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Kor J Oral Maxillofac Pathol 2006 ; 30(3) : 173-182 수산화인회석과 anorganic bovine bone mineral 에서배양된정상인조골세포의생체적합성연구 윤병만, 천재식 *, 오충훈 단국대학교치과대학구강생리학교실 ABSTRACT A Study about Biocompatibility of Normal Human Osteoblast Cultured on Hydroxy Apatite and Anorganic Bovine Bone Mineral Byeong Man Yoon, Gae Sig Chun*, Chung Hun Oh Department of Oral Physiology, Colloge of Dentistry, Dankook University This experiment was performed to study the biocompatibility of xenograft materials (ABBM. coralline HA). Both autogenous bone grafts and allogenic banked bone were frequently and successfully used to promote regeneration of parts of skeleton. The use of these types of grafts were limited by the cost of donor site operation for autogenous boneor by fear of the risk of infection of allogenic materials. Another type of graft is xenograft which include ABBM and coralline HA. For investigating the biocompatibility, generally many investigators used cancer cell lines or animal cell lines. But cancer cell lines and animal cell lines had functioned different metabolism from normal human cell. So the experiment used normal human osteoblast for compare the biocompatibility of ABBM with coralline HA which were fixed in 24 well base contained culture medium. After 1st, 3rd, 7th, 14th, 28th s, the culture medium were taken out and checked the concentrations ofcalcium(), inorganic phosphate() and alkaline phosphatase(). In another method, histologic samples were investigated after 8weeks of xenograft materials implantated on rabbit's tibia, the bone was cut and made undecalcified ground samples and checked with fluorecent microscope, polarizing microscope, reflection electron microscope and electron probe microanalysis. The statistical results of concentrations (,, ) of materials in the culture medium have decreasedby 's, which meant that xenograft materials were effective for bone remodelling. The concentrations in the culture medium of ABBM were lower than that of coralline HA, that meant that biocompatibility of ABBM were superior than that of coralline HA. Histologic samples showed that ABBM had better bone remodelling effect than coralline HA. ABBM showed good alizarin red marking lines, more deposition of,, and dense color of bone around newly formed osteon and bone trabeculae. it was concluded that ABBM was more biocompatible than corallineha in vivo and in vitro test. Key words : Biocompatibility, Normal human osteoblast, Hydroxy apatite, Anorganic bovine bone mineral I. 