Calcium Phosphate-Based Osteoinductive Materials - Chemical

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Chem. Rev. 2008, 108, 4742–4753

Calcium Phosphate-Based Osteoinductive Materials Racquel Zapanta LeGeros* Department of Biomaterials and Biomimetics, New York University College of Dentistry, 345 East 24th Street, New York, New York 10010 Received July 2, 2008

Contents 1. Introduction 2. Bone and Its Properties 3. Calcium Phosphate-Based Biomaterials 3.1. Dental and Medical Applications 3.2. Composition 3.3. Properties of CaPs That Mimic Properties of Bone (or Bone Mineral) 4. Osteoinductive Properties of Calcium Phosphate-Based Biomaterials 4.1. “Intrinsic” Osteoinductivity 4.2. Engineered or Programmed Osteoinductivity 4.3. Challenges 5. Summary 6. Acknowledgments 7. References

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1. Introduction This review focuses on calcium phosphate-based bone substitute materials that are used (or can be used) for teeth or bone replacement, bone repair, augmentation, or regeneration. This review will also include some properties of bone (e.g., interconnected porosity, biodegradability, bioactivity, osteoconductivity) that are being mimicked in the manufacture of calcium phosphate-based biomaterials and some of the reported factors and strategies that can make the calcium phosphate-based biomaterials acquire osteoinductive properties. Archaeological findings showed that attempts to replace missing teeth date back to the prehistoric period. The materials used then included shells, corals, ivory (from elephant tusks), metals, and human (from corpses) and animal bones.1 Because of the practice of cremation in many societies, not much is known about prehistoric materials used to replace bones lost to accident or disease. Presently, autografts (bones obtained from another anatomic site in the same subject) remain the gold standard for bone repair, substitution, and augmentation followed by allografts (bones from another subject, such as processed cadaver bones). Autografts and allografts while having the important advantage of being osteogenic or osteoinductive (i.e., inducing bone formation), suffer from several disadvantages. With autografts the drawbacks include additional expense and trauma to the patient, possibility of donor site morbidity, and limited availability. In the case of allografts, in addition to limited supply and high cost, potential viral transmission and immunogenicity are of serious concern.2 Because of the high cost and limited availability of autografts * To whom correspondence should be addressed. Phone: (212) 998-9580. Fax: (212) 995-4244. E-mail: [email protected].

Racquel Zapanta LeGeros received her Ph.D. degree from New York University. She is currently a Professor and Associate Chair of the Department of Biomaterials and Biomimetics at New York University College of Dentistry. Her pioneering work was on substitution in the apatite structure and effect on properties. Her research interests includes biologic and synthetic apatites and related calcium phosphates, calcium phosphatebased biomaterials in the form of granules, scaffolds, cements, and coatings, and implant surface modifications. Her current research is on the development of calcium phosphate-based biomaterial for prevention of bone loss induced by diseases (e.g., osteoporosis), therapy (e.g., radiation), condition (e.g., mineral deficiency, immobility), and recovery of bone loss. She is married to Dr. John P. LeGeros and mother of Bernard, David, Katherine, and Alessandra.

and allografts, there is a great need to develop synthetic alternative biomaterials for bone replacement, repair, and augmentation. Current commercial substitute materials to replace or repair teeth and bones include metals, polymers (natural or synthetic), corals, human bones (processed cadaver bones), animal bones (processed cow bones), corals and coral derived, synthetic ceramics (calcium phosphates, calcium sulfates, calcium carbonate, bioactive glasses), and composites.3-28 It is interesting to note that several of the materials used in prehistoric times are similar to the materials used presently (e.g., coral and coral derived, animal bone derived, metals). Generally, depending on the ability to stimulate bone tissue, materials for tooth or bone repair or replacement are classified as bioinert or bioactive.3,4 Bioinert materials do not stimulate bone formation but instead stimulate formation of fibrous tissue and therefore do not directly bond to bone and thus form a weak biomaterial-bone interface.4,29 Bioactive materials stimulate bone tissue formation and therefore directly bond with bone and thus form a uniquely strong biomaterial-bone interface.3-5,11,24 Bioinert materials include metals (e.g., titanium or titanium alloys, stainless steel, cobalt-chromium, Co-Cr, alloys), some synthetic polymers (e.g., PEEK, Teflon-type), and some ceramics (e.g., alumina,

10.1021/cr800427g CCC: $71.00  2008 American Chemical Society Published on Web 11/12/2008

