Excretion of resorption products from bioactive glassimplanted in rabbit muscle

William Lai,1 Jonathan Garino,2 Catherine Flaitz,3 Paul Ducheyne1,2

1Center for Bioactive Materials and Tissue Engineering, Department of Bioengineering, University of Pennsylvania,Philadelphia, Pennsylvania 191042Department of Orthopaedic Surgery, University of Pennsylvania, Philadelphia, Pennsylvania 191043Division of Oral and Maxillofacial Pathology, Dental Branch, University of Texas, Houston, Texas 77030

Received 5 April 2005; revised 6 April 2006; accepted 11 April 2005Published online 4 August 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.30425

Abstract: Bioactive glass granules were implanted in theparaspinal muscle of rabbits to determine the pathway of thesilicon released from bioactive glass in vivo. We traced andquantified the silicon released by obtaining 24-h urine andblood samples for up to 6 months after implantation. Fur-thermore, local muscle tissue as well as the following organswere resected for chemical and histopathological analyses:brain, heart, kidney, liver, lung, lymph nodes, spleen, andthymus. The urinary silicon of the group with implantedgranules was significantly higher than in the sham-operated,control group. The average excretion rate was 2.4 mg/day,and as such, 100% of the implanted silicon was excreted in19 weeks. No elevated concentrations of silicon were found

at the implant sites or in the other organs at sacrifice, that is,24 weeks. The histological appearance of all organs wasnormal throughout. The concentrations of silicon measuredin the urine were well below saturation and because nosignificant increase in silicon was found in any organ, in-cluding kidney, the increased silicon excretion rate waswithin the physiological capacity of rabbits. Therefore, it canbe concluded that the resorbed silica gel is harmlessly ex-creted in soluble form through the urine. © 2005 WileyPeriodicals, Inc. J Biomed Mater Res 75A: 398–407, 2005

Key words: bioactive glass; degradation; excretion; resorp-tion

INTRODUCTION

Bioactive glass (BG) granules of a narrow size range(45S5, 300–355 �m) implanted in bone tissue fully reactin situ to form internal silica-gel cores with a calcium–phosphate-rich surface. In a unique process, the internalsilica-gel core degrades and what remains is an externalcalcium phosphate shell. Inside the excavated granules,osteoprogenitor cells differentiate and form new bonetissue that has not originated from the external surface ofthe granule or from preexisting bone.1 Although thedegraded material is no longer found at the local implantsite, the path of removal of the resorption products hasnot yet been traced. To address this need, we devised arabbit model to quantify the resorption, transport, andexcretion of the silica gel.

Although the specific reaction pathways of BGtransformation in situ have not been determined, the

resorption of bioactive glass and other calcium—phos-phate ceramics are well documented in vivo.2–5 A re-view paper summarizes the physicochemical phenom-ena that occur at the bioactive ceramic surface.6 Thedissolution of silicon from BG is also documented byin vitro studies modeling the excavation of BG.7

Silicon, next to oxygen, is the most prevalent ele-ment on Earth (26%) and crystalline silica in the formof quartz is the most abundant mineral in the Earth’scrust. Although silica is a major component in ourenvironment, it is not a major component of our tis-sues. Animals, including humans, over time haveevolved mechanisms to remove silicon from their bod-ies to maintain trace concentrations of silicon. In ani-mals, silicon is most abundant in connective tissue,hair, feathers, skin, tendons, muscle, and bone. Siliconis a constituent of certain glycosaminoglycans andpolyuronides, where it occurs firmly bound to thepolysaccharide matrix.8 In most tissues, silicon con-centrations in humans range from 0.6 ppm for serum,41 ppm for muscle, and to a high of 57 ppm for lungtissue. In other animals, the silicon concentrations aresimilar to humans, �1 ppm in serum and 25 ppm infemurs for both rats and monkeys.9–11

Correspondence to: P. Ducheyne; e-mail: ducheyne@seas.upenn.edu

Contract grant sponsor: the Veterans Administration; con-tract grant number: A2141RC

© 2005 Wiley Periodicals, Inc.

In addition to the many studies documenting thatbioactive glass bonds to bone and promotes bonegrowth, the biocompatibility of this material has longbeen established.12–15 However, in experiments withsilica-containing ceramic powders injected into mice, asurprising result revealed rapid death in mice by ne-phrosis.16,17 The mortality rate was directly propor-tional to the silica content of the glass.18 In this articlewe also address the relevance of these data with re-spect to the clinical use of BG by tracing the siliconrelease from BG and examining a variety of tissues forsilicon accumulation using chemical and histopatho-logical analyses.

With knowledge of trace silicon chemistry in thebody and previous bioactive glass resorption studies,we propose the following hypothesis to describe theremoval pathway of silicon from BG granules in vivo:(1) the bioactive glass is constantly reacting with itssurroundings, and the silica gel is removed by a com-bination of silicon dissolution into the circulating in-terstitial fluid and active degradation by phagocyticaction of macrophages contacting the silica matrixsurface; (2) the silicon-containing resorption productdiffuses into the local tissue and then enters the blood-stream or lymph to be distributed to other parts of thebody in soluble form;19–21 (3) because there are noknown physiological processes specific for the absorp-tion and metabolism of soluble silicon, the silicon willfinally be filtered out of the blood by the kidney;21–24

during this resorption, the silicon concentration in theorgans and body fluids remains within physiologi-cally safe ranges. This article provides the data sup-porting this hypothesis.

