Preparation and Bioactive Characteristics of Porous Borate Glass Substrates

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Processing of Biomaterials



P W A U T I O N AND BIOACI'IVE CHARACTERISTICS OF POROUS BORATE GLASSSUBSTRATES

Mohamed N.mentofMaterialsScienceand Engineering, and Materials Research Center,Rob, MO 5409

Nicholas W. Marion, GwendolenC. Reilly, and JeremyJ. Mso,Univcraity ofIllinois atChicago,DepartmentofBiocnghemingandTissUeJhgbeering aboratory,Chicago, IL.60607

ABSTIWCTWhereas silicate-based bioactive glasses and glass-cerarmc8 have been widely investigated

for bone mpak or as d o l d e for cell-basad bone tim mginaerin tcotllt data have demon-

strated that silica-h borate glasses also exhibit bioactive behavior. The objectivesofthis

studywcrc to fabricate porous, thresduamai d ubstrates of a borate glass and to investigate thebiocompatibilityoftheborate glass substratesby n vim cell culturewitbhuman mesenchyrrmlstem cells (hMSCs) and MnSGduivcd osteoblasts @MScObs). Borate glass particles wthsizes 212-355 pm wcrc loosely compacted and then sintered at 600°C to form porous disc-

shaped eubstratee@orosity n 40 ). Partialor nearly complete conversionof the glass subaratato a calcium phosphate (Ca-P)ma te d was achieved by soaking the substrates for 1 day or 7days in a 0.25 molar K2 HP O4 solution at 37°C and at pH of 9.0. Bone marrow derived hMSCsand hMScObe seeded in he samplca both adheredto he porouscoIlstNct8whereas hMSGObsmarkedly synthesized alknline phosphatase, an early ostcogenic marker. These data indicatestrong bioactive charactenstics forthe borate glass constNct3and the potential u8c of the con-

structs for bane isnreenginceling.

Wen Liang, and Delbert E. Day,Univcraity of Missouri-Rolla, Depart-

INTRODUCTION

Cutain compositions of glasses, g cs, and ceramics referred to as bioactiveceramics,have been widely investigated for healing bone d e f e due o their ability to enhance

bone finmation and to bond to surrounding ti ssue [l-51. Cell-seedbd bioactive d c s rc also

of interest as potential scaffoldsforbone issue engineering [6,7l. Hydroxyapatite and tricalcium

phosphate ceramics, composedof the same ions as bone, re biocompatiileand produce no sys-

temic toxicity or immunologicald o n s but theymrbslowly or undergo littleco1IvQsionto

a bone-like material fter implantation [8,9]. Many bone regenemtion applications equire grad-

ual resorptiollof the implanted biomaterials and collcumnt replacemat of the biomaterials bythe host bone.

Bioactive glasses are Superior to the less reactive ceramicsin that they an ostcoinductiveas

opposed to osteocoaductive. Furthumurc, the dissolution and COllVQSiollof bioactive glasses to

a calcium phosphate (Ca-P)material seema to induce bone cell differentiation [lo]. A character-

istic featun of bioactive g l am s is the time-depcndcnt modification of the surface, resulting inthe fomation of a calcium phosphate (Ca-P)ayer through which a bond with the surrouudiug

tissue is established I 1,121. It has been suggested that the fomration of a Ca-P aya n vim isindicative of amaterial's bioactivepotential in viw [4,5,13].

Since the report of its bone bonding propexties in 1971 by Hench er d. 14], the bioactive

glass u x l d 535, referred to as Bioglass , with the compositionof 45% Sit& 6%P&

24.5% Na20, and 24.5%CaO (by weight), has fcccivdd moat interest for biological applications[4,5]. B i d v e lasses baaed on he45S5 compositionarc amactivescaffold materialsbecause

dvances in Bioceramics and BiocompositesMineo Mizuno

Copy right020 05 The American Ceramic Society





tially converted borate glass substrates (denoted pBG) and the fully converted rnbstmtes (denoted Ca-P)wcrc preparedby immersing the porous glass substmta for 1 day and 7 days, xe-Spectively,intheK m , olution.