Calcium Phosphate-Based Osteoinductive Materials

zirconia, carbon). Bioactive materials include natural polymers (e.g., collagen, demineralized bone matrix), calcium phosphates (synthetic or derived from biologic materials such as corals, algae, bovine bone), calcium carbonate (natural or synthetic), calcium sulfates, and bioactive glasses (silica or nonsilica based). The rational for the development of calcium phosphatebased (CaP) biomaterials is their similarity in composition to the bone mineral6-8 (a calcium phosphate in the form of carbonate apatite) and similarities in some properties of bone that include biodegradability, bioactivity, and osteoconductivity. Interconnecting porosity, another important bone property, can be introduced in the manufacture of CaP biomaterials by addition of porogens. However, in spite of these desirable properties, CaP biomaterials have low fracture strength and are not suitable for load-bearing areas.6-8 Depositing CaP coatings on orthopedic and dental implants combine the bioactivity of CaP and the strength of the metal. A very important property of bone is the osteoinductivity that allows the bone to repair and regenerate itself (up to a point). The osteoinductive property of bone is due to bone morphogenetic proteins (BMPs) and osteogenic proteins (e.g., collagen, osteonectin, osteopontin, bone sialoprotein) present in the extracellular matrix.30-37 CaP biomaterials are not osteoinductive (does not form bone de novo, for example, forming bone in nonosseous sites).11 However, some CaP biomaterials have been described to have ‘inherent’ osteoinductive property, while others can be ‘engineered’ to have this property. This will be briefly discussed in section 3.

2. Bone and Its Properties Bone is a mineralized matrix, a complex composite of biopolymer and biomineral. The biopolymer consists of matrix proteins, mostly collagen (type 1) with some minor but important noncollagenous proteins (e.g., proteoglycans), minor amounts of lipids and osteogenic factors (e.g., bone morphogenetic proteins, BMPs).30-33 Bone is formed by a series of complex events rigorously orchestrated by different types of bone cells interacting with each other and with the extracellular matrix. The bone cells include (1) osteoblasts, (2) osteoclasts, (3) osteocytes, and (4) bone-lining cells. Osteoblasts are responsible for production and mineralization of the bone matrix; osteoclasts maintain the bone matrix; osteoclasts are responsible for bone resorption. Cell attachment, proliferation, and differentiation are important activities involved in bone formation. The osteoblast (bone-forming) cells attach, proliferate, and differentiate, leading to production of matrix proteins that include collagen (mostly type 1), osteopontin (OSP), bone sialoprotein (BSP), osteonectin (ONN), osteocalcin (OSC), fibronectin (FN), and BMPs before mineral deposition.30-37 BMPs and matrix proteins induce bone formation in vitro and in vivo. Collagen, OSP, and ONN have been shown in vitro to nucleate apatite formation and also inhibit or modulate apatite crystal growth. Important physicochemical properties of bone include (1) interconnecting porosity, (2) biodegradability, (3) bioactivity, (4) osteoconductivity, and (5) osteoinductivity. Pore size ranges from 10 to 50 µm and 100 to 300 nm in cortical bone and 200 to 600 µm in trabecular bone. The size and interconnection of bone porosity is essential for vascularization, diffusion of nutrients and cells, and tissue ingrowths. The bone architecture and composition allows cell attach-

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ment, migration, proliferation, and differentiation, promoting bone formation, repair, and regeneration. The bone mineral was identified as an apatite based on its similarity to the X-ray diffraction profiles of mineral apatites and their similarities in composition with each other (principally calcium and phosphate ions).38-40 The mineral of teeth and bones was idealized as calcium hydroxyapatite (HA), Ca10(PO4)6(OH)2.41 However, biologic apatites contain minor and trace elements and are therefore not pure HA. The most important minor elements are carbonate (CO3), magnesium (Mg), and sodium (Na). Systematic studies on synthetic carbonate-substituted apatites and biologic apatites using combined analytical methods (X-ray diffraction, infrared spectroscopy and chemical analyses)42-46 led to the conclusion that biologic apatites should be considered as carbonate hydroxyapatite,47-49 CHA, approximated by the formula (Ca, Na, Mg)10(PO4,HPO4,CO3)6(OH,Cl,F)2 (compared to pure hydroxyapatite, HA, Ca10(PO4)6(OH)2). Bone apatite crystals are irregularly shaped platelets of variable lengths and widths (30-45 nm) and thickness (average about 5 nm) oriented with their c axis parallel to one another and lies along the collagen fibrils.50 Biologic apatites of enamel have considerably larger crystal size (about 2000 nm) compared to that of either bone or dentin apatite, as indicated by the well-defined diffraction peaks in the XRD profile of enamel apatite47 and much broader diffraction peaks of either bone or dentin apatite (Figure 1). The concentrations of Mg and CO3 in enamel apatite are much lower than those in either dentin or bone apatite.22,47,51 The mineral phase of some fish enameloids (e.g., shark enameloid) have fluoride (F-) ions replacing the hydroxyl (OH) groups in the apatite structure.52 Differences in composition affect the lattice parameters of the apatite hexagonal structure (a- and c-axis dimensions), crystal size, and solubility of the biological and synthetic apatites22,47 as demonstrated in the differences in crystallite size and solubility of the biologic apatites in enamel, dentin, and bone.22,47,51-55 For example, substitution of CO32-(for PO43-) or Mg2+ or Sr2+ (for Ca2+) in the apatite structure causes a decrease in crystallite size and an increase in solubility, while incorporation of fluoride ions (Ffor OH- substitution in the apatite structure) causes an increase in crystallite size and decrease in solubility.22,42,43,52-55 Other calcium phosphates also occur in biologic systems, usually in pathologic calcifications (e.g., dental calculus, urinary stones, soft-tissue calcifications) or diseased states (e.g., dental caries).22,56,57 Biologic nonapatitic calcium phosphates include amorphous calcium phosphate, Cax(PO4)y · zH2O (ACP), dicalcium phosphate dihydrate, CaHPO4 · 2H2O (DCPD), octacalcium phosphate, Ca8H2(PO4)6 · 5H2O (OCP), Mg-substituted tricalcium phosphate, (Ca,Mg)3(PO4)2 (β-TCMP or Mg-TCP), and calcium pyrophosphate dihydrate, Ca2P2O7 · 2H2O (CPPD), as summarized in Table 1. However, while only CHA is present in normal calcified tissues (teeth and bones), several types of CaPs coexist in pathologic calcifications. For example, DCPD, OCP, β-TCMP (or Mg-TCP), and CHA coexist in human dental calculus. Formation of different types of calcium phosphates in both synthetic and biologic systems depends on the solution pH, temperature, and composition. In biologic as well as synthetic systems, calcium phosphates can transform from one form to another depending on the pH and composition of the synthetic solution or biologic microenvironment.22,57 For example, ACP DCPD, and OCP can transform to CHA in

