Biomaterials 22 (2001) 135}150
`
`Resorbable bioceramics based on stabilized calcium phosphates.
`Part II: evaluation of biological response
`S. Langsta! *, M. Sayer, T.J.N. Smith , S.M. Pugh
`Department of Physics, Queen+s University, Stirling Hall, Rm 206A, Kingston, Ont., Canada K7L 3N6
` Millenium Biologix Inc., 785 Midpark Drive, Suite 200, Kingston, Ont., Canada K7M 7G3
`Received 4 December 1998; received in revised form 28 January 2000; accepted 13 April 2000
`
`Abstract
`
`Synthetic materials capable of being remodelled in vivo by the same processes responsible for natural bone turnover have long been
`sought for use as an arti"cial bone substitute. These materials must ideally combine osteoinductive capacity with the ability to
`withstand random dissolution at normal physiological pH, while being resorbed by natural cell-mediated processes. Resorbable
`calcium phosphate based coatings and bulk ceramics have been developed which promote the uniform deposition of new mineralized
`bone matrix thus enabling rapid integration with the surrounding host bone tissue in vivo. Furthermore, a critical result of this study
`is the determination that the silicon-stabilized calcium phosphate ceramics are essentially insoluble in biological media but are
`resorbed when acted upon by osteoclasts. In vitro biological testing and preliminary in vivo testing show that the important features
`of this new biomaterial are a characteristic calcium phosphate phase composition and a unique microporous morphology.  2000
`Elsevier Science Ltd. All rights reserved.
`
`Keywords: Bioceramic; Resorbable; Orthopedics; Hydroxyapatite; Tricalcium phosphate; Osteoclast
`
`1. Introduction
`
`The development of new bone biomaterials continues
`to attract considerable academic and commercial interest
`due in part to the need for a synthetic bone replacement
`that addresses the limitations of current orthopedic tech-
`niques. In fact, a diverse and large number of roles exist
`for bone biomaterials that are capable of incorporation
`into the natural process of bone remodeling. These in-
`clude in vitro bone cell assays [1}3], in vivo resorbable
`bone cements [4,5], implantable coatings which enhance
`the bonding of natural bone to the implant [6], implant-
`able prostheses, bone grafts and bone repair agents [7,8].
`Inherent in each of these applications is the need to
`promote both osteoblastic activity, and complementary
`osteoclastic resorption inherent in bone remodeling. In
`order to avoid the problems associated with random
`
`* Corresponding author. Tel.:#1-613-533-600074793; fax:#1-613-
`533-6463.
`E-mail address: sarah@physics.queensu.ca (S. Langsta!).
`
`dissolution which include uncontrolled physical degrada-
`tion, particulate release and long-term durability, the
`materials need to remain essentially insoluble only to be
`removed by speci"c cell activity.
`Silicon-stabilized calcium phosphate based coat-
`ings and bulk ceramics have been developed that
`not only act as suitable substrates for bone mineraliz-
`ation by osteoblasts but are essentially insoluble in
`biological media and are resorbed when acted upon
`by osteoclasts [9]. This paper describes the in vitro
`and in vivo biological response elicited by silicon-
`modi"ed calcium phosphate bone biomaterials. An
`analysis of dissolution data as compared with established
`calcium phosphate compounds provides further evidence
`for the unique behaviour observed in vitro and in
`vivo. By examination of thin "lms made using a number
`of di!erent sintering conditions and bulk ceramics
`made from commercial powders, the biological activity
`can be related to two unique characteristics of the
`colloidal powders: the presence of a mixture of sili-
`con stabilized ♡-TCP (Si-♡TCP) and HA, and a micro-
`porous morphology based on interconnected particles
`[10}12].
`
`0142-9612/01/$ - see front matter  2000 Elsevier Science Ltd. All rights reserved.
