Enhanced bioactivity of

Bio-Medical Materials and Engineering 13 (2003) 115–124 IOS Press 115 Enhanced bioactivity of a poly(propylene fumarate) bone graft substitute by augmentation with nano-hydroxyapatite Kai-Uwe Lewandrowski a , Shrikar P Bondre b , Donald L Wise b and Debra J Trantolo b,∗ a Orthopaedic b Cambridge Research Laboratories, Massachusetts General Hospital, Boston, MA 02114, USA Scientific, Inc., 180 Fawcett Street, Cambridge, MA 02138, USA Received 31 May 2002 Abstract The bioactivity of a nano-hydroxyapatite-augmented, bioresorbable bone graft substitute made from the unsaturated polyester, poly(propylene fumarate), was analyzed by evaluating biocompatibility and osteointegration of implants placed into a rat tibial defect Three groups of eight animals each were evaluated by grouting bone graft substitutes into 3-mm holes that were made into the anteromedial tibial metaphysis of rats Thus, a total of 24 animals was included in this study Two different formulations varying as to the type of hydroxyapatite were used: Group – nano-hydroxyapatite, Group – micronhydroxyapatite, with a Group control defect remaining unfilled Animals of each of the three groups were sacrificed in groups of eight at postoperative week three Histologic analysis revealed best superior biocompatibility and osteointegration of bone graft substitutes when nanohydroxyapatite was employed At three weeks, there was more reactive new bone formation in this group when compared to the micron-hydroxyapatite group The control group showed incomplete closure of the defect This study suggested that nano-hydroxyapatite may improve upon the bioactivity of bone implant and repair materials The model scaffold used in this study, poly(propylene fumarate), appeared to provide an osteoconductive pathway by which bone will grow in faster Clinical implications of the use potential advantages of nano-hydroxyapatite on bone repair and orthopaedic implant design are discussed Keywords: Bioactivity, nanohydroxyapatite, bioresorbable, bone graft substitute Introduction We have previously demonstrated the development of a resorbable bone repair material that does not contain biological material (either collagen or protein) [8] This material is made from the unsaturated polyester poly(propylene fumarate) (PPF) It can be mixed with cancellous autograft and crosslinked in the presence of a hydroxyapatite filler and effervescent foaming agents The porous bone graft substitute formulation can then be grouted directly in a void created by removal of a cyst or infected bone, or resulting from trauma It is generally assumed that a material with such properties would initially provide structural support to the defect site Thereafter, as the implant degrades, the net result of newly formed bone plus residual implant, the “repair-composite”, must continue to provide support to the defect reconstruction, while * Corresponding author: Debra J Trantolo, Ph.D., Cambridge Scientific, Inc., 180 Fawcett Street St., Cambridge, MA 02138, USA Tel.: 617 576 2663; Fax: 617 547 2663; E-mail: dtrantolo@aol.com 0959-2989/03/$8.00  2003 – IOS Press All rights reserved 116 K.-U Lewandrowski et al / Bone graft substitute made from poly(propylene fumarate) yielding to the establishment of native bone Ideally, such biodegradable bone graft substitute materials should sustain bony ingrowth over time as they degrade to better resemble native bone by addressing both biological and mechanical outcomes However, bony ingrowth is often superficial and limited to the periphery of the implanted bone graft substitute material Nano-hydroxyapatite (Nano-HAp) has recently been introduced to enhance the three-dimensional distribution and growth of cells within a porous scaffold Nano-HAp/collagen composites were tested utilizing organ culture techniques [2] In addition, tissue responses to nano-hydroxyapatite/collagen composites implanted in a marrow cavity were analyzed [3] This carbonate-substituted hydroxyapatite, with low crystallinity and nanometer size, were found to be bioactive, as well as biodegradable The clinical application of new forms of hydroxyapatite having improved material properties would be significant if bony ingrowth could be promoted beyond the commonly