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LeGeros Table 2. Calcium Phosphate-Based Biomaterials: Commercial calcium deficient apatite (CDA) hydroxyapatite (HA) Ca10(PO4)6(OH)2

HA/polyethylene HA/CaSO4 coralline HA (derived from coral) bovine bone Ap (unsintered) bovine bone apatite (sintered) tricalcium phosphate (β-TCP) Ca3(PO4)2 biphasic calcium phosphates, BCP (HA + β-TCP)

BCP/collagen BCP/fibrin BCP/silicon CHA/collagen

Figure 1. X-ray diffraction profiles of biologic apatites from (A) bone, (B) dentin, and (C) enamel. The sharper diffraction peaks in C compared to either B or A indicate that enamel apatite crystals are much larger compared to either bone or dentin apatite crystals. Table 1. Calcium Phosphates in Biologic Systems calcium phosphates amorphous calcium phosphates, ACP dicalcium phosphate dihydrate, DCPD octacalcium phosphate, OCP Mg-substituted tricalcium phosphate, β-TCMP carbonate hydroxyapatite, CHA carbonate fluorapatite, CFA calcium pyrophosphate dihydrate, CPPD

occurrence soft-tissue calcifications dental calculus, dental caries dental calculus, urinary stone dental calculus, soft tissue calcifications dental calculus, urinary stone, mineral phases of enamel, dentin, cementum, bone, fish enameloids fish enameloids joints

neutral or basic pH in the presence of HCO3- or CO3 2ions or to β-TCMP in acid, neutral, or basic pH in the presence of Mg2+ ions, or (F,OH)-apatites in the presence of F- ions.22

3. Calcium Phosphate-Based Biomaterials As stated in the Introduction, the principal rationale for developing calcium phosphate-based biomaterials and recommending their use as bone substitute materials in dentistry (e.g., in oral maxillofacial reconstruction, tooth replacement) and medicine (e.g., orthopedics) is the similarity of these

Osteogen (Impladent, NY) Calcitite Ostegraf (Ceramed,CO) Bioroc (Depuy-Bioland, France) HAPEX (Gyrus, TN) Hapset (LifeCore, MINN) Interpore, ProOsteon (Interpore, CA) BioOss(EdGeitslich, Switzerland) Endobon (Merck, Germany) Osteograf-N (Ceramed, CO) Vitoss (Orthovita, PA) MBCP (Biomatlante, France), Triosite (Zimmer, IN) Osteosynt (Einco Ltd., Brazil) Tribone (Stryker, Europe) Allograft (Zimmer, IN) Tricos (Baxter BioScience,France) FlexHA (Xomed, FL) Healos (Orquest Inc., CA)

materials to the composition of bone mineral, a calcium phosphate in the form of carbonate apatite nanocrystals. Carbonate apatite prepared at low temperature (25 or 37 °C) have similar morphology and size to that of bone apatite (Figure 2). In addition, CaP biomaterials are similar to bone mineral in its biodegradability, bioactivity, and osteoconductivity. Interconnecting porosity similar to that of bone can be introduced during the manufacture. While other materials (e.g., calcium carbonate, CaCO3; calcium sulfate, CaSO4 · 2H2O; silica-based bioactive glasses from the system CaO-Na2O-SiO2) are also bioactive, biodegradable, and osteoconductive, they do not have the chemical composition similar to that of the bone mineral.