`(cid:20)(cid:3)(cid:82)(cid:73)(cid:3)(cid:20)(cid:25)
`PII: S 0 1 4 2 - 9 6 1 2 ( 0 0 ) 0 0 1 3 9 - 3
`
`MILLENIUM EXHIBIT 2051
`Baxter Healthcare Corp. et al. v. Millenium Biologix, LLC
`IPR2013-00582, -00590
`
`

`

`S. Langstaw et al. / Biomaterials 22 (2001) 135}150
`
`136
`
`2. Experimental
`
`2.1. Materials preparation
`
`(1)
`
`Microporous calcium phosphate (mHA) thin "lms
`were prepared by dip coating quartz discs in an alkaline
`colloidal suspension of HA created according to the
`following chemical reaction [13]:
`#3NHHPO
`#7NHOH
`5Ca(NO)
`#10NHNO
`#6HO
`B Ca(OH)(PO)
`The mHA thin "lms were allowed to air dry and then
`sintered in an air atmosphere for 1 h forming an Si}mHA
`thin "lm. Samples were created at a variety of sintering
`temperatures and environments to produce "lms with
`a variety of material properties [13]. Biological testing
`was performed in an attempt to correlate biological act-
`ivity with material properties. The "rst set of samples
`were sintered at 800, 850, 900, 950 and 10003C for 1 h and
`10003C for 5 min to create a set of samples with di!erent
`surface morphologies and phase compositions. A second
`set of samples examined in more detail the e!ect of
`sintering duration on surface morphology and biological
`activity. These samples were sintered at 10003C for 1,
`60 min and 8 h. All samples were sintered in a Lindberg
`Model 894 box furnace. For each sintering temperature
`under consideration, 20 identical samples were produced:
`1 for GA-XRD and SEM analysis, 1 for TEM analysis, 16
`for biological testing with cells, 1 for a a &no cell' control
`and 1 as a retained sample.
`To create an mHA powder from the colloidal suspen-
`sion, the centrifuged and decanted precipitate was dried
`for approximately 5 h at 1003C and sintered for 1 h in an
`open alumina crucible in air at a temperature of 10003C.
`The resultant product was ground to a "ne powder.
`Ceramic pellets were formed from previously sintered
`powders using a small amount of 2.5% solution of poly-
`vinyl alcohol mixed into the sintered powder as a binding
`agent. The powders were uniaxially pressed into pellets
`with a pressure of 1;10 N/m [15 000 psi]. The "nal
`pellets were "red in air at 5503C for 1 h to remove the
`binder and then sintered at 10003C for 1 h to create
`ceramic components with the desired characteristics. The
`pellet density was approximately 1.5 g/cm and the pellet
`exhibited uniform microporosity throughout the struc-
`ture.
`The role of silicon in promoting the unique bioactive
`properties was also investigated. Following the proced-
`ures for the formation and aging of the colloidal suspen-
`sion of HA outlined above, the colloid was processed to
`the stage of reducing the volume by centrifugation. In
`order to retain the colloidal sol characteristics of the
`reaction, silicon was introduced as a sol}gel metal}
`organic precursor,
`either
`tetrapropyl orthosilicate
`(Si(OCH) or TPOS) or
`tetraethyl orthosilicate
`
`Table 1
`List of materials used for experimental samples and reference standards
`
`Materials
`
`Source/preparation technique
`
`Commercial HA
`♡-TCP
`♢-TCP
`
`Calcium silicate
`
`cHA
`♡-TCP
`♢-TCP
`
`CaSiO
`
`Aldrich C28 939-6 LotC04302TQ
`Supelco Inc C3-3910 LotC200792
`Fluka C21218 AnalysisC357352/1
`14996
`Aldrich C37 266-8 LotC00714LN
`
`Microporous HA
`
`mHA
`
`Si-TCP#mHA
`
`Si}mHA
`
`Powder prepared from the thermal
`processing of the colloid in Eq. (1)
`Powder prepared from the thermal
`processing of the colloid in Eq. (1),
`where Si is the introduced additive
`
`Isotonic saline
`solution
`
`ISS
`
`Celline2+*CS203-10D
`
`♡-MEM Gibco C12571-048
`
`♡-minimal essential
`medium
`Fetal bovine serum FBS
`Penicillin G
`Gentamicin
`Fungizone
`AA
`Ascorbic acid
`♢-Glycerophosphate ♢GP
`
`Gibco C26140-079
`Sigma-Aldrich CP-3032
`Sigma-Aldrich CG-1397
`Sigma-Aldrich CA-9528
`Sigma-Aldrich CA-4544
`Sigma-Aldrich CC-9891
`
`(Si(OCH) or TEOS), in an organic carrier, either
`2-methoxyethanol (CHOCHCHOH or 2Me) or 2-4
`pentanedione (CHCOCHCOCH or ACAC). The ef-
`fect of metal-organic precursor and organic carrier are
`discussed thoroughly in Part I of this series [14]. The
`precipitates with introduced additives were dried and
`"red as discussed to form a silicon doped microporous
`calcium phosphate compound (Si}mHA). Si}mHA pow-
`ders and pellets were subsequently prepared in the same
`manner as the undoped ceramics.
