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Material-dependent bone induction by calcium phosphate ceramics: a 2.5-year study in dog
Biomaterials 22 (2001) 2617}2623
Material-dependent bone induction by calcium phosphate ceramics: a 2.5-year study in dog Huipin Yuan *, Zongjian Yang , Joost D. de Bruijn , Klaas de Groot , Xingdong Zhang Institute of Materials Science and Technology, Sichuan University, Chengdu, China Biomaterials Research Group, Leiden University, Leiden, Netherlands IsoTis N.V., Prof. Bronkhorstlaan 10, 3723 MB Bilthoven, Netherlands
Abstract Bone induction by di!erent calcium phosphate biomaterials has been reported previously. With regard to (1) whether the induced bone would disappear with time due to the absence of mechanical stresses and (2) whether this heterotopically formed bone would give rise to uncontrolled growth, a long-time investigation of porous hydroxyapatite ceramic (HA), porous biphasic calcium phosphate ceramic (TCP/HA, BCP), porous -tricalcium phosphate ceramic (-TCP) and porous -tricalcium phosphate ceramic (-TCP) was performed in dorsal muscles of dog, for 2.5 years. Histological observation, backscattered scanning electron microscopy observation and histomorphometric analysis were made on thin un-decalci"ed sections of retrieved samples. Normal compact bone with bone marrow was found in all HA implants (n"4) and in all BCP implants (n"4), 48$4% pore area was "lled with bone in HA implants and 41$2% in BCP implants. Bone-like tissue, which was a mineralised bone matrix with osteocytes but lacked osteoblasts and bone marrow, was found in all -TCP implants (n"4) and in one of the four -TCP implants. Both normal bone and bone-like tissues were con"ned inside the pores of the implants. The results show that calcium phosphate ceramics are osteoinductive in muscles of dogs. Although the quality and quantity varied among di!erent ceramics, the induced bone in both HA and BCP ceramics did neither disappear nor grow uncontrollably during the period as long as 2.5 years. 2001 Elsevier Science Ltd. All rights reserved. Keywords: Bone; Calcium phosphates; Soft tissue implantation; Osteoinductive biomaterials
1. Introduction Bone is one of the few connective tissues that have a large amount of inorganic components, mainly, calcium phosphates, which, through a complicated process of biomineralisation, are deposited within bone matrix and, therefore, give bone its excellent mechanical properties [1]. In addition to the roles in the construction of bone, calcium phosphates in bone play important roles in the homeostasis of calcium and phosphorous in body #uids and may also play roles in bone's own physiology. It was thought that calcium phosphates might stimulate osteogenesis and since the 1890s, they have been used medically in bone repair [2}4]. However, when calcium * Correspondence address: Biomaterials Research Group, Leiden University, IsoTis N.V., Prof. Bronkhorstlaan 10, 3723 MB Bilthoven, Netherlands. Tel: #31-30-2295251; fax: #31-30-2280255. E-mail address: [email protected] (H. Yuan).
phosphates were "rst used as medicines, no positive result was obtained until Albee found that tricalcium phosphate stimulated bone formation in the 1920s [2}5]. A great progress in the medical use of calcium phosphates was made between the 1970s and the 1980s. Osteoconduction, which means a guided bone formation on a biomaterial surface and the formation of chemical bonding between the newly formed bone and biomaterials, was found, when calcium phosphates were introduced into bioglasses and glass ceramics or were applied as hydroxyapatite ceramic, tricalcium phosphate ceramic and biphasic calcium phosphate ceramic [2}4,6}8]. Since then, a series of calcium phosphatebased biomaterials including bioglass, glass ceramics, di!erent calcium phosphate ceramics, calcium phosphate cements, calcium phosphate coatings and bioactive composites with calcium phosphates have been developed [2,7}14]. Extensive fundamental studies and clinical applications have demonstrated that calcium
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phosphate-based biomaterials are biocompatible and osteoconductive [3,4,8,11}13,15}17]. It appeared that osteoconduction is a common phenomenon of all calcium phosphate-based biomaterials [18], however, the roles of calcium phosphates in osteogenesis were not well understood through osteoconduction studies in bony sites because of the regeneration of bone itself. When tested in soft tissues, none of the calcium phosphate-based biomaterials showed osteoinductivity till 1990, in Japan, Yamasaki found bone formation induced by a hydroxyapatite ceramic under the subcutis of dogs [19]. Later, similar results were reported by Ripamonti in South Africa [20], by Zhang et al. in China [21], by Vagervik [22], by Toth [23] in USA, by Klein in Netherlands [24] and by various other