3.1. Dental and Medical Applications The first application of a calcium phosphate material for bone repair was reported in 1920 by Albee and Morrison,58 who used a chemical reagent marked “tricalcium phosphate”. The first dental application was reported by Nery et al.59 more than 50 years later using a synthetic porous material obtained by sintering a ‘tricalcium phosphate reagent’ that was originally described by the authors as “tricalcium phosphate” or “TCP” but later demonstrated to consist of a mixture of HA and β-TCP.10 Such mixtures of HA and β-TCP is now referred to as biphasic calcium phosphates, BCP.12,60 Current dental and medical applications of CaP biomaterials include repair of periodontal defects, augmentation of alveolar bone, sinus lifts, tooth replacement, repair of large bone defects caused by tumors, spine fusion, and ear and eye implants.4-27,61-68 CaPs are used as scaffolds in tissue engineering for bone or dentin regeneration69-73 or delivery systems for drugs73-77 or antibacterial agents.78-81 CaPs are used in the form of injectable cements17,82-84or as coatings on titanium and titanium alloy implants85-89 to combine the bioactivity of the CaP and the strength of the metal.87-90 CaP (HA, BCP) are used as abrasives (instead of alumina) to roughen metal implant surfaces.90,91



Figure 2. Transmission electron microscopic (TEM) images of (A) bone apatite crystals and (B) synthetic carbonate apatite prepared at 37 °C. (Reprinted with permission from ref 91. Copyright 1998 Woodhead Publishing.)

Figure 3. Scanning electron microscopic (SEM) images showing the difference in crystal sizes and microporosity among (A) synthetic HA, (B) coralline HA, and (C) precipitated calcium deficient apatite, CDA. HA was prepared by precipitation and sintering at 1100 °C, coralline HA by hydrothermal conversion of coral (CaCO3) in (NH4)2HPO4 at 365 °C, 200 psi, and CDA by precipitation at 95 °C.

3.2. Composition Commercialization of hydroxyapatite (HA) and beta-tricalcium phosphate (β-TCP) started in the early 1980s6-9 and of biphasic calcium phosphates (BCPs) in the 1990s.12 On the basis of composition, current commercial calcium phosphates for bone and tooth repair are classified as (1) calciumdeficient apatite, CDA (i.e., Ca/P molar ratio less than the stoichiometric value of 1.67 for pure HA), (2) hydroxapatite (HA), Ca10(PO4)6(OH)2, (3) beta-tricalcium phosphate (βTCP), Ca3(PO4)2, and (4) biphasic calcium phosphate (BCP), an intimate mixture of HA and β-TCP of varying HA/βTCP weight ratios (Table 2). Experimental calcium phosphatebased biomaterials include substituted apatites,92-95 substituted tricalcium phosphates,18,98,99 calcium phosphate glasses (CPGs),21,100 and other CaPs such as OCP.86,101 CDA can be prepared by precipitation6-8,22,42,43,108 or hydrolysis of either CaHPO4 · 2H2O (DCPD)22,77 or CaHPO4 (DCP).22,42 HA is prepared by precipitation, hydrolysis, or hydrothermal methods22,42,108 at high pH then sintering at 1000-1200 °C. Coral-derived HA (coralline HA) is prepared by the hydrothermal reaction of coral (CaCO3) and (NH4)2HPO4 at 375 °C and 200 psi.106 Bovine-bone-derived apatite is obtained by removing the organic phase with or without subsequent sintering at 1000 °C.10,14,22-24 BCP is obtained by sintering CDA above 900 °C with the resulting HA/β-TCP weight ratio depending on the Ca/P ratio of the CDA before sintering.12,107 Substituted apatites or substituted βTCPs are prepared by precipitation, hydrolysis, hydrothermal, or solidstate reactions.12,22-24,42,107 The crystal size of synthetic apatite is controlled by the solution composition, reaction pH, and temperature (37-100 °C) and subsequent sintering temperature (900-1200 °C).22-24 For example, apatite nanocrystals similar to bone or dentin apatite crystals can be obtained at 25-37 °C (Figure 2), while larger crystals, similar to enamel apatite, are obtained at 80-95 °C.22-24,42,43 The difference in preparation conditions (e.g., coralline HA obtained by hydrothermal method vs

synthetic HA obtained by precipitation and sintering at 1000-1200 °C) also affect crystal size, e.g., coralline HA vs synthetic HA (Figure 3). Comparison of XRD profiles of commercial bovine-derived apatite (nonsintered) and commercial HA is shown in Figure 4.

3.3. Properties of CaPs That Mimic Properties of Bone (or Bone Mineral) Protein Adhesion Synthetic apatites readily facilitate protein adhesion as evidenced by its use in chromatography.108 Protein adhesion to synthetic surfaces is important for cell binding, proliferation, and differentiation.

Interconnecting Porosity Interconnecting macroporosity (Figure 5) is introduced in synthetic HA, β-TCP, or BCP by adding porogens (e.g., naphthalene, H2O2, polymeric porogens) or using the foaming method.105,109 Microporosity depends on sintering temperature or sintering program. Thus, CaP sintered at 1200 °C shows significantly less microporosity than that sintered at 1000 °C and a dramatic change in crystal size12,107 (Figure 6). In the case of coralline HA or bovine-derived apatites, the porosity of the original biologic material (coral or bovine bone) is preserved during processing (Figure 5).

