Implanted octacalcium phosphate is more resorbable thanb-tricalcium phosphate and hydroxyapatite

S. Kamakura,1 Y. Sasano,2 T. Shimizu,3 K. Hatori,4 O. Suzuki,5 M. Kagayama,2 K. Motegi1

1Division of Stomatology and Maxillofacial Surgery, Tohoku University Graduate School of Dentistry,Sendai 980-8575, Japan2Division of Oral Molecular Biology, Tohoku University Graduate School of Dentistry, Sendai, Japan3Division of Periodontics and Endodontics, Tohoku University Graduate School of Dentistry, Sendai, Japan4Division of Stomatognathic Physiology and Prosthodontics, Tohoku University Graduate School of Dentistry,Sendai, Japan5Research and Development Center, JGC Corporation, Oarai, Japan

Received 16 April 2001; accepted 11 May 2001Abstract: Our previous studies have suggested that syn-thetic octacalcium phosphate (OCP) could be resorbed andreplaced by newly formed bone if implanted in rat skulldefects. We hypothesized that the implanted OCP is moreresorbable than other commonly used bone graft substitutesof calcium phosphate compounds, such as hydroxyapatite(HA) and b-tricalcium phosphate (b-TCP). To test the hy-pothesis, the present study was designed to compare histo-morphometrically resorption of the implanted OCP, HA,and b-TCP, which were kept in the experimental cranialdefect of rats for a long term. A full thickness of standard-ized trephine defect was made in the rat parietal bone, andthe same volume of granules of OCP, HA, and b-TCP wereimplanted into the defect. Five specimens of each group

were fixed 6 months after implantation. The percentage ofremaining implants (r-Imp%) and newly formed bone (n-Bone%) in the defect was analyzed histomorphometrically.The statistical analysis showed that the r-Imp% of OCP wassignificantly lower than that of HA and b-TCP. In contrast,the n-Bone% of OCP was significantly higher than that ofHA and b-TCP. The present study has shown that the im-planted OCP in the rat cranial defect is more resorbable thanthe implanted b-TCP and HA, whereas the implanted OCPenhances bone formation more than the implanted b-TCPand HA. © 2001 John Wiley & Sons, Inc. J Biomed Mater Res59: 29–34, 2002

Key words: octacalcium phosphate; b-tricalcium phosphate;hydroxyapatite; bone repair; bone defect

INTRODUCTION

In oral and maxillofacial surgery, a variety of bonegrafts and implants have been used to fill the largebone defects resulting from trauma, neoplasms, or in-fection.1,2 Synthetic calcium phosphates such as hy-droxyapatite [Ca10(PO4)6(OH)2; HA] and b-tricalciumphosphate [b-Ca3(PO4)2; b-TCP] are commonly usedbone graft substitutes.3–6 It is known that HA orb-TCP implants possess good tissue compatibility,and new bone is formed directly on the implants with-out no intervening cellular component.7,8 It has beenreported that TCP is more bioresorbable than HA ce-ramics, which usually show minimal resorption.9,10

Octacalcium phosphate [Ca8H2(PO4)65H2O; OCP]has been supposed to be a precursor phases of bio-logical apatite in bone tissue,11–13 and has been shownto be converted to apatitic phase if implanted into thesubperiosteal region of mice.14 Our previous studieshave indicated that the implantation of the syntheticgranules of OCP causes newly bone formation.14–18

Furthermore, the implanted OCP can serve as a corefor initiating bone formation and shows the osteoin-ductive ability as well as the osteoconduction if im-planted in the critical-sized calvarial defects of rats.19

OCP is physicochemically resorbable more than HAor b-TCP under the physiological condition.20 Wehave shown that the implanted OCP is resorbed by themultinucleated giant cells (MNGCs), which share ul-trastructural features with osteoclasts.21

We hypothesized that the implanted OCP is moreresorbable than other commonly used bone graft sub-stitutes of calcium phosphate compounds, such as HAand b-TCP. To test the hypothesis, the present studywas designed to compare histomorphometrically re-

Correspondence to: S. Kamakura; e-mail: kamakura@mail.cc.tohoku.ac.jp

Contract grant sponsor: Ministry of Education, Culture,Sports, Science, and Technology of Japan; contract grantnumbers: 11671974, 11671794, 13671893, 13672076.