MATERIALS

Seven hundred fifty milligrams of 45S5 bioactive glassgranules (300–355 �m; 45% SiO2, 24.5% CaO, 24.5% Na2O,6% P2O5 by weight) were loaded directly into bilateral in-tramuscular sites in the back of New Zealand White rabbitsweighing approximately 4 kg. A total of 1500 mg of BGgranules reflects a relevant dose used in a human clinicalcase, where the granules would be used as a bone graftsubstitute (30 cc/70 kg body mass). The intramuscular im-plant site was chosen because it was assumed that the re-sorption rate of the Si would be enhanced in muscle tissue incomparison to implantation in a bone site with slower fluidturnover and healing rates.25 Studies in bone have beenconducted, though, and are reported separately.26

METHODS

The surgical procedure was performed under sterile con-ditions with a protocol approved by the Institutional Animal

Care and Use Committee at the University of Pennsylvania.NIH guidelines for the care and the use of laboratory ani-mals (NIH Publication #85-23 Rev. 1985) have been ob-served. The main, long-term experiment followed twoshorter term experiments described below. In this mainexperiment, a shallow incision (approximately 1 cm indepth) was made in the lower paraspinal muscle mass of thespinotrapezius, parallel with the muscle fibers. By spreadingthe fibers apart in the muscle bundle, a defect was formed,1 � 5 cm in area. The sterile bioactive glass granules werereceived in specially designed syringes (Orthovita, Inc.,Malvern, PA), and the granules were loaded dry, directlyinto the defect. The layers were then sutured closed withresorbable sutures. One nonresorbable suture at the implan-tation site was placed to mark the defect site for later exci-sion after healing. The rabbits were placed into a plasticmetabolic chamber immediately after surgery, and at latertime points described below.

The completely plastic metabolic chamber (Plas-Labs,Lansing, MI) is designed to sequester the rabbits in a con-trolled environment, and can separate the feces from urineusing a sieve. The plastic construction can be cleaned easily,and acid washing between uses minimizes contamination ofthe samples. In this study, the rabbits were placed in thechambers for a 24-h period to collect urine. Sufficient foodand water were provided so the rabbits were not placedunder undue dietary stress. Urine samples were obtained ona weekly basis during the first month and then on a bi-weekly basis for the remainder of the experiment.

Blood samples were also obtained on a weekly basis. Aftercoagulation at room temperature, the whole blood wasstored in a refrigerator. At the end of each experiment, therabbits were euthanized and the following organs wereremoved for chemical analysis: brain, heart, kidney, liver,lung, popliteal lymph nodes, spleen, and thymus. The loca-tion of the initial incision was located and a sample ofmuscle tissue was excised for analysis. The tissues werefrozen in a deep freezer, �70°C, until digestion. The endpoint of the experiment was determined by monitoring theurinary silicon excretion rate of the implanted group and therabbits were euthanized when the rates in the experimentaland control groups were comparable.

The tissue and blood samples were chemically digested inconcentrated nitric acid using a microwave digester (modelMDS-81D, CEM Corporation, Indian Trail, NC). Each tissuerequired a specially tailored digestion protocol, but in gen-eral, approximately 1–5 g of tissue were placed in 3–10 g ofnitric acid inside a Teflon vessel and were heated for aperiod of 1 to 2 h in the microwave. To minimize siliconcontamination, all samples were stored in plasticware and astrict regimen of acid washing was followed. The concentra-tions of elemental Si was determined by flame atomic ab-sorption spectrophotometry (AAS, Perkin-Elmer 5100, Nor-walk, CT). A student’s t-test was used to determine thestatistical significance of the differences between the im-planted and the control/sham groups. A normal distribu-tion for the data was assumed given the following results ofa Shapiro-Wilk W test: sham rabbit urinary Si excretion rate,p � 0.145, n � 61; sham muscle tissue Si concentration, p �0.501, n � 15.

Urinary creatinine excretion was measured to comparekidney function between control and implanted groups.

EXCRETION OF RESPORTION PRODUCTS FROM BIOACTIVE GLASS 399

Creatinine is a natural metabolite found in the urine, whichis proportional to a body’s muscle mass. Its excretion ratereflects kidney function, with a decreased rate suggestingimpairment. The creatinine concentrations were determinedby a colorimetric assay (Sigma Diagnostics Kit 555-A) andare reported in units of mg creatinine/day/kg body mass.Because the rabbits are all the same weight, their creatinineexcretions should all be similar. Therefore, when reportingthe silicon urinary excretion rate, it was not necessary tonormalize to creatinine excretion.

Samples of the heart, kidney, liver, lung, spleen, andthymus were saved for histopathological analysis. The tis-sues were processed for routine histologic examination andsections were stained with hematoxylin and eosin. A mini-mum of three distributed tissue sections was reviewed forall the organs.

Sequence of implantation experiments

In preparation for the main, long-term implantation ex-periment, two pilot experiments were designed to assesspertinent parameters for the implantation model. Both theseexperiments, the Short-Term Implantation and the Effect ofImplant Geometry experiments, are presented in this articlebecause they provide useful information regarding the re-sorption process.