Biodegradability In vitro biodegradation is determined by suspending the material in acidic buffer and monitoring the release of Ca2+ ions with time.54,110,111 The acidic buffer, to some extent, mimics the acidic environment during osteoclastic activity (bone resorption).112 In vitro or in vivo degradation of CaPs depends on their composition, particle size, crystallinity (reflecting crystal size), porosity, and preparation condi-


Figure 4. X-ray diffraction profiles of (A) synthetic HA and (B) bovine bone apatite. The difference in the sharpness of the diffraction peaks indicates the vast difference in their crystallite sizes. Synthetic HA was prepared by precipitation and sintering at 1000 °C. Bovine bone apatite was prepared by removing the organic component at 37 °C.

tions.113 Such experiments have demonstrated that the degradation or rate of dissolution proceeds in the following decreasing order: β-TCP . bovine bone Ap (unsintered) . bovine bone Ap (sintered) > coralline HA > HA. In the case of BCPs, degradation depends on the HA/ β-TCP ratio: the higher the ratio, the lower the degradation rate.107 Comparing different synthetic CaPs (unsintered), the solubility decreases in the order ACP > DCPD > OCP > CDA. Incorporation of different ions apatite can increase (e.g., CO3-2, Mg2+, or Sr2+) or decrease (e.g., F-) the solubility of the apatite.22,51-55 The solubility of β-TCP is decreased by incorporation of either Mg2+ or Zn2+ ions.12,114 In vitro the effect of composition observed for solution-induced or cell-induced degradation is similar. For example, osteoclastic resorption and the rate of dissolution was observed to be greater for CHA compared to CFA.93,115

Bioactivity Bioactivity, the property of the material to directly bond with the new forming bone, was first observed and described by Hench et al. in special silica-based bioactive glasses.3 In contrast, an unmineralized fibrous tissue forms at the interface of the new bone and bionert materials.4 For example, direct bone attachment is observed on a plasma-sprayed HA-coated Ti alloy surface, while fibrous tissue encapsulates the uncoated surface.116 The Ti alloy surface grit blasted with apatitic abrasive showed direct bone attachment, while the fibrous tissue interface was observed on the surface grit blasted with alumina (Figure 7), suggesting that grit blasting with bioactive apatitic abrasive rendered the surface more bioactive than grit blasting with bioinert alumina.91

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The newly formed bone bonds directly with bioactive materials through a carbonate apatite (CHA) layer at the bone-material interface.5,119 HA nanocrystals (similar in dimensions to bone apatite nanocrystals) growing on HA ceramic implanted in nonosseous site (Figure 8) was first reported by Heughebaert et al.117 In vitro a greater amount of CHA nanocrystals was observed on the coralline HA surface compared to that on the synthetic dense HA surface after immersion in fetal bovine serum.118 Presently, in vitro bioactivity (assumed to predict in vivo bioactivity) is usually tested by immersing the material in simulated body fluid (SBF) with electrolyte composition similar to that of serum.119 In vitro CHA formation on CaP surfaces occurs in the presence of proteins (e.g., in serum)118 or the absence of proteins (e.g., in mineralizing solution or in SBF).119 In vitro CHA formation on CaP-based materials in SBF (pH 7.4) or other calcifying or mineralizing solution or in serum is a precipitation process as evidenced by the uptake of calcium and phosphate ions from the serum118 and from the SBF. However, formation of CHA on CaP ceramic surfaces in vivo in nonosseous sites or osseous sites (Figure 8) is a cell-mediated dissolution/precipitation process.120,121 In vivo the cellular activity (e.g., by macrophages or osteoclasts) associated with acidic environment results in partial dissolution of the CaP ceramic, causing liberation of calcium (Ca2+) and phosphate (HPO42-, PO43-) ions onto the microenvironment. The liberated ions increase the supersaturation condition of the biologic fluid, causing precipitation of CHA incorporating Ca2+, HPO42-, PO43-, and other ions (Mg2+, Na+, CO32-) present in the biologic fluid (Figure 10).120,121 Infrared spectroscopic analyses demonstrated that these nanocrystals were intimately associated with an organic component (probably proteins) that may also have originated from the biologic fluid or serum.117,118 Bone apatite nanocrystals are also intimately associated with organic component. The population of the CHA nanocrystals on the surface of CaP material appear to depend on its dissolution property. For example, when coralline HA and synthetic HA were immersed in FBS, much more CHA nanocrystals were observed associated with the coralline HA compared with those with synthetic HA.118 The higher solubility of coralline HA compared to synthetic HA may be due to its smaller size (Figure 3) and its CO3 and Mg contents. These ions incorporated in synthetic apatites were shown to increase their solubilities. In vivo BCPs with lower HA/βTCP were associated with greater population of the CHA nanocrystals: 15HA/85βTCP. 60HA/40βTCP.120 Biodegradation of BCP depends on the HA/β-TCP ratio: the higher the ratio, the less the biodegradation. This is explained by the greater solubility of β-TCP hich is hence more easily biodegraded than HA. Therefore, the amount of CHA formed associated with CaP biomaterials in vitro and in vivo is directly related to the solubility of the material.

Osteoconductivity Osteoconductivity, when referring to biomaterials, is the ability of the material to serve as a scaffold or template to guide formation of the newly forming bone along their surfaces.122 In vivo the CHA layer that forms on CaP biomaterial surfaces adsorbs circulating proteins (from the biologic environment) on which bone cells attach, migrate, proliferate, and differentiate, leading to matrix production



Figure 5. (A) Bovine bone-derived HA. (B and C) Biphasic calcium phosphate, BCP. The original interconnecting macroporosity in bone was preserved in A. Macroporsity in B and C was introduced using porogens before sintering. C shows the presence of concavities.