authors [25}38]. In the last decade, bone induction has been found in coral-derived hydroxyapatite ceramic, synthetic hydroxyapatite ceramic, biphasic calcium phosphate ceramic, tricalcium phosphate ceramic, calcium pyrophosphate ceramic and calcium phosphate cement in rabbits, goats, pigs, dogs, monkeys, baboons and even in humans [19}38]. The evidence suggested that calcium phosphates not only stimulate bone formation in bony sites, but also induce bone formation in soft tissues. Bone induction by calcium phosphate biomaterials is both material- and animal-dependent [27,28,33,35}37]. Further studies on the material factors related to osteoinductivity of calcium phosphate biomaterials revealed that the chemistry, the geometry and the porous structures of calcium phosphate biomaterials are important for bone induction [27,33,35}37], and strongly suggested that calcium phosphate biomaterials can be endowed with osteoinductivity by the optimisation of the materials themselves [35,36] rather than by tissue engineering in which biomaterials were made to be osteoinductive by introducing bone morphogenetic proteins (BMPs) or osteogenic cells [39}41]. It is thus possible that osteoinductive calcium phosphate biomaterials can be made available for clinical use in orthopaedic and dental surgery in the near future, but some questions need to be answered before considering clinical use of osteoinductive calcium phosphate biomaterials. The "rst and important issue is their safety. Osteoinductive calcium phosphate biomaterials are biomaterials that induce cell di!erentiation and bone morphogenesis in soft tissues where normally no bone formation occurs. So, whether this heterotopically induced bone would give rise to uncontrolled growth must be answered. Secondly, whether the induced bone would disappear with time due to the absence of mechanical stresses in soft tissues is also important because these stresses play important roles in bone formation and bone metabolism [42]. To address these questions, a long-time investigation is necessary and therefore in this study, calcium phosphate
ceramics were implanted in the dorsal muscles of dog for 2.5 years. Moreover, material factors, which were related to the quality and quantity of calcium phosphateinduced bone, were further studied.
2. Materials and methods Porous calcium phosphate ceramics were made from wet-synthetic calcium phosphate apatite powders with various Ca/P ratios, i.e. 1.67 (HA), 1.61 (BCP), 1.5 (TCP), by foaming with 5% H O and sintering at 11003C for 3 h. Porous ceramics, with microporous structures on their macropore surface, with di!erent phase components of HA (HA ceramic), 37% -TCP/63% HA (BCP ceramic), -TCP (-TCP ceramic) and -TCP (-TCP ceramic), were obtained by di!erent cooling procedures after sintering [28,36,37]. After sterilisation, the ceramics were implanted as cylinders (5 mm in diameter, 6 mm in length) in the dorsal muscles of dogs. X-ray di!raction (XRD) analysis of the chemistry and scanning electron microscopy observation of the porous structures of di!erent calcium phosphate ceramics, together with the osteoinductivity in early stage (before 180 days), have been reported earlier [28,29,31,33,34,36,37]. One dog (male, adult, 13 kg), with four HA cylinders, four BCP cylinders, four -TCP cylinders and four -TCP cylinders in its dorsal muscles, was left for a long-time investigation. After two and a half years, the dog was sacri"ced with an overdose of pentobarbital sodium. The implants were harvested with surrounding tissues and "xed in 4% buffered formaldehyde (pH"7.4) immediately. The "xed implants were washed with phosphate bu!er solution (PBS), dehydrated in a series of ethanol solutions (70, 80, 90, 95, 100 and 100%) and embedded in methyl methacrylate (MMA). Thin un-decalci"ed sections (20 m) were made (KDG 95, IsoTis N.V., Netherlands) and stained with methylene blue and basic fuchsin for histological examination. Some sections were coated with carbon for backscattered scanning electron microscopy observation (BSE) (Philips SEM 525). The amount of bone formed in HA implants and BCP implants was determined by histomorphometric analysis of the sections. Brie#y, using KS400 image system (Zeiss), the area of bone (bone marrow was not included), pores and calcium phosphate ceramic were semi-automatically measured, respectively (Objective, X10). As shown in Fig. 1, three images were chosen in each section. For each implant, at least four sections were analysed. Then the porosity (in area) of the implant and the percentage of bone to the pore of each implant of HA and BCP were obtained. Regarding to the di!erence of porosity and percentage of bone to the pore, Student's t-test was made between HA and BCP implants (n"4).
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Fig. 1. A schematic diagram of the images in histomorphometric analysis.