`In order to make a comparison with materials pre-
`pared using previously recognized processing methods,
`bulk ceramics were created by physical mixing and sub-
`sequent "ring of commercial powders [14]. Table 1 out-
`lines the commercial products utilized during the course
`of this work.
`
`2.2. Materials analysis
`
`X-ray di!raction (XRD) spectra of thin "lm ceramics
`were acquired using a glancing angle (GA-XRD) tech-
`nique with an angle of incidence ♫"23, whereas powders
`were examined using a conventional ♫}2♫ geometry. The
`source was a 12 kW Rigaku rotating anode XRD gene-
`rator "tted with a Cr target for improved peak resolu-
`tion. The glancing angle geometry signi"cantly reduced
`interference from the substrate. The percent phase com-
`position for each material was calculated using integ-
`rated intensities of peaks distinguishable as HA, ♡-TCP
`or ♢-TCP (see Part 1 of this series [14]). Scanning
`
`(cid:21)(cid:3)(cid:82)(cid:73)(cid:3)(cid:20)(cid:25)
`
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`S. Langstaw et al. / Biomaterials 22 (2001) 135}150
`
`137
`
`electron microscopy (SEM) was performed, using
`a JEOL JSM 840 with a 10 kV accelerating voltage, to
`assess the surface and bulk morphology [13,15]. Trans-
`mission electron microscopy results are published else-
`where [14,16].
`Dissolution studies were performed to determine the
`solubility of the various calcium phosphate ceramics in
`a physiological environment. Calcium release from ce-
`ramic pellets into a solution of isotonic saline solution
`(ISS) incubated at 373C for times up to and including 10
`days was studied. Comparisons were made between
`Si}mHA, mHA, cHA, ♡-TCP, and ♢-TCP ceramics. Ce-
`ramic pellets, 12 mm OD;1 mm, were created, dried
`and "red as described above. Post-sintering mass for
`each pellet was recorded. Eleven incubation times were
`chosen for this study: 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 days.
`Twenty-two pellets were created for each powder type to
`provide duplicate samples for each incubation time.
`Each pellet was placed in a clean, dry vial and immer-
`sed in 20 ml of ISS and incubated at 373C. The samples
`were agitated twice daily by shaking the sample tray
`containing the individual vials. At the speci"ed incuba-
`tion time, 19 ml of the solution were extracted from the
`sample vial and "ltered through a 0.2 ♯m syringe "lter to
`remove any particulate matter. Each sample was diluted
`to a total volume of 39 ml with ISS and sent for atomic
`absorption spectroscopy (AAS) calcium analysis. A nega-
`tive control (ISS only) was processed for the duration of
`the 10 days in the same manner.
`AAS provided a calcium concentration (in mg/l) for
`each sample vial. As no calcium was detectable in any of
`the negative controls, the concentration of calcium in
`solution was determined directly from the average of the
`reported calcium concentration values (each sample was
`run in duplicate). The total mass of calcium dissolved in
`solution was calculated and normalized to the initial
`mass of the ceramic.
`
`2.3. In vitro biological assessment
`
`Both thin "lm and bulk ceramic samples were sterili-
`zed in an ethylene oxide (EtO) environment prior to
`initiating osteoclast (!DEX) resorption and osteoblast
`(#DEX) bone mineral deposition studies.
`Supplemented medium (SM) refers to ♡-MEM supple-
`mented with 15% FBS, 0.1 mg/ml Penicillin G, 50 ♯g/ml
`Gentamicin, and 0.3 ♯g/ml Fungizone. Fully supple-
`mented medium (FSM) refers to SM supplemented with
`50 ♯g/ml AA, and 5 mM ♢GP. Fully supplemented me-
`dium with dexamethasone (FSM#DEX) refers to FSM
`supplemented with 10\ M dexamethasone. A standard
`antibiotic stock solution (10;AB) was created by dis-
`solving the following products in ♡-MEM to produce
`concentrations of: 1.0 mg/ml Penicillin G, 500 ♯g/ml
`Gentamicin, 3.0 ♯g/ml Fungizone, 50 ♯g/ml AA, and
`5 mM ♢GP.
`
`On day 0, the femurs were excised from young adult
`male Wistar rats (115}135 g) after cervical dislocation.