서론 악안면영역에서골조직은구강조직의기능유지에매우중요하며상실된조직이나병적인조직을인공대치물로회 * Correspondence:Gae Sig Chun, Department of Oral Physiology, School of Dentistry, Dankook University, Anseodong, San 29, Cheonan, Chungnam, 330-714, Korea, Tel:041-550-1916, Fax:041-550-1916, mobile : 016-677-2070, E-mail : dkphy@dankook.ac.kr 복시키고자하는노력이끊임없이계속되어왔다. 1960년대후반에소개된골유착 (Osseo-intergration) 의개념 1) 은치의학분야에서재건, 보철및외과술식에많은변화를유발하였다. 골유착된임프란트에서의장기적인예측이가능한결과를얻기위해서는식립하려는치조골부위에효율적인골질과골양이필요하다고하였다. 이상적인임프란트의식립은힘들며종종기능적이고심미적인문제가발생되기도한다. 이런기능적이고심미적인

문제점을보완하기위하여골유도재생술또는조직유도재생술등이사용되고있다. 이러한시술에는골대체물질이사용되어야하는데재료의원천에따라자가골이식, 동종골이식, 이종골이식, 합성골이식등으로분류된다. 이식재료등을이용한골수복시의문제점은수혜부의생활력, 공여부골의제한된혈류공급, 해부학및구조적문제점, 골흡수증가등이있고 2), 유전적상이성및내재된질병의감염으로인한면역학적반응도깊은관계를갖게된다 3,4). 자가골이식이나동종골이식은골격의재형성을촉진시키는데성공적으로자주사용되며이런형태의이식은자가골이식시공여부의시술경비나동종골이식시감염 (HIV, 간염 ) 등의위험때문에제한되어시술되고있다. 이러한시술시제한된결과로합성골생산을초래하였다. 현재까지알려진합성골로는합성수산화인회석 (HA), 삼차칼슘인회석 (TCP), 생활성유리세라믹 (BG) 등이있으며 5,6) 이러한물질은매식후흡수는양호하나골전도효과는제한적인것으로알려져있다 7). 따라서보완적으로시행되는방법으로는다른형태의골이식시이종골을사용하는것이다. 그런데최근까지이종골의이식방법을주로사용하지않은이유는재료내의유기물질에의한면역학적반응에대한가능성때문이었다. 최근에이러한반응을최소화하는방법이개발되었는데이종골에서유기물질을제거하고무기질의원래의구조와성분을보존하게처치하여사용하는방법으로 Anorganic bovine bone mineral(abbm. Bio-oss) 을소의뼈에서유기물질을제거하고순수하게변화하지않은무기물질로구성된골대체제가개발되었다 8). 반면에골대체제나골이식물질로수산화인회석의사용은 1920 년 Albee 에의해삼차인산칼슘이골손실부에이식되면골형성이유발된다다고하였다. 약 50년후, Levitt 등 9) (1969) 과 Monroe 등 (1971) 10) 은인산ceramic 이나불화칼슘아파타이트등을개발하여치의학이나의학분야에사용을제의하였다. 그후 Levin 등 (1974) 11) 은삼차인산칼슘을치주조직이손상된개에게처음으로치의학분야에적용하였다. 인산칼슘 ceramic 은조성성분에따라수산화인회석, β -TCP. biphasic calcium phosphate(bcp) 등으로구분되며세분하면합성수산화인회석과자연수산화인회석으로분류된다. Jarcho(1976) 12) 에의해개발된밀도가높은수산화인회석 ceramic 인 Durapatite는 Alveograf(Cook-waite) 와 lcite(lcitek. Inc) 로시판되고있다. 이는기본상태에서침적및가온으로합성한것이며산호에서추출된천연수산화인회석은 Interpore-200(interpore. Inc) 으로시판되며산호의탄산칼슘에인산암모늄을가한후 375 의열에 의해변형시킨천연수산화인회석으로 ABBM과같이이종골의분류에속할수있다. ABBM 에관한연구에서시험관내에서골과생체물질의표면에관한연구와동물을이용한실험이진행되어왔으나가끔원료인소의광우병등의문제가제기되었다. 따라서같은이종골이면서감염의문제가제기되지않고시험관내에서의연구가미미한산호에서추출된수산회인회석과정상인골세포를이용한생체적합성및가토에서골조직의조직변화를비교연구하고자본실험을시행하였다. II. 연구재료및방법 1) 수산화인회석제작산호 (Porties) 를분쇄하여채에통과시켜크기가 0.25-0.84mm범위의분말을채득한후 Ammonium phosphate(sigma. USA) 를가하여 Roy와 Linnehan방법 (CO 3+(NH4) 2 HPO 4 10(PO 4) 6(OH) 2) 으로제작한후 X- ray회절 (DMAZ-ⅢA, Rigaku, Japan) 로수산화인회석을확인하였다. 2) Anorganic Bovine Bone (ABBM) 천연골석회질인 Anorganic bovine bone ABBM,.Bio-oss, (osteohealth. Swiss) 를입자굵기가 0.25-1.00 mm인것을사용하였다. 