In the short-term implantation experiment, there were atotal of nine rabbits: five rabbits that received granules andfour control rabbits that were not operated upon. The gran-ules were loaded in a small bolus in a defect area of 1 � 1cm. After a period of 6 weeks the rabbits were sacrificed andthe organs and tissues were resected for chemical analysis.The duration to sacrifice was determined by considering thatin the preliminary histological study, the granules appearedexcavated after 1 to 3 months in bone tissue.1

With the results of the brief experiment above, anotherexperiment was designed to examine the effect of implantgeometry. Two syringes of BG granules were loaded intotwo sizes of muscle defects a short defect—1 � 1 cm, and along defect—1 � 5 cm. There were three sham, three short,two long, and one control rabbit. The sham rabbits receivedsame incisions as the long implants but no BG was im-planted. Blood and urine samples were obtained and theorgans harvested for silicon analysis. The duration untilsacrifice was determined by monitoring the Si excretionrates of the different treatment groups. The creatinine con-centration in urine was also measured every 3 weeks.

Using the experience gained from the previous twoexperiments, the final long-term implantation imple-mented the “long” geometry and the experiment wasprolonged until all of the implanted silicon was removed.There were seven sham and seven implant rabbits. Bloodand urine samples were obtained and the organs har-vested for silicon analysis. The duration of the experimentwas determined by comparing the Si excretion rates of thetwo groups. Creatinine concentrations in urine were mea-sured weekly for 1 month.

RESULTS

Short-term implantation

The urinary excretion rates of silicon from controland implanted groups are presented in Figure 1 (datain Table IV): they were not significantly different untilweek 5 and 6 (p � 0.05, power � 0.80). The concen-trations of silicon measured in the urine were 60 �11ppm (2.14 � 0.39 mM) ranging from 34 to 85 ppm,well below silicon saturation in urine (�180 ppm).27

There was no correlation in urinary silicon concentra-tions between the implanted and control groups.

It appears that measurably more silicon was ex-creted in the implant group starting sometime be-tween the second and third week, thus representingthe removal of silicon from the implanted BG. Theincrease was about 3 mg/day (10–7 mg/day at week3, from Fig. 1). The increased rate remained constantand did not appear to increase or decrease. There wasa total of 315 mg of silicon implanted in the rabbit.(1500 mg of bioactive glass implanted; 45% of bioac-tive glass is silica; 46.7% of silica is Si.) At 3 mg/day,13% of the silicon was removed over the last 2 weeks.

Figure 1. Urinary Si excretion rate for control and implantgroups (n � 4, 5, respectively). There was a significantelevated excretion rate for the implant group compared tocontrols for weeks 5 and 6 (p � 0.0076, 0.025, respectively,power � 0.88, 0.93, respectively). Week 0 was sampled theday before surgery and Week 1 was sampled immediatelyafter surgery. The basal rate is the mean of all control datapoints.

400 LAI ET AL.

Table I compares the silicon concentrations of theharvested organs from both groups. The appendixwas analyzed because it is a large lymphoid tissuefound in the peritoneum. No significant accumulationof silicon was found in any of the peripheral organs.After locating the original bolus of BG granules, mus-cle tissue adjacent to the implant and not including thegranules was saved for analysis. Elevated silicon wasonly found in the muscle tissue surrounding the im-plant. No significant differences in blood silicon con-centrations were found between implanted and con-trol groups (data not shown).

Effect of implant geometry

The silicon excretion rates of the control and shamrabbits were comparable and no significant differenceswere found. Figure 2 (data in Table V) shows the daily Siexcretion rate in mg/day for the different groups over aspan of 23 weeks. The sham and control data is plottedtogether. The basal silicon excretion rate is the average ofall control data and was used in the calculation of totalsilicon excreted from the implants.

Because the error bars are significantly large, a qual-itative analysis is required. The “long” rabbits appearto have a higher excretion rate than the “short” rab-bits. By calculating the area bounded by the implantcurve and the basal excretion rate, the total siliconexcreted was determined for each rabbit. The “short”rabbits excreted 65% and the “long” rabbits excreted91% of the silicon from the granules.

To evaluate the kidney function of the rabbits, uri-nary creatinine was measured. Creatinine is a naturalmetabolite found in the urine that is proportional tothe body’s muscle mass. Its excretion rate reflects kid-ney function, with a decreased rate reflecting impair-ment. Creatinine excretion rates were relatively con-stant and stable over three different treatment groupssampled at several time points during the experiment

(Fig. 3). Because creatinine excretion rates were similarfor all rabbits, we conclude that the surgical procedureand the increased silicon excretion did not adverselyaffect kidney function.

No significant differences in blood silicon concen-trations were found among all rabbits. The results ofthe chemical analyses of the organs are presented inTable II. The only significant difference betweengroups was found in the muscle tissue close to the“short” implant. The muscle surrounding the “short”implant was significantly different from the controlmuscle (p � 0.024) and the muscle surrounding the“long” implant (p � 0.034).

Long-term implantation

Figure 4 (data in Table VI) shows the silicon excre-tion over a period of 23 weeks. Using the same meth-ods as described before, it was calculated that 318 � 32mg of the theoretical 315 mg of the silicon from the BGwas excreted in the urine over 19 weeks.

Table III shows the concentrations of silicon in var-ious tissues. Again, no significant differences werefound between groups. Creatinine excretion rateswere similar and stable over the first month of theexperiment when the silicon excretion rate reached its

TABLE IConcentrations of Si in Organs Harvested

from Control and Implanted Groups

TissueControl

ppm of SiImplantedppm of Si p-Value

Appendix 11.2 � 0.99 10.3 � 0.70 0.506Brain 2.80 � 0.12 2.71 � 0.10 0.581Heart 2.88 � 0.10 2.94 � 0.09 0.672Kidney 5.16 � 1.05 4.46 � 0.94 0.637Liver 3.46 � 0.78 2.45 � 0.70 0.371Lung 2.46 � 0.08 2.23 � 0.07 0.080Muscle 7.0 � 5.8 29.1 � 5.2 0.024Thymus 3.8 � 0.38 2.4 � 0.31 0.066

All concentrations are in parts per million wet weight. Theonly significant difference between groups was found in themuscle tissue close to the implant.