`Following four 10 min washes in 10;AB and one rinse
`in SM, the epiphyses were removed and the marrow
`#ushed into test tubes using 10 ml of FSM (for osteoclast
`cultures) or FSM#DEX (for osteoblast cultures) per
`femur. This #ushing was repeated, from the other end of
`the diaphysis, using the "rst 10 ml washout. After the
`second washout, 5 ml of FSM or FSM#DEX were
`added, producing 15 ml of available cell explant suspen-
`sion per femur (30 ml per animal).
`All osteoclast cultures were performed using the pri-
`mary cell culture suspension. Each culture substrate con-
`tained within a single well of a 24-well tissue culture tray
`was directly inoculated with 1 ml of the cell explant
`suspension. Both the thin "lm samples and the bulk
`ceramic pellets were 12.7 mm in diameter and provided
`the same surface area, thus minimizing variations in
`cell-seeding density. Cultures were incubated at 373C, in
`100% humidity and 7.5% CO in air. Incubation was
`continued for the following ten days with a re-feed (1 ml
`FSM/substrate) occurring on days 1, 4, 6, and 8. Cultures
`were terminated on day 11 by either cell "xation followed
`by tartrate-resistant acid phosphatase (TRAP) staining
`or cell removal. Cell removal was accomplished by the
`addition of 1 ml 6% NaOCl with 5.2% NaCl in DDHO
`to each well followed by extensive rinsing with DDH0.
`This termination procedure preserved the surface mor-
`phology of the thin "lm and served to e$ciently remove
`organic debris, thus permitting easy visualization and
`measurement of resorption events. Osteoclast culture "x-
`ation was accomplished using Karnovsky's "xative.
`&No-cell' control samples, cultured in FSM, were per-
`formed in the same tray to identify any e!ects inherent in
`the cell culture system that were not directly attributable
`to the presence of cells. TRAP staining was not per-
`formed on the no-cell control samples.
`Resorption was identi"ed by characteristic three-di-
`mensional pits visible in the surface of the ceramic. For
`thin "lm samples, these resorption events were assessed
`as being essentially two-dimensional due to the thickness
`of the "lm ((1.0 ♯m). The amount of resorption was
`approximated by calculating the surface area of the re-
`sorption pits and normalizing it to the surface area of the
`disk. This function was performed by the Microst instru-
`ment (a computer controlled image analysis system de-
`signed by Millenium Biologix Inc.). Resorption on bulk
`ceramics was identi"ed and graded using SEM analysis.
`For cell staining, fresh TRAP stain was prepared as
`follows. A solution (C1) was created through the addi-
`tion of 2.5 ml veronal bu!er (3.09 g of sodium veronal
`mixed with 2.04 g of sodium acetate in 115 ml of pre-
`boiled distilled water), 6.25 ml pre-boiled distilled water,
`and 0.5 ml of substrate solution (10 mg of naphthol AS-
`BI phosphate in 1 ml N, N-dimethyl formamide). A sec-
`ond solution (C2) was created through the addition of
`
`(cid:22)(cid:3)(cid:82)(cid:73)(cid:3)(cid:20)(cid:25)
`
`

`

`138
`
`S. Langstaw et al. / Biomaterials 22 (2001) 135}150
`
`17.5 mg of sodium nitrate into 0.4 ml of pararolsarilin-
`HCl stock solution (1g of pararosanilin gently heated in
`20 ml of pre-boiled distilled water and 5 ml of concen-
`trated HCl to below 703C and then cooled to room
`temperature prior to "ltering). Solutions C1 and C2
`were then mixed together to create a straw coloured,
`cloudy mixture. Tartaric acid (14.3 mg) was added and
`the "nal solution allowed to sit for 5 min. This "nal
`solution was "ltered, and the pH adjusted to 5.0 using 1 N
`NaOH.
`Prior to TRAP staining, the "xed cultures were rinsed
`once with non-phosphate-bu!ered saline and twice with
`distilled water. The cultures were then rinsed with the
`TRAP stain and then incubated for 50 min at 373C in the
`TRAP stain. After incubation, the cell cultures were rin-
`sed in distilled water and stored in 70% ethanol. The
`cultures were then analysed using a light microscope.
`Osteoclasts appear as red, multi-nucleate cells whereas
`activated macrophages appear as red mononucleate cells.
