreign250.mbody reaction. One month after transplantation,the interface was visible, there were abundant os-Fig.2 New bone(NB) showing osteon structureteoblasts on the surface of osteoid, mesenchymalforming bone trabecul(BT) and narowcell hyperplasia and incursion was found in materi-als. New bone that showed an irregular wovencavity(MC) after six months implantationbone structure appeared on the surfaces and inpores of implanted β -TCP. The new woven bone中国煤化工cervation indicatedoften formed masses in shape together with chon-that-uble fluorescencedrocytes and mesenchymal cells, lined by a layer ofbanCNMHGbone forming areosteoblast on their surfaces, and generally locatedfourHinterface be-at periphery of the masses. New capillaries ap-tween β -TCP ceramics and bone after one month.Vol. 13 No. 1()steogenesis process of tricalcium phosphate ceramics in vivo●67.As the time went on, β - TCP particles were surrounded by fluorescence which became moremarkedly. and fluorescence appeared clearly amongβ- TCP particles in the center of defect section. β-TCP ceramics degraded and were dispersed by newformation bone(Fig. 3). The great mass of materi-Borsais was substituted by bone tissue. While at thesame period，the control group did not appear fluo-rescent phenomenon.BoNGFig, 4 EPMA pho:ograph cf mate;ial'sgrarule after six mcr.th mplanle tion阳TCF2S0AmFig. 3 Double fluorescent cycle directly embracedβ -TC particles in center of defect areaafter six months implantation,SEM morphology demonstrated that, the in.terface among particles changed obviously after im-plantation. The original connection among parti-cles broke up. The particles were separated andFig.5 SEM photograph oftheir external form became irregular or incom-new bone tissue directly connect with β -TCPplete. Five months after implantation, we foundceramics after six month implantationthat the crystal granules of material have alreadyaltered their formation under EPMA. Unlike regu-4 DISCUSSIONlar crystal shape these granules' pointedness hadied away and their edge has been softened. Mean-while the material's granules began to get touchThe results showed that the synthetic porous βwith the bone firmly(Fig. 4) and life element e--TCP ceramics are capable of inducing osteogene-merged inside the material(point a) such as C, N,sis.‘ This ability depends on biodegradation andS and so on(Table 1). New bone formation bind-bioactivity of the ceramic, which are improved bying with grafted material closely increases withlttle crystal size， low-temperature sintering,time(Fig. 5). The materials entrely lost their orig-phase composition, porous structure and the addi-inal shape and combined with bone tissue as ation of binder. The biodegradation process iwhole after six months. Some particles could becaused by the action of living systems. The dissolu-found in the Haversian system of new bone. Thetion process in body fluid and the absorptiontypical structure of spongy bone formed.process mediated by cells. On one hand, the parti-cles of β -TCP ceramics can be dissolved continu-Table 1 FElement analysis of point by EDXAously or be degraded by cells(e. g. phagocytes orElementMassMoleosteoclasts)which stretch out tiny processes tofraction/ % ;fraction/%wrap the grains and phagocytize them into their()28. 6146. 98body，then phagolysosome in plasma releaseNa1. 221. 394g0.740.80hydrolytize enzymes to the particulates and makep25. 6321. 75:he |中国煤化工-ial circumstance as0.320. 26ceramics to be de-:a43. 1728. 3graHCN M H Ge acid products of0. 310.52.metadonsm(e. g. lactate and cltrate) and acid hy-Total100drolytic enzymes in tissue fluid, parts of implanta-●68.Trans. Nonferrous Met. Soc. ChinaFeb. 2003tion arca become acidulous and it will promote thedegradation process of p -TCP.5 CONCLUSIONSThe products ( including calcium ions andphosphate ions), which are degraded from the im-Histological specificities of bone formation andplanted materials, enter blood circulation irmmedi-biodegradation of β -TCP ceramics are found, andately and take part in the matabolism of organs andporous β - TCP ceramics can serve as a flling mate-tissues and are excreted with urine and feces. Mostrial for bone defects and can facilitate new bones toof them are stored in the calcium-pool of body andgrow into its pores and defective area. It not onlyare reused by bony tissue to form new bone.can play the role of bracket for bone growing butTetracycline that is an innocuous and non-ra-also can degrade and afford osteoblasts enough cal-dioactive fluorescence dyestuff can enter into newcium and phosphate ions. So it will induce the newbone tissue selectively, and combine with the calci-bone growing, incorporate in the process of boneum of the hydroxyapatite(HA) in new bone tissuetissue forming, make full use of bone conduction forby forming steady chelating-ligand. With excitat-the repair of bone defects, finally integrate with theeded by a certain wavelength ultraviolet radiation,host bone and become a portion of the organic newthe un decalcified sections can appear olivine fluo-bone. Thus β-TCP ceramic is a degradable biocom-rescence, which reveals the growth position of newpatible artificial bone material which has the ability ofbone tissue. The results of tetracyeline -labeled ex-inducing osteogenesis. In a word, β -TCP ceramic is aperiment further show that osteogensis is very ac-unique substitute material for bone transplantationtive, and β-TCP ceramics are degraded and dis-with wide application perspective.persed by new formation bone. 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