Figure 6. SEM of BOP sintered at (A) 105 000 and (B) 1200 °C. Note the presence of microporosities in A and not in B.

Figure 7. Bone growth and attachment on Ti alloy cylinder grit blasted with apatitic abrasive on one side (A) and alumina abrasive on the other side (B). The side grit blasted with apatitic abrasive showed direct bone attachment (A), while the side grit blasted with alumina showed indirect bone attachment through a nonmineralized fibrous layer. (Reprinted with permission from ref 91. Copyright 1998 Woodhead Publishing.)

and biomineralization. The newly forming bone follows the outline of the bioactive CaP surface (Figure 10). All bioactive materials are also osteoconductive. Bioactive (and therefore osteoconductive) biomaterials, unlike bioinert materials, actively participate in the dynamic activity occurring at the bone-biomaterial interface.

Cellular Response to CaPs Cells attach to and engulf the CaPs, causing them to biodegrade in vitro and in vivo123 (Figure 11). CaPs allow osteoblast cells to attach, proliferate, and differentiate. Differentiating osteoblast cells produce collagen (type 1),


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Figure 8. Formation of carbonate hydroxypatite (CHA) on CaP surfaces under different conditions: (A) on HA after immersion in simulated body fluid; (B) on coralline HA after immersion in fetal bovine serum; (C) associated with HA implanted in nonosseous sites; and (D) associated with BCP implanted in sites. Biodegradation of HA and BCP is evident in C and D. Deposition is observed on A and B.

Figure 9. Schematic representation of the dissolution/precipitation process involved in formation of CHA on CaP surfaces in vivo. Acid environment caused by cellular (macrophages, osteoclasts) activity causes partial dissolution of CaP, causing increased supersaturation of the biologic or physiological fluid, causing precipitation of CHA incorporating 003 and other ions and organic molecules (protein).

Figure 10. Osteoconductive property SEM images showing new bone (NB) growing along the surfaces of Mg-substituted tricalcium phosphate (β-TCMP) after implantation in osseous site.

alkaline phosphatase, proteoglycans (decorin, lumican, biglycan), and matrix proteins (osteocalcin, osteopontin, bone

sialoprotein) known to signify bone formation27,28,34 as shown in Figure 12. Cellular response is affected by the composition of CaP. For example, zinc (Zn) from zinccontaining tricalcium phosphate (Zn-TCP)18,114,124 or fluoride (F) from F-apatite125 or carbonate-F-apatite (CFA)93,115 has been shown to inhibit osteoclastic activity. On the other hand, F in FAP125 or Mg or Zn and/or F or combination of the three ions in carbonate apatite matrix126 was shown in vitro to promote collagen production and phenotypic expression of proteoglycans and matrix proteins associated with bone mineralization (Figure 12). Factors that affect cellular response to CaPs include surface topography (roughness),127,128 composition,93,116,125,126 and particle size.129



Figure 11. SEM (A-C) and TEM (D) images showing cellular response to CaP: (A) proliferation of chondrocytes on BCP, (B) attachment on coralline HA, and phagocytosis of HA particles (C) in vitro and (D) in vivo.

4.1. “Intrinsic” Osteoinductivity

Figure 12. Response of human osteoblast cells to carbonate hydroxyapatite of different composition:51-54 production of collagen type I (col 1), expression of alkaline phosphatase (AP), matrix protein, osteocalcin (OSO), and proteoglycans (lumican, decorin, biglycan).

4. Osteoinductive Properties of Calcium Phosphate-Based Biomaterials Osteoinductivity is the ability of the material to induce de novo bone formation without the presence of osteogenic factors. The osteoinductive property of a material is usually demonstrated by bone formation after implantation in nonosseous sites (e.g., subcutaneously or in intramuscular sites). Induction of bone formation by demineralized bone matrix, DBM (bone decalcified with hydrochloric acid), after implantation in muscles of different animals was first reported by Urist in 1965.130 Urist et al. later131 demonstrated that proteins (specifically, bone morphogenetic protein, BMPs) that were originally present in the DBM were the osteoinductive factors. This conclusion was confirmed by others.132-134 A recent review by Wozney133 and de Bruijn et al.134 summarized the bone formation cascade associated with BMPs as follows: “chemotaxis of undifferentiated mesenchymal cells f cell proliferation, differentiation into chondroblasts and chondrocytes f formation of cartilaginous extracellular matrix f maturation and subsequent mineralization of hypertropic chondrocytes f removal of calcified cartilage by osteoclasts f production of bone matrix by osteoblasts f bone remodeling. Osteoinductivity may be ‘intrinsic’ or ‘engineered’.