Cell Cultureon P o m Borate G b sSubstrates

Human bone mamm derived meacnchymal stem cells (hMscS) wae isolalcd from bonem w amples (AllCells, Berkeley, CA) singa RosateSep kit (Stem CellTechnologies, Inc.,Vancouver, BC, Canada). ThehMscswere grown in monolayex in cell culturemedia cmsisthg

of 89% DMEM, 0%FBS, % penicillin-Streptomycin (basalcell culture media). Afta4 days

non-adherent cellswerc rcmovcd and the media was changed cvcry 4 days. Cellswere passaged

up to four times each time upond u c n c y . Upon he 4 passage,50% of the hMSCs werc ex-

p g / d L-asmbic acid-2-phosphate). Upon exposun to ostmgaic supplement, hMSCs differ-entiatedinto osteoblasticcells (hMscobs) [30-321, whereas the other 50% hMSCs continued

The hMSCs and hMSC-Obs wae seeded 30,000 cells per cm? on porous substmtcs of the

converted borateglass (Ca-P),and incubated oran additional14 days. Live cell assay was then

perfbrmad using Promega (Madison,WI)CellTim960 A& One Solution Cell proliferation

posed& ostsogenicsupplementedmedium(basalcellculturemadia,100nM 50

incubation n basal culture~ l c t cediawithoutosteogeniceupplemcnt.

~ V c r t C dorate glass (BGX the partially converted borate glass @BG), or thc completely

Assay, Which quantified cell viability thtough NADH activity Using 3-(4J-dimethyt-2-yr)5-(3-c a r b o x y m e t h o x y p h e n y l ~ 2 - ( ~ ~ ~ h ~ y l ) - 2 h e r salt (MTS). The ~ C C

valuui for MTS comtate with a live cell number as documentedin thc product informationsheer.Alkaline phogphataseactivity(AP) as assayed by Napthol as-biphosphate, fastredviolet

salt, andN,N dimethylfonnamidesolution (Sigma-AIdrichCo.,St. ouis,MO).

RESULTS AND DISCUSSION

Figure 1 shows an optical micrograph of the surface of a porousborate glass mbstmtc pro-duced by sintcring.The touchingparticlesaxe bonded at the necks,providing enhanced strength

without significantflow of the glass into the pores. The reductionof the porosity of the sub-

strata during sintcringwasnegligible.Computer imagingof optical mimgqhs i n d i d that

the sul#ltrateshad a porosity of 4045% and a median pore sizeof 1W150pa The porosity e8-

timated by computer imaging was in agreement with the value detnmmed from he measureddensity of the substrate and the density of the fidly dense glass (2.58 g/cm?. X-ray dithctionshowedthat the glasn in the porous substrate remained amoqthous attcr sintaing.

Figure 1. Optical micrograph of the waface of a

inga loosely compactedmass ofparticles(212-

355 pm) for 10min at600°C.

porous borate glass substrateproducedby sin*-

5



The weight loss data for the porous boxate glass substratesduring their conversion to Ca-P inGHPOI solution are shown in Fig. 2 as a h a i o n of time. Gmversion of the glass to Ca-P, asindicated by the maximum weight loss (ma s% ), was completed aAa appmximately 7 days.The conversion of the borate glass to Ca-P is believed to involve dissolution of the glass into thesm und in g liquid and precipitation of calcium and phosphate ions onto the surface of the sub

strate [33]. Assuming that al l of the sodium and borate ions from the glass go into solution andall of the calcium ions go into the formation of a Ca-P materhl with the composition ofstoichiomeaic hydmxyapatite, cS1o(po4)s(0H)z,them the theoreticelweight logs should be 69%.

The discrepancy between the maximum measurtd weight low and the theoretical weight lossmay be due to incomplete convasion of the glass,some alcium ions remainhg in solution, thefonnation of a noastoichiomeaic hydmxyapatite with a CdP ratio lower than he stoichiometricvalue of 1.67, or a combination of all three factors. Chemical analysis of the Ca-P material

formed by the conversion of similar borate glssaea rmder the same conditions indicated that theCa/P ratio was well below 1.67 [34].

