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*XPDJHOODQ**MHVWZLąĪąF\PZDSĔDQLRQRZ\PSR- Gellan gum (GG) is an anionic calcium-binding polysac-
lisacharydem wytwarzanym przez bakterie (Sphingomonas charide produced by bacteria (Sphingomonas elodea) during
elodeaZSURFHVLHIHUPHQWDFMLWOHQRZHM:VNáDG**ZFKR-
G]ąSRZWDU]DMąFHVLĊMHGQRVWNLWHWUDVDFKDU\GRZH]EXGRZD-
ne z glukozy, kwasu glukuronowego i ramanozy w stosunku
>@**MHVWV]HURNRZ\NRU]\VW\ZDQDZSU]HP\ĞOH
VSRĪ\ZF]\PRUD]IDUPDFHXW\F]Q\P>@RVWDWQLRVWRVRZD-
QDMHVWWDNĪHMDNRPDWHULDáGRUHJHQHUDFMLFKU]ąVWNL>@
**VWRVRZDQRWHĪZFKDUDNWHU]HUXV]WRZDQLDGRLQĪ\QLHULL
WNDQNRZHMMDNRQRĞQLNOHNyZRUD]PDWHULDáZVWU]\NLZDQ\
=DOHWą**MHVWQLVNLNRV]WZ\VRNDELR]JRGQRĞüELRGHJUD-
GRZDOQRĞü QLVND WRNV\F]QRĞü SURGXNWyZ UR]NáDGX RUD]
WRĪHQLHMHVWWRSURGXNWSRFKRG]HQLD]ZLHU]ĊFHJR-HGQą
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>@:SURZDG]RQ\FKRVWDWQLREDGDQLDFKSRVWXOXMHVLĊ
ZSURZDG]HQLHGRĪHOXID]\QLHRUJDQLF]QHMZSRVWDFLQDQR-
F]ąVWHN DSDW\WRZHJR IRVIRUDQX ZDSQLD PRJąFHJR VZRMą
VWUXNWXUąSU]\SRPLQDüQDQRVWUXNWXUĊWNDQNLNRVWQHM>@
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aerobic fermentation. It consists of repeating tetrasacharide
XQLWVRIJOXFRVHJOXFXURQLFDFLGDQGUKDPQRVHLQD
ratio [1,2]. It is widely used in the food and pharmaceutical
industries [3] and more recently it has been used as a hydro-
gel biomaterial for cartilage regeneration [4-6]. GG has been
applied as a tissue engineering scaffold for controlled drug
delivery and as an injectable material. Further advantages
of GG are its low cost, high biocompatibility, biodegradabilty
and the low toxicity of its by-products, as well as the fact
that it is not animal-derived, avoiding regulatory concerns.
One of the main disadvantages concerning the application of
GG as an alternative strategy for bone regeneration is its low
mechanical strength [1,3,7]. Many studies have explored the
reinforcement of hydrogels by the use of inorganic phases,
such as apatitic calcium phosphate (CaP) nanoparticles,
because they closely resemble the nanostructured mineral
phase of bone tissue [8].
The mineralization of GG with CaP minerals can be
LQNXEDFMĊZUR]WZRU]HJOLFHURIRVIRUDQXZDSQLD&D*3SU]\
MHGQRF]HVQHMREHFQRĞFLZĪHOXHQ]\PX±IRVIDWD]\DOND-
licznej (ALP). ALP katalizuje uwalnianie grupy fosforanowej
]&D*3FR]ZLĊNV]DORNDOQHVWĊĪHQLHMRQyZIRVIRUDQRZ\FK
L XPRĪOLZLD SUHF\SLWDFMĊ QLHUR]SXV]F]DOQ\FK VROL IRVIRUD-
nowych [8,9].
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PLQHUDOL]RZDQHM JXP\ JHOODQ Z FKDUDNWHU]H ]DVWRVRZDĔ
GRWNDQNLNRVWQHME\á\EDGDQHSU]H]'RXJODVDLZVS [9,10].
:SLHUZV]HMSUDF\SRGDWQRĞüQDPLQHUDOL]DFMĊRUD]V]W\Z-
QRĞü ĪHOL ]  ** SRSUDZLDQR SU]H] ZSURZDG]HQLH
achieved by the incorporation of an enzyme, alkaline
phosphatase (ALP), during incubation in solutions contain-
ing calcium glycerophosphate (CaGP). The ALP uses the
CaGP as a substrate, cleaving the phosphate and, thereby
increasing its local concentration, enabling the precipitation
of insoluble phosphate salts [8,9].
Physical and biological properties of enzymatically min-
eralized GG with a focus on bone regeneration applications
were studied by Douglas et al [9,10]. Mineralizability and
VWLIIQHVVRI**JHOVZDVHQKDQFHGE\WKHLQFRUSRUD-
tion of polydopamine (PDA) [9]. In a second study CaP
SROLGRSDPLQ\3'$>@:GUXJLHMSUDF\PDWHULDáPLQHUDOL]R- and magnesium phosphate (MgP) mineral formation in
wano fosforanem wapnia oraz fosforanem magnezu (MgP) **K\GURJHOVZDVSHUIRUPHGE\LQFXEDWLRQLQ&D*3
SRSU]H]LQNXEDFMĊ&D*3RUD]JOLFHURIRVIRUDQLHPDJQH]X and magnesium glycerophosphate (MgGP) solutions [10].