Table 1 Bone formation induced by calcium phosphate ceramics implanted in muscles of dog for 2.5 years Materials
Bone incidence
Porosity (%)
% bone in pores
HA BCP -TCP -TCP
4/4 4/4 4/4 1/4
51$4 41$4 n/a n/a
48$4 41$2 n/a n/a
t-test of porosity between HA and BCP: p"0.03; t-test of bone ratio between HA and BCP: p"0.09. Bone-like tissues.
3. Results None of the implanted materials was fully degraded after two and a half years of implantation. All implants were traced and harvested in the sites where they were implanted. The implants were encapsulated in a thin layer of connective tissues. The soft tissues around the implants, i.e. the muscles, were normal. Histologically, calci"ed tissues were found in all HA implants (n"4), all BCP implants (n"4), all -TCP implants (n"4) and one of the four -TCP implants (Table 1). Calci"ed tissue was always found inside the implants and was not detected on the outer surface of the implants or in the soft tissues away from the implants. In HA implants, the calci"ed tissue was normal mature bone with obvious bone remodelling (Fig. 2A). Osteocytes, osteoblasts and mineralised bone matrix were clearly observed (Fig. 2A). Bone marrow was found in the core of the implants (Fig. 2A). The bone bonded to HA ceramic directly (Figs. 2A and 3A). BSE observation showed that, well-mineralised bone matrix with os-
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teocytes was formed in the pores, the bone contacted directly with pore surface, and Haversian systems were also formed (Fig. 3A). As in HA implants, the calci"ed tissue in BCP ceramic implants was also normal mature bone. Mineralised bone matrix, osteoblasts, osteocytes, bone remodelling, bone marrow and Haversian systems were observed, and the bone bonded to BCP ceramic directly (Figs. 2B and 3B). The calci"ed tissue in -TCP implants was mineralised bone matrix with osteocytes (Figs. 2C and 3C), but osteoblasts, the bone remodelling process and bone marrow were not observed (Fig. 2C). This calci"ed tissue could not be de"ned as normal bone and so was termed `bone-like tissuea in this study. Demineralisation of the bone-like tissue was observed (Fig. 2C). In one of the four -TCP implants, calci"ed tissue was found. The calci"ed tissue was the same as found in -TCP implants. In the other three -TCP implants, only loose "brous tissues were observed inside the pores (Fig. 2D). The normal bone formed in both HA and BCP implants was remodelled and is still being remodelled (Fig. 2A and B). The amount of bone remained stable, through bone remodelling. In HA implants, 48$4% pore area was occupied by bone and in BCP implants, bone "lled 41$2% pore area (Table 1). Cell-mediated degradation was not obvious in the tested implants.
4. Discussion Bone induction by porous HA, BCP and -TCP ceramics used in this study was found in our earlier studies [29,33,36,37]. In HA and BCP ceramics, bone formation was observed 45 days after implantation and normal mature bone was found after a longer time. In -TCP ceramic, normal bone was found on day 45, but after a longer time, the bone tissue was replaced by bone-like tissue [33,37]. No bone induction was found in -TCP ceramic before 150 days in our previous studies [33,37]. In this study, after two and half years, normal mature bone with bone marrow was observed in HA ceramic implants and BCP ceramic implants. Bone-like tissue was found in -TCP ceramic implants, and at a low incidence (one in four implants), bone-like tissue was also found in -TCP ceramic implants. The bone-like tissue, as suggested in our previous investigation [37], was a transformation of normal bone formed at an early stage. It is likely that TCP ceramics (both -TCP and -TCP) induced normal bone formation at a certain time period and the normal bone reversed into bone-like tissue after a longer period. Hence, the present results indicate that calcium phosphate ceramics, no matter whether the ceramic is HA,
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Fig. 2. Histological observation of calcium phosphate ceramics implanted in muscles of dog for 2.5 years: (A) mature bone with bone marrow in HA ceramic; (B) mature bone with bone marrow in BCP ceramic; (C) bone tissue in -TCP ceramic (arrow: demineralisation); (D) "brous tissues in pores of -TCP ceramic (un-decalci"ed sections, methylene blue and basic fuchsin staining, bar"100 m).
