7/30/2019 2012 Bio Matter 0031 r

1/13

www.lndbioin.om Biomttr 23162-1

Biomttr 3:1, 23162; Jnury/Fbrury/Mr 2013; 2013 Lnd Bioin

ReseaRch papeR ReseaRch papeR

*Correspondence to: Niina Ahola; Email: niina.ahola@tut.

Submitted: 08/08/12; Revised: 11/21/12; Accepted: 12/07/12

Citation: Ahola N, Mnnist N, Veiranto M, Karp M, Rich J, Emov A, et al. An in vitro study o composites o poly(L-lactide-co--caprolactone), -tricalcium

phosphate and ciprofoxacin intended or local treatment o osteomyelitis. Biomatter 2013; 3:23162; http://dx.doi.org/10.4161/biom.23162

Introduction

The objective o the work was to develop ciprooxacin releasingand bioabsorbable bone deect fller material that also containsa ceramic component to aid bone healing. In this study, the invitro drug release and degradation behavior o biodegradable cip-rooxacin releasing bone flling material, which has high -TCPcontent o up to 60 wt%, or enhanced osteoconductivity, wasinvestigated. Additionally, the eect o the released antibioticagainst a common osteomyelitis causing bacteria Pseudomonasaeruginosawas tested using the bioluminescence method. Therequired length o the controlled antibiotic delivery is rom three

to six months. This length o time is considered adequate in thetreatment o osteomyelitis.1 Furthermore, the degradation o thefllers should occur within a similar time rame.

Osteomyelitis is a severe complication that is challenging totreat. It is caused by bacteria, commonlyStaphylococcus aureus,Pseudomonas aeruginosa or Staphylococcus epidermidis and leadsto bone destruction.2 Traditionally, osteomyelitis is treated by

Otomyliti i btril di tt n bom roni, nd trtmnt otn inlud urgil ortion to rmov

intd bon. T im o ti tudy w to dvlo nd invtigt in vitro bon lling omoit mtril tt rlirooxin to kill ny rmining btri nd ontin biormi to l t bon to l. Tr omoit o oly(L-

ltid-o--rolton),-trilium ot nd irooxin wr omoundd uing twin-rw xtruion nd

trilizd by gmm irrdition. Drug rl nd dgrdtion o t omoit wr invtigtd in vitro or 52 wk.

T omoit wit 50 wt% o-Tcp d t mot romiing irooxin rl rol. T rmi omonnt

lrtd t drug rl tt ourrd in tr obying rt-ordr kinti. Inibition zon tting uing

bioluminn owd tt t rld irooxin d ft in rditing ommon otomyliti uing

btri Pseudomonas aeruginosa. During t in vitro dgrdtion tt ri, molr wigt o t olymr mtrix o

t omoit drd ridly. additionlly, 1h-NMR nlyi owd tt t olymr d bloky trutur nd t

omonomr rtio ngd during ydrolyi. T ttd omoit owd grt otntil to b dvlod into bon

llr mtril or t trtmnt o otomyliti or otr bon rltd intion.

An in vitro study o composites opoly(L-lactide-co--caprolactone), -tricalciumphosphate and ciprofoxacin intended or local

treatment o osteomyelitisNiin aol,1,2,* Noor Mnnit,1 Minn Virnto,1,3 Mtti Kr,4 Jn Ri,5 alxndr emov,4 Jukk sl5

nd Minn Kllomki1,2

1Drtmnt o Biomdil enginring; Tmr Univrity o Tnology; Tmr, Finlnd; 2BioMdiT; Tmr, Finlnd; 3Biort Ltd.; Tmr, Finlnd;4Drtmnt o cmitry nd Bionginring Tmr; Univrity o Tnology; Tmr, Finlnd; 5Drtmnt o Biotnology nd cmil Tnology;

sool o cmil Tnology; alto Univrity; eoo, Finlnd

Keywords: controlled drug delivery, ciproloxacin, antibiotic, biodegradable, composite, poly(L-lactide-co-caprolactone)

surgical debridement o the inected tissues ollowed by a longcourse o intravenous or parenteral antibiotics.3-6 Fractures, espe-cially open ones, implant suraces, and external racture fxationsare examples o situations that are known to enhance bacterialadhesion. These conditions, i let untreated, may lead to a bio-flm ormation and osteomyelitis. These problems have beenaddressed in numerous reviews.7-9

The surgical debridement o the inected bone in the treatingo osteomyelitis creates a deect in the bone called a dead space.Because bacteria may remain in the surrounding tissues, antibiot-ics are also needed in the treatment. Adequate concentrations othe antibiotic on the site o the dead space are difcult to achieve

due to the poor circulation o blood in the inected bone tissue.Local delivery o the antibiotics provides an efcient way o deliv-ering the drug in situ and achieving therapeutic levels o the drug.One o the greatest advantages in local drug therapy is that sys-temic adverse eects are avoided.10,11 The challenge is to keep thedrug concentration at the therapeutic level and not to exceed toxiclevels. Previous studies have shown that with local treatment, the

7/30/2019 2012 Bio Matter 0031 r

2/13

23162-2 Biomttr Volum 3 Iu 1

-caprolactone monomer contents o the processed samples wereanalyzed rom two parallel samples o each manuactured com-posite and the amounts o both monomers were very low. Theaverage L-lactide monomer content ater processing was 0.05-mol% and caprolactone monomer content below 0.02-mol%.This indicates that no monomers were generated during process-ing in any o the studied composites. Because there were no di-

erences in the monomer contents o the manuactured materials,it can be assumed that the monomers did not cause dierences inthe hydrolytic degradation behavior o the studied composites. 25