3) 세포배양일차배양된정상인조골세포 (Normal Human osteoblast. NHOST. CSDB. N. I. H. USA) 는 10%FBS(FBS) 을포함하는 DMEM(Dulbeco's Minimum essential Medium, Hyclone U.S.A) 과이배양액에 0.05mML-ascorbic acid-2-phosphate(a.a; wako. Japan), 10mM β -glycerophosphate(β-gp; SIGMA. USA) 를첨가한배양액 (FBS+) 을사용하였다. 24well(NUNC, Denmark) 를사용하여실험실에서제작된천연수산화인회석 (HA, 국산 ) 및시판 A.B.B.M (Bio-oss. osteohealth. Swiss) 를 well 의바닥에밀착시키고각 well 당 2 104 개세포가포함되어있는 2 ml배양액을분주하고 2-3일마다배양액을교환해주었다. 174

Table 1. Mean and standard devication of,, concentration in FBS control. 7.45(± 0.14) 6.32 (±0.14) 2.30 (±0.11) 2.33 (±0.04) 2.37 (±0.06) 3.37 (±0.23) 2.91 (±0.12) 1.10 (±0.04) 1.17 (±0.04) 5.80 (±0.05) 19.67(±2.34) 15.56(±0.68) 5.74 (±0.37) 1.17 (±0.36) 6.33 (±0.45) Table 2. Mean and standard devication of,, concentration in FBS+ control. 8.68 (±0.23) 7.05 (±0.31) 2.68 (±0.09) 2.24 (±0.17) 2.61 (±0.30) 8.52 (±0.49) 6.81 (±0.33) 2.63 (±0.23) 2.24 (±0.11) 2.18 (±0.09) 19.50(±1.05) 16.94(±2.28) 6.36 (±0.88) 5.13 (±0.74) 5.21 (±0.58) Table 3. Mean and standard devication of,, concentration in FBS+HA 10.19 (±0.27) 8.69 (±0.27) 6.14 (±0.62) 3.29 (±0.42) 2.48 (±0.07) 4.68 (±0.12) 3.9 7 (±0.10) 2.71 (±0.21) 1.53 (±0.15) 1.12 (±0.05) 27.38 (±1.67) 21.25 (±1.37) 16.79 (±1.41) 9.02 (±0.85) 5.98 (±0.44) Table 4. Mean and standard devication of,, concentration in FBS++HA. 12.40 (±0.33) 10.38(±1.36) 6.12 (±0.51) 4.17 (±0.18) 2.65 (±0.12) 12.00 (±0.57) 12.84 (±1.71) 6.29 (±1.00) 5.02 (±0.49) 3.18 (±0.24) 27.38 (±1.08) 24.60 (±3.51) 13.72(±0.90) 8.89 (±0.78) 5.49 (±0.52) Table 5. Mean and standard devication of,, concentration in FBS +ABBM. 8.85 (±0.40) 8.38 (±0.31) 5.64 (±0.15) 4.28 (±0.37) 2.63 (±0.08) 9.90 (±0.13) 9.02 (±0.14) 5.49 (±0.06) 3.43 (±0.10) 2.40 (±0.03) 22.78 (±0.93) 19.50 (±1.64) 12.75 (±1.16) 8.67 (±0.74) 6.29 (±0.29) Table 6. Mean and standard devication of,, concentration in FBS++ ABBM. 10.02 (±0.44) 8.88 (±0.25) 6.61 (±0.16) 4.99 (±0.25) 3.05 (±0.08) 9.69 (±0.39) 8.47 (±0.48) 6.68 (±0.30) 4.56 (±0.40) 3.10 (±0.30) 22.59 (±1.67) 22.33 (±0.820 15.78 (±0.58) 10.60 (±083) 6.79 (±0.29) 175

Table 7. Anova test of concetration. () FBS++ HA FBS+ HA FBS++ Bio-oss FBS+ Bio-oss FBS++ HA -0.5868 p=0.0611-0.03373 p=0.9136-0.7883 p=0.0124 FBS+ HA 0.5531 p=0.0772-0.2015 p=0.5170 FBS++ Bio-oss -0.7546 p=0.0166 FBS+ Bio-oss Table 8. Anova test of concetration. () FBS++ HA FBS+ HA FBS++ Bio-oss FBS+ Bio-oss FBS++ HA -.25231 p=0.001-1.3680 p=0.0003 0.7145 p=0.0516 