Figure 2. Urinary Si excretion rate for different implantgeometries. Three sham and one control rabbits are plottedtogether. There were three short rabbits and two long rab-bits. The basal rate is the average of all sham and controlpoints. Measurable silicon excretion ended for the shortgroup at 13 weeks and for the long group at 19 weeks. Week0 was sampled before surgery and Week 1 was sampledimmediately after surgery.

EXCRETION OF RESPORTION PRODUCTS FROM BIOACTIVE GLASS 401

maximum, 38.3 � 5.1 mg/day/kg for sham rabbitsand 36.8 � 6.5 mg/day/kg for implant rabbits.

Histopathology

The concentrations of Si measured in the urine werewell below saturation. The creatinine excretion rates

also remained normal for all rabbits over the initialphase of the experiment, which indicated no impair-ment of kidney function. Reinforcing these findings,the histopathological analysis of the various organsverified the normal appearance of the kidney includ-ing heart, liver, lung, spleen, and thymus for all rab-bits.

Figure 3. Creatinine excretion rates normalized for bodymass for sham (n � 3), implant (n � 5), and control (n � 1)rabbits. The three sham rabbits and the five rabbbits im-planted with BG did not have a reduced creatinine excretionrate compared to the control rabbit. Because the excretionrates were similar, kidney function was not adversely af-fected by the surgical procedure or the elevated silicon ex-cretion rate of the implanted rabbits.

TABLE IIConcentrations of Si in Organs Harvested from Control/

Sham Rabbits, Short Implants, and Long Implants

TissueControl/Sham

ppm of SiShort

ppm of SiLong

ppm of Si

Appendix 13.4 � 1.2 12.3 � 0.87 11.5 � 1.2Brain 2.9 � 0.56 2.8 � 0.23 2.8 � 0.80Heart 3.1 � 0.30 3.0 � 0.25 2.99 � 0.57Kidney 4.99 � 1.15 5.21 � 0.87 5.11 � 1.35Liver 3.21 � 0.98 3.41 � 1.01 2.95 � 1.34Lung 2.11 � 0.56 2.31 � 0.71 1.99 � 0.89Muscle 7.69 � 2.9 38.67 � 10.7 13.43 � 6.0Thymus 5.1 � 1.51 3.9 � 0.94 4.6 � 1.8

All concentrations are in parts per million wet weight. Theonly significant difference between groups was found in themuscle tissue close to the short implant. The short implantwas significantly different than the control (p � 0.024) andthe long implant (p � 0.034).

Figure 4. Urinary silicon excretion rates for the long-termstudy with “long” implants. Total silicon excreted by theimplant rabbits after 19 weeks � 318 mg (100%). n � 7 forboth sham and implant groups. Basal excretion rate is theaverage of all sham data points. Week 0 was sampled beforesurgery and Week 1 was sampled immediately after sur-gery.

TABLE IIISilicon Concentrations in Various Tissues and Organs

after 24 Weeks for Implanted and Sham Rabbits(Long-Term Study)

TissueSham ppm

of SiImplantedppm of Si p-Value

Blood 0.80 � 0.22 0.83 � 0.14 0.770Brain 2.71 � 0.23 2.74 � 0.25 0.804Heart 2.98 � 0.16 2.87 � 0.16 0.198Kidney 5.34 � 0.87 5.90 � 1.71 0.455Liver 2.42 � 0.17 2.38 � 0.14 0.648Lung 2.29 � 0.14 2.35 � 0.25 0.609Lymph node 3.19 � 0.59 3.04 � 0.68 0.682Muscle 6.70 � 0.86 6.99 � 1.36 0.633Thymus 2.82 � 0.59 2.88 � 0.99 0.920

All concentrations are in parts per million wet weight. Nosignificant differences were found between groups in any ofthe tissues.

402 LAI ET AL.

DISCUSSION

Implanting a clinically relevant dose of BG in softtissue with a putative faster release than in bone tissueled to dissolution of silicon into the local muscle tissueand subsequent diffusion into the bloodstream. The sil-icon-containing product was filtered by the kidney andwas finally excreted into the urine. After 19 weeks as thesilicon was removed from the body; no accumulation ofsilicon was found in any of the major organs.

Rates of silicon transport

Figure 5 is a diagram of the pathway of silicontransport from bioactive glass granule to excretion.

The various K variables are the conceptual rate con-stants for the silicon-containing resorption product(SiRP) mass flux between compartments. The shadedcircles represent the transformed bioactive glass gran-ules with a silica-rich gel center and calcium–phos-phate-rich outer layer. Kf represents rate of dissolutionof silica into fluid surrounding the granules, whichincludes the resident interstitial fluid and the localenvironment elaborated by activated macrophages ad-jacent to the glass. Kt represents the diffusion andconvection of SiRP into the local tissue compartment,which in the present experiment is the back muscle ofthe rabbit. Kb represents the diffusion of SiRP into thecirculatory system by way of the blood or lymph. TheSiRP is systemically distributed throughout the bodyuntil it is filtered out by the kidney. Kk represents the

Figure 5. Model diagram of excretion of silicon-containingresorption product (SiRP) from implanted bioactive glassgranules. K are the conceptual rate constants of SiRP trans-port to the different compartments as denoted by the ar-rows.

