Evaluation of Pluronic Polyols as Carriers for Grafting Materials: Study in Rat Calvaria Defects

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  • J Periodontol February 2002

    Evaluation of Pluronic Polyols as Carriersfor Grafting Materials: Study in RatCalvaria DefectsEdward B. Fowler,* Michael F. Cuenin, Steven D. Hokett, Mark E. Peacock,James C. McPherson III, Thomas R. Dirksen, Mohamed Sharawy, and Michael A. Billman

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    Background: Pluronic polyols are a family of non-ionic sur-factants currently used as drug carriers for antibiotic, anti-inflam-matory, and anti-neoplastic agents. Therapeutic administrationof non-ionic surface-active agents is known to facilitate early col-lagen synthesis and microcirculation, thus promoting woundhealing. The purpose of this study was to determine the in vivoeffects of pluronic polyols combined with either an allograft oran alloplast on the healing of critical-sized calvarial defects.

    Methods: One hundred fifty (150) adult (95 to 105 days old)male Sprague-Dawley rats weighing between 375 and 425 gwere randomly and evenly assigned to each of 15 separate treat-ment groups and anesthetized, and 8 mm calvarial critical-sizeddefects were created. Pluronic F-68 (F-68) or pluronic F-127 (F-127) was administered either topically or systemically and inconjuction with demineralized bone powder (DBP), tricalciumphosphate (TCP), or non-grafted controls. Pluronic polyols areeasily mixed with either DBP or TCP to improve handling ease.Calvaria were harvested at 12 weeks postsurgery and evalu-ated histomorphometrically, by contact radiography with sub-sequent densitometric analysis, through energy spectrometryutilizing a scanning electron microscope, and by fluorescentmicroscopy.

    Results: There was a significant difference in the percentageof bone fill among the control, TCP, and DBP only groups, P

  • Pluronic Polyols as Bone Graft Material Carriers Volume 73 Number 2

    administration; 3) as compared to each other; 4) whencombined with either demineralized bone powder ortricalcium phosphate; and 5) as suitable graft carriers.

    MATERIALS AND METHODSOne hundred fifty (150) adult male Harland Sprague-Dawley rats (Rattus norvegicus), approximately 95days old, weighing between 375 and 425 g were ran-domly and evenly divided into 15 treatment groups:group 1, control; group 2, control + topical F-127;group 3, control + topical F-68; group 4, control + sys-temic F-127; group 5, control + systemic F-68; group6, TCP; group 7, TCP + topical F-127; group 8, TCP+ topical F-68; group 9, TCP + systemic F-127; group10, TCP + systemic F-68; group 11, DBP; group 12,DBP + topical F-127; group 13, DBP + topical F-68;group 14, DBP + systemic F-127; and group 15, DBP+ systemic F-68. Animals were anesthetized by intra-muscular injection of xylazine (2.5 mg/kg bodyweight) and ketamine# (44 mg/kg body weight) andmaintained as needed for appropriate anesthesia. Theywere stabilized in a stereotactic device, the fur overeach animals cranium shaved, and the site preparedwith a betadine scrub. Under sterile conditions, a mid-line incision was made from the middle of thenasofrontal area to the external occipital protuberance;full-thickness skin flaps were reflected, and a stan-dardized 8 mm diameter calvarial critical-sized osseousdefect (CSD) was created as described by Schmitzand Hollinger11 (Fig. 2). Great caution was exercisedto avoid injury to the underlying sagittal sinus, duramater, and brain. Twenty-five additional Sprague-Daw-ley rats were sacrificed by carbon monoxide inhalationand both femoral shafts immediately harvested by ster-ile technique. Demineralized bone powder (DBP) wasprepared in a milling device** and evaluated by scan-ning electron microscopy to confirm smooth, ovoidparticles of 170 280 in diameter.12 Commerciallyavailable pluronic polyols, F-68 and F-127, wereadministered either systemically or topically in com-bination with either DBP or commercially available sin-tered beta-tricalcium phosphate (TCP) and carefullyplaced into the defects in amounts sufficient to fill tothe level of the external surface of the osseous rim. Theperiosteum was repositioned and closed as the firstlayer with interrupted 5-0 vicryl sutures, while skin

    was closed by a continuous running, locking 5-0 vicrylsuture. If animals displayed any signs of discomfort,they were given 2.5 mg/kg of butorphanol every 6hours until these signs disappeared.

    Each animal in groups 4, 9, and 14 received 8 ml/kgbody weight of an isotonic 12 mM F-127 solution insterile saline immediately after surgery. Each animalin groups 5, 10, and 15 received 8 ml/kg body weightof an isotonic 12 mM F-68 solution in sterile salineimmediately after surgery and continuing at a rate of3 times daily for 4 consecutive days. The different dos-ing regimen is due to the half-life of the pluronics.Oxytetracycline (10 mg/kg body weight) and alizarinred (30 mg/kg body weight), fluorescent compoundsincorporated as osteoid mineralizes, were injected intra-muscularly to labeling new bone growth.13-18 Oxy-tetracycline was injected twice daily for 4 consecu-tive days following surgery and a second series givenfrom days 56 to 59. Alizarin red was injected twicedaily from days 28 to 31.