0J*3>@2EHFQRĞü0J3RUD]3'$Z]PLQHUDOL]RZD- The presence of MgP as well as PDA in mineralized samples
Q\FKĪHODFKVSU]\MDáDDGKH]MLLSRSUDZLDáDSU]HĪ\ZDOQRĞü promoted attachment and vitality of osteoblastic MC3T3-E1
NRPyUHN RVWHREODVW\F]Q\FK 0&7( QLH SRZRGXMąF
MHGQRF]HĞQLHSRJRUV]HQLDF\WR]JRGQRĞFLPDWHULDáX
&HOHPQLQLHMV]\FKEDGDĔE\áROHSV]H]UR]XPLHQLHSUR-
cesu enzymatycznej mineralizacji GG, przede wszystkim
ZNRQWHNĞFLHUR]ORNRZDQLDID]\PLQHUDOQHMRUD]MHMVWDELOQRĞFL
ZREUĊELHPDWHULDáX:W\PFHOXĪHOHRUyĪQ\FKVWĊĪHQLDFK
cells and did not negatively impact the materials’ cytocom-
patibility.
The aim of this study was to gain a deeper understand-
ing of mineralization process of GG with CaGP in terms of
mineral phase distribution and its stability within the material.
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DQDOL]RZDQRLFKZáDĞFLZRĞFL]DUD]SRPLQHUDOL]DFMLDWDNĪH
po 7 dniowej inkubacji w wodzie. Dodatkowo przeprowadzo-
QREDGDQLDELRORJLF]QH]Z\NRU]\VWDQLHPNRPyUHNRVWHR-
EODVWRSRGREQ\FK0*KRGXMąFMHQDSRZLHU]FKQLRUD]QD
SU]HNURMDFK]PLQHUDOL]RZDQ\FKPDWHULDáyZ%DGDQLDWHPLDá\
QDFHOXVWZLHUG]HQLHF]\REHFQRĞüID]\PLQHUDOQHMZSá\ZD
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mineralized hydrogels and the properties of the materials
were analyzed prior to and after incubation in water for
7 days. Moreover osteoblast-like MG-63 cells were cultured
on the outer surfaces as well as on the cross sections of
mineralized materials to ascertain if differences in mineral
phase concentrations affect cell adhesion and viability.
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3U]\JRWRZDQLHĪHOX containing ALP (P7640, Sigma-Aldrich) were prepared by
+\GURĪHOH ** *HO]DQŒ &0 * 6LJPD$OGULFK heating a GG solution to 90oC and addition of CaCl2 as
]DZLHUDMąFH$/3 3 6LJPD$OGULFK SU]\JRWRZDQR DFURVVOLQNHUWRUHDFKD¿QDO&D&O2FRQFHQWUDWLRQRI
przez ogrzanie wodnego roztworu GG do temp. 90oC oraz ZY>@$IWHUFRROLQJWRo&$/3PJPOZDVDGGHG
dodanie CaCl2ZYMDNRF]\QQLNDVLHFLXMąFHJR>@ the solution was cast on glass Petri dishes at room tem-
3RVFKáRG]HQLXGRo&GRGDQR$/3PJPODUR]WZyU perature. After gelation cylindrical hydrogels samples (12 or
Z\ODQRQDSá\WNL3HWULHJRLSR]RVWDZLRQRGRRFKáRG]HQLD 4 mm in diameter, 4 mm in height) were cut out with a hole
3R ĪHOLILNDFML ] ĪHOL SU]\ SRPRF\ Z\FLQDND Z\FLĊWR F\OLQ- SXQFK6L[W\SHVRIWKHVDPSOHVZHUHSUHSDUHG
GU\F]QH SUyENL  L  PP ĞUHGQLF\  PP Z\VRNRĞFL **ZLWKPJPO$/3DQGZLWKRXW$/3
3U]\JRWRZDQRV]HĞüURG]DMyZSUyEHN**
]PJPO$/3RUD]EH]$/3
8 8ZDOQLDQLH$/3 $/3UHOHDVHVWXGLHV
$E\RFHQLüXE\WHNELDáND]ĪHOXSUyENLĪHOL**PP To evaluate the protein loss, GG disks (4 mm in diameter
ĞUHGQLF\ L  PP Z\VRNRĞFL LQNXERZDQR Z  —O ZRG\
GHVW\ORZDQHM'ODNDĪGHJRSXQNWXF]DVRZHJR—OHOXDWX
SU]HQRV]RQRGRGRáNDSá\WNLGRáNRZHMLGRSHáQLDQR—O
odczynnika BCA (5 ml kwasu bicynchoninowego, 20 mg
bezwodnego siarczanu (II) miedzi - oba z Sigma-Aldrich
DQGPPLQKHLJKWZHUHLQFXEDWHGLQ—ORIZDWHURYHU
different periods of time. At each time point the water was
UHPRYHG DQG  —O ZHUH DGGHG WR RQH ZHOO RI D ZHOO
SODWH—ORI%&$UHDJHQWPOELFLQFKRQLQLFDFLGVROXWLRQ
20 mg of copper II sulfate anhydrous pure - both from Sigma-
L—O+22$EVRUEDQFMĊUR]WZRUXPLHU]RQRSU]\QP $OGULFKDQG—O+2O) were then added and the absorb-
SRPLQLQNXEDFML'RVSRU]ąG]HQLDNU]\ZHMNDOLEUDF\M- ance was measured at 570 nm after 30 min of incubation.