BCP, -TCP or -TCP, and if the architectural properties are that (1) the macropores are interconnected and big enough for soft tissues and blood vessels to grow in and (2) the macropore surface has microporous structures [33,36,37], can induce bone formation in muscles of dogs, although the quality of bone in di!erent calcium phosphate ceramics varied with time. The observed normal mature bone with bone marrow in HA ceramic and BCP ceramic implanted for 2.5 years suggested that calcium phosphate-induced bone does not disappear even in an environment without mechanical stresses. One important reason for the existence of bone after a long-time implantation may be the co-presence of bone marrow in the porous calcium phosphate ceramics. Mesenchymal cells in bone marrow can form bone because of which they are often used in bone tissue engineering approaches [40,41]. The induced bone was seen inside the implants but not observed on the outer surface. This indicates that hetero-
topic bone formation induced by calcium phosphates is controllable and limited to the geometry of the implants. Di!erent bone substitutes have been developed from calcium phosphates. The various qualities of bone formation in HA, BCP, -TCP and -TCP ceramics, including the long-term tissue responses of calcium phosphate biomaterials need to be addressed in more detail. An important question is why the early-formed normal bone in -TCP and -TCP ceramics reversed to bonelike tissue? The high dissolution rate of -TCP and -TCP ceramics might be a reason. Generally, TCP ceramics have higher dissolution rates than HA ceramic, and the dissolution rate is even higher when the ceramics have micropores [4,42}45]. When implanted in vivo, -TCP ceramic dissolved and the dissolution increased with time. Before bone formation, the pores of the implants were "lled with soft tissues with blood vessels, the dissolved products of -TCP were taken away and free calcium and phosphorous did not concentrate in local
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Fig. 3. BSE observation of calcium phosphate ceramics implanted in muscles of dog for 2.5 years: (A) normal mature bone in HA ceramic (arrow: Haversian system); (B) normal mature bone in BCP ceramic (arrow: Haversian system); (C) bone tissue in -TCP ceramic.
sites. However, after a longer time, when a lot of bone was formed in the pores, the soft tissues with blood vessels decreased and the free calcium and phosphorous were not taken away immediately. The local high concentration of calcium and phosphorus could be too high to allow survival of osteoblasts, osteoclasts and osteocytes, thus being harmful to bone remodelling [37]. If so, normal osteogenesis and bone remodelling are expected to cease, in agreement with our results [37]. The acidic microenvironment caused by the dissolution of -TCP ceramic made the mineralised bone matrix demineralise. In -TCP ceramic which has a higher dissolution rate than -TCP ceramic, the high local concentration of dissolved ingredients may be harmful to the cells in soft tissues, which could explain that at most times no bone formation was found at early stage and only a low incidence of bone-like tissue was found after a long time. None of the HA, BCP, -TCP and -TCP ceramics was fully degradable, even -TCP and -TCP ceramics,
which usually are considered as resorbable biomaterials, were still present after 2.5 years in vivo. Cell-mediated degradation of the ceramics was not obviously observed, moreover the high dissolution rates of TCP ceramics were found to be harmful for bone formation and even detrimental to the formed bone when -TCP ceramic was implanted in bony site for a longer time [44,46,47]. So, a resorbable calcium phosphate biomaterial is not recommended for bone induction. Although more bone was found in HA implants (48$4% bone in pores) than in BCP implants (41$2% bone in pores), the di!erence was not statistically signi"cant ( p"0.09'0.05). Meanwhile, if a signi"cant di!erence was found, the di!erence would be due to di!erent porosities of HA and BCP implants. In this study, HA ceramic had a higher porosity (51$4%) than BCP ceramic (41$4%) ( p"0.03(0.05) (Table 1). Biocompatibility and osteoconductivity are excellent pro"les of calcium phosphate biomaterials for bone
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repair and replacement. Now that the osteoinductive property of certain calcium phosphate biomaterials is gradually accepted, biocompatible, osteoconductive and osteoinductive calcium phosphate biomaterials can be synthesised. Our results presented herein indicate that the induced bone in certain calcium phosphate biomaterials does not disappear for a long time in vivo and remains stable inside the implants. The results also suggest the safety and e$cacy of osteoinductive calcium phosphate biomaterials for clinical use. However, variations among various calcium phosphate biomaterials must be studied in order to improve osteoinductive calcium phosphate biomaterials further.
5. Conclusion The quality and quantity of bone induced by calcium phosphate ceramics varied in di!erent ceramics in the period as long as 2.5 years. In HA and BCP ceramics, the induced bone was normal with bone marrow, but in -TCP ceramic and -TCP ceramic, the initially induced bone turned into bone-like tissue. The formed bone was con"ned inside the implants and the amount of bone remained stable through bone remodelling in HA and BCP ceramics. The results suggest not only the safety and e$cacy of osteoinductive calcium phosphate biomaterials for clinical use, but also the possibility of further optimising and improving osteoinductive calcium phosphate biomaterials.