`Osteoblast cultures were performed using cells derived
`from the young adult male Wistar rats and cultured as
`previously described by Maniatopoulos et al. [17]. On
`Day 0, the cell explant suspension in FSM#DEX was
`obtained as described above and 15 ml of the suspension
`was pipetted into a T75 #ask with a vented cap. The
`culture was incubated at 373C, in 100% humidity and
`7.5% CO in air. The cells were re-fed using 15 ml of
`SM#DEX per #ask on days 1 and 4. On day 5 the
`adherent cells were removed from the culture #ask using
`a trypsinization procedure and were resuspended in
`FSM#DEX to create a cell suspension with a concen-
`tration of 5;10 cells/ml. The samples were then in-
`noculated with 1 ml/well of this cell suspension. The cells
`were subsequently re-fed with 1 ml/well of FSM#DEX
`on days 6, 8, 11, 13, 15 and 18. On day 19, the cell cultures
`were "xed using Karnorvsky's "xative and stored at 43C
`in 0.1 M cacodylate bu!er until critical-point drying for
`SEM analysis.
`
`2.4. In vivo biological assessment
`
`Male Wistar rats, weighing 100}125 g, were anaesthet-
`ized and the lateral aspect of the thigh shaved and scrub-
`bed with iodine and alcohol. An incision was made in the
`lateral aspect of the knee, exposing the muscles vastus
`lateralis and adductor. These muscles were moved aside
`using a blunt probe. The lateral cortex of the femur was
`exposed and the periosteum was scraped. A round dental
`burr, with continuous saline irrigation, was used to create
`a cylindrical hole perpendicular to the long axis of the
`femur. The cylindrical implants (1.5 mm O.D.;2.0 mm
`length) were tapped into the drilled hole and "t #ush with
`the cortical surface of the femur. After implantation, the
`muscles were allowed to return to their natural arrange-
`ment and the skin was sutured closed. A single implant
`was placed in each femur.
`
`Implants were left in place for three and six weeks, after
`which termination was performed by cardiac perfusion
`with Karnovsky's "xative. The femora were dissected to
`reveal the implant site and subsequently remained in
`Karnovsky's "xative for another 5 days. One femur from
`each animal was then freeze fractured by immersion in
`liquid nitrogen and examined in the scanning electron
`microscope (SEM). In this procedure, the femur was
`notched by cutting with a rotating abrasive diamond
`disk (61T from Shofu Inc., Japan) on a dental hand piece
`(Emesco Dental Corp., NY, USA). The minimal notches
`removed anterior, posterior and medial bone in the plane
`of the implant site, but did not remove bone adjacent to
`the implant itself. The purpose of the notches was to
`create a stress riser on immersion in liquid nitrogen.
`Immersion resulted in the preferential fracture of the
`sample across the plane of the implant. Additionally,
`a second fracture line bisected the proximal portion of
`the sample along the mid-femoral long axis. In this way,
`"ve fractured implant faces were created. Following de-
`hydration,
`stub
`critical point drying from CO,
`mounting and gold coating, the sample was observed by
`Scanning Electron Microscopy (SEM-Hitachi Model
`S-570).
`The second femur was decalci"ed in a solution of 1%
`formic acid and 10% sodium citrate (pH"5). The
`sample was then dehydrated in a graded ethanol: twice
`for 1 h in 70%, twice for 1 h in 80%, three times for 1 h in
`95%,
`three times for 1 h in 100%, 1 h in 100%
`ethanol/toluene (1 : 1), overnight in cedarwood oil, three
`times
`for 1 h in methyl salicylate, 1 h in methyl
`salicylate/wax (1 : 1), and "nally wax 1, wax 2, and wax
`3 under vacuum. The sample was oriented, embedded
`and the block cooled. Six micron thick serial sections
`were cut on an AOmicrotome and stained for histologi-
`cal evaluation under light microscopy.
`
`3. Results
`
`3.1. Calcium phosphate thin xlms on quartz
`
`Six di!erent sintering temperatures were evaluated
`during the course of this work: 800, 850, 900, 950 and
`10003C for 1 h and 10003C for 5 min. The resultant phase
`composition was analyzed using GA-XRD and quanti-
`"ed using the method outlined above (see Table 2). Dur-
`ing sintering, the HA thin "lm decomposes according to
`the following reaction, creating a mixture of hydroxyapa-
`tite (HA) and silicon-stabilized tri-calcium phosphate
`(Si}TCP) [14]:
`#CaO#HO
` 3Ca(PO)
`2Ca(OH)(PO)
`It has been previously determined that the phase com-
`position is a function of sintering temperature alone [13].