CaP biomaterials are generally known to be osteoconductive but not osteoinductive.22 However, several CaP materials have been reported to have the ability to form bone in nonbony sites of different animals without addition of osteogenic factors. These CaP materials have included porous synthetic HA,135-138 coralline HA,139,140 β-TCP,141 porous BCP,142 calcium phosphate cements,143,144 and OCP coatings on Ti alloy.86,145 Since this osteoinductive property was observed in some CaP materials but not in others of similar composition, these materials were described to have ‘intrinsic’ osteoinductivity. This inductive phenomenon for some CaP materials was attributed to the topography, geometry, composition, macropore size, and percent porosity of the CaP. Such geometry was believed to allow entrapment and concentration of circulating bone growth factors (BMPs) and osteoprogenitor cells imparting osteoinductive properties to the CaP materials. More recently, combination of interconnecting macro- and microporosities146-151 and concavities146 (Figures 5 and 6) were demonstrated to be important features of CaPs because these features allow adsorption, entrapment, and concentration of circulating BMPs and osteogenic factors and/or osteoprogenitor cells, thus imparting osteoinductive properties to these materials. Ripamonti146 demonstrated that BMPs were concentrated on the concavities of the HA scaffolds. Zhang et al.136 demonstrated that HA with pore dimensions of 75-550 µm and 60% porosity promoted more bone formation in nonosseous (muscle) and osseous sites. Yuan et al.147 demonstrated that HA with microporosity on the macropore walls implanted in dog’s dorsal muscle induced bone formation compared to BCP without microporosity. LeNihouannen et al.,142 using microporous/macroporous BCP, demonstrated bone growth not only inside the pores (as shown by others using macroporous HA) but also on the outer surface of the BCP particles after 6 months implantation in sheep’s dorsal muscles (Figure 13). Ripamonti146 also reported that demonstration of inductive property of certain


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Figure 13. Osteoinductivity. (A) Histologic and (B) polarized pictures of new bone (NB) formation inside macroporous BCP (BCP) granule after implantation in goat muscle for 6 months. (A) New bone is shown in the lighter color.142 (Photos courtesy of Prof. G. Daculsi.)

CaPs depends on the animal models: “highly reproducible in primates, minimal in dogs and absent in rabbits and rodents”. This animal dependency of osteoinductivity is believed to be due to the difference in the amount of circulating growth factors in each of the species. In addition, implantation time may also be a factor: porous and nonporous HA and calcium phosphate cement (CPC) implanted intramuscularly in rabbit showed bone formation after 1 year.143 OCP coated on titanium implants was also reported to exhibit osteoinductive property.86,148 This may be due not to the OCP composition but to the microporosities between OCP crystals and OCP,22 bundles that allow entrapment and concentration of bone growth factors (BMPs) and/or osteogenic proteins. The amount of carbonate hydroxyapatite (CHA) formed on the BCP surface depends on the HA/β-TCP ratio in the BCP: the lower the ratio, the greater the amount of CHA formed due to the preferential dissolution of the β-TCP.120 The CHA forms by a dissolution/precipitation process.120,121 The Ca2+ and phosphate (HPO4- and PO43-) ions released from the dissolving β-TCP (due to the acid environment created by the macrophages and osteoclasts) increase the supersaturation of the biologic fluid (containing electrolytes and proteins), leading to precipitation of CHA (Figures 8 and 9) intimately associated with an organic entity (probably, protein). OCP when exposed to carbonate-containing solution (e.g., biologic fluid) can easily transform to CHA.22 The CHA layer that forms on the CaP (or any bioactive material) after implantation facilitates adhesion of proteins on which the osteoprogenitor cells can attach, proliferate, differentiate, and produce extracellular matrix that eventually leads to biomineralization or bone formation. The so-called ‘intrinsic’ osteoinductive property of CaP materials is based on their geometry, topography, interconnecting macroporosity, and microporosity (e.g., combination of Figures 5 and 6A), which allows entrapment and concentration of circulating BMPs in the biologic fluid. In addition, the CHA layer (bone apatite-like) associated with protein forming on the surface of CaP materials as a result of dissolution/precipitation processes is recognized by osteoprogenitor cells as bone apatite (CHA intimately associated with protein); these cells then attach, proliferate, and differentiate, producing extracellular matrix that leads to bone formation. It should be noted that certain CaP materials with architectural features described above as having ‘inherent’ osteoinductivity cannot be compared with osteoinductive materials like demineralized bone matrix or autografts or allografts. These latter materials originally contained the

osteogenic factors (e.g., BMPs) which are later released. The CaP materials, by virtue of their architecture, acquired the osteogenic factors circulating in the environment (osseous or nonosseous sites) and later release these factors that eventually resulted in bone formation.