QHMXĪ\WRSLĊüUyĪQ\FKVWĊĪHĔ$/3PJPOPJPO )RUWKHVWDQGDUGFXUYH¿YHGLIIHUHQWFRQFHQWUDWLRQVRI$/3
PJPOPJPODQGPJPO ZHUHSUHSDUHGPJPOPJPOPJPOPJPO
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&D*3 Z\PLHQLDQR FR GUXJL G]LHĔ 3R PLQHUDOL]DFML ĪHOH
SU]HP\WR ZRGą GHMRQL]RZDQą D QDVWĊSQLH LQNXERZDQR
ZZRG]LHGHMRQL]RZDQHMG]LHĔZFHOXZ\SáXNDQLDSR]R-
VWDáRĞFL &D*3 MDN UyZQLHĪ  GQL Z FHO RFHQ\ VWDELOQRĞFL
fazy mineralnej.
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The samples were incubated in 0.1 M CaGP (50043,
Sigma-Aldrich) solution at room temperature over 14 days.
Mineralization medium was changed every other day.
After mineralization, the gels were rinsed with UHQ-water
and subsequently incubated in UHQ-water for 1 day with
the aim of removing residual CaGP and 7 days to assess
mineral phase stability.
3UyENLK\GURĪHOLQ SRGGDQRWHVWRPPHFKDQLF]Q\P 0HFKDQLFDOWHVW
QDĞFLVNDQLHQDXQLZHUVDOQHMPDV]\QLHZ\WU]\PDáRĞFLRZHM +\GURJHOVDPSOHVQ ZHUHVXEMHFWHGWRFRPSUHVVLYH
=ZLFN7HVWSU]HSURZDG]RQR]V]\ENRĞFLąĞFLVNDQLD testing in a universal testing machine (Zwick 1435). Samples
PPPLQ%D]XMąFQDX]\VNDQHM]DOHĪQRĞFLQDSUĊĪHQLH- ZHUHWHVWHGZLWKFRPSUHVVLQJVSHHGRIPPPLQ%DVHG
RGNV]WDáFHQLH PRGXá <RXQJD Z\]QDF]DQR ] OLQLRZHJR on the obtained force - strain curve Young’s modulus was
fragmentu krzywej (zakres w przedziale 0,2 do 5 N). GHWHUPLQHGIURPWKHOLQHDUGHSHQGHQFHIURPWR1

6XFKDPDVD 'U\PDVV
$E\R]QDF]\ü]DZDUWRĞüID]\PLQHUDOQHMZ\]QDF]RQR To determine formed mineral content mass change was
VXFKHMPDV\3UyENLZDĪRQRQDPRNUR]DUD]SRPLQHUDOL- measured. Samples were weighed after incubation (m1)
zacji (m1) oraz po suszeniu w temp. 37°C przez okres 2 dni and after drying at 37°C for 2 days (m2). The dry mass
(m2). Procent suchej masy (dmp) wyznaczono w oparciu SHUFHQWDJHGPSZDVFDOFXODWHGDFFRUGLQJWRWKHIRUPXOD
RQDVWĊSXMąF\Z]yUGPS P2P1Â GPS P2P1Â

%DGDQLDELRORJLF]QH &HOOFXOWXUH
3U]HNURMH SUyEHN QLH]PLQHUDOL]RZDQ\FK ± ** RUD] Cross-sections of the samples (non-mineralized GG and
]PLQHUDOL]RZDQ\FK ± **$/3 X]\VNDQR SU]H] SU]HFLĊ- PLQHUDOL]HG**$/3ZHUHSUHSDUHGE\FXWWLQJWKHVDP-
FLH F\OLQGU\F]QHM SUyENL QD Syá UyZQROHJOH GR SRGVWDZ\ ples into halves parallel to the bases of the cylinders. The
F\OLQGUD 3UyENL Z\VWHU\OL]RZDQR SRG 89 SU]H]  PLQ samples were sterilized under UV light for 20 min (10 min
 PLQ ] NDĪGHM VWURQ\ D QDVWĊSQLH XPLHV]F]RQR from each side), placed in 24-well plates and incubated at
ZSá\WNDFKGRáNRZ\FKLLQNXERZDQRSU]H]KZoC 37oC for 2 h in minimal essential medium (DMEM, ATTC),
Z PHGLXP KRGRZODQ\P '0(0$77& ]  )%6  VXSSOHPHQWHG ZLWK  )%6  SHQLFLOLQVWUHSWRP\FLQ
SHQLFLOLQ\VWUHSWRP\F\Q\ L  P0 /JOXWDPLQ\ 1DVWĊSQLH DQG  P0 /JOXWDPLQH$IWHUZDUGV  —O RI 0* FHOO
—O]DZLHVLQ\NRPyUHN0*[4NRPyUHNQDQLH-
VLRQRQDSRZLHU]FKQLĊNDĪGHMSUyENL3RPLQGRGDQR
—O'0(0.RPyUNLKRGRZDQRSU]H]RNUHVRUD]GQL
w temp. 37o&L&22:FHOXRFHQ\SU]HĪ\ZDOQRĞFLGR
NDĪGHJRGRáNDGRGDQRPOUR]WZRUXEDUZQLNDPO3%6
suspension (3x104 cells) were added on the surface of each
VDPSOH$IWHUPLQ—ORI'0(0ZHUHDGGHGDQGWKH
cells were cultured for 1 and 7 days at 37o&DQG&22.