© 2001 John Wiley & Sons, Inc.

sorption of the implanted OCP, HA, and b-TCP,which were kept in the experimental cranial defect ofrats for a long term.

MATERIALS AND METHODS

Animals

Fifteen 12-week-old male Wistar rats weighing from 250to 300 g were used. They were obtained from the SLC Corp.(Kotoh, Shizuoka, Japan) and kept under a standard light-dark schedule and relative humidity. Stock diet and tap wa-ter were available ad libitum. All procedures were approvedby the Animal Research Committee of Tohoku University.

Preparation of implants

OCP was prepared according to the method described byLeGeros.22 HA was synthesized by the method previouslydescribed,14 which was modified by the method of Morenoet al.23 Particle sizes between 300 and 500 mm were used forimplantation. The sieved granules of OCP and HA weresterilized by being heated to 120°C for 2 h. Our previousstudy has shown that the heating does not affect physicalproperties such as the crystalline structure or the specificsurface area of the granules of OCP and HA.14,24 b-TCP wasobtained from Olympus Optical Co. (Tokyo, Japan), whichwas prepared according to the mechanochemical method.The autoclaved b-TCP of particle sizes between 250 and 500mm were used for implantation.

Implantation procedure

The experimental rats were anesthetized with intraperito-neal sodium pentobarbital (50 mg/kg) supplemented byether inhalation. A skin incision was made aseptically, andthe dissection was carried down to the calvarium. Perioste-um of the calvarium was ablated, and a full-thickness ofstandardized trephine defect, 4 mm in diameter, in the pa-rietal bone was made under continuous saline buffer irriga-tion. Extreme care was exercised to avoid injury to duramater. Five defects of each groups were treated with OCP,b-TCP, or HA. The created defects were filled with the samevolume of granules of implants. After the defects weretreated, the ablated periosteum and skin were repositionedand sutured, respectively. Experimental rats were fixed 6months after the implantation of OCP, b-TCP, or HA.

Tissue preparations

The rats were anesthetized by intraperitoneal sodium pen-tobarbital (50 mg/kg) and fixed with 4% paraformaldehyde

in 0.1M phosphate-buffered saline (PBS), pH 7.4, by perfu-sion through the aorta. The implants were resected togetherwith the surrounding bones and tissues and kept in the samefixative overnight at 4°C and decalcified in 10 % EDTA in0.01M PBS, pH 7.4, for 6 weeks at 4°C. The samples weredehydrated in a graded series of ethanol, embedded in par-affin, and serially sectioned coronally with 6 mm thickness.The sections including the center of the defect were ex-tracted and stained with hematoxylin and eosin. Photo-graphs were taken with a photomicroscope (AX-80, Olym-pus Optical Co., Tokyo, Japan).

Quantitative micrograph analysis

Light micrographs of the sections stained with hematoxy-lin and eosin were used for the histomorphometric measure-ment.25 Photographs projecting the overall defect weretaken from the each specimen. The micrographs werescanned at 1350 dots per inch by a film scanner (LS-1000,Nikon, Tokyo, Japan).

The percentage of remaining implants in the defect (r-Imp%) was calculated as area of remaining implants/area ofthe defect originally created by trephination × 100 (Fig. 1).Likewise, the percentage of newly formed bone in the defect(n-Bone%) was calculated as area of newly formed bone/area of the defect originally created by trephination × 100(Fig. 1). The r-Imp% and n-Bone% were quantified on acomputer (Power Macintosh 7300/166, Apple Japan Inc., To-kyo, Japan) using the version 1.62 of NIH Image public do-main software.