seen superficial penetration of 250 to 300 micrometers [8] Prevailing interest in nano-particle technology and the potential of this technology to possibly overcome some of the shortcomings associated with conventional hydroxyapatite has motivated this in vivo evaluation of bioactivity of nano-hydroxyapatite augmented PPF-scaffolds In this study, we report on the result of a comparative histologic and histomorphometric analysis of bone ingrowth after implantation of nano- and micron-hydroxyapatite using poly(propylene fumarate) scaffolds into a rat tibia defect model Results indicate enhanced healing with nano-hydroxyapatite composites over micron-sized composites Materials and methods 2.1 Materials and formulations The general formulation used for the study is shown in Table PPF (Mw ∼ 5,000 by GPC) was synthesized from equimolar fumaric acid and propylene glycol in the presence of p-toluene sulfonic acid [5, 6] 1-Vinyl-2-pyrollidinone (VP), benzoyl peroxide (BP), hydroquinone (HQ), and N-N-dimethyl-ptoluidine (DMPT) were purchased from Aldrich (USA) and used as received Sodium bicarbonate (SB), and citric acid (CA) were purchased from Fisher Scientific (USA) The liquid component (part II) consisting of VP, accelerator DMPT, and distilled water was added to the dry powdered mixture (part I) consisting of PPF, HA, SB, BP initiator, and CA to form a viscous putty-like paste resulting in a crosslinked polymer The accelerator, DMPT, at a concentration of 0.03% w/w, gave a working time of about 90 seconds The reaction of CA/SB with water produces carbon Table General composition of the two-part formulation PPF HA SB CtA BP Total Part I (wt., mg (wt.%)) 1179.5 (47.2) 341.5 (13.7) 51.3 (2.1) 43.4 (1.7) 52.5 (2.1) 1668.2 (66.8) VP DMPT H2 O Total Part II (wt., mg (wt.%)) 380.0 0.65 450.0 845.65 (15.2) (0.03) (18.0) (33.2) PPF: poly propylene fumarate, HA: hydroxyapatite, BP: benzoyl peroxide, SB: sodium bicarbonate, CtA: citric acid, VP: vinyl pyrollidinone, DMPT: dimethyl-para-toulidene, H2 O: distilled water K.-U Lewandrowski et al / Bone graft substitute made from poly(propylene fumarate) 117 dioxide, the blowing agent responsible for pore formation and expansion The stoichiometry requires a : mole ratio of CA : SB with a CA : SB weight ratio of 1.00 : 1.31 [1] PPF foam with pore sizes of 100–300 microns appeared desirable for bone cell ingrowth The reaction of CA/SB with water produces carbon dioxide, the blowing agent responsible for foam formation and expansion The stoichiometry requires a : mole ratio of CA : SB with a CA : SB weight ratio of 1.00 : 1.31 The moles of CO2 , which can be generated per gram of material, depend on the loading of CA/SB in the foaming cement A 0.15% CA/SB loading would produce a 25% expansion at 37◦ C and atm based on the above stoichiometry In addition to the blowing agent, the PPF formulation was crosslinked using vinyl pyrollidinone in the presence of an osteoconductive HA filler using techniques described previously [1] The hydroxyapatites used in this study were: nano HA (group 1, median particle size = 40 nanometers, as produced and characterized by Professor Ying at MIT) and 10 µm HA sintered, spherical micron-HA (group 2, median particle size = 26 microns, commercially available from CAM Implants, The Netherlands) [10,13–18] The nano HA was compared to the micron HA and empty defects (Group 3), which were left to spontaneously heal All HA preparations used in this study have been characterized using X-ray diffraction (XRD) to investigate the crystalline purity and size, photoacoustic Fourier transform infrared (PA-FTIR) spectroscopy to substantiate the molecular structure, and transmission electron microscopy (TEM) to determine the particle size and porosity 2.2 In vivo animal studies and group design Three groups were tested in the rat tibial metaphysis implantation model according to Gerhart et al [4] using either type of HA available (Groups and 2) and the unfilled control (Group 3) NIH guidelines for the care and use of laboratory animals (NIH Publication #85-23 Rev 1985) were observed Adult male Sprague Dawley rats weighing approximately 200 g were used as the animal model (Zivic Miller, Zelienople, PA, USA) Animals were anesthetized using an intramuscular