To assess cell viability, 1 ml of staining solution (20 ml of
3%6—ORIFDOFHLQDQG—ORISURSLGLXPLRGLGHERWKIURP
—ONDOFHLQ\L—OMRGNXSURSLG\Q\6LJPDLLQNXERZDQR Sigma) was added to each well and incubated for 20 min at
przez 20 min w temperaturze pokojowej. Po tym czasie wy- room temperature. Afterwards, the samples were observed
EDUZLRQHSUHSDUDW\REVHUZRZDQR]DSRPRFąPLNURVNRSX
fluorescencyjnego (Axiovert, Zeiss).
:\QLNL
5<6SU]HGVWDZLDXZDOQLDQLH$/3]SUyEHN**$/3
w czasie 3 dni inkubacji w wodzie. Najbardziej intensywny
proces uwalniania ALP zaobserwowano w pierwszych go-
G]LQDFKLQNXEDFML3RJRG]LQDFKRNRáR$/3]RVWDáR
XZROQLRQH]ĪHOXDSRGQLDFKSUDNW\F]QLHFDáH$/3]R-
VWDáRZ\SáXNDQHSU]H]ZRGĊ3UyENL]DZLHUDMąFH**
Z\ND]\ZDá\ WHQGHQFMĊ GR V]\EV]HJR XZDOQLDQLD$/3 QLĪ
ĪHOHL**
XQGHUDÀXRUHVFHQFHPLFURVFRSH$[LRYHUW=HLVV
5HVXOWV
FIG. 1 shows concentration of ALP released from GG-
ALP samples during 3 days. The most intensive release
RI$/3 ZDV REVHUYHG LQ WKH ¿UVW KRXU RI WKH H[SHULPHQW
After 2 h ~RI$/3ZDVUHOHDVHGDQGDIWHUGD\VSUDFWL-
cally all the ALP introduced was leached out. The samples
ZLWK**VKRZHGWKHWHQGHQF\WRUHOHDVH$/3IDVWHU
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FIG. 2 presents cross-sections of mineralized gels with
different concentrations of GG before and after 7-day in-
cubation in water. In every case, the mineral phase was
located mostly inside the gel while the regions close to
the surface were poorly mineralized. There were no visual
differences in terms of mineral phase distribution just after
the mineralization process between hydrogels with different
5<6 $%& 3UyENL SR  GQLRZHM LQNXEDFML Z ZRG]LH GG concentrations (FIG. 1 A,B,C). On the other hand the
5<6  '() VWDá\ VLĊ EDUG]LHM SU]H]URF]\VWH MHVW WR samples after incubation in water for 7 days (FIG. 1 D,E,F)
V]F]HJyOQLHZLGRF]QHZSU]\SDGNXEU]HJyZSUyENL** seemed to be more transparent. This was especially visible
(RYS. 1 F).
5<6SU]HGVWDZLDZ\QLNLSRPLDUyZPRGXáyZ<RXQJD
QLH]PLQHUDOL]RZDQ\FKL]PLQHUDOL]RZDQ\FKĪHOL**SU]HG
RUD]SRGQLDFKLQNXEDFMLZRG]LH'ODSUyEHNQLH]PLQH-
UDOL]RZDQ\FK PRGXá <RXQJD Z]UDVWDá ZUD] ]H Z]URVWHP
VWĊĪHQLD ** SUyENL ]DZLHUDMąFH  ** E\á\ RNRáR 
UD]\V]W\ZQLHMV]HRGSUyEHN**0RF]HQLHZZRG]LH
QLH ZSá\ZDáR QD ZáDĞFLZRĞFL PHFKDQLF]QH W\FK ĪHOL 3R
PLQHUDOL]DFML PRGXá\ ZV]\VWNLFK PDWHULDáyZ E\á\ Z\ĪV]H
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FIG. 3 shows the results of Young’s modulus of non-
mineralized and mineralized GG samples prior and after
incubation in water for 7 days. For the samples without
ALP Young’s modulus increased with GG concentration.