Conversion of the borate glass to Ca-P tarts at the surface and moves inward [33]. By con-

trolling the timeof d o n n the K3HPO4 sobion, substr tes with different ratios of the sur-rounding Ca-P layer to the borate glass corecaa be produced. Constructs meted for 1 day con-

sisted of an inwnnected mass of composite particles, with a thin surface layer of the glass

converted to Ca-P. The thickness of the Ca-P ayer, estimated from the weight loss data was 40-50 pm. Subs- reacted in the K-4 solution for 7 dayswere almost fully COIlverted to Ca

P, and consisted of an intacaMectedmass of Ca-Pparticlea.Figure3 shows SEM micrographs

ofthe sutfaces of the three typesofporous substratesused in the present work in cell cultm ex-perimenb.The unconverted borate glass BG)substratehas mooth surfacee charactenstic of thespheroidized glass particles, whereas the cmmucts of the partially collvcttcd glass (PBG) andthe fully converted glass (Ca-P) have less smooth surfaces.High resolution SEM, erformed inrelated work 1351, indicated that the Ca-P materialwas highly porous, with tinepores on he or-

der of several tens of nanometers.

80I

70

40

3

L 20

10

0 212-355-Th.omE-m'

20 40 60 8 100 120 140 160 180

Ro8c tkn ime (h)

Figure 2. Weight loss of porous borate glass substrates as a function of time in 0.25 molarKzHPO4 solution at 37OC and a pH value of9.0. Conversion of the glasa to a calcium phosphate(Ca-P) material in the solution is accompanied by a weight loss. The estimated theoretical weightloss is shown by he horizontal dottad ine.

6



Fipn3. SEM micrographsof the ofthe surfaces

periments: (A) uconvutcdborate glass (BG); (B)

CoIlVertcdborateglass (Ca-P)ormedby reaction

ofborateglass substratesused in cellCUMve ex-

partidycollvettcd borate glass@BG) formed byreaction for 1 day in& r n 4 aolution;(c) y

for I days inK5Hpo4 solutim

The difI;ennces in the conditionof the borate glass substratesmay influence the interaction

with cells. However, hemost fkvorable condition of the borateglass farcellular interaction is, atprcsu~t, nclear. The unconverted borate glass (BG) with its gmooth maEace initially may notprovide hvorabk sites for cell attachment and significant di ssol ut i onof calcium, sodium andborate ionswill occur hitiallyinto he surrounding fluidas the glass aubcereactswith the fluid.For constructs of the partiaUy converted glass (PBG), the porous Ca-P surhxa may provide

morc favorable sites for cellattachment. Dissolutionof calcium, sodiumandborate ions into themmmd ing fluid is stillcxpcctd to occur but at a lower rate han fortheunconverted glass. Thefblly collvcrtcd mmamcts (Ca-P) rovide maEace sites similarto that ofthepBG ccmtwts,but

almost no dissolutionof sodiumand borate ions into the surrounding fluidwill occur due to the

The unamvuted borate glass mhlmtca (BG) dismtegratcdduring cell culture experiments,

presumably due to d o n s of the glass with the cell culhm medium. However, the pnr&idy

tai ned theiroriginalcylindrical shape hroughout the experiments. Live cell number (MIS) as-

absenceofanysignificantquantityofborateglass in the SUbQtrate.

CoIlVgtcd substretes @BG) and the IbUy CollVQtcd SU~&WCS(Ca-P) intact and main-

sayad after 14 days verified the cell viability of both mscs and hMscobs cultured on thePBG andCa-P8 u b a t ~ .hehMscIleaded OIL the PBG~CIIIPWad ~ @ & ~ t l yhighercell

viability than IMSCa seeded on he Ca-P emplatea 4). The data show a similar trend forhMscobs seeded an he pBG andCa-P templatesbut the difference s not significant due to thewider Mliability of tbe data for Ca-P templatea. The higher cell viability of the hMSC on hepBG substmlea may indicate that pBG dmulata cell f uncti on.As ou t l i d earlier, a key differ-ence between the pBG and Ca-P mbsmtcs is the potential for dissolution of calcium, sodium,and borate ions &om the underlyingborate glass core of thepBG templatesinto thecum me-dium. hemechanismby which these ions mayinfluencecell f unct i on is not clear at present butmay be imporcant for determmngtheOptimumcanditionoftheborateglasssubstmtafortissueenginemkgapplications.For cells seeded on thepBG substrates,he data in Fig. 4 ale0 ndicated