Statistical analysis

Histomorphometric data were analyzed by a computersoftware package (Excel 5.0, Microsoft Co.). All values arereported means ± standard deviation (SD). Unpaired t testswere used to compare mean values among the OCP-,b-TCP–, and HA-treated groups. Statistical significance wasconsidered p < 0.05.

RESULTS

Histological examination

In the OCP-treated animals, the created defectswere filled with newly formed bone [Fig. 2(a)]. Newlybone formation was observed at the margin of thedefect, along the side of dura mater, and on the im-planted OCP [Figs. 2(a) and 3(a)]. The remaining OCPin the defect was surrounded by newly formed bonewithout intervening cellular components [Fig. 3(a,b)],and no remaining OCP was seen in the connective

30 KAMAKURA ET AL.

tissue. The new bone was formed concentricallyaround the implanted OCP and was barely distin-guishable from the host bone [Fig. 3(a)]. There was noinflammatory cell infiltration at the implantation site.

In the b-TCP–treated animals, newly formed bonewas observed at the margin of the defect, along theside of dura mater, and on the implanted b-TCP [Figs.2(b) and 3(c)]. The newly formed bone matrix wascompact and barely distinguishable from the hostbone [Fig. 3(c)]. There was no inflammatory cell infil-tration at the implantation site. The remaining b-TCPin the defect was seen both in the newly formed boneand in the connective tissue. The remaining b-TCP inbone was directly in contact with the newly formedbone [Fig. 3(d)].

In the HA-treated animals, newly bone formationwas observed at the margin of the defect, along theside of dura mater, and on the implanted HA [Figs. 2(c)and 3(e)]. The newly formed bone matrix was compact

[Fig. 3(e)]. There was no inflammatory cell infiltrationat the implantation site. The remaining HA in the de-fect was both in the newly formed bone and in theconnective tissue. The remaining HA in bone was di-rectly in contact with the newly formed bone [Fig.3(f)].

Histomorphometric examination

The percentage of remaining implants in the defect(r-Imp%) ± SD within the OCP, b-TCP, and HAgroups was 6.58 ± 1.46, 23.4 ± 6.82, and 39.0 ± 6.86,respectively (Fig. 4). The difference of mean value ofthe r-Imp% between the OCP-treated group and theother two groups and that of the r-Imp% between theb-TCP-treated group and the HA-treated group weresignificant (Fig. 4). The percentage of new bone in the

Figure 2. Overview of histological sections of the skull defect treated with (a) OCP, (b) b-TCP, and (c) HA. (a) In theOCP-treated animals, the created defect is mainly filled with newly formed bone. (b) In the b-TCP–treated animals, newlyformed bone is observed at the margin of the defect (.), along the side of dura mater. (c) In the HA-treated animals, newlyformed bone is observed at the margin of the defect (.), along the side of dura mater. .: defect margin,. Bars = 1 mm.

Figure 1. Histomorphometric measurement of newly formed bone (n-Bone%) and remaining implants (r-Imp%) in thedefect. The whole area of the defect originally created by trephination is defined on histological sections. The n-Bone% iscalculated as area of newly formed bone/area of the defect originally created by trephination × 100. Likewise, the r-Imp% iscalculated as area of remaining implants /area of the defect originally created by trephination × 100.

31RESORBABILITY OF IMPLANTED OCP IN BONE DEFECT

defect (n-Bone%) ± SD within the OCP, b-TCP, andHA groups was 63.3 ± 7.52, 42.8 ± 13.6, and 24.2 ± 7.82,respectively (Fig. 5). The difference of mean value ofthe n-Bone% between the OCP-treated group and theother two groups and that of the n-Bone% betweenb-TCP–treated group and HA-treated group were sig-nificant (Fig. 5).