`sintered at 10003C for 5 min show
`The samples
`
`(2)
`
`(cid:23)(cid:3)(cid:82)(cid:73)(cid:3)(cid:20)(cid:25)
`
`

`

`S. Langstaw et al. / Biomaterials 22 (2001) 135}150
`
`139
`
`Table 2
`Summary of sintering study biological results
`
`Temp (3C)
`
`% HA ($5%)
`
`% Si-TCP ($5%)
`
`Particle size range (♯m)
`
`8003C for 60 min
`8503C for 60 min
`9003C for 60 min
`9503C for 60 min
`10003C for 60 min
`(10003C for 5 min
`
`94.0
`97.0
`62.2
`39.6
`33.7
`56.9
`
`6.0
`3.0
`37.8
`60.4
`66.3
`43.1
`
`(0.1
`0.1}0.3
`0.2}0.5
`0.3}0.8
`0.3}1.1
`0.3}0.8
`
`Average Microst
`(!NC control)
`
`NA
`1.1$0.6
`1.2$0.5
`1.5$0.7
`4.1$2.6
`0.7$0.4
`
`Fig. 1. Summary of SEM micrographs of thin "lms sintered at (a) 8003C for 1 h, (b) 8503C for 1 h, (c) 9003C for 1 h, (d) 9503C for 1 h, (e) 10003C for 1 h
`and (f) 10003C for 5 min.
`
`a 43.1$5% conversion level, which suggests that the
`samples did not completely attain the required 10003C
`processing. As indicated above, the surface morphology
`of the thin "lms samples was determined at high magni"-
`cation using a scanning electron microscope (SEM).
`Fig. 1 illustrates the e!ect of sintering temperature on
`"lm surface morphology. The unique surface morpho-
`logy can be described as an interconnected microstruc-
`ture composed of granules. As the sintering temperature
`is increased from 800 to 10003C, the size of the granules
`increases. The micrograph of the sample sintered at
`9003C appears to be a combination of the 850 and 9503C
`"lms. This is interesting as the transformation from HA
`to Si}TCP begins to occur at 9003C (Table 2).
`It appears that increasing sintering time increases
`granule size uniformity. The sample sintered at 10003C
`
`for 5 min shows areas of large, well-connected granules
`(smaller than those observed in the sample sintered at
`10003C for 60 min) as well as some areas of smaller, more
`separated granules. It is important to note that, accord-
`ing to the GA-XRD results (Table 2), the samples did not
`completely attain the required 10003C processing and
`therefore, these smaller, more separated granules may be
`a result of a decreased sintering temperature as well as
`a decreased sintering time.
`During cell culture, samples were examined using
`a light microscope equipped with phase optics. Multinuc-
`leate giant cells were identi"ed on the surface of these
`mHA thin "lms (Fig. 2(a)). TRAP staining of these sam-
`ples reveals that these cells are TRAP#, multinucleate
`giant cells (osteoclasts) (Fig. 2(b)). The standard Si}mHA
`thin "lms on quartz (sintered at 10003C for 60 min) have
`
`(cid:24)(cid:3)(cid:82)(cid:73)(cid:3)(cid:20)(cid:25)
`
`

`

`140
`
`S. Langstaw et al. / Biomaterials 22 (2001) 135}150
`
`Fig. 2. Light micrographs of cultured Si}mHA thin "lms: (a) during cell culture illustrating a multinucleated giant cell and (b) after "xation and TRAP
`staining illustrating the presence of TRAP#multinucleated giant cells.
`
`Fig. 3. Summary of samples sintered at 10003C for 60 min: (a) pre-biotest, (b) after acellular biotest, (c) after biotest illustrating areas of resorption and
`(d) a higher magni"cation of the resorption area. Arrow denotes an area of partial resorption at the periphery of the resorption pit.