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Material-dependent bone induction by calcium phosphate ceramics: a 2.5-year study in dog Huipin Yuan *, Zongjian Yang , Joost D. de Bruijn , Klaas de Groot , Xingdong Zhang Institute of Materials Science and Technology, Sichuan University, Chengdu, China Biomaterials Research Group, Leiden University, Leiden, Netherlands IsoTis N.V., Prof. Bronkhorstlaan 10, 3723 MB Bilthoven, Netherlands
Abstract Bone induction by di!erent calcium phosphate biomaterials has been reported previously. With regard to (1) whether the induced bone would disappear with time due to the absence of mechanical stresses and (2) whether this heterotopically formed bone would give rise to uncontrolled growth, a long-time investigation of porous hydroxyapatite ceramic (HA), porous biphasic calcium phosphate ceramic (TCP/HA, BCP), porous -tricalcium phosphate ceramic (-TCP) and porous -tricalcium phosphate ceramic (-TCP) was performed in dorsal muscles of dog, for 2.5 years. Histological observation, backscattered scanning electron microscopy observation and histomorphometric analysis were made on thin un-decalci"ed sections of retrieved samples. Normal compact bone with bone marrow was found in all HA implants (n"4) and in all BCP implants (n"4), 48$4% pore area was "lled with bone in HA implants and 41$2% in BCP implants. Bone-like tissue, which was a mineralised bone matrix with osteocytes but lacked osteoblasts and bone marrow, was found in all -TCP implants (n"4) and in one of the four -TCP implants. Both normal bone and bone-like tissues were con"ned inside the pores of the implants. The results show that calcium phosphate ceramics are osteoinductive in muscles of dogs. Although the quality and quantity varied among di!erent ceramics, the induced bone in both HA and BCP ceramics did neither disappear nor grow uncontrollably during the period as long as 2.5 years. 2001 Elsevier Science Ltd. All rights reserved. Keywords: Bone; Calcium phosphates; Soft tissue implantation; Osteoinductive biomaterials
1. Introduction Bone is one of the few connective tissues that have a large amount of inorganic components, mainly, calcium phosphates, which, through a complicated process of biomineralisation, are deposited within bone matrix and, therefore, give bone its excellent mechanical properties [1]. In addition to the roles in the construction of bone, calcium phosphates in bone play important roles in the homeostasis of calcium and phosphorous in body #uids and may also play roles in bone's own physiology. It was thought that calcium phosphates might stimulate osteogenesis and since the 1890s, they have been used medically in bone repair [2}4]. However, when calcium * Correspondence address: Biomaterials Research Group, Leiden University, IsoTis N.V., Prof. Bronkhorstlaan 10, 3723 MB Bilthoven, Netherlands. Tel: #31-30-2295251; fax: #31-30-2280255. E-mail address: [email protected] (H. Yuan).
phosphates were "rst used as medicines, no positive result was obtained until Albee found that tricalcium phosphate stimulated bone formation in the 1920s [2}5]. A great progress in the medical use of calcium phosphates was made between the 1970s and the 1980s. Osteoconduction, which means a guided bone formation on a biomaterial surface and the formation of chemical bonding between the newly formed bone and biomaterials, was found, when calcium phosphates were introduced into bioglasses and glass ceramics or were applied as hydroxyapatite ceramic, tricalcium phosphate ceramic and biphasic calcium phosphate ceramic [2}4,6}8]. Since then, a series of calcium phosphatebased biomaterials including bioglass, glass ceramics, di!erent calcium phosphate ceramics, calcium phosphate cements, calcium phosphate coatings and bioactive composites with calcium phosphates have been developed [2,7}14]. Extensive fundamental studies and clinical applications have demonstrated that calcium
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phosphate-based biomaterials are biocompatible and osteoconductive [3,4,8,11}13,15}17]. It appeared that osteoconduction is a common phenomenon of all calcium phosphate-based biomaterials [18], however, the roles of calcium phosphates in osteogenesis were not well understood through osteoconduction studies in bony sites because of the regeneration of bone itself. When tested in soft tissues, none of the calcium phosphate-based biomaterials showed osteoinductivity till 1990, in Japan, Yamasaki found bone formation induced by a hydroxyapatite ceramic under the subcutis of dogs [19]. Later, similar results were reported by Ripamonti in South Africa [20], by Zhang et al. in China [21], by Vagervik [22], by Toth [23] in USA, by Klein in Netherlands [24] and by various other authors [25}38]. In the last decade, bone induction has been