7



that the ogteogeaic cells had significantly higher cell viability th nWCSThe hMSCs wereinitially seeded at a hi* density than the hMSWb8 (9,000 cell8 per comttw, versus 3,000cells per construct, respectively) Chre to the fkct thatMSCe prolifaatedmom rapidly thanMSC

seededcellnumbet- * to MSCS gmwn on Ca-P. Future studia will investigate apop

tosie and prolifidon ofMSCs and their diffaentiatedostcoblastson theborate slaps substrates.Active alkaline phosphatase wan produces by the cells wthin the borate glass substrates as

i n d i d b y he dark redstain (Fig. 5). Higheralkalinepboephataee activitywas seen in hMSC

cates that a combiuatim of Ogtaogenic supplementswth a b i d w orate glass substrate willhave a positive effecton ostaogenc differentiation.

Ob during the p t. The data presentad in 4 plOe the MIS absor bance per

Obs eampleS@IC, D) compatcd to the uadifferentiatcd, h M S C S (Fig. SA, B). This hdi-

Figure 4. Live cell assay using

bolic indicator ofhMSCs and

vextedborateglass @=) andCa-P substrates.n4, = Stu-

light abmbamc as an meta-

hMsc-obs onpartially con-

dents T-test p <0.05.

Figure 5. Photographs of (A)human mesenchymalstem cells

(hMSCs) cultured on substratesofpartiaUy converted borate glass

(PBG), (B) hMSCs cultured onsubstrates of fully collvcrtcdbo-rate (Ca-p), (C) hMSC d e

rivedosteobbts (hMSC-Obs)

@)hMSG0bSCulnved Ca-Pcultured on pBG substratw, and

substrates. ll samples stained for

alLalinep h o s p b activityus-ing the sameprobcol.Red (dark)stain ndicateshigh alkalinephos-phatsse activity.

8



CONCLUSIONSPorous bioactive borate glass substrates, pnpand by sintuing a loosely colllpacted mass of

paxticles, were conditioned in 0.25 molar K D O , solution at 37°C to convert a controlledBmoullt of the glass to a calcium phosphate (Ca-P) material. The cytocompah bitityof p o wsubstr tes consisting of the ul l col l vett td glass (BG), thepartially convated glass (PBG), and the

stem cells (hA4SCs) and hMSC derived osteobtasEs (hMsGobs). The hMscs seedad on pBGsubstrates had a higher metabolic activi tyand cell viability than on Ca-P substrates. For pBG

iblly collvcrtcd glass (a-P), WBS n ~ & p t d by tnvim cell culturewib human I W S C Z I ~

substram hMscob8 had significantly highercell viabiity thanWCS Aural i nephosphataseacti vi ty on the pBG and ca-P mbstmawith h M s c o b s revealed the ability of these mataialsto support osteogeniccells. The data suggest thenecessityfor additional in vim and k viw in-vcstigations of the potential ofbioactiveborate glass as a cell-acconrreodatmg d o l d or boneti ssueengineering. Inparticular, thepBG co118t~ct onsistingof anetworkofborate glasa parti-

cles surrounded by a Ca-P layer, had thehighestcellviablity forboth cell types and may repre-

sent a more fivorable condition oftheborate glass forbone tissue cngincuing.

ACKNOWLEDGEh4ENTSThepnsentedrc8cBTch was suppolted by a UniversityofMitwmi Research Board Grant (to

M.N.R.), a Biomedical JhgineeringReseatch Grant from theWhitalrnFowlationRG-01-0075,IR GrantonBiotechnologyjointly Grom the Univusity of I l l inoh at Chicago) and the Univer-sity of Illinois at Urbana-gu, and by Research Grants DE13964, DE15391, and

EB02332 from theNational nsti tutesof Health(to J.J.M.).

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Preparation and Bioactive Characteristics of Porous Borate Glass Substrates