injection of ketamine HCl (100 mg/kg) and xylazine (5 mg/kg) The rats were also given an intramuscular prophylactic dose of penicillin G (25,000 U/kg), and the surgical site was shaved and prepared with a solution of Betadine (povidone-iodine) and alcohol (Dura-Prep; 3M Health Care, St Paul, MD, USA) A 1.5 cm longitudinal incision was made in the anterior left hind leg, and the tibial metaphysis exposed A 3-mm hole was made in the anteromedial tibial metaphysis of rats The formulations, mixed prior to surgery to the consistency similar to a paste or putty, were implanted into the prepared tibial defect site with a spatula The PPFbased grout cured in situ and after the implantation of the bone grout, the soft tissues and skin were closed in layers with running absorbable sutures A single formulation was implanted in eight animals Thus for the three groups in the study a total to 24 animals were used All the animals were sacrificed, three weeks postoperatively 2.3 Methods of evaluation Evaluation was done by high-resolution radiographs taken immediately postoperatively and at threeweek intervals until sacrifice using a specimen X-ray unit (Microfocus 50E6310F/G; Xerox, Rochester, NY, USA) Radiographs were taken with minimal exposure (32 kvp, sec), and mammography film (Cronex Microvision; Dupont Medical Products, Wilmington, DE, USA), cassettes (MR Detail; Agfa Richfield Park, NJ, USA) and screens (Mammoray; Agfa) were employed Following sacrifice, 10-mmlong segments of the tibial bone including the section that was implanted with a bone graft substitute were harvested The specimens were processed for histologic analysis by fixation in 10% buffered formalin Specimens, which included residual bone graft material, were decalcified in EDTA and paraffin 118 K.-U Lewandrowski et al / Bone graft substitute made from poly(propylene fumarate) embedded Longitudinal sections (5 µm thick) of the total specimen were then cut and stained with hematoxyline and eosin In addition, slides were stained with the von Kossa method to visualize calcium crystals Slides were examined for resorptive activity and new bone formation at the implantation site, as well as for inflammatory responses Histomorphometric evaluation of new bone formation around the different types of grafts was done by acquiring images of serial longitudinal hematoxyline and eosin stained sections of the specimen using a CCD video camera system (TM-745; PULNiX, Sunnyvale, CA, USA) that was mounted on a Zeiss microscope Images were digitized and analyzed using Image Pro Plus software For each specimen, the area of newly formed bone surrounding the implant and within the implant was measured This measurement was standardized against the total area occupied by the implant in the same section A minimum of five sections obtained from different levels of the specimen was included for this analysis The spacing between sections of adjacent levels was typically 300 micrometers, allowing an approximate absolute volume of the newly formed bone, which is given as an average percentage rate (mean ± standard deviation) of these volume measures for each bone specimen to be obtained To compare the extent of new bone formation around the implant at its metaphyseal site between the experimental groups and the control group, the recovery index was determined It was defined as the volume ratio of newly formed bone and the volume of the whole implant based on eight animals per study group They are thus given as average percentage rates 2.4 Statistical analysis Differences in the remodeling indices were analyzed for statistical significance by employing an ANOVA test A p-level of 0.05 was considered statistically significant Results In the control group, new bone formation in the metaphyseal defect made in the rat tibia was absent There was some periosteal bone formation at the cortical drill hole site, but the remainder of the defect in the tibial metaphysis was filled primarily with bone marrow and fatty tissue In the micron-hydroxyapatite group, the implant remained structurally stable and did not disintegrate There was no histologic evidence of implant dissolution or active cellular resorption from the recipient site Although there was some moderate infiltration with