`
`a unique interconnected surface morphology as deter-
`mined by SEM (Fig. 1 and Fig. 3(a)). The microstructure
`of these ceramics is discussed in part one of this series
`[14]. Changes in surface morphology that occur during
`biological testing both in the presence and absence of
`cells are shown in Fig. 3(b)}(d) [1,2,18]. The arrow in
`Fig. 3(d) points to a region where partial resorption has
`occurred around the edges of the resorption pit. Random
`dissolution, arising from the presence of an acellular
`culture medium, is not observed (Fig. 3(b))*while mul-
`
`tiple discrete surface resorption events, identi"able as
`resorption pits, occur only in the presence of osteoclasts
`(Fig. 3(c) and (d)) [15]. The regular margins of these
`resorption pits correspond closely to the size and shape
`of the ru%ed borders normally produced by osteoclasts
`in the resorptive phase. Activated macrophage resorp-
`tion events, characterized by punctate pits, were also
`visible but in small numbers [19]. Resorption levels were
`quanti"ed using a Microst2+ image analysis system [20]
`and found to increase with the concentration of the
`
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`S. Langstaw et al. / Biomaterials 22 (2001) 135}150
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`141
`
`Table 3
`Newman}Keuls range test results to determine which samples possessed di!erent mean resorption values
`
`Samples being
`compared
`
`1000(60), (1000(5)
`1000(60), 850(60)
`1000(60), 900(60)
`1000(60), 950(60)
`950(60), (1000(5)
`950(60), 850(60)
`950(60), 900(60)
`900(60), (1000(5)
`900(60), 850(60)
`850(60), (1000(5)
`
`Mean di!erence
`
`Critical value (5%)
`
`Signi"cance (S/NS)
`
`Critical value (1%)
`
`Signi"cance (S/NS)
`
`3.43
`3.23
`2.95
`2.62
`0.82
`0.62
`0.33
`0.48
`0.28
`0.20
`
`2.36
`2.26
`2.11
`1.91
`2.11
`1.91
`1.59
`1.91
`1.59
`1.59
`
`S
`S
`S
`S
`NS
`NS
`NS
`NS
`NS
`NS
`
`2.88
`2.77
`2.64
`2.44
`2.64
`2.44
`2.14
`2.44
`2.14
`2.14
`
`S
`S
`S
`S
`NS
`NS
`NS
`NS
`NS
`NS
`
`S*signi"cant, NS*not signi"cant.
`
`Si}TCP phase. Table 2 summarizes the sintering pro"le,
`the phase composition, the range of particle sizes com-
`prising the surface morphology and the average amount
`of resorption determined using the Microst system. It
`should be noted that samples sintered at 8003C for
`60 min possessed a di!erent optical opacity than the
`other samples, which precludes the use of Microst for
`these samples.
`ANOVA statistical analysis was performed to deter-
`mine if these means were statistically di!erent [21,22].
`ANOVA results indicate that there is a statistical di!er-
`ence between the means [22]. The Newman}Keuls
`Range Test was then used to determine which samples
`produced statistically di!erent results [21]. These results
`are summarized in Table 3.
`Scanning and transmission electron microscopy in-
`dicated that the "lm morphology continues to evolve
`during sintering. From the results summarized in Tables
`2 and 3 above, evidence appeared indicating that both
`the phase composition and surface morphology may be
`important factors in determining osteoclastic response.
`As the samples sintered for only 5 min at 10003C did not
`represent the same phase composition as the samples
`sintered for 60 min at 10003C and since only samples
`sintered at 10003C showed signi"cant levels of resorp-
`tion, further analysis on samples sintered for di!erent
`times at 10003C was performed.
`To further analyse the e!ect of surface morphology,
`a set of three sample types was created by sintering at
`10003C for 1, 60 and 480 min. In particular, the 1 min
`exposure was created with speci"c attention to attaining
`the required thermal pro"le. Twenty samples were cre-
`ated for each sample type, with 16 samples being sent for
`biological testing to determine osteoclastic resorption.
`All three sample types had equivalent phase composition
`(approximately 75$5% Si}TCP and 25$5% HA). As
`each sample type exhibited di!erent thin "lm optical
`properties, the use of Microst to analyze resorption levels
`was precluded for these samples. SEM micrographs were
`
`taken of the "lms prior to biotest, after acellular biotest
`and after biotest (Figs. 3}5).
`While all of the "lms had the same phase composition
`within the stated 5% uncertainty, distinct di!erences in
`surface morphology were observed. The samples sintered
`at 10003C for only 1 min had most of the resorption
`occurring as partial resorption with a substantial amount
`of partial resorption around the periphery of the resorp-
`tion events. As indicated by the cross-sectional TEM
`analysis (Fig. 6), "lms sintered for short times develop an
`elaborate bilayer structure composed of a microporous
`upper layer of large grains and a more dense underlying
`layer of small grains. The underlying interfacial layer
`appears to be di$cult for cells to resorb. This results in
`partial resorption, where a layer of material remains at
`the bottom of the resorption pit either covering the entire
`base or in areas of the periphery of the resorption pit. It is
`possible that the di!erent chemistry present in the inter-
`facial layer limits osteoclastic activity in this "ne grained
`region [13].