DISCUSSION

The present study indicated that the percentage ofremaining implants in the defect (r-Imp%) of the OCP-treated group was significantly lower than that of theb-TCP– or the HA-treated group, and the r-Imp% ofthe b-TCP–treated group was significantly lower than

Figure 3. Histological examination of the skull defects treated with (a,b) OCP, (c,d) b-TCP, and (e,f) HA. (a,b) In OCP-treatedanimals, newly formed bone (B) is observed at the margin of the defect (.) and on the implanted OCP (*). The newly formedbone (B) surrounds directly the implanted OCP (*) without intervening cellular components. The newly formed bone matrixis compact and barely distinguishable from the host bone. The remaining OCP (*) in the defect is surrounded by the newlyformed bone (B). (c,d) In the b-TCP–treated animals, the newly formed bone (B) directly contacts the implanted b-TCP (*)without intervening cellular components. The newly formed bone matrix is compact and barely distinguishable from the hostbone (.). The remaining b-TCP (*) in the defect is surrounded by the newly formed bone (B) and the connective tissue. (e,f)In the HA-treated animals, the newly formed bone (B) directly contacts the implanted HA (*) without intervening cellularcomponents. The remaining HA (*) in the defect is surrounded by the newly formed bone (B) and the connective tissue: Bars= (a,c,e) 300 mm; (b,d,f) 200 mm.

32 KAMAKURA ET AL.

that of the HA-treated group. It suggests that the im-planted OCP is more resorbable than the b-TCP or theHA, and the implanted b-TCP is more resorbable thanHA. Compared with b-TCP and HA, OCP has highersolubility.20,26,27 As a precursor of biological apatite,OCP converts to HA by either of two mechanisms: (i)in situ hydrolysis of OCP and/or (ii) dissolution ofOCP followed by HA precipitation.13,27 Our previousstudy reported that the implanted OCP is surroundedby the MNGCs, which share some ultrastructuralcharacteristics with osteoclasts, and frayed where it isin contact with the surface of the MNGCs, whereasthe surface of the implanted HA associated with theMNGC is smooth and not frayed.21 It has been re-ported that the resorption pits were observed onpreincubated OCP disks but not on HA and b-TCPdisks, when incubated in osteoclastic cell culture.28

The present study supported those previous reports,demonstrating that OCP is more resorbable thanb-TCP or HA.

The histological observation showed that the im-planted b-TCP and HA remained in both connectivetissue and newly formed bone, whereas the implantedOCP remained only in the newly formed bone. It sug-gests that the OCP might be resorbed faster in theconnective tissue than in the bone tissue. The remain-

ing OCP in the bone tissue could be resorbed beingassociated with bone remodeling.

The present study indicated that the percentage ofnew bone in the defect (n-Bone%) of the OCP-treatedgroup was significantly higher than that of the b-TCP–or the HA-treated group, and the n-Bone% of b-TCP–treated group was significantly higher than that ofHA-treated group. It suggested that the OCP implantenhances bone formation more than b-TCP and HA,and the b-TCP implant enhances bone formation morethan HA. It is known that calcium phosphates possessgood tissue compatibility.7,8,29 Our previous studieshave reported that the OCP implantation enhancesbone formation15–18 and results in faster bone forma-tion than hydroxyapatite.14 We have shown that theimplanted OCP serves as a core for initiating boneformation and causes the osteoinductive property inthe rat cranial defect,19 whereas b-TCP and HA do nothave such properties. The implanted OCP initiatesmultiple ossification points in the bone defect wherethe new bone is concentrically formed around the im-planted OCP. Consequently, the bone repair could befastened by fusing the multiple ossification pointswith each other and with the margin of the defect,which results in bone repair to a larger extent than theother calcium phosphates.

The present study has demonstrated that the im-

Figure 4. Histomorphometrical examination of remainingimplants in the defect (r-Imp%) caused by OCP, b-TCP, andHA. The r-Imp% ± SD within the OCP, b-TCP, and HAgroup is 6.58 ± 1.46, 23.4 ± 6.82, and 39.0 ± 6.86, respectively.The difference of mean value of the r-Imp% between OCP-treated and other two groups and that of the r-Imp% be-tween b-TCP–treated group and HA-treated group are sig-nificant. Data are means ± SD of five specimens. Statisticalanalysis is performed using an unpaired t test. **: p < 1 ×10−2; ***: p < 5 × 10−3; .: p < 5 × 10−4.