6DPSOHVFRQWDLQLQJ**ZHUHDSSUR[LPDWHO\WLPHV
PRUHULJLGWKDQ**6RDNLQJLQZDWHUGLGQRWLQÀXHQFH
mechanical properties of those gels. After mineralization,
Young’s modulus of every material was higher, although the
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Zmiany procentu suchej masy po inkubacji w wodzie Dry mass changes after mineralization and after incuba-
przedstawiono na RYS. 4. Zanotowano spadek suchej masy tion in water are presented in FIG. 4. There was a drop in
RNRáRGODZV]\VWNLFKSUyEHNQLH]DZLHUDMąF\FK$/3SR
LQNXEDFMLZZRG]LH6XFKDPDVDSRPLQHUDOL]DFMLE\áDRNRáR
NURWQLHZ\ĪV]DQLĪSU]HGPLQHUDOL]DFMąGODZV]\VWNLFKSUy-
EHNLZ\QRVLáDRNRáR1LH]DQRWRZDQRLVWRWQ\FKUyĪQLF
SRGZ]JOĊGHPSURFHQWXVXFKHMPDV\SRPLĊG]\SUyENDPL
R UyĪQ\P VWĊĪHQLX ** 3R LQNXEDFML Z ZRG]LH ZV]\VWNLH
SUyENL Z\ND]\ZDá\ ]QDF]ąF\ VSDGHN VXFKHM PDV\ GR
SR]LRPXRNRáRMHGQDNQDGDOQLHVWZLHUG]RQRLVWRWQ\FK
VWDW\VW\F]QLHUyĪQLFGODSUyEHNRUyĪQ\PVWĊĪHQLX**
GU\PDVVDSSUR[LPDWHO\RIWKH**VDPSOHVZLWKRXW
ALP after soaking in water. Dry mass after mineralization
was approximately four times higher than before mineraliza-
WLRQIRUHYHU\VDPSOHDQGDFFRXQWHGIRUDSSUR[LPDWHO\
7KHUHZHUHQRVLJQL¿FDQWGLIIHUHQFHVLQGU\PDVVHVDIWHU
mineralization between the samples with different concentra-
WLRQVRI**$IWHULQFXEDWLRQLQZDWHUDVLJQL¿FDQWGHFUHDVH
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between the samples with various GG concentrations were
VWLOOQRWVLJQL¿FDQW
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LSU]HGRUD]SRLQNXEDFMLZZR
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**$/3 DQG QRQPLQHUDOL]HG ** JHO
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2EUD]\ EDUZLHQLD OLYHGHDG NRPyUHN KRGRZDQ\FK /LYHGHDGVWDLQLQJRIWKHFHOOVFXOWXUHGRQWKHVXUIDFH

QD SRZLHU]FKQL PDWHULDáyZ MDN UyZQLHĪ QD SU]HNURMDFK of the materials as well as on their cross-sections are pre-
SU]HGVWDZLRQRQD5<6ĩ\ZHNRPyUNL]DEDUZLRQHQD sented in FIG. 5. Live (stained in green) cells were found on
]LHORQRE\á\REHFQHQDZV]\VWNLFKEDGDQ\FKPDWHULDáDFK
zmineralizowanych oraz niezmineralizowanych. Jednak
REDW\S\PDWHULDáyZFKDUDNWHU\]RZDá\Z\UDĨQHUyĪQLFHZ
PRUIRORJLLURVQąF\FKQDQLFKNRPyUHN1DPDWHULDOHQLH]PL-
QHUDOL]RZDQ\FKNRPyUNLZ\ND]\ZDá\WHQGHQFMĊGRZ]URVWX
all samples studied, both mineralized as well as non-miner-
alized. However there were pronounced differences in mor-
phology between these groups. Cells on non-mineralized
GG gels had a tendency to grow in clusters (FIG. 5 A,B,F,G)
whereas on mineralized materials cells were more evenly
ZSRVWDFLNODVWUyZ5<6$%)*SRGF]DVJG\QDPD- separated on day 1 (FIG. 5 C,D,H,I). However their spread-
WHULDOH]PLQHUDOL]RZDQ\PNRPyUNLSRGQLXE\á\]QDF]QLH ing was reduced compared to that on TCPS (FIG. 5 E).
EDUG]LHM UyZQRPLHUQLH UR]PLHV]F]RQH 5<6  &'+, After day 7 (FIG. 5 H,I) the cells were spreading over the
-HGQDN Z SRUyZQDQLX ] 7&36 NRPyUNL WH E\á\ ]QDF]QLH surface and their morphology was similar to that on TCPS
VáDELHM UR]SáDV]F]RQH 5<6  ( 3R  GQLDFK NRPyUNL (FIG. 5 E,J). Higher cell density was observed on the
SU]\MPRZDá\ EDUG]LHM UR]SáDV]F]RQą L Z\GáXĪRQą SRVWDü cross-section of mineralized samples (FIG. 5 I) than on the
DLFKPRUIRORJLDE\áD]EOLĪRQDGRW\FKURVQąF\FKQD7&36 material surface (FIG. 5 H).