PMNs, this seemed consistent with postoperative inflammatory changes In addition, there was new bone formation, which at three weeks postoperatively appeared tightly packed around the implant without excessive fibrous or inflammatory tissue There was osteoclastic and osteoblastic activity at the surface of the implant suggesting that the bone surrounding it was undergoing active remodeling Although the bone surrounding the implant bone graft underwent active remodeling, the implant remained structurally intact (Figs 1, 2) Micron-hydroxyapatite crystals were easily demonstrated with the von Kossa stain New bone formation and large round cells whose morphologic appearance was consistent with osteoblasts were noted in close proximity to the micron-HA crystal (Fig 3) In the nano-hydroxyapatite group, the implant surface stimulated a more vigorous inflammatory response with infiltration by PMNs and macrophages In addition, there appeared to be more new bone formation around the implants Similar to Group 2, in this group there was also no histologic evidence of implant dissolution or active cellular resorption from the recipient site (Figs 4, 5) In contrast to the K.-U Lewandrowski et al / Bone graft substitute made from poly(propylene fumarate) 119 Fig Photomicrograph of longitudinal section (H&E) of a rat tibia procured at three weeks postoperatively: The drill hole defect was filled with a PPF scaffold containing micron-sized hydroxyapatite showing some reactive bone formation around the PPF-based implant (10×) Note the width of the newly formed bone margin surrounding the implant is on the order of 50 to 150 µm Fig Photomicrograph of longitudinal section (H&E) of a rat tibia procured at three weeks postoperatively: The drill hole defect was filled with a PPF scaffold containing micron-sized hydroxyapatite This high power photograph (20×) shows micron-size HA (black arrows) within newly formed bone Multiple osteoblast depositing osteoid onto the implant are seen (white arrows) micron-hydroxyapatite group, no HA crystals were stainable with the von Kossa technique in the nanohydroxyapatite group (Fig 6) Similarly, as in the micron-HA group, large cells with a round nucleus, positioned towards the interface with the implant, were present Osteoids appeared to be secreted on the implant material Histomorphometry showed that the amount of new bone formed around the different types of grafts used in this study was significantly higher in the nano-hydroxyapatite group than in the control group (no implant; p < 0.002) and in the micron-HA group (p < 0.025) Although both formulations were 120 K.-U Lewandrowski et al / Bone graft substitute made from poly(propylene fumarate) Fig Photomicrograph of longitudinal section (von Kossa stain) of a rat tibia procured at three weeks postoperatively: The drill hole defect was filled with a PPF scaffold containing micron-sized hydroxyapatite This high power photograph (20×) confirms the presence of micron-size HA (black arrows) within newly formed bone Round cells with large peripheral nuclei consistent with osteoblasts are noted to deposit osteoid onto the implant (white arrows) Fig Photomicrograph of longitudinal section (H&E) of a rat tibia procured at three weeks postoperatively: The drill hole defect was filled with a PPF scaffold containing nano-sized hydroxyapatite showing vigorous reactive bone formation around the PPF-based implant (10×) There is also some new bone within the implant Note the width of the newly formed bone margin surrounding the implant is on the order of 100 to 350 µm equally osteoconductive, as measured by the implant area covered by newly formed woven bone, a wider margin of newly formed bone was noted around nano-hydroxyapatite implants In addition, more new bone was found within these types of implants As a result, the remodeling index was higher in the nano-hydroxyapatite group when compared to the micron-hydroxyapatite group (see Table 2) K.