Drug release rom the materials. The initial ciprooxacincontents o the composites were measured as 7.0 0.4 wt% orPLCL + C, 8.1 0.1 wt% or PLCL + TCP50 + C, and 7.5 0.2 wt% or PLCL + TCP60 + C. The cumulative ciprooxa-cin release rom the studied materials is presented as Figure 1A,where the release occurring in three phases can be noticed. Thefrst phase was the burst in the beginning o the drug release thatlasted or one day. This is the phase when the drug molecules atthe surace or near the surace are released.27,31 Although initialburst, which is typical or drug releasing polymeric materials, has

been claimed as unwanted phenomenon,28,29 it may be benefcialwhen the target is to destroy remaining bacteria ater the surgicaldebridement o inected tissue. I the burst is moderate, it mayhelp to achieve the required antibiotic concentration in the tissueand eradicate bacteria or to prevent the attachment o bacteria tothe operated bone or implant.10,30

The second phase was rom the day 1 until day 32, whenthe ciprooxacin release rom PLCL + C was very slow andthe release rom the composites with 50 and 60 wt% o-TCP occurred steadily. The second phase was diusion con-trolled31 and obeyed the frst-order kinetics (R2 values 0.99or both o the composites containing 50 wt% and 60 wt%

o

-TCP). In frst order kinetics, the release rate o the drugis dependent on the concentration and obeys Equation 1:

lnA = -kt + lnA0

( 1 )

where A is the drug load at the time t, A0

is the initial drug load,and k is the rate constant. This equation results in a straight linewhen the logarithm o the remaining drug in the sample is plot-ted against time.

The third phase o the ciprooxacin release star ted around day32 o the release tests, where the release rom PLCL + C andPLCL + TCP50 + C accelerated. This can be seen as a change inthe release profle at 32 d in Figure 1A. There was, however, no

notable change in the release profle at this point or the compos-ite with 60-wt% o-TCP. This was probably due to the porousstructure o the composite caused by the high -TCP contentthat enabled a high diusion rate o ciprooxacin out o the poly-mer matrix. In this case, the polymer degradation did not seemto have much o an eect on the ciprooxacin release. Ater thephase change at 32 d, all the materials obeyed frst-order kinet-ics (R2 values 0.99 or all the tested materials) but with dierentslopes than seen in the second phase.

It was noticed that the -TCP content o the compositeshad a considerable eect on the ciprooxacin release (Fig. 1A).

systemic drug concentrations in the blood or other tissues are sig-nifcantly lower than in the surrounding local tissues.11-17

Local biodegradable and antibiotic releasing systems havebeen studied both in vitro and in vivo11,12,15,16,18-22 and reviewed bymany research groups.10 Koort et al. have studied ciprooxacinreleasing bone deect fllers with osteoconductive ceramic com-ponent in a localized osteomyelitis rabbit model and the results

have been promising. Ciprooxacin was ound to penetrate bonewell and higher local concentrations o ciprooxacin could beachieved than by using systemic administration.10,13,14,23

Mkinen24 has proposed a new clinical treatment algorithm inthe treatment o osteomyelitis based on osteoconductive materi-als that release antibiotics locally. In this algorithm, the surgicaldebridement and the antibiotic treatment o the resulting deadspace in the bone are perormed in one operation. Ater treat-ment, no surgical removal o the antibiotic releasing implantsor bone grating is required due to the bioabsorbable and osteo-conductive nature o the implants. The fllers developed in thecurrent study may provide the osteoconductive and antibioticreleasing materials that Mkinen has proposed. However, there

is need to test the most promising composites urther in vivo toprove their efcacy in living tissues.

Results and Discussion

The eect o processing and sterilization on the materials. Thecomposite materials were manuactured using twin-screw extru-sion and the resulting composites had ceramic particles and cipro-oxacin antibiotic evenly distributed in the polymer matrix due tothe efcient mixing in the extrusion process. The composites aredenoted PLCL + C [Poly(L-lactide-co--caprolactone) (PLCL)with 8 wt% o ciprooxacin in eed], PLCL + TCP50 + C [PLCL

with 50 wt% o

-tricalcium phosphate (

-TCP) and 8 wt%o ciprooxacin in eed] and PLCL + TCP60 + C (PLCL with50 wt% -TCP and 8 wt% o ciprooxacin in eed). Processingcaused only minor degradation in the composites. The weightaverage molecular weight (M

w) o the raw material was measured

as 245,000 g/mol and the number average molecular weight (Mn)

150,000 g/mol. The processing o the composites caused a slightdecrease both in the M

wand M

n. The decrease in the M

wwas

8% or PLCL + C and 4% and 3% or the PLCL + TCP50 +C and the PLCL + TCP60 + C respectively and the decrease inthe M

nwas 12% or the PLCL + C and negligible (below 1%)

or both PLCL + TCP50 + C and the PLCL + TCP60 + C.Polydispersity (PD) was slightly increased or PLCL + C (rom

1.6 to 1.7) but no change was observed or PLCL + TCP50 + Cand PLCL + TCP60 + C. As was expected, the sterilization usinggamma irradiation with the measured dose o 2935 kGy causedconsiderable degradation to all the composites. Gamma irradia-tion is known to aect polymers and cause degradation. 25,26 Thedecrease was 20%, 45% and 50% or the PLCL + C, the PLCL+ TCP50 + C, and the PLCL + TCP60 + C respectively, whencompared with the initial M

wo the raw material.

The residual monomer content o the raw material was 0.08wt% or the L-lactide monomer and below detection limit (