5<6(-:LĊNV]ąJĊVWRĞüNRPyUHN]DQRWRZDQRGOD
SU]HNURMyZ]PLQHUDOL]RZDQ\FKSUyEHN5<6,ZSRUyZ-
QDQLX]NRPyUNDPLURVQąF\PLQDSRZLHU]FKQL5<6+
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L)+RUD]QDSU]HNURMDFK%'L*,QLH]PLQHUDOL]RZDQ\FKĪHOL**$%)*RUD]]PLQHUDOL]RZDQ\FKĪHOL**$/3
&'+,'RGDWNRZRSU]HGVWDZLRQRNRPyUNLQDPDWHULDOHRGQLHVLHQLD7&36SRGQLX(RUD]GQLDFK-
),*/LYHGHDGVWDLQLQJRI0*FHOOVDIWHUGD\$(DQGGD\V)-&HOOZHUHFXOWXUHGRQWKHVXUIDFHV
$&DQG)+DQGFURVVVHFWLRQV%'DQG*,RI**VDPSOHVQRQPLQHUDOL]HG$%)*DQGPLQHUDOL]HG
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'\VNXVMDZ\QLNyZ 'LVFXVVLRQ
7HVW\PHFKDQLF]QHQDĞFLVNDQLHK\GURĪHOLSRGQLRZHM Compressive testing of the hydrogels after 14 days
PLQHUDOL]DFML Z &D*3 Z\ND]Dá\ Z]URVW PRGXáX <RXQJD incubation in mineralization solution (CaGP) revealed an
ZV]\VWNLFK SUyEHN ]DZLHUDMąF\FK$/3 5<6  %\áR WR increase in Young’s modulus of every sample containing
VSRZRGRZDQH IRUPRZDQLHP VLĊ ID]\ PLQHUDOQHM &D3 ALP (FIG. 3). It is due to formation of a mineral phase
ZK\GURĪHODFKZREHFQRĞFLHQ]\PX±$/3FRZ\ND]DQR (CaP) in hydrogels in the presence of ALP as previously
MXĪZHZF]HĞQLHMV]\FKEDGDQLDFK>@2EHFQRĞüPLQHUD- reported in other studies [8,9]. The presence of minerals
áyZ]RVWDáDSRWZLHUG]RQDSRSU]H]SRPLDUSURFHQWXVXFKHM was demonstrated by the increase in dry mass percentages
PDV\5<65R]PLHV]F]HQLH&D3]RVWDáRXZLGRF]QLRQH of the samples (FIG. 4). Distribution of CaP was visualized
SRSU]H]SU]\JRWRZDQLHSU]HNURMyZ]PLQHUDOL]RZDQ\FKĪHOL by preparing cross-sections of mineralized samples. It was
:\ND]DQRĪHQDMZLĊNV]D]DZDUWRĞüID]\PLQHUDOQHM]QDMGR- found that the majority of the minerals were concentrated
ZDáDVLĊZHZQąWU]ĪHOXSRGF]DVJG\SRZLHU]FKQLDLEU]HJL inside the gels, whereas the surface and edges of the sam-
SUyENLE\á\]PLQHUDOL]RZDQHZQLHZLHONLPVWRSQLX5<6 ples were only mildly mineralized (FIG. 2).
:HGáXJQDV]HMKLSRWH]\QLHMHGQRURGQHUR]ORNRZDQLHID]\ Our hypothesis is that inhomogeneous distribution of CaP
&D3MHVWVSRZRGRZDQHZ\SáXNLZDQLHP$/3]SRZLHU]FKQL may be attributed to leaching out of ALP from the surface
PDWHULDáXZF]DVLHPRF]HQLDZUR]WZRU]HGRPLQHUDOL]DFML of the samples during soaking in mineralization solution,
&D*3 FR VSUDZLD ĪH W\ONR ZHZQĊWU]QD F]ĊĞü ĪHOX ]D- as a result of which only the inner part of the sample con-
FKRZXMHZ\VWDUF]DMąFąLORĞüHQ]\PXQLH]EĊGQąGRSHáQHM WDLQVDVXI¿FLHQWDPRXQWRIHQ]\PHWREHIXOO\PLQHUDOL]HG
PLQHUDOL]DFML : QDV]\P HNVSHU\PHQFLH Z\ND]DQR ĪH In our experiment it was found that more than half of the
SRQDGSRáRZD$/3MHVWXZDOQLDQD]ĪHOXGRUR]WZRUXSRG- amount of ALP was released from the samples during the
czas pierwszych 2 h inkubacji w wodzie (RYS. 1). Podobna
NLQHW\NDSURFHVXZ\P\ZDQLDE\áDSUH]HQWRZDQDGODJXP\
JHOODQRLQQ\PVWĊĪHQLX$/3>@
'UXJLP]MDZLVNLHPRGSRZLHG]LDOQ\P]D]PLDQĊVWĊĪH-
QLD&D3ZREUĊELHĪHOXMHVWUR]SXV]F]DQLHID]\PLQHUDOQHM
ZUR]WZRU]HZRGQ\P3RGF]DVSURFHVXSU]\JRWRZDQLDSUy-
EHNLQNXEDFMDZZRG]LHMHVWQLH]EĊGQ\PHWDSHPSR]ZDODMą-
F\PQDZ\SáXNDQLHSR]RVWDáRĞFL&D*3]H]PLQHUDOL]RZDQH-
JRPDWHULDáX1DV]HEDGDQLDSRND]XMąĪHID]D&D3QLHMHVW
FDáNRZLFLHVWDELOQDZUR]WZRU]HZRGQ\PQDFRZVND]XMH
]QDF]ąF\ VSDGHN PRGXáX <RXQJD RUD] SURFHQWX VXFKHM
masy po 7 dniowym okresie inkubacji w wodzie (RYS. 3 i 4).