`Osteoblast cultures (#DEX) were performed to deter-
`mine if the thin "lms provided a suitable substrate for
`bone mineralization. Fig. 7(a) illustrates the surface of the
`thin "lm after osteoblast bone mineral deposition. A col-
`lagen sheet has been deposited on the surface of the "lm
`and preliminary mineralization has begun. Fig. 7(b) is
`a high magni"cation micrograph of a section where the
`overlying bone mineral deposit has been pulled away
`from the surface of the substrate. A dense mineral layer
`(see arrow) can be seen between the substrate and the
`overlying collagen sheet.
`
`3.2. Materials analysis of silicon-modixed calcium
`phosphate bulk ceramics
`
`Dissolution studies were performed to determine the
`solubility properties of di!erent calcium phosphate
`ceramics in a simulated acellular, physiological environ-
`ment. The average rate of dissolution (illustrated in Fig. 8),
`
`(cid:26)(cid:3)(cid:82)(cid:73)(cid:3)(cid:20)(cid:25)
`
`

`

`142
`
`S. Langstaw et al. / Biomaterials 22 (2001) 135}150
`
`Fig. 4. Summary of samples sintered at 10003C for 1 min: (a) pre-biotest, (b) after acellular biotest, (c) after biotest illustrating areas of resorption and
`(d) a higher magni"cation of the partial resorption area. Arrow denotes an area of partial resorption.
`
`Fig. 5. Summary of samples sintered at 10003C for 480 min: (a) pre-biotest, (b) after acellular biotest, (c) after biotest illustrating areas of resorption and
`(d) a higher magni"cation of the resorption edge. Arrow denotes an area of partial resorption at the periphery of the resorption pit.
`
`(cid:27)(cid:3)(cid:82)(cid:73)(cid:3)(cid:20)(cid:25)
`
`

`

`S. Langstaw et al. / Biomaterials 22 (2001) 135}150
`
`143
`
`Fig. 6. Cross-sectional TEM image of an Si}mHA thin "lm sintered at
`(10003C for 5 min.
`
`Fig. 7. SEM micrograph illustrating the surface of a Si}mHA thin "lm
`after osteoblast cell culture. The arrow indicates a dense mineral layer
`between the substrate and the overlying collagen sheet.
`
`was determined by the slope of a linear least-squares "t
`to the cumulative calcium dissolution vs. dissolution time
`graph. After a one day stabilization period Si}mHA, ♢-
`TCP, mHA and cHA showed less than 0.02% calcium
`dissolution per day over the 10 day period. Although the
`dissolution rate of ♡-TCP was initially quite low, it quick-
`ly rose. This resulted in a dissolution rate of &0.03% per
`
`Fig. 8. Dissolution rate of calcium phosphate ceramic pellets as deter-
`mined by the slope of a linear least-squares "t to the cumulative calcium
`dissolution Vs. dissolution time graph.
`
`day. In contrast, the dissolution of Si}mHA was unique
`with an initial release of calcium upon introduction
`into the ISS and essentially no further dissolution
`over the remainder of the 10 day period. This re-
`sulted in an on-going average dissolution rate of
`&0.006% per day.
`The surface morphology of the pellets was assessed by
`comparing SEM micrograph results with that obtained
`for the thin "lm samples. The uniformity of the morpho-
`logy throughout the pellet interior was assessed by imag-
`ing a fracture surface. Pellets made from mHA or
`Si}mHA powders possessed a morphology similar to the
`interconnected, microporous surface morphology of the
`standard thin "lms (Fig. 1(e)) with the microporosity
`continuing throughout the ceramic (Fig. 9). The pellets
`were comprised of particles approximately 0.2}1.0 ♯m
`in size.
`Pellets created using cHA powder mixed with TPOS,
`to e!ect Si addition, had a signi"cantly di!erent surface
`morphology (Fig. 10(a) and (b)). These pellets appear to
`be aggregates of very large particles (1}10 ♯m) with jagged
`edges and little or no inter-connectivity present. At high-
`er magni"cations where the characteristic surface mor-
`phology of the standard thin "lms is best identi"ed, the
`large irregular particles exhibit little or no undulations in
`their surface (Fig. 10(b)). When cHA, ♢-TCP and CaSiO
`were mixed at pH"10, the resultant pellets appeared to
`be aggregates of particles. The particle size varied be-
`tween 0.1 and 2.0 ♯m with no apparent interconnected-
`ness (Fig. 10(c) and (d)). Furthermore, pellets created
`from a mixture of cHA and ♢-TCP appeared to have the
`characteristi