-U Lewandrowski et al / Bone graft substitute made from poly(propylene fumarate) 121 Fig Photomicrograph of longitudinal section (H&E) of a rat tibia procured at three weeks postoperatively: The drill hole defect was filled with a PPF scaffold containing nano-sized hydroxyapatite This high power photograph shows (20×) newly formed bone deposited onto the implant Multiple osteoblast are seen (white arrows) Newly formed woven bone (black arrows) and neovascularization (gray arrow) is present in close proximity to the implant Fig Photomicrograph of longitudinal section (von Kossa stain) of a rat tibia procured at three weeks postoperatively: The drill hole defect was filled with a PPF scaffold containing nano-sized hydroxyapatite This high power photograph (20×) does not reveal the presence of nano-sized HA Round cells with large peripheral nuclei consistent with osteoblasts are noted between the implant and the newly formed bone (black arrows) Table Histomorphometric analysis of new bone formation Groups Empty defect Micron-HA-PPF implant Nano-HA-PPF implant Recovery index [%] 12.3 ± 6.9 34.3 ± 10.6 48.3 ± 14.9 122 K.-U Lewandrowski et al / Bone graft substitute made from poly(propylene fumarate) Discussion Hydroxyapatite, Ca10 (PO4 )6 (OH)2 , is an attractive and widely-utilized, bioceramic material for orthopaedic and dental implants because it closely resembles native tooth and bone crystal structure Though hydroxyapatite is a common bioceramic, applications for its use have been limited by its processability and architectural design conceptualization Conventional processing lacks compositional purity and homogeneity Because hydroxyapatite is difficult to sinter, dense hydroxyapatite structures for dental implants and low-wear, orthopaedic applications typically have been obtained by high-temperature and/or high-pressure sintering with glassy sintering aids, which frequently induce decomposition to undesirable phases with poor mechanical stability and poor chemical resistance to physiological conditions Thus, conventionally formed hydroxyapatite necessitates expensive processing and compromises structural integrity due to the presence of secondary phases Existing methods require high forming and machining costs to obtain products with complex shapes Furthermore, typical conventional hydroxyapatite decomposes above 1200◦ C resulting in a material with poor mechanical stability and poor chemical resistance Recent attention has been focused on nanocrystalline or nanocomposite materials for mechanical, optical and catalytic applications By designing materials from the cluster level, crystallite building blocks of less than 10 nm are possible Various nanocrystalline ceramics for structural applications have been rigorously investigated in the 1990’s Siegel (1996) [11] discussed nanophase metals and ceramics noting that although many methods exist for the synthesis of nanostructured materials, including chemical or physical vapor deposition, gas condensation, chemical precipitation, aerosol reactions, and biological templating Nano-hydroxyapatite could eliminate some of the disadvantages associated with the use of convetional hydroxyapatite It is more homogeneous and of higher purity In the case of bioegradable polymers, better mechanical properties of copolymer composites have been demonstrated when nanoapatite particles were used Liu et al hydrothermally synthesized acicular nano-apatite (Nap) which was used as filler to make composites with a polyethylene glycol/poly(butylene terephthalate) (PEG/PBT) block copolymer (Polyactive 70 : 30) [9] The Nap had a particle diameter of 9–25 nm and a length of 80–200 nm The mechanical properties and the physiochemical characteristics of the composites, such as Young’s modulus, swelling degree in water and the calcification behaviour, were determined Nap had a strong ability to promote the calcification of composites when incorporated into Polyactive 70 : 30 In the dry state, Nap had a prominent stiffening effect for Polyactive 70 : 30 If replicable in an aqueous environment, such reinforcement of the polymer by Nap would have immediate applicability for orthopaedic implant and repair materials Investigators analyzed the effect of nano-hydroxyapatite on osteogenic cells in vitro and noted increased bioactivity Du et al [2] produced a porous nHAC composite in sheet form, which convolved to be a three-dimensional scaffold [2] Using organ culture techniques, they developed three-dimensional osteogenic cells/nHAC constructs in vitro The porous nHAC scaffold