    All animals were sacrificed at 12 weeks postsurgeryand decapitated. The cranium was removed and fixedin 70% ethanol until ready for evaluation by high-res-olution contact radiography, providing microscopicdetails of mineralized tissue sections.19 The externalcalvaria was placed in direct contact with the radi-ographic film,## radiographed at 35 kV, 2.5 mA with

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    Figure 2.View of the Rattus norvegicus calvarial critical-sized defect. Note theunderlying sagittal sinus and dura mater.

    Figure 1.Chemical structure of the generic pluronic polyol compound. EO =ethylene oxide unit; PO = polyoxypropylene (PO) base.

    Xylazine, Mobay Corporation, Shawnee, KS.# Aveco Corporation, Fort Dodge, IA.** Tekmar A-10 Analytic Micromill, Cincinnati, OH. BASF Corporation, Mount Olive, NJ. Peri-Oss, Mitzer Inc., Columbus, OH. Ethicon Inc., a Johnson & Johnson Company, Somerville, NJ. Stadol, Bristol Laboratories, New York, NY. Sigma Chemical Co., St. Louis, MO.## Kodak Diagnostic Film, Ektascan B, Eastman Kodak Co., Rochester, NY.

    1051_IPC_AAP_553082 2/11/02 8:40 AM Page 192

  • J Periodontol February 2002 Fowler, Cuenin, Hokett, et al.

    an exposure time of 15 seconds, andfilms batch processed.19-21 Imageswere digitized by computer scan-ning*** and defect radiodensity eval-uated by a computer program viaa personal computer with an 8-bitcolor display.22-25 Each radi-ograph was digitized using 50 pix-els in 8-bit images and convertingthe optical densities into 256 graylevels or bin frequencies. A rectan-gular, standardized region of interest(ROI) was drawn on all radiographs.Next, the ROIs were drawn with aninteractive image processing softwarepackage. An area histogram wascalculated for each ROI and con-verted to a cumulative percent his-togram (CPH) by dividing the 256bin frequencies by the sum of all binfrequencies and multiplying by 100.The CPH is plotted with the calcu-lated value as the y-axis and the graylevel as the x-axis.24 Analysis of dig-itized radiographic images was then accomplishedusing a standard statistical software package.

    Each calvaria sample was divided longitudinally toyield 2 halves. One half was decalcified and evaluatedby histology to determine the bone fill across the coro-nal plane.26 Samples were placed in a 1:1 solution ofsodium citrate and formic acid (50.0 g sodium citrate+ 250 ml distilled water: 125 ml formic acid + 125 mldistilled water) for 72 to 84 hours until the sample wasnon-mineralized. Samples were washed for 4 to 8 hoursunder running water and returned to 70% ethyl alco-hol until embedding in paraffin. Multiple 5 to 6 sec-tions were cut from each sample, and representativesections were stained with hematoxylin and eosin.Quantitative analysis was accomplished by viewingunder a microscope connected to a video camera andcomputer. A digitizer was used to trace the defectoutline versus new bone formation, and an averagepercentage of bone fill was determined.

    The second half of each calvaria sample wasembedded in methyl-methacrylate and ground toapproximately 50 m in thickness (unpublished data).These unstained sections were examined under anultraviolet light microscope with appropriate filters forthe yellow-green fluorescence of oxytetracycline andthe red fluorescence of alizarin red.15 Three repre-sentative calvaria from each group were randomlyselected and embedded in methyl-methacrylate forSEM evaluation. Energy spectrometry provided forelemental analysis, such that the calcium-to-phos-phorus ratios from areas of new bone within thedefects could be determined and compared to cal-

    cium-to-phosphorus ratios of the old bone at sitesdistant from the defects.

    Statistical ModelThe histomorphometric and densitometric data wereevaluated by analysis of variance, followed by the Stu-dent-Newman-Keuls test. The calcium-phosphorusratios were compared using Fishers exact test.