4.2. Engineered or Programmed Osteoinductivity Independent of geometry, architecture, or unique porosity, CaPs or CaP-based composite scaffolds combined with osteoprogenitor cells (stem cells, marrow cells, dental pulp cells, chondrocytes), bone growth factors (BMPs), and bioactive proteins (e.g., collagen, Ops, fibrin) or peptides (based on amino acid fragments of bone sialoprotein structure) have been shown to promote enhanced bone formation.69-73,130-134,150-157 The commonly used peptide is the amino acid sequence Arg-Gly-Asp (RGD).70,146,151,157 The rationale for combining CaPs or grafting metal implants with bioactive proteins is to enhance cell adhesion, differentiation, matrix formation, and biomineralization.26,70,151,157 In tissue engineering, BCP seeded with dental pulp cells was shown to promote dentin formation.69 Chondrocytes were observed to proliferate, mature, and differentiate on BCP.158 Adult mesenchymal stem cells seeded on BCPs were shown to facilitate bone formation depending on the HA/ βTCP ratio: 20/80 . 60/40 . 100HA or 100βTCP.71,72 The structure, geometry, macroporosity, microporosity, particle size, and composition are important features of CaP materials to enhance their efficacy as carriers of the osteogenic factors for bone formation. Porosity was found to be more important than the composition of the scaffold as a property of growth factor carrier.158 De Bruijn et al.148 demonstrated that tantalum cylinders coated by coprecipitating octacalcium phosphate (OCP) with rhBMP2 implanted in intramuscular sites in dogs showed osteoinductive properties, while the uncoated cylinders did not. Engineered osteoinductivity by grafting bone growth factors, osteogenic proteins, or peptides is especially important to apply to metal implants used in orthopedic and dentistry. This will allow accelerated bone formation and enhanced osseointegration (direct bone bonding) of the implants with bone,134,152 minimizing implant loosening that could lead to implant failure. It is projected that CaP materials with ‘intrinsic’ or engineered osteoinductive properties will be able to replace autografts and allografts in bone repair and bone regeneration.


4.3. Challenges For the CaP materials with ‘inherent’ osteoinductivity, the challenge is to determine the appropriate architecture (appropriate combination of microporosity and macroporosity) in fabricating the CaP materials to optimize the ability to entrap and concentrate osteogenic factors (growth factors and/ or osteoprogenitor cells) and then be able to fabricate CaP materials with such architecture reproducibly. For CaP materials with ‘engineered’ osteoinductivity, the challenge is to determine the appropriate scaffold or carrier, the appropriate dosage of the osteogenic factors (BMPs, Ops, RGDs), and the appropriate controlled release of these factors for maximum efficiency.

5. Summary Calcium phosphate-based bone substitute materials (CaPBSMs) in different forms (granules, blocks, composites) or as cements or coatings on orthopedic and dental implants are used in many medical and dental applications. They can also be used as scaffolds in tissue engineering for dentin or bone regeneration. CaPs are similar to bone in composition and having bioactive (ability to directly bond to bone, thus forming a uniquely strong interface) and osteoconductive (ability to serve as a template or guide for the newly forming bone) properties. Interconnecting porosity (macroporosity and microporosity) similar to that of bone can be introduced by chemical or physical methods. The bioactive property promotes formation of a carbonate hydroxyapatite layer, which attracts protein to which cells bind or adhere, proliferate, and differentiate, leading to matrix production and biomineralization or formation of new bone. CaP materials, by themselves, are not osteoinductive (i.e., do not have the ability to induce de novo bone formation as evidenced by bone formation nonskeletal sites such as subcutaneous or intramuscular sites). However, osteoinductive properties can be introduced to CaP materials by two methods: (1) designing the CaPs with appropriate geometry, topography, combined appropriate macroporosity/microporosity and concavities that will allow the entrapment and concentration of circulating growth factors or osteoprogenitor cells responsible for bone formation or (2) combining CaP with growth factors (BMPs, mesenchymal cells) or bioactive proteins (collagen, OPs, or peptides based on osteonectin and bone sialoprotein). In the latter case, microporosity/ macroporosity, composition, and particle size affect the efficacy of the CaP scaffold or carrier. Introducing osteoinductive property to CaP materials will enhance their application in bone repair and regeneration for medical and dental applications. Two ways of introducing osteoinductive properties to CaPBSMs include (1) designing the CaPs with appropriate geometry, topography, combined macroporosity/microporosity and concavities or that will entrap and concentrate the circulating growth factors or (2) combining CaP with growth factors (BMPs, mesenchymal cells) or bioactive proteins (collagen, OPs, or peptides based on osteonectin and bone sialoprotein). In the latter case, microporosity/macroporosity, composition, and particle size affect the efficiency of the CaP scaffold or carrier. The concept of introducing osteoinductive properties to biomaterials is an exciting one. However, it has the appropriate architectural features of the materials or scaffolds with ‘intrinsic’ osteoinductivity, and the reproducibility of


producing these features in the manufactured is yet to be determined. In the case of ‘engineered’ osteoinductivity, the properties of the scaffolds or carriers, the mode of incorporating the osteogenic factors, their dosage, and controlled release rate have yet to be determined and optimized. Nevertheless, such materials would potentially replace the use of autografts and allografts with their attendant shortcomings.

6. Acknowledgments The author gratefully acknowledges the professional collaboration of Profs. G. Daculsi, J. P. LeGeros, A. M. Gatti, C. Frondoza, A. Ito, Y.-K. Lee, R. Kijkowska, C. Texeira, R. Rohanizadeh, and T. Sakae, Drs. D. Mijares and I. Orly, the technical assistance of Ms. F. Yao, and the support of research grants from NIH/NIDCR and NIH/NIBIB for some of the author’s work cited here.

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