was noted to provide a microenvironment resembling that seen in vivo, and cells within the composite eventually acquired a threedimensional polygonal shape In another study, Du et al [3] investigated the tissue response to a nanohydroxyapatite/collagen composite implanted in a marrow cavity Histologic and scanning electron microscopic evaluation demonstrated the material to be bioactive as well as biodegradable At the interface of the implant and marrow tissue, solution-mediated dissolution and giant cell mediated resorption led to the degradation of the composite Bone formation at the periphery of the implant was also evident The process of implant degradation and bone substitution appeared to be reminiscent of bone remodeling K.-U Lewandrowski et al / Bone graft substitute made from poly(propylene fumarate) 123 The ultimate objective of this animal study in rats was to establish the utility of nano-hydroxyapatite use in an osteoconductive grout while demonstrating biocompatibility in the absence of foreign body reactions and evaluating the effect on the bone healing/repair process The PPF-based resorbable bone graft substitute presented here was expected to be osteoconductive because the hydroxyapatite filler has been successfully employed in similar model evaluations In this initial biocompatibility study in rats, we report on the histomorphology of the osteointegration process of a PPF-based bioresorbable bone graft material augmented either with conventional, micron-HA or nano-HA Two types of HA particle sizes were investigated in this study: (a) sintered, spherical nano-HA (median particle size = 40 nanometers), and (b) sintered, spherical micron-HA (median particle size = 26 microns) These formulations were tested in an animal model developed and utilized by Gerhart et al [4] It was straightforward and easily exploitable experimentally as a useful screening model It allowed facile evaluation of the grouting process and easy visualization of tissue bonding, in addition to allowing a comparative histologic assessment of degradation and bone ingrowth Results of this study showed absent new bone formation in negative controls, which were not filled with any graft material At three weeks, there was more reactive new bone formation without complete closure of the defect in the nano-HA) group than in the micron-HA group These histologic observations were supported by histomorphometric measurements of new bone formation that demonstrated significant increases when PPF was used in combination with nano-HA (Table 1) For the time period analyzed in this rodent study (three weeks), there was no evidence of implant failure or disintegration In addition, it was clearly evident that these PPF-based bone graft substitutes were extremely osteoconductive showing both ingrowth of newly formed woven bone with concurrent neovacularization This is consistent with earlier reports, which have suggested that osteoconductive properties of biopolymers can be improved by the addition of hydroxyapatite [7,12] The accompanying inflammatory responses appeared more pronounced in the nano-HA group than in the micron-HA group This is consistent with reports by Du et al who noted similar histologic observations [3] It remains unclear, however, what the basis of this sustained inflammatory responses is and whether or not it is of any significance for the bone healing/repair process On the basis of these observations, this study suggested that the osteoconductive properties of a PPF-based bone graft material containing 13.7% HA can be further improved by utilization of nanohydroxyapatite Rapid bony ingrowth and healing could be facilitated by accelerated bone formation around and within a biodegradable scaffold Further investigation of nano-hydroxyapatite should focus on designing bone graft materials that are structurally superior to established bone graft substitute materials and capable of stimulating in vivo ingrowth, whose temporal sequence is synchronized with the sequence of histologic events of the bone healing Acknowledgements The authors wish to thank Dr Joseph Alroy, DVM, Associate Professor in Pathology, Tufts University Schools of Medicine and Veterinary Medicine for his assistance in the histologic analysis of this study This work was supported in part by NIH/NIDR Grant No R43 DE13881-02 (to Debra J Trantolo), and NIH/NIAMS Grant AR 45062 (to Kai-Uwe Lewandrowski) 124 K.