Figure 5. Histomorphometrical examination of newlyformed bone in the defect (n-Bone%) caused by OCP, b-TCP,and HA. The n-Bone% ± SD within the OCP, b-TCP, and HAgroups is 63.3 ± 7.52, 42.8 ± 13.6, and 24.2 ± 7.82, respec-tively. The difference of mean value of the n-Bone% betweenOCP-treated and other two groups, and that of the n-Bone%between b-TCP–treated group and HA-treated group aresignificant. Data are means ± SD of five specimens. Statisti-cal analysis is performed using an unpaired t test. *: p < 5 ×10−2; . .: p < 5 × 10−5.

33RESORBABILITY OF IMPLANTED OCP IN BONE DEFECT

planted OCP in the rat cranial defect is more resorb-able than the implanted b-TCP and HA, whereas itenhances bone formation more than the implantedb-TCP and HA. OCP could be a bone substitute that ismore suitable than other commonly used calciumphosphates.

References

1. Kent JN, Misiek DJ, Kinnebrew MC. Alloplastic materials foraugmentation in cosmetic surgery. In: Peterson LJ, editor. Prin-ciples of oral and maxillofacial surgery. Philadelphia: JB Lip-pincott; 1992. p 1769–1817.

2. Schmitz JP, Hollinger JO. The critical size defect as an experi-mental model for craniomandibulofacial nonunions. Clin Or-thop 1986;205:299–308.

3. Nunes CR, Simske SJ, Sachdeva R, Wolford LM. Long-termingrowth and apposition of porous hydroxylapatite implants. JBiomed Mater Res 1997;36:560–563.

4. Nicholas RW, Lange TA. Granular tricalcium phosphate graft-ing of cavitary lesions in human bone. Clin Orthop 1994;306:197–203.

5. McAndrew MP, Gorman PW, Lange TA. Tricalcium phosphateas a bone graft substitute in trauma: preliminary report. J Or-thop Trauma 1988;2:333–339.

6. Vanassche BJ, Stoelinga PJ, de Koomen HA, Blijdorp PA,Schoenaers JH. Reconstruction of the severely resorbed man-dible with interposed bone grafts and hydroxylapatite. A 2–3year follow-up. Int J Oral Maxillofac Surg 1988;17:157–160.

7. Spector M. Anorganic bovine bone and ceramic analogs ofbone mineral as implants to facilitate bone regeneration. ClinPlast Surg 1994;21:437–444.

8. Rawlings CE 3rd. Modern bone substitutes with emphasis oncalcium phosphate ceramics and osteoinductors. Neurosur-gery 1993;33:935–938.

9. Schmitz JP, Hollinger JO, Milam SB. Reconstruction of boneusing calcium phosphate bone cements: a critical review. J OralMaxillofac Surg 1999;57:1122–1126.

10. Duguy N, Petite H, Arnaud E. Biomaterials and osseous re-generation. [in French]. Ann Chir Plast Esthet 2000;45:364–376.

11. Brown WE, Smith JP, Lehr JR, Frazier AW. Crystallographicand chemical relations between octacalcium phosphate andhydroxyapatite. Nature 1962;196:1050–1055.

12. Nelson DGA, McLean JD. High-resolution electron microscopyof octacalcium phosphate and its hydrolysis products. CalcifTissue Int 1984;36:219–232.

13. Brown WE, Eidelman N, Tomazic B. Octacalcium phosphate asa precursor in biomineral formation. Adv Dent Res 1987;1:306–313.

14. Suzuki O, Nakamura M, Miyasaka Y, Kagayama M, Sakurai M.Bone formation on synthetic precursors of hydroxyapatite. To-hoku J Exp Med 1991;164:37–50.