found in coral-derived hydroxyapatite ceramic, synthetic hydroxyapatite ceramic, biphasic calcium phosphate ceramic, tricalcium phosphate ceramic, calcium pyrophosphate ceramic and calcium phosphate cement in rabbits, goats, pigs, dogs, monkeys, baboons and even in humans [19}38]. The evidence suggested that calcium phosphates not only stimulate bone formation in bony sites, but also induce bone formation in soft tissues. Bone induction by calcium phosphate biomaterials is both material- and animal-dependent [27,28,33,35}37]. Further studies on the material factors related to osteoinductivity of calcium phosphate biomaterials revealed that the chemistry, the geometry and the porous structures of calcium phosphate biomaterials are important for bone induction [27,33,35}37], and strongly suggested that calcium phosphate biomaterials can be endowed with osteoinductivity by the optimisation of the materials themselves [35,36] rather than by tissue engineering in which biomaterials were made to be osteoinductive by introducing bone morphogenetic proteins (BMPs) or osteogenic cells [39}41]. It is thus possible that osteoinductive calcium phosphate biomaterials can be made available for clinical use in orthopaedic and dental surgery in the near future, but some questions need to be answered before considering clinical use of osteoinductive calcium phosphate biomaterials. The "rst and important issue is their safety. Osteoinductive calcium phosphate biomaterials are biomaterials that induce cell di!erentiation and bone morphogenesis in soft tissues where normally no bone formation occurs. So, whether this heterotopically induced bone would give rise to uncontrolled growth must be answered. Secondly, whether the induced bone would disappear with time due to the absence of mechanical stresses in soft tissues is also important because these stresses play important roles in bone formation and bone metabolism [42]. To address these questions, a long-time investigation is necessary and therefore in this study, calcium phosphate
ceramics were implanted in the dorsal muscles of dog for 2.5 years. Moreover, material factors, which were related to the quality and quantity of calcium phosphateinduced bone, were further studied.
2. Materials and methods Porous calcium phosphate ceramics were made from wet-synthetic calcium phosphate apatite powders with various Ca/P ratios, i.e. 1.67 (HA), 1.61 (BCP), 1.5 (TCP), by foaming with 5% H O and sintering at 11003C for 3 h. Porous ceramics, with microporous structures on their macropore surface, with di!erent phase components of HA (HA ceramic), 37% -TCP/63% HA (BCP ceramic), -TCP (-TCP ceramic) and -TCP (-TCP ceramic), were obtained by di!erent cooling procedures after sintering [28,36,37]. After sterilisation, the ceramics were implanted as cylinders (5 mm in diameter, 6 mm in length) in the dorsal muscles of dogs. X-ray di!raction (XRD) analysis of the chemistry and scanning electron microscopy observation of the porous structures of di!erent calcium phosphate ceramics, together with the osteoinductivity in early stage (before 180 days), have been reported earlier [28,29,31,33,34,36,37]. One dog (male, adult, 13 kg), with four HA cylinders, four BCP cylinders, four -TCP cylinders and four -TCP cylinders in its dorsal muscles, was left for a long-time investigation. After two and a half years, the dog was sacri"ced with an overdose of pentobarbital sodium. The implants were harvested with surrounding tissues and "xed in 4% buffered formaldehyde (pH"7.4) immediately. The "xed implants were washed with phosphate bu!er solution (PBS), dehydrated in a series of ethanol solutions (70, 80, 90, 95, 100 and 100%) and embedded in methyl methacrylate (MMA). Thin un-decalci"ed sections (20 m) were made (KDG 95, IsoTis N.V., Netherlands) and stained with methylene blue and basic fuchsin for histological examination. Some sections were coated with carbon for backscattered scanning electron microscopy observation (BSE) (Philips SEM 525). The amount of bone formed in HA implants and BCP implants was determined by histomorphometric analysis of the sections. Brie#y, using KS400 image system (Zeiss), the area of bone (bone marrow was not included), pores and calcium phosphate ceramic were semi-automatically measured, respectively (Objective, X10). As shown in Fig. 1, three images were chosen in each section. For each implant, at least four sections were analysed. Then the porosity (in area) of the implant and the percentage of bone to the pore of each implant of HA and BCP were obtained. Regarding to the di!erence of porosity and percentage of bone to the pore, Student's t-test was made between HA and BCP implants (n"4).
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Fig. 1. A schematic diagram of the images in histomorphometric analysis.