    RESULTSHistomorphometryResults fell into 1 of 3 major groups depending uponwhether the defect was grafted and by the graft type(Fig. 3). The percentage of bone fill in the control onlygroup (group 1, 39.7 14.4%) was not significantly dif-ferent from the TCP only group (group 6, 28.5 7.8%).There was a significant stimulation of bone formationin the DBP only group (group 11, 66.9 11.9%) com-pared to either group 1 or group 6, P

  • Pluronic Polyols as Bone Graft Material Carriers Volume 73 Number 2

    margins or became embedded within new bone (Fig.4B); however, defect centers were generally not col-lapsed due to the presence of TCP particles. While iso-lated particles or groups of particles were found eithercompletely or partially surrounded by new bone, TCPparticles were usually encapsulated by fibrous mate-rial. Among the TCP groups, only group 9, receivingsystemic F-127, was statistically different (P

  • J Periodontol February 2002 Fowler, Cuenin, Hokett, et al.

    icant stimulation in the percentage of bone filling thedefect when DBP only was used as an alloplast, com-pared to either control only or TCP alone. There wasno statistically significant difference between the modesof polyol administration within treatment groups, exceptbetween group 6 (TCP only) and group 9 (TCP + sys-temic F-127). Although not significant, there was less

    bone fill using tricalcium phos-phate alloplast than using noimplant material. The general dif-ferences between using no graftmaterial and using TCP might beexplained by the fact that the TCPparticle occupied the spaces thatmay have healed with bone; thus,the non-grafted sites had a trendtowards a higher percentage ofbone fill than the defects graftedwith alloplast. Additionally, sinceTCP particles occupied a spaceacross the defect, yet did not sig-nificantly stimulate additionalbone formation, the animals in theTCP groups had a greater defectvolume compared to control ani-mal groups. In control groups, thedefect volume in the center wasminimal due to the collapse of theperiosteum and endosteum uponthemselves. This would result in alower percentage of bone fill in the

    TCP groups, even if the TCP groups formedthe same or slightly more bone than the con-trols.

    General trends noted during the densito-metric analysis included: 1) defects graftedwith either an alloplast or an allograft resultedin more radiodense healing, and 2) the modeof pluronic polyol administration did notresult in statistically significant differenceswithin the various treatment groups, exceptbetween groups 11 (DBP only) and 13 (DBPplus topical F-68). New bone formation,assessed by fluorescent microscopy usingoxytetracycline and alizarin red, yielded dif-fuse fluorescence at the wound margin andaround alloplast and allograft. Fluorescentbands marked bone apposition. Demineral-ized bone particles may act as a nidus fornew bone formation, while tricalcium phos-phate particles were noted in close associa-tion with new bone.

    Demineralized bone powder resulting ingreater fill is consistent with reports by Mel-lonig et al.,27 who stated that decalcifiedfreeze-dried bone allografts were highly

    osteogenic. Additionally, it was demonstrated in a crit-ical-sized defect in rat calvaria that bone matrix pro-vided the greatest bone volume of new trabeculae (cal-cified plus osteoid) (unpublished data). The presentinvestigation confirmed that demineralized bone pow-der allograft results in better defect resolution thanalloplasts and non-grafted defects, and that either filler

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    Figure 7.Energy spectrometry (calcium-phosphorous ratio) results.

    Figure 6.Densitometry (80th percentile) results.

    1051_IPC_AAP_553082 2/11/02 8:40 AM Page 195

  • Pluronic Polyols as Bone Graft Material Carriers Volume 73 Number 2

    had better fill alone, although not significant, com-pared to fill mixed with pluronic polyols.

    One interesting observation during this study was thefinding of osteocytes within the implanted, demineral-ized bone powder particles. This discovery was a con-sistent finding in all the animals where DBP was uti-lized. This was in stark contrast to recent studies whichnoted that demineralized freeze-dried bone allograft(DFDBA) reacted as dead particles,28 nonviablebone chips,29 and provided minimal to no osteoin-duction.30 These differences might be related to thecompletely different models: human extraction sock-ets,28 dogs with implants and membranes for guidedtissue regeneration,29 and athymic mice, respectively.30

    An additional difference was the fact that this study uti-lized allogenic demineralized bone powder, as opposedto human demineralized freeze-dried bone powder,which would be a xenograft in the dog and mice mod-els studied.

    We found that tricalcium phosphate particles wereoften embedded in new bone growth at the defect mar-gins; however, those in the defect center were usuallyencased in fibrous tissue. This is consistent with Morsand Kaminski31 who reported that TCP needs to be inclose association with bone when used. Osteoid isknown to form around and within tricalcium phosphate,but TCP probably does not serve as a nidus for newbone formation within osseous defects.32 This researchalso confirms that TCP results in a denser fill radio-graphically, but had less histologic bone fill than non-grafted defects.

    From the gross evaluation, histomorphometry, densi-tometry, elemental analysis, and fluorescent microscopy,the general trend was that the mode of administrationof the pluronic polyols did not result in a significantchange in bone wound healing. Topical versus sys-temic pluronic polyol administration produced resultsthat were not statistically different. Pluronic polyolsinhibited the regeneration process only in group 9.Both Fitzpatrick et al. (unpublished data) and Sha-han33 found that pluronic polyols were beneficial inshort-term soft tissue healing, but after approximately1 week, healing was independent of polyol use. Sub-jectively, pluronic polyols improved graft handling.Future work should investigate ways in which pluronicpolyols can be used to enhance b...

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