15. Sasano Y, Kamakura S, Nakamura M, Suzuki O, Mizoguchi I,

Akita H, Kagayama M. Subperiosteal implantation of octacal-cium phosphate (OCP) stimulates both chondrogenesis andosteogenesis in the tibia, but only osteogenesis in the parietalbone of a rat. Anat Rec 1995;242:40–46.

16. Sasano Y, Kamakura S, Homma H, Suzuki O, Mizoguchi I,Kagayama M. Implanted octacalcium phosphate (OCP) stimu-lates osteogenesis by osteoblastic cells and/or committed os-teoprogenitors in rat calvarial periosteum. Anat Rec 1999;256:1–6.

17. Kamakura S, Sasano Y, Nakamura M, Suzuki O, Ohki H,Kagayama M, Motegi K. Initiation of alveolar ridge augmen-tation in the rat mandible by subperiosteal implantation ofoctacalcium phosphate. Arch Oral Biol 1996;41:1029–1038.

18. Kamakura S, Sasano Y, Homma H, Suzuki O, Kagayama M,Motegi K. Implantation of octacalcium phosphate (OCP) in ratskull defects enhances bone repair. J Dent Res 1999;78:1682–1687.

19. Kamakura S, Sasano Y, Homma H, Suzuki O, Kagayama M,Motegi K. Implantation of octacalcium phosphate nucleatesisolated bone formation in rat skull defects. Oral Dis2001;7:259–266.

20. Brown WE, Mathew M, Tung MS. Crystal chemistry of octa-calcium phosphate. Prog Crystal Growth Charact 1981;4:59–87.

21. Kamakura S, Sasano Y, Homma-Ohki H, Nakamura M, SuzukiO, Kagayama M, Motegi K. Multinucleated giant cells re-cruited by implantation of octacalcium phosphate (OCP) in ratbone marrow share ultrastructural characteristics with osteo-clasts. J Electron Microsc (Tokyo) 1997;46:397–403.

22. LeGeros RZ. Preparation of octacalcium phosphate (OCP): adirect fast method. Calcif Tissue Int 1985;37:194–197.

23. Moreno EC, Gregory TM, Brown WE. Preparation and solubil-ity of hydroxyapatite. J Res Nat Bur Stand 1968;72A:773–782.

24. Suzuki O, Nakamura M, Miyasaka Y, Kagayama M, Sakurai M.Maclura pomifera agglutinin-binding glycoconjugates on con-verted apatite from synthetic octacalcium phosphate im-planted into subperiosteal region of mouse calvaria. BoneMiner 1993;20:151–166.

25. Kamakura S, Sasano Y, Nakajo S, Shimizu T, Suzuki O, KatouF, Kagayama M, Motegi K. Implantation of octacalcium phos-phate combined with transforming growth factor-b1 enhancesbone repair as well as resorption of the implant in rat skulldefects. J Biomed Mater Res 2001;57:175–182.

26. Oonishi H, Hench LL, Wilson J, Sugihara F, Tsuji E, KushitaniS, Iwaki H. Comparative bone growth behavior in granules ofbioceramic materials of various sizes. J Biomed Mater Res 1999;44:31–43.

27. Bigi A, Boanini E, Borghi M, Cojazzi G, Panzavolta S, Roveri N.Synthesis and hydrolysis of octacalcium phosphate: effect ofsodium polyacrylate. J Inorg Biochem 1999;75:145–151.

28. Doi Y, Shibutani T, Moriwaki Y, Kajimoto T, Iwayama Y. Sin-tered carbonate apatites as bioresorbable bone substitutes. JBiomed Mater Res 1998;39:603–610.

29. Langstaff S, Sayer M, Smith TJ, Pugh SM, Hesp SA, ThompsonWT. Resorbable bioceramics based on stabilized calcium phos-phates. Part I: rational design, sample preparation and materialcharacterization. Biomaterials 1999;20:1727–1741.

34 KAMAKURA ET AL.