TABLE IVData for Figure 1 in Tabular Form

Week ControlControl

SE ImplantImplant

SE

0 6.46 0.812 6.58 0.7261 8.25 0.929 5.40 0.8312 7.11 0.707 6.58 0.6333 7.40 1.045 9.76 0.9354 7.30 0.698 8.47 0.6245 6.17 0.554 9.33 0.4966 6.60 0.561 9.54 0.458

Units are mg of silicon excreted per day. The error barsrepresent the standard error (SE) of the data. n � 4 forcontrols and n � 5 for implanted rabbits. The basal excretionrate was the mean of all control data points in the figure, 7.04mg/day.

TABLE VData for Figure 2 in Tabular Form

WeekSham/Control

SH/CONSE Short

ShortSE Long

LongSE

0 6.42 1.289 6.91 0.164 6.50 2.3001 7.42 0.791 8.97 0.463 8.79 2.2682 8.10 0.169 10.57 1.405 11.06 3.1053 7.47 0.890 10.35 1.236 10.87 0.9804 7.35 0.849 9.24 1.587 11.32 1.6306 6.89 0.632 8.12 0.393 10.07 1.7108 7.36 1.487 8.90 0.630 9.82 1.458

11 7.70 1.891 7.94 1.750 9.65 1.73313 7.38 0.731 7.64 0.665 9.84 1.00715 6.13 1.415 7.00 0.983 8.60 0.64819 7.38 1.553 6.08 0.800 7.09 0.83823 6.99 0.745 7.76 1.148 7.91 0.597

Units are mg of silicon excreted per day. The error barsrepresent the standard error (SE) of the data. n � 4 forsham/controls, n � 3 for for short rabbits, and n � 2 for longrabbits. The basal excretion rate was the mean of all controland sham data points in the figure, 7.22 mg/day.

TABLE VIData for Figure 4 in Tabular Form

Week ShamSham

SE ImplantImplant

SE

0 6.80 0.840 6.28 1.2141 7.09 0.832 10.68 1.2872 7.60 0.320 11.36 1.3633 7.32 0.912 11.04 0.8404 7.16 0.848 11.64 0.6076 7.32 0.728 10.32 0.6808 7.82 0.440 10.12 1.042

11 7.04 0.520 9.86 1.12013 6.89 0.734 10.07 0.68315 7.24 0.618 8.85 0.89119 7.22 0.360 7.30 1.10323 7.43 0.783 7.68 0.821

Units are mg of silicon excreted per day. The error barsrepresent the standard error (SE) of the data. n � 7 for shamrabbits and n � 7 for implanted rabbits. The basal excretionrate was the mean of all control and sham data points in thefigure, 7.24 mg/day.

EXCRETION OF RESPORTION PRODUCTS FROM BIOACTIVE GLASS 403

rate of SiRP entering the kidney’s glomerular filtrate.In the renal tubules, solute is reabsorbed and thefiltrate with SiRP is concentrated into urine. Ku repre-sents the rate of SiRP being excreted in the urine.

From the chemical analysis of kidney tissue at earlyand late time points, we can conclude that Ku is veryfast and is greater than Kk, because there is no accu-mulation of silicon in the kidney. There are no knownphysiological mechanisms for reabsorption of siliconin the mammalian kidney, and thus, the excretion ofSiRP is expected.

From the blood data we can also conclude that Kk

must be fast and greater than Kb because there is noaccumulation of silicon in the blood. The kidney con-stantly filters the blood, and the renal clearance ofSiRP must be much greater than the mass flux of SiRPinto the blood. Provided that the silicon is completelyexcreted through the urine and not reabsorbed in therenal tubules, then the clearance of silicon from theblood can approach the maximum clearance rate, orthe renal plasma flow. Measured by the clearance ofp-aminohippurate, the renal plasma flow is approxi-mately 60 mL/min out of a total plasma volume of 155mL for 4 kg rabbits.28

There is accumulation of silicon in the local tissue atearly time points, so Kb must be less than Kt for theinitial release of SiRP. Therefore, the rate-limitingsteps of the SiRP removal and the rates of interest areKf, Kt, and Kb.

Kf, the rate of silica dissolution into the surroundingfluid around the implant, is a complex process af-fected by many different factors. Initially when un-treated BG is implanted and is exposed to the aqueousenvironment, the silica dissolution is at its highestrate. However, as the glass transforms a protectivelater of calcium phosphate forms over the silica-gel,which impedes silica dissolution. So with time, Kf

decreases.Kt, the rate of diffusion and convection into the

local tissue of SiRP, also changes with time. As partof the healing and remodeling process, fibrous scartissue can form around a foreign object to encapsu-late and sequester it, causing Kt to decrease withtime. The rate of SiRP entering the bloodstream, Kb,is a function of the healing process like Kt. As thelocal tissue at the defect site heals, the vasculaturearound the wound increases to bring nutrients andallow remodeling cells to access the site. With moreblood vessels at the implant site, Kb increases withtime.

At the end of the experiment, no accumulation ofsilicon was detected in the local tissue, indicating thatKb is ultimately greater than Kt and Kf. This observa-tion is explained by the analysis above. As timepasses, Kf and Kt decrease while Kb increases.