FBS+ HA 1.1551 p=0.0019-3.2376 p=0.001 FBS++ Bio-oss 2.0825 p=0.0001 FBS+ Bio-oss Table 9. Anova test of concetration. () FBS++ HA FBS+ HA FBS++ Bio-oss FBS+ Bio-oss FBS++ HA 0.1738 p=0.8419-0.319 p=0.0222-2.0160 p=0.0222 FBS+ HA -0.5656 p=0.5165-2.1898 p=0.0132 FBS++ Bio-oss -1.6241 p=0.0644 FBS+ Bio-oss Table 10. T-Test Varible Method Variances DF t Value Pr > ㅣtㅣ CA pooled Equal 58-0.76 0.4477 Satterhwaite Unequal 36.8-4.78 <.0001 pooled Equal 58-0.01 0.9959 176

Fig. 1. Linear regression Curve of concetration. Fig. 2. Linear regression Curve of concetration. Fig. 3. Linear regression Curve of concetration. 4) (calcium), (Inorganic phosphate), (Alkaline phosphatase) 측정법 1, 3, 7, 14, 28일기준으로정상인조골세포가배양된배양액을수거하여자동생화학분석기 (Hitachi 747, 일본 ) 을사용하여,, 의를측정하였다. 는 OCPC(O-Cresolphthalein complex), 는 Fiske - Subbarow sample 방법으로, 는 IFCC(international federation of clinical chemistry) 방법으로측정한후각각의통계적비교를위하여 t- test, 분산분석 (ANOVA test) 및선형회귀분석 (Linear regression analysis) 를하였다. 177

5) 천연수산화인회석및 A.B.B.M 의신생골형성연구 III. 연구결과 체중 3kg정도의뉴질랜드가토 (Newzeland white rabbit) 5마리에 30mg / kg으로 Nembutal(Somnopentyl, Pitman - Moors 사. USA) 를귀정맥을통해정맥마취후가토경골을노출시킨후동통을억제하기위하여경골에국소마취 (Lidocaine. 광명약품한국 ) 를시행한후치밀골을천공하였다. 직경 5mm깊이 6mm의골결손부두곳을형성하였고, 천연 HA(0.25-0.84 mm ) 및 ABBM(0.25-1.0 mm ) 을매식하고봉합후염증예방을위해 2일간 Terramycin( 한국화이자. 한국 ) 를 100mg /1개체를근육주사하였다. 7주째 Alizarin red(sigma. 미국 ) 를 50mg / kg으로근주하였다. 8주후실험동물을희생시켜매식재가매식된경골주위를절단하여 100μm두께의비탈회골표본을제작하였다. 제작된표본을형광현미경 (Nikon fluophot. 일본 ). 편광현미경 (Nikon OPtiPhot-PDL 일본 ). 반사전자현미경 (REM. JEOL. Superprobe 733. 일본 ), 및전자탐침미세분석 (JEOL, JXA- 8800 electrone Probe Microanalysis, 일본 ) 으로검경하였다. 배양액및재료에따른,, 의변화변화를표시하였다 (Table 1, 2, 3, 4, 5, 6). 선형회귀분석에서,, 모두시간이갈수록유의성있는감소를보여 HA나 ABBM 모두골형성에효과가있는것으로나타났다 (Fig. 1, 2, 3). 정상인조골세포배양시 ABBM 과 HA에서.. 의변화를 Anova test 로처리한결과전체 18개비교군에서 8개비교군만유의성있는차이를나타냈으며나머지는차이는있지만유의성은없었다 (Table 7, 8, 9). 정상인조골세포배양시 ABBM 과 HA에서.. 의변화를 Anova test 로처리한결과유의성있는결과중에 ABBM 이 HA보다우세한것으로나타났다. 배양액성분에따른.. 의차이는없는것같았다 (Table 10). 8 주조직소견에게 ABBM에는뚜렷한골석회화소견이보이며주변으로치밀한골주형성을나타내고인의침착도많았으나 HA주변은osteon 발달도미약하며 HA주변골주형성도 ABBM 에비해미약하며인의함량도 ABBM 보다낮게나타났다 (Fig. 4, 5, 6, 7, 8, 9, 10, 11). Fig. 4. Fluorecent marking( ) around new osteon on ABBM. (Fluorecent microscope, x100), Fig. 5. Fluorecent marking( ) around new osteon on coral HA. (Fluorecent microscope, x100), Fig. 6. High density bone color in the experimental area of ABBM. (Polarizing microscope, x20), Fig. 7. Low density bone