8E\WHNID]\PLQHUDOQHM]RVWDáUyZQLHĪZ\ND]DQ\QDSU]H-
¿UVWKRILQFXEDWLRQLQZDWHU),*6LPLODU$/3UHOHDVH
kinetics was measured previously for GG with different
concentration [9].
The second phenomenon affecting mineral concentration
in the samples is CaP dissolution in aqueous media. During
the manufacturing procedure, one day incubation in water
is applied to remove residual CaGP from the mineralized
material. Our results show that the CaP phase is not com-
pletely stable in the aqueous environment, which is indicated
by substantial loss in Young’s modulus and dry mass after
7 days incubation in water (FIG. 3 and 4). Mineral phase
loss was also demonstrated on sample cross-sections
(FIG. 2 D,E,F). Although mass loss was independent of the
NURMDFKĪHOL5<6'()&KRFLDĪXE\WHNPDV\QLHE\á GG concentration (FIG. 4), Young’s modulus decrease was
]DOHĪQ\RGVWĊĪHQLDĪHOX5<6VSDGHNPRGXáX<RXQJD WKHPRUHSURQRXQFHGIRUWKDQIRU**VDPSOHV
E\á EDUG]LHM Z\UDĨQ\ GOD ĪHOL  QLĪ  ** 5<6  (FIG. 3). Such results suggest that by changing GG con-
:\QLNWHQVXJHUXMHĪHSRSU]H]]PLDQĊVWĊĪHQLD**PRĪ- centration it is possible to reduce ALP as well as mineral
OLZHMHVWRJUDQLF]HQLHXWUDW\$/3RUD]&D3]ĪHOXMHGQDN release from the samples, but further studies are needed.
]DJDGQLHQLHWRZ\PDJDGDOV]\FKEDGDĔ
12 1DV]HEDGDQLDNRPyUNRZHZ\ND]Dá\UyĪQLFĊZ]DFKR- Our biological results show that cells’ behavior is differ-
ZDQLXVLĊNRPyUHNSRPLĊG]\ĪHODPLQLH]PLQHUDOL]RZDQ\PL ent if cells are cultured on non-mineralized or mineralized
a zmineralizowanymi (RYS. 5). W pierwszym przypadku
NRPyUNLPLDá\NV]WDáWRNUąJá\E\á\VáDERUR]SáDV]F]RQHFR
VXJHUXMHLFKVáDEąDGKH]MĊGRSRGáRĪD3RGQLDFKNRPyUNL
QDW\PPDWHULDOHWZRU]\á\NODVWU\1DWRPLDVWQD]PLQHUDOL]R-
ZDQ\P**NRPyUNLE\á\EDUG]LHMUyZQRPLHUQLHUR]ORNRZDQH
na powierzchni po 1 dniu a po 7 dniach ich morfologia przy-
**K\GURJHOV),*,QWKH¿UVWFDVHWKHFHOOVDUHURXQG
poorly spread, which suggests weak adhesion. After 7
days they form clusters. On the other hand, on mineral-
ized GG the cells are evenly distributed on day 1 and after
day 7 their well-spread morphology resembles that of cells
on TCPS (FIG. 5). Similar results were already reported in
SRPLQDáDPRUIRORJLĊNRPyUHNURVQąF\FKQD7&365<6
3RGREQHZ\QLNL]RVWDá\MXĪRSXEOLNRZDQHSU]H]'RXJODVD
LZVSNWyU]\EDGDOLĪHOH**SU]HGRUD]SRSURFHVLH
PLQHUDOL]DFML>@1DV]HEDGDQLDZ\ND]XMąMHGQDNLVWQLH-
QLHFLHNDZHJR]MDZLVNDPLDQRZLFLHOLF]EDNRPyUHNURVQą-
F\FKQDSU]HNURMDFKPDWHULDáyZE\áD]QDF]ąFRZLĊNV]DRG
OLF]E\NRPyUHNURVQąF\FKQDSRZLHU]FKQL3U]\F]\QąWHJR
PRĪHE\üXWUDWDPLQHUDáyZLHNVSR]\FMDPDWU\F\SROLPHUR-
ZHMĪHOX**NWyUDQLHVSU]\MDDGKH]MLNRPyUHN
:QLRVNL
3RSU]H]LQNXEDFMĊZUR]WZRU]H&D*3ĪHOL]DZLHUDMąF\FK
$/3Z\WZRU]RQRZLFKZQĊWU]XID]ĊPLQHUDOQą0LQHUDOL-
]DFMDHQ]\PDW\F]QDGRSURZDG]LáDGRZ]URVWXV]W\ZQRĞFL