Silicon excretion

With the knowledge of approximate saturation con-centrations of silicon in urine, for example, 180 ppm(guinea pig),27 we can estimate the maximum urinaryexcretion rate (mass flux) of silicon from a rabbit. Theaverage 24-h urine volume is 130 ml/kg (ranging from20–350 mL/kg).28 In our study, the 24-h total urinevolume ranged from 30 to 350 mL for 4-kg rabbits.Using the literature data, the calculated maximumsilicon excretion rate for 4 kg rabbits is 93.6 mg/day.The maximum excretion rate we observed was ap-proximately 12 mg/day (from Fig. 4).

The shape of the release rate curves suggests a sat-urated excretion, which can be described by the fol-lowing simple mass flux equation:

Jb � DeffCt � Cb)

Jb is the mass flux of dissolved silicon in the localtissue adjacent to the implant diffusing into the blood-stream. Deff is the effective diffusion coefficient. Ct isthe saturation concentration of silicon in the local tis-sue. Cb is the concentration of silicon in the blood.Because Ku and Kk are both much greater than Kb, wecan assume that Jb is also equal to the urinary siliconexcretion rate that was measured in this experiment.Cb was also measured, and it remained constantthroughout the experiment at approximately 0.8 ppm.So for the purposes of the equation, the blood com-partment can be treated as a sink. The above equationresembles Fick’s law of diffusion at a steady state.However, for the purposes of this discussion, theequation above does not describe a steady-state sys-tem and is a tool to discuss the various parameters andhow they affect SiRP transport.

The unknowns, Deff and Ct, are both dependent onthe properties of the tissues surrounding the implant.The wound-healing process is dynamic and the mus-cle around the implant undergoes scarring and re-modeling over the duration of the experiment. Relatedto Kb, Deff is also dependent on the total effectivesurface area contacting the cellular millieu and acces-sibility to the vasculature. The Ct is related to Kt andchanges over time as the BG surface reacts in situ andis transformed by cells.

Initially, the silicon excretion rate is at its highestand then decreases to a lower plateau. It is possiblethat as fibrous tissue encapsulates the implant, the Deffdecreases, which then inhibits the excretion of silicon.Ct must also be at its highest at the beginning ofimplantation when the silicon dissolution is at its max-imum.

Comparing data from the long-term study and thetwo initial studies, we notice a higher excretion rate inthe “long” implant geometry. The effective surfacearea for dissolution and diffusion is increased in the 1�5 cm2 defect versus the 1 �1 cm2 defect. This re-

404 LAI ET AL.

sulted in a higher silicon mass flux rate from theimplant site (�4.3 vs. 3.3 mg/day at maximum) and ahigher total silicon excretion.

Alternate pathway to excretion

When the blood and lymph enter the liver, there isanother possible pathway for the silicon-containingresorption product to be removed. The SiRP can beexcreted by the liver into the bile, which in turn, entersthe small intestine. Aftering entering the gastrointes-tinal tract, the SiRP can be absorbed by the intestinesto reenter the blood or it can be excreted into the feces.

After 6 weeks and 23 weeks of implantation we didnot find accumulation of silicon in the liver or theblood. The trace amount of silicon that is secreted intothe bile must be very low or nonexistent, and if someis secreted through the bile, it is likely reabsorbedthrough the small and large intestines into the blood-stream.

Measurements of silicon excretion rates in the feceswere problematic because of the high silicon contentin the feces. The measured normal rabbit fecal excre-tion of silicon for 1 day is approximately 4.1 � 1.2g/day with a large variation dependent on theamount of feed the rabbit ate. The normal rabbit uri-nary excretion of silicon was three orders of magni-tude less at 7.2 mg/day.

Form of the silicon-containing resorption product

Measurable levels of Si were found in the urine.Thus, the Si-release product in the blood must berelatively small for it to pass through kidney filtration.In humans, the size of molecules passing throughfiltration is approximately 18 Å.29 The increased uri-nary Si measured here supports the hypothesis thatthe silica gel from the reacted BG granules is dissolvedinto the blood stream and removed by the kidney.

Silicic acid, Si(OH)4, in food and beverages, hasbeen reported to be readily absorbed across the intes-tinal wall and rapidly excreted in the urine.19,30 Inplasma and red cells, Baumann found that the concen-tration of silica was the same (0.4–0.5 ppm), and waspresent as a monomer. When 50 mg of soluble silicawas given to humans in drinking water, it was allexcreted in the urine in 10 h; the concentration in urineranged from 200 to 600 ppm at its peak, depending onurine volume, and it was still all monomeric. Theelimination rate was constant when measured as mi-crograms per minute, regardless of urine volume andthe rate was proportional to the amount not yet ex-creted (first order rate constant). The concentration in

blood reached 2 to 3 ppm, and if 300 mg of solublesilica was given, it reached 6 ppm. Baumann alsoshowed that monomeric Si(OH)4 penetrates all bodyliquids and tissues at concentrations less than its sol-ubility (1.7 ppm, which is 1% of Si solubility in water).However, Policard et al.21 suggested that polymericmolecules of silicic acid containing up to four to fivesilicon–oxygen units characterize the transport formof silicic acid in blood.

Previous studies

In a previous study,1 the BG granules were partiallyexcavated after 1 month. After 3 months the granulesappeared fully excavated and bone tissue formed inthese particles. In the present study, the silicon wascompletely excreted after 5 months. The slower rateobserved here can have been caused by differences inthe animal models and implantation sites. Further-more, the previous study used histological evaluationsto determine the time to excavation and did not quan-tify the release. By virtue of these hitherto unknownphenomena, it was not designed to address transportand excretion of BG degradation products.