color in the experimental area of coral HA. (Polarizing microscope, x20), Fig. 8. High density bone trabecule(pale gray) around ABBM. (Reflection electrone microscope, x50), Fig. 9. Low density bone trabecule(dark gray) around coral HA. (Reflection electrone microscope, x50), Fig. 10. EPMA analysis of,, in ABBM. (EPMA, x25), Fig. 11. EPMA analysis of,, in coral HA (EPMA, x25) 178

IV. 총괄및고찰 악안면영역에서손상된경조직을회복시키는방법은여러가지가있으나이중에서치아및치아를지지하는악골의손상시치과용임플란트를사용하기도한다. 치과용매식술에관해서는고대이집트나마야문명에서도볼수있으며 13) 최근의임플란트와유사한물질을생산하여시술한기록은 19세기후반에서 20세기초에걸쳐나타났다 14-15). 초기연구에서는임플란트를구성하는물질과악골조직간의생물학적적합성이있어야한다고인식하였다. 임플란트의생물학적적합성판단에서는섬유성결합및골유착성결합등을요구하다가 1939 년 Stock 가골과임플란트사이에결체조직이없는결합 16) 을발표한이후 1960 년대에 Branemark 1) 에의해골유착성결합으로되었다. 골유착성결합은정상적으로재형성된골과임플란트의표면사이에결체조직의개재가없는접촉이라정의되고있지만골과임플란트사이에 100% 유착이발생하지않는다. Johansson 과 Albrektsson 17) 은토끼에게나사형임플란트를매식하였을때골유착은 3개월후 50% 6개월후 65% 1년후 85% 정도라고보고하였다. 1985 년 Deputter 등 18) 은임플란트의고정과유지에두가지방식이있음을보고하였으며기계적인것과생물학적인것이라했다. 기계적유지는티타늄이나티타늄 Alloy 같은금속의표면형태에의한것이며생물학적인것은 HA같은생물학적물질에의해자연치와유사하게골과의직접적결합을말한다. 많은학자들이 HA도포임플란트가초기에골재형성속도가빠르고성숙속도가빠르다고보고하였다. 19) 골유착된임플란트가장기적으로성공하기위해서는임플란트의매식부위에충분한골질과골량이필요하게된다. 부족한골질과골양을보완하기위하여골이식재라는생체재료를사용하게된다. 생체재료는생체에적용되었을때생물학적으로잘받아들여지는재료를말하며생체에서얻어진생물학적인재료및인공적인이물성형재료로분류된다. 이재료들을원천에따라서세분하면자가골, 동종골, 이종골, 합성골등으로나눌수있다. 자가골의경우가가장이상적이기는하지만환자에게서직접채취하기에수혜부가공여부보다큰경우필요한양의골조직확보가곤란하며수술시간과경비가많이필요하다는단점이있다. 자가골이식의대체방법중에동종골이식이있으나개체간의유전학적차이점에의하여이식물의항원성을줄이기위하여다양한처리방법을사용해야하는점외에도처리과정의시간과경제적인문제를포함하고있다. 이외에도이종골이식재가있 으며이종골이식재는동물특히소나송아지의뼈를사용하며면역반응이문제가되므로항원성을없애기위하여유기질을제거한것을많이사용한다. 본연구에사용된 ABBM 은 Bio-oss란상표로시판되는제품이며송아지뼈를무균적으로채취하여단백질등의유기질을제거한것이다. Bio-oss 는매우높은골전도성을갖는것으로설명되고있으며그이유는인간의골과매우유사하기때문인것이다. 이외에도천연상태에서구할수있는이종골 HA는산호충에서구할수있다. 산호에서추출한 HA는특정산호류 (Porties) 의 CO3를인산암모늄존재하에열처리변환하여제작한것이다. HA의다양한연구로 HA 입자크기나내공의크기및압축강도를치밀골과비교하거나 HA에이물질을혼합하여강도및성능개선에대한연구등이진행되어왔다. 20,21) 본연구에서는입자의크기가적절하다고생각되는 ABBM 0.25-1.00 mm를사용하였다. 이식재이식후문제가되는것은이식재에대한생체조직의반응인데이를확인하기위하여는일차적으로생체와유사한환경하에서세포배양방법을통하여생체재료에대한세포의성장등을확인할수있다. 22-24) HA가피복된임플란트재료와골원세포간의상호작용에대하여연구한학자도있다. 25) 일반적인연구들은동물세포주및종양세포주를사용하였는데이런세포주들은정상인의세포와대사등이차이가많이나므로정상인간세포와같은효과를기대하기는어려워정상인조골세포에서의효능에관한요구가증가되고있으나정상인조골세포를이용한 ABBM 이나산호HA 의생체적합성을비교한연구는전무한형편이다. 본연구는이종골이식재인산호 HA와 ABBM을배양액에고정시킨후정상인조골세포의성장과정을골형성에관여하는물질을분석하여시행하였다. ABBM 과산호HA를배양한배양액속의석회화에관계되는물질인.. 등의는실험기간중유의성있는감소를보이며선형회귀분석에도뚜렷하게감소하는경향을나타냈다. 이는 Toguet 26) 등이행한연구와동일하게나타났는데이는시간이갈수록신생골의형성및성숙이진행되면서배양액속의석회화물질의가감소하는것같았다. 석회화물질의감소양을비교하기위해서 ABBM 과자연 HA의배양액내석회화물질의를 Anova test 로비교한결과절반정도에서 ABBM 이우세한것으로나타나골재형성에산호HA 보다는 ABBM 이효과가우수한것으로나타났으나 ABBM은제약회사의생산품이고산호HA는실험실에서추출한것이라서공정상에의한차이인지재료의원천인소뼈와산호의차이인지는구별하기힘들었다. 179