Table 1 Bone formation induced by calcium phosphate ceramics implanted in muscles of dog for 2.5 years Materials
Bone incidence
Porosity (%)
% bone in pores
HA BCP -TCP -TCP
4/4 4/4 4/4 1/4
51$4 41$4 n/a n/a
48$4 41$2 n/a n/a
t-test of porosity between HA and BCP: p"0.03; t-test of bone ratio between HA and BCP: p"0.09. Bone-like tissues.
3. Results None of the implanted materials was fully degraded after two and a half years of implantation. All implants were traced and harvested in the sites where they were implanted. The implants were encapsulated in a thin layer of connective tissues. The soft tissues around the implants, i.e. the muscles, were normal. Histologically, calci"ed tissues were found in all HA implants (n"4), all BCP implants (n"4), all -TCP implants (n"4) and one of the four -TCP implants (Table 1). Calci"ed tissue was always found inside the implants and was not detected on the outer surface of the implants or in the soft tissues away from the implants. In HA implants, the calci"ed tissue was normal mature bone with obvious bone remodelling (Fig. 2A). Osteocytes, osteoblasts and mineralised bone matrix were clearly observed (Fig. 2A). Bone marrow was found in the core of the implants (Fig. 2A). The bone bonded to HA ceramic directly (Figs. 2A and 3A). BSE observation showed that, well-mineralised bone matrix with os-
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teocytes was formed in the pores, the bone contacted directly with pore surface, and Haversian systems were also formed (Fig. 3A). As in HA implants, the calci"ed tissue in BCP ceramic implants was also normal mature bone. Mineralised bone matrix, osteoblasts, osteocytes, bone remodelling, bone marrow and Haversian systems were observed, and the bone bonded to BCP ceramic directly (Figs. 2B and 3B). The calci"ed tissue in -TCP implants was mineralised bone matrix with osteocytes (Figs. 2C and 3C), but osteoblasts, the bone remodelling process and bone marrow were not observed (Fig. 2C). This calci"ed tissue could not be de"ned as normal bone and so was termed `bone-like tissuea in this study. Demineralisation of the bone-like tissue was observed (Fig. 2C). In one of the four -TCP implants, calci"ed tissue was found. The calci"ed tissue was the same as found in -TCP implants. In the other three -TCP implants, only loose "brous tissues were observed inside the pores (Fig. 2D). The normal bone formed in both HA and BCP implants was remodelled and is still being remodelled (Fig. 2A and B). The amount of bone remained stable, through bone remodelling. In HA implants, 48$4% pore area was occupied by bone and in BCP implants, bone "lled 41$2% pore area (Table 1). Cell-mediated degradation was not obvious in the tested implants.
4. Discussion Bone induction by porous HA, BCP and -TCP ceramics used in this study was found in our earlier studies [29,33,36,37]. In HA and BCP ceramics, bone formation was observed 45 days after implantation and normal mature bone was found after a longer time. In -TCP ceramic, normal bone was found on day 45, but after a longer time, the bone tissue was replaced by bone-like tissue [33,37]. No bone induction was found in -TCP ceramic before 150 days in our previous studies [33,37]. In this study, after two and half years, normal mature bone with bone marrow was observed in HA ceramic implants and BCP ceramic implants. Bone-like tissue was found in -TCP ceramic implants, and at a low incidence (one in four implants), bone-like tissue was also found in -TCP ceramic implants. The bone-like tissue, as suggested in our previous investigation [37], was a transformation of normal bone formed at an early stage. It is likely that TCP ceramics (both -TCP and -TCP) induced normal bone formation at a certain time period and the normal bone reversed into bone-like tissue after a longer period. Hence, the present results indicate that calcium phosphate ceramics, no matter whether the ceramic is HA,
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Fig. 2. Histological observation of calcium phosphate ceramics implanted in muscles of dog for 2.5 years: (A) mature bone with bone marrow in HA ceramic; (B) mature bone with bone marrow in BCP ceramic; (C) bone tissue in -TCP ceramic (arrow: demineralisation); (D) "brous tissues in pores of -TCP ceramic (un-decalci"ed sections, methylene blue and basic fuchsin staining, bar"100 m).