-U Lewandrowski et al / Bone graft substitute made from poly(propylene fumarate) References [1] S.P Bondre, K.U Lewandrowski, M.V Cattaneo, V Hasirci, J.D Gresser, D.L Wise, W.W Tomford and D.J Trantolo, Biodegradable foam coating of cortical allografts for improved osteoconduction, Tissue Engineering 6(3) (2000), 217– 227 [2] C Du, F.Z Cui, X.D Zhu and K de Groot, Three-dimensional nano-HAp/collagen matrix loading with osteogenic cells in organ culture, J Biomed Mater Res 44(4) (1999), 407–415 [3] C Du, F.Z Cui, Q.L Feng, X.D Zhu and K de Groot, Tissue response to nano-hydroxyapatite/collagen composite implants in marrow cavity, J Biomed Mater Res 42(4) (1998), 540–548 [4] T.N Gerhart, R.D Roux, P.A Hanff, G.L Horowitz, A.A Renshaw and W.C Hayes, Antibiotic-loaded biodegradable bone cement for prophylaxis and treatment of experimental osteomyelitis in rats, J Orthop Res 11 (1993), 250–255 [5] J.D Gresser, S.-H Hsu, H Nagaoka, C.M Lyons, D.P Nieratko, D.L Wise, G.A Barabino and D.J Trantolo, Analysis of a vinyl pyrrolidone/poly(propylene fumarate) resorbable bone cement, J Biomed Mater Res 29 (1995), 1241–1247 [6] J.D Gresser, D.J Trantolo, H Nagaoka, D.L Wise, D.E Altobelli, M.J Yaszemski and G.E Wnek, Bone cement Part 1: Biopolymer for avulsive maxillofacial repair, in: Human Biomaterials Applications, D.L Wise, J.D Gresser, D.J Trantolo and M.J Yaszemski, eds, Humana Press, Inc., Totowa, New Jersey, 1996, pp 169–187 [7] S Higashi, T Yamamuro, T Nakamura, Y Ikada, S.H Hyon and K Jamshidi, Polymer-hydroxyapatite composites for biodegradable bone fillers, Biomaterials (1986), 183–187 [8] K.U Lewandrowski, M.V Cattaneo, J.D Gresser, D.L Wise and D.J Trantolo, Effect of a poly(propylene fumarate) foaming cement on healing of critical size bone defects, Tissue Engineering 5(4) (1999), 305–316 [9] Q Liu, J.R de Wijn, K de Groot and C.A van Blitterswijk, Surface modification of nanoapatite by grafting organic polymer, Biomaterials 19(11–12) (1998), 1067–1072 [10] M.L Panchula and J.Y Ying, Enhanced transformation and sintering of transitional alumina through mechanical seeding, in: Nanostructured Materials: Science and Technology, G.M Chow and N.I Noskova, eds, Kluwer, Netherlands, 1998, pp 319–333 [11] R Siegel, Recent progress in nanophase materials, in: B Minerals, Metals and Materials, Suryanarayana et al., eds, 1996 [12] M Saito, A Maruoka, T Mori, N Sugano and K Hino, Experimental studies on a new bioactive bone cement: hydroxyapatite composite resin, Biomaterials 15 (1994), 156–160 [13] T Sun and J.Y Ying, Synthesis of microporous transition-metal-oxide molecular sieves by a supramolecular templating mechanism, Nature 389 (1997), 704–706 [14] Y Ying, Designer materials through nano processing, in: Frontiers of Engineering, National Academy Press, Washington, DC, 1996, pp 23–27 [15] Y Ying, J.B Benziger and A Navrotsky, The structural evolution of colloidal silica gels to Ceramics, J Am Ceram Soc 76(10) (1993), 2561–2570 [16] J.Y Ying, C.P Mehnert and M.S Wong, Synthesis and applications of supramolecular-templated mesoporous materials, Angew Chem Int Ed 38(1) (1999), 56–77 [17] Z Zhang, D Gekhtman, M.S Dresselhaus and J.Y Ying, Processing and characterization of single-crystalline ultrafine bismuth nanowires, Chem Mater 11(7) (1999), 1659–1665 [18] L Zhang, T Sun and J.Y Ying, Oxidation catalysis over functionalized metalloporphyrins fixated within ultralarge-pore transition metal-doped silicate supports, Chem Commun (1999), 1103–1104  ... the potential of this technology to possibly overcome some of the shortcomings associated with conventional hydroxyapatite has motivated this in vivo evaluation of bioactivity of nano-hydroxyapatite... stoichiometry requires a : mole ratio of CA : SB with a CA : SB weight ratio of 1.00 : 1.31 The moles of CO2 , which can be generated per gram of material, depend on the loading of CA/SB in the foaming cement... some of the disadvantages associated with the use of convetional hydroxyapatite It is more homogeneous and of higher purity In the case of bioegradable polymers, better mechanical properties of