이식재의크기는혈관발달에매우중요하다. 크기가너무크면조직치유를방해한다고했다. 6) 따라서이식된산호HA 나 ABBM은 0.25-1.00 mm정도를선택하여정상인조골세포가충분히부착할수있는크기를정하였다. 본연구에사용된 ABBM은소뼈로구성된천연물질로치주결손부를채우는데효력이있다고하여이종골이식재로사용되고있다. 27,28) 동물실험결과천연해면골과유사한공극을갖는 ABBM 은가토결손부나비글견의상악동에이식시골전도가유발되어신생골형성및침착이발생한다고하였다. 20,29) 산호HA를이용하여골이식시 50-70% 의성공률을보인다는보고도있어 30) 가토의경골에 ABBM과산호HA를이식하고 7주후 Alizalin red 를투여후 8주째의조직소견을검경하였다. 형광현미경에서는골원주변의석회화물질의침착을알려주는형광물질의표지가 ABBM 에서뚜렷하였고편광현미경상에서골의밀도가높은청색이 ABBM에서나타났다. 반사전자현미경에서는골주의형성이 ABBM에서뚜렷하였고 EPMA 상의석회화물질의침착정도도높았다. 석회화물질의배양액내의에서는부분적인차이만보이다조직소견상에서는확실하게나타난것으로보아골결손시이식재로사용되는 ABBM 과산호HA의비교는 ABBM 이우수한것으로나타났다. 이원인은종에따른차이인지생산공정의차이인지를구별할수없었다. V. 결론악안면영역에서손상된골조직을회복시키기위하여사용하는이식재중에서골재형성효과를비교하기위하여소뼈에서추출된 ABBM과산호에서추출된산호HA를사용하였다. 24well의바닥에 ABBM과산호HA를부착하고배양액 (FBS. FBS+) 에정상인조골세포를 28일까지배양하여 1일째, 3일째, 7일째, 14일째, 28일째채취하여골의석회화물질 (,, ) 들의를측정하여비교분석하였다. 조직학적소견을확인하기위하여가토경골에 ABBM 과산호 HA를이식후 8주후제거하여비탈회골표본을제작하여검사하였다. 2가지연구에서얻어진결과및결론은다음과같다. 1. 선형회귀분석에서배양액내의,, 가시간이갈수록감소하여 ABBM 이나산호HA 모두골재형성에효과가있는것으로나타났다. 2. 정상인조골세포배양시 ABBM 과산호HA에서,, 변화를 ANOVA test 로분석한결과 ABBM 이산호HA에비해골재형성이우수한것으로나타났다. 3. 배양액성분에따른,, 의차이는없는것같았다. 4. 8주조직소견에서 ABBM 이산호HA에비하여골석회화, Osteon 주변의신생골형성, 신생골주형성및석회화물질의함량에서우세하게나타나골재형성이더잘되는것같다. 상기와같은결과에서골손실부의회복을유도하기위하여사용하는이종골이식재료중 ABBM 이산호 HA보다효과가우세한것으로나타났으나 ABBM은제약사에서생산된제품으로제작상의차이에대한의문도있다. VI. 참고문헌 1. Branemark P-I et al : osseointegrated implants in the treatment of edentulous jaw : experience from a 10-year period, Scand : J. Plast. Reconstr. Surg 11(suppp 16). 1977. 2. Glowacki J, Mulliken JB : Demineralized bone implants. Clin Plst Surg 12 : 233-41, 1985. 3. Buck BE, Malinin TI, Brown MD : Bone transplantation and human immunodeficiency virus. An estimate of risk of acquired immunodeficency syndrome(aids). Clin Orthop 240 : 129-36, 1989. 4. Binderman I, Fin N : Bone substitute-organic, inorganic, and polymeric cell material interactions. In : Yamamura, Hench LL, Wilson J, editors. CRC handbook of bioactive materials. Boca Raton : CRC Press, p45-61, 1990. 5. Le Geros RZ : lcium phosphate materials in restorative dentistry ; a review. Adv Dent Res 2 : 164-80, 1988. 6. Schpers EJC, Ducheyne P, Barbier L, Schepers S : Bioactive glass particles of narrow size range : A new materials for the repair of bone defects. Implant Dent 2 : 151-6, 1993. 7. Schpers EJC, Declercq M, Ducheyne P, 180

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