BCP, -TCP or -TCP, and if the architectural properties are that (1) the macropores are interconnected and big enough for soft tissues and blood vessels to grow in and (2) the macropore surface has microporous structures [33,36,37], can induce bone formation in muscles of dogs, although the quality of bone in di!erent calcium phosphate ceramics varied with time. The observed normal mature bone with bone marrow in HA ceramic and BCP ceramic implanted for 2.5 years suggested that calcium phosphate-induced bone does not disappear even in an environment without mechanical stresses. One important reason for the existence of bone after a long-time implantation may be the co-presence of bone marrow in the porous calcium phosphate ceramics. Mesenchymal cells in bone marrow can form bone because of which they are often used in bone tissue engineering approaches [40,41]. The induced bone was seen inside the implants but not observed on the outer surface. This indicates that hetero-
topic bone formation induced by calcium phosphates is controllable and limited to the geometry of the implants. Di!erent bone substitutes have been developed from calcium phosphates. The various qualities of bone formation in HA, BCP, -TCP and -TCP ceramics, including the long-term tissue responses of calcium phosphate biomaterials need to be addressed in more detail. An important question is why the early-formed normal bone in -TCP and -TCP ceramics reversed to bonelike tissue? The high dissolution rate of -TCP and -TCP ceramics might be a reason. Generally, TCP ceramics have higher dissolution rates than HA ceramic, and the dissolution rate is even higher when the ceramics have micropores [4,42}45]. When implanted in vivo, -TCP ceramic dissolved and the dissolution increased with time. Before bone formation, the pores of the implants were "lled with soft tissues with blood vessels, the dissolved products of -TCP were taken away and free calcium and phosphorous did not concentrate in local
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Fig. 3. BSE observation of calcium phosphate ceramics implanted in muscles of dog for 2.5 years: (A) normal mature bone in HA ceramic (arrow: Haversian system); (B) normal mature bone in BCP ceramic (arrow: Haversian system); (C) bone tissue in -TCP ceramic.
sites. However, after a longer time, when a lot of bone was formed in the pores, the soft tissues with blood vessels decreased and the free calcium and phosphorous were not taken away immediately. The local high concentration of calcium and phosphorus could be too high to allow survival of osteoblasts, osteoclasts and osteocytes, thus being harmful to bone remodelling [37]. If so, normal osteogenesis and bone remodelling are expected to cease, in agreement with our results [37]. The acidic microenvironment caused by the dissolution of -TCP ceramic made the mineralised bone matrix demineralise. In -TCP ceramic which has a higher dissolution rate than -TCP ceramic, the high local concentration of dissolved ingredients may be harmful to the cells in soft tissues, which could explain that at most times no bone formation was found at early stage and only a low incidence of bone-like tissue was found after a long time. None of the HA, BCP, -TCP and -TCP ceramics was fully degradable, even -TCP and -TCP ceramics,
which usually are considered as resorbable biomaterials, were still present after 2.5 years in vivo. Cell-mediated degradation of the ceramics was not obviously observed, moreover the high dissolution rates of TCP ceramics were found to be harmful for bone formation and even detrimental to the formed bone when -TCP ceramic was implanted in bony site for a longer time [44,46,47]. So, a resorbable calcium phosphate biomaterial is not recommended for bone induction. Although more bone was found in HA implants (48$4% bone in pores) than in BCP implants (41$2% bone in pores), the di!erence was not statistically signi"cant ( p"0.09'0.05). Meanwhile, if a signi"cant di!erence was found, the di!erence would be due to di!erent porosities of HA and BCP implants. In this study, HA ceramic had a higher porosity (51$4%) than BCP ceramic (41$4%) ( p"0.03(0.05) (Table 1). Biocompatibility and osteoconductivity are excellent pro"les of calcium phosphate biomaterials for bone
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repair and replacement. Now that the osteoinductive property of certain calcium phosphate biomaterials is gradually accepted, biocompatible, osteoconductive and osteoinductive calcium phosphate biomaterials can be synthesised. Our results presented herein indicate that the induced bone in certain calcium phosphate biomaterials does not disappear for a long time in vivo and remains stable inside the implants. The results also suggest the safety and e$cacy of osteoinductive calcium phosphate biomaterials for clinical use. However, variations among various calcium phosphate biomaterials must be studied in order to improve osteoinductive calcium phosphate biomaterials further.
5. Conclusion The quality and quantity of bone induced by calcium phosphate ceramics varied in di!erent ceramics in the period as long as 2.5 years. In HA and BCP ceramics, the induced bone was normal with bone marrow, but in -TCP ceramic and -TCP ceramic, the initially induced bone turned into bone-like tissue. The formed bone was con"ned inside the implants and the amount of bone remained stable through bone remodelling in HA and BCP ceramics. The results suggest not only the safety and e$cacy of osteoinductive calcium phosphate biomaterials for clinical use, but also the possibility of further optimising and improving osteoinductive calcium phosphate biomaterials.
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