The Schepers study1 involved a dog model with amandibular implantation site with much less BG im-planted. Herein rabbits were used. In general, theserodents have faster healing rates than dogs. Further-more, the muscle implantation site used here healsfaster than a bone site in the previous study. It ispossible that the ability of the rabbit to heal quickerand form fibrous scar tissue enclosing the foreignmaterial may have impeded the resorption productsfrom being removed faster, where the scar tissue im-pedes normal fluid flow from the implant to the vas-culature. Regardless, the previous dog study did notchemically trace the excavated silica gel. Thus, thepossible slow diffusion of dissolved silicon in to thevasculature of bone tissue in this study cannot beassessed. Recent chemical analysis of BG particles im-planted in the same dog model31 show that the trans-formed BG particles contained low levels of siliconthat can continue to be released, even after 3 and 6months.

The rate of silicon excretion observed here was alsomuch slower than the dissolution of silicon in vitro, asmeasured by Radin et al.7 The authors immersed 45S5BG granules (300–355 �m) in fluid with plasma elec-trolytes and serum proteins exchanging the solutiondaily. All of the silicon release occurred within the firstweek. These in vitro experiments model one compart-ment of the overall removal pathway, Kf into the fluidsurrounding the granules (Fig. 5), and with a highfluid flow environment the silicon dissolution is en-hanced. The measured silicon excretion also includes

EXCRETION OF RESPORTION PRODUCTS FROM BIOACTIVE GLASS 405

Kt and Kb, which slow down the silicon removal asdescribed before.

Previously published experiments with silica-con-taining bioceramics include studies by Kawanabe etal.16,17 These authors injected mice and rats intraperi-toneally with massive amounts of several bioceramicpowders (5000 mg/kg body weight, of Bioglass, Cer-avital, A-W Glass Ceramic) of large specific surfacearea (�0.05 m2/g). (Compare to present study: 375mg/kg body weight, �0.007 m2/g.) These animalsdied by nephrosis. It was concluded that the largequantity of silicon eluted from the powder was ab-sorbed from the peritoneum, and that the silicon con-centration of the glomerular filtrate and urine in-creased due to reabsorption of water in the proximaltubules until polymerization occurred and silica de-posits formed.32 Kawanabe et al. observed the depo-sition of silicon in the renal tubules, which led toepithelial degeneration and overall nephrotoxicity.Along similar lines, the work of Nagase et al.18 withintraperitoneal injections (5000 mg/kg body weight)of fine powders of silica containing calcium phosphateglasses (specific surface area: �3 m2/g) showed thatmortality in mice were directly proportional to thesilica content of the glass. Their results showed thatlarge amounts of dissolved silica from glass, mono-meric, or low molecular silicic anion can be toxic.

Chemical analysis by Kawanabe et al.17 of the sys-temic distribution of silicon in brain, heart, liver, lung,and blood revealed no significant differences betweencontrols and mice with intramuscular injections of BGafter 24 h. There was a significant increase in siliconconcentration in the kidney but with no apparent tis-sue damage. Even at the high dose, the authors believethat BG powders administered intramuscularly orsubcutaneously do not increase the silicon in the glo-merular filtrate and urine to degree that leads to po-lymerization in the renal tubules. However, in thesame study,17 when the BG powders were injectedinto the peritoneum, the abundant blood circulationand absorptive surface area in a large hydrated vol-ume promoted the rapid adsorption and accumulationof silicon in the kidney and liver, causing severe renaldamage. In contrast, in the present study, no systemicaccumulation of the silicon was detected except in themuscle at early time points. With a clinically relevantdose and a BG effective surface area two orders ofmagnitude less than in experiments by Kawanabe16

and Nagase,18 a much slower silicon dissolution ratefrom the BG (Kf) and subsequent diffusion into theblood was observed (Jb). Under these conditions thecritical concentration at which silicon polymerizes(180 ppm) is never reached.27 Meanwhile, the kidney’sability to excrete the SiRP continues and no accumu-lation of silicon takes place.

The systemic accumulation of silicon from Si-con-taining bioactive ceramics depends on the rate of dis-

solved silica that diffuses into the tissue and into theblood. Furthermore, the rate of dissolution is depen-dent on dose, site of implantation, effective surfacearea, and chemical composition of the silica. Givensilicon biology, the rate of dissolution is typicallysmaller than the rate of excretion. As seen in thepresent experiments, the silicon release rates from theBG granules were well within physiological toler-ances.

CONCLUSION

In a rabbit model with intramuscular implants ofbioactive glass granules, significantly increasedamounts of silicon were excreted in the urine. Theconcentrations of silicon found in the urine were wellbelow saturation and no accumulation of silicon wasfound in the major organs after silicon excretion hadhalted. Over a period of 5 months, the silicon compo-nent of a clinically relevant dose of bioactive glass wasexcreted safely in the urine as shown by the chemicaland histological analyses. Therefore, the results sup-port the hypothesis that the silicon diffuses into thelocal tissue and enters the bloodstream or lymph to besubsequently filtered by the kidney.

We gratefully acknowledge the generous donation of thebioactive glass from Orthovita Inc. (Malvern, PA).

References

1. Schepers E, Declercq M, Ducheyne P, Kempeneers R. Bioactiveglass particulate material as a filler for bone lesions. J OralRehabil 1991;18:439–452.