oraz procentu suchej masy wszystkich mineralizowanych
PDWHULDáyZ 3URFHV WZRU]HQLD VLĊ ID]\ PLQHUDOQHM E\á
QLHUyZQRPLHUQ\ Z REUĊELH SUyENL VLOQLHMV]D PLQHUDOL]D-
FMD ]DFKRG]LáD ZHZQąWU] QLĪ QD SRZLHU]FKQL +\GURĪHOH
RZ\ĪV]\PVWĊĪHQLX**E\á\EDUG]LHMSRGDWQHQDWZRU]HQLH
VáDELHM ]PLQHUDOL]RZDQHM SRZLHU]FKQL FR E\áR Z\QLNLHP
V]\EV]HJRXZDOQLDQLD$/3=PLQHUDOL]RZDQ\ĪHOZVSLHUDá
DGKH]MĊRUD]Z]URVWNRPyUHNDE\áRWRV]F]HJyOQLHZLGRF]QH
QDSU]HNURMDFKPDWHULDáyZWR]QDF]\ZPLHMVFDFKJG]LH
VWĊĪHQLHID]\&D3E\áRQDMZ\ĪV]H
3RGVXPRZXMąF ]MDZLVNR QLHMHGQRURGQHM PLQHUDOL]DFML
VSRZRGRZDQHM XWUDWą HQ]\PX RUD] SyĨQLHMV]\P Z\SáXNL-
ZDQLHPID]\PLQHUDOQHMSRZLQQR]RVWDüXZ]JOĊGQLRQ\SU]\
Z\WZDU]DQLX]PLQHUDOL]RZDQ\FKHQ]\PDW\F]QLHPDWHULDáyZ
do dobudowy tkanki kostnej.
the studies of Douglas et al. who studied GG samples of
FRQFHQWUDWLRQSULRUDQGDIWHUHQ]\PDWLFPLQHUDOL]DWLRQ
[9,10]. However in our recent experiments we also found
DYHU\LQWHUHVWLQJSKHQRPHQRQQDPHO\DVLJQL¿FDQWO\KLJKHU
number of cells on mineralized GG material cross-sections
than on the outer surfaces. This can be attributed to surface
mineral loss, leaving only the GG polysaccharide hydrogel
matrix, which does not promote cell adhesion.
&RQFOXVLRQ
CaP mineral formation in GG hydrogels was induced by
incorporation of ALP and subsequent incubation in CaGP
solution. Enzymatic mineralization led to an increase in
stiffness and dry mass of all materials. Mineral formation
ZDVLQKRPRJHQHRXVZLWKLQWKHVDPSOHVPRUHPLQHUDOZDV
formed inside than on the outer surface. The samples with
higher GG concentration were more prone to form mildly
mineralized surfaces due to mineral and ALP loss. Mineral-
ized gels promoted cell attachment and growth, especially
in the case of cell culturing on sample cross-sections, i.e.
where the concentration of mineral phase was the highest.
In summary, the processes of inhomogeneous miner-
alization caused by enzyme and mineral loss should be
considered in order to produce materials for bone tissue
engineering by enzymatic means.
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ST8/00129).
3LĞPLHQQLFWZR
>@ , *LDYDVL / 0 +DUYH\ DQG % 0F1HLO Gellan gum. Critical
Reviews in Biotechnology 20.3 (2000) 177-211.
>@ 7 2NDPRWR . .XERWD 6ROJHO WUDQVLWLRQ RI SRO\VDFFKDULGH
gellan gum. Carbohydrate Polymers 30.2 (1996) 149-153.
[3] A.M. Fialho, L.M. Moreira, A.T. Granja, A.O. Popescu, K. Hoffmann,
,6i&RUUHLD2FFXUUHQFHSURGXFWLRQDQGDSSOLFDWLRQVRIJHOODQ
current state and perspectives. Applied Microbiology and Biotech-
nology 79.6 (2008) 889-900.
>@-(5ROGDQ+\GURJHOV,QWURGXFWLRQDQG$SSOLFDWLRQVLQ%LRORJ\
and Engineering, Lousiana Tech University Dept. of Biological
Sciences, 2003.
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