2. Griss P, Werner E, Heimke G, Raute-Kreinsen U. Comparativeexperimental investigations with bioglass (L. L. Hench) andAl2O3-ceramic coated with mod. bioglass. II. Results of exper-iments with loaded implants. Arch Orthop Trauma Surg 1978;92:199–210.

3. Winter M, Griss P, de Groot K, Tagai H, Heimke G, van DijkHJ, Sawai K. Comparative histocompatibility testing of sevencalcium phosphate ceramics. Biomaterials 1981;2:159–160.

4. Klein CP, Driessen AA, de Groot K, van den Hooff A. Biodeg-radation behavior of various calcium phosphate materials inbone tissue. J Biomed Mater Res 1983;17:769–784.

5. Livingston TL, Ducheyne P, Garino J. In vivo evaluation of abioactive scaffold for bone tissue engineering. J Biomed MaterRes 2002;62:1–13.

6. Ducheyne P. Stimulation of biological function with bioactiveglass. MRS Bull 1998;23:43–49.

7. Radin S, Ducheyne P, Falaize S, Hammond A. Transformationof bioactive glass granules into Ca-P shells in vitro. J BiomedMater Res 2000;49:264–272.

8. Schwarz K. A bound form of silicon in glycosaminoglycansand polyuronides. Proc Natl Acad Sci USA 1973;70:1608–1612.

9. Dobbie JW, Smith MJB. The silicon content of body fluids. ScottMed J 1982;27:17–19.

406 LAI ET AL.

10. Hamilton EI, Minski MJ, Cleary JJ. The concentration anddistribution of some stable elements in healthy human tissuesfrom the United Kingdom: An environmental study. Sci TotalEnviron 1973;1:341–374.

11. LeVier RR. Distribution of silicon in the adult rat and rhesusmonkey. Bioinorg Chem 1975;4:109–115.

12. Hench L, Splinter R, Greenlee T, Allen W. Bonding mecha-nisms at the interface of ceramic prosthetic materials. J BiomedEng 1971;2:117–141.

13. Ito G, Matsuda T, Inoue N, Kamegai T. A histological compar-ison of the tissue interface of bioglass and silica glass. J BiomedMater Res 1987;21:485–497.

14. Wilson J, Pigott GH, Schoen FJ, Hench LL. Toxicology and bio-compatibility of bioglasses. J Biomed Mater Res 1981;15:805–817.

15. Hench LL, Wilson J. Biocompatibility of silicates for medicaluse. Ciba Found Symp1986;121:231–246.

16. Kawanabe K, Yamamuro T, Nakamura T, Kotani S. Effects ofinjecting massive amounts of bioactive ceramics in mice.J Biomed Mater Res 1991;25:117–128.

17. Kawanabe K, Yamamuro T, Kotani S, Nakamura T. Acutenephrotoxicity as an adverse effect after intraperitoneal injec-tion of massive amounts of bioactive ceramic powders in miceand rats. J Biomed Mater Res 1992;26:209–219.

18. Nagase M, Abe Y, Chigira M, Udagawa E. Toxicity of silica-containing calcium-phosphate glasses demonstrated in mice.Biomaterials 1992;13:172–175.

19. Baumann H. Behavior of silicic acid in human blood and urine.Hoppe-Seylers Z Physiol Chem 1960;320:11–20.

20. Iler RK. The chemistry of silica: Solubility, Polymerization,colloid and surface properties, and biochemistry. New York:Wiley; 1979.

21. Policard A, Collet A. On the first stages of experimentalchanges in the kidney caused by silicic acid. Study with anelectron microscope. J Urol Med Chir 1960;65:585–600.

22. Scheel LD, Fleischer E, Klemperer FW. Toxicity of silica. I.Silica solutions. AMA Arch Ind Hyg Occup Med 1953;8:564–573.

23. Gye WE, Purdy WJ. The poisonous properties of colloidalsilica. Br J Exp Pathol 1922;3:75–94.

24. Policard A, Collet A, Moussard DH, Pregermain S. Depositionof silica in mitochondria: an electron microscopic study. J Bio-phys Biochem Cytol 1961;9:236–238.

25. Blencke BA, Alletsee P, Bromer H, Pfeil E. Studies on soft tissuereactions to implantation of glass ceramics. Arch Orthop Un-fallchir 1975;82(2):135–146.

26. Lai W, Garino J, Ducheyne P. Silicon excretion from bioactiveglass implanted in rabbit bone. Biomaterials 2002;23:213–217.

27. Sauer F, Laughland DH, Davidson WM. Silica metabolism inguinea pigs. Can J Biochem Physiol 1959;37:183–191.

28. Kozma C, Macklin W, Cummins LM, Mauer R. Anatomy,Physiology, and biochemistry of the rabbit. In: Weisbroth SH,Flatt RE, Kraus AL, editors. The biology of the laboratoryrabbit. New York: Academic Press; 1974. p 50.

29. Berne RM, Levy MN. Physiology. St. Louis: C.V. Mosby; 1988.30. Baumann H. The determination of silicic acid in biological

material. Hoppe-Seylers Z Physiol Chem 1960;319:38–51.31. Schepers E, Barbier L, Huygh A, Ducheyne P. Analysis of

chemical transformation of bioactive glass particles after im-plantation. Sixth World Biomater Congress Trans 2000;2:644.

32. Settle WR, Sauer F. Demonstration of siliceous deposits in thekidney of the guinea pig. Am J Vet Res 1960;21:709–711.

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