Effectable application of vascular endothelial growth factor tocritical sized rat calvaria defects

Yutaka Yonamine, DDS,a Takashi Matsuyama, DDS, PhD,a Takahiro Sonomura, DDS, PhD,b

Hironobu Takeuchi, DDS, PhD,a Yasushi Furuichi, DDS, PhD,c Masanori Uemura, DDS, PhD,b

Yuichi Izumi, DDS, PhD,d,e and Kazuyuki Noguchi, DDS, PhD,a Kagoshima, Sapporo, andTokyo, JapanKAGOSHIMA UNIVERSITY, HEALTH SCIENCES UNIVERSITY OF HOKKAIDO, AND TOKYO MEDICALAND DENTAL UNIVERSITY

Objective. An early vascular response for angiogenesis is essential for the normal progression of bone defect healing.Vascular endothelial growth factor (VEGF) is a potent inducer of angiogenesis. The aim of this study was to evaluatethe effects of a poly (L,D-lactic-co-glycolic acid) (PLGA) membrane with VEGF encapsulated into PLGA microsphereson bone regeneration at bone defects in rat calvaria.Study Design. Microspheres of PLGA incorporating VEGF165 (VEGF microspheres) were prepared, and critical-sizebone defects were created in rat calvaria. The VEGF microspheres, PLGA microspheres, or VEGF microspheres plusPLGA membrane were applied to the defects. Bone regeneration was evaluated using image analysis based on softradiographic and histologic examination.Results. Mature thick bone regeneration was observed in selected sites at bone defects that had been applied withVEGF microspheres/PLGA membrane compared with those that had been applied with the other treatments.Conclusion. A combination of VEGF microspheres and a PLGA membrane effectively enhances bone

regeneration. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;109:225-231)

Bone is a highly vascularized tissue with the unique abil-ity to remodel and repair during bone fracture.1 However,segmental bone defects and nonunions resulting fromtrauma, resection, or pathology present significant clinicalchallenges for reconstructive and maxillofacial sur-geons.2,3 Bone grafting techniques including autograftsand/or allografts hold significant limitations for bone re-pair, including lack of sufficient material, risk of diseasetransmission,4 and/or cell-mediated immune responses.5

Recent advances in the field of regenerative medicinehave led to the possibility of successfully repairing and

aDepartment of Periodontology, Kagoshima University GraduateSchool of Medical and Dental Sciences.bDepartment of Anatomy for Oral Sciences, Kagoshima UniversityGraduate School of Medical and Dental Sciences.cDepartment of Periodontology and Endodontology, Health SciencesUniversity of Hokkaido.dSection of Periodontology, Department of Hard Tissue Engineering,Graduate School of Medical and Dental Sciences, Tokyo Medical andDental University.eGlobal Center of Excellence Program, International Research Centerfor Molecular Science in Tooth and Bone Diseases, Tokyo Medicaland Dental University.Received for publication May 22, 2009; returned for revision Aug 19,2009; accepted for publication Sep 5, 2009.1079-2104/$ - see front matter© 2010 Mosby, Inc. All rights reserved.

doi:10.1016/j.tripleo.2009.09.010

restoring function in damaged or diseased tissues.6-8

One of the key issues of bone tissue engineering ap-proaches is to generate the microvascular network toprovide oxygen and nutrients for growth and differen-tiation.3,9 Angiogenesis is a fundamental process forosseous formation and repair, and an early vascularresponse is essential for the normal progression offracture healing. Vascular endothelial growth factor(VEGF), which is a potent growth factor that inducesangiogenesis, has been shown to be involved in osteo-genesis and bone repair.10 Geiger et al.11 have shownthat angiogenesis and osteogenesis can be promoted usinga local plasmid gene transfer technology with collagensponges that are soaked with a human VEGF165 plasmid.

A variety of porous scaffold polymers, naturally de-rived or synthetic matrices, have shown the potentialfor bone tissue engineering applications by offeringnew approaches to cell and growth factor delivery.12-15

However, the use of these biomaterials has been limitedowing to physical and chemical instability. Accord-ingly, further new approaches to tissue engineering mayproduce additional benefits in regenerative medicine.

Guided bone regeneration (GBR) technique uses bar-rier membranes to protect bone augmentation sites fromnonosteogenic tissue ingrowth.16,17 However, humanclinical trials of GBR have proven that membrane-

protected bone regeneration takes considerable time to

225

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reach the desired clinical outcome.18-20 Clinically, aprolonged healing period of 6 or more months is aserious problem. Nevertheless, there are currently nopractical approaches to shorten the healing period ofGBR. Therefore, new techniques that accelerate boneregeneration by stimulating bone cell proliferation anddifferentiation should be developed.

The objective of the present study was to investigatethe effect of VEGF encapsulated into poly(L,D-lactic-co-glycolic acid) (PLGA) (VEGF microspheres) and aPLGA membrane on bone regeneration at critical-sizebone defects in rat calvaria.

MATERIAL AND METHODSMaterials

The PLGA (molecular weight 10,000, lactide moi-ety/glycolide moiety ratio 75/25) was purchased fromWako (Tokyo, Japan). Recombinant human VEGF165

(rhVEGF165) was obtained from PeproTech EC (Lon-don, U.K.). Dichloromethane, bovine serum albumin(BSA), poly (vinyl alcohol) (PVA), and sodium heparinwere purchased from Sigma (St. Louis, MO).

AnimalsTwenty male Wistar rats weighing 400-500 g were

purchased from Kyudo (Fukuoka, Japan). Animal selec-tion and management, surgical protocol, and preparationfollowed routines approved by the Institutional AnimalCare and Use Committee of Kagoshima University.

Preparation of PLGA microspheres andencapsulation of VEGF into microspheres

PLGA 75/25 was used to prepare microspheres incor-porating a growth factor, using a double emulsion (water/oil/water) process, as previously described.21 One micro-gram of VEGF165 was added to 500 �L of the organicsolution of 500 mg PLGA in dichloromethane and stirredfor 5 minutes. The first emulsion was mixed with 500 �Lethyl acetate solution, and then 200 mL 0.3% PVA wasadded and stirred for 80 minutes. After centrifugation at1,000 rpm, the microspheres were frozen and dried.

In vitro VEGF release from VEGF microspheresThe microspheres (10 mg) were placed in Dulbecco

Modified Eagle Medium including 2% BSA in 24-wellplates. After incubation for 0, 1, 2, 4, 7, 10, 14, and 21days at 37°C, each supernatant was collected andstocked at �80°C. The levels of VEGF released intothe supernatant were measured using human VEGFELISA kits (Biosource, Camarillo, CA) according tothe manufacturer’s instructions.

In vivo surgical procedureTwenty male Wistar rats were divided into 4 groups

(group 1: sham surgery; group 2: graft with VEGF

microspheres; group 3: graft with a PLGA membraneand PLGA microspheres; group 4: graft with a PLGAmembrane and VEGF microspheres). Briefly, animalswere anesthetized using chloral hydrate (70 mg/100 gbody wt.). The contoured calvarial bone was exposedby periosteal elevator. Critical-size bone defects (8 mmin diameter) were created in the calvaria using a dentaltrephine and a round bur, avoiding perforation of thedura mater (Fig. 1, A). In group 1, animals receivedsham surgery (control group). In group 2, the defectwas filled with VEGF microspheres. In group 3, PLGAmicrospheres on a PLGA membrane were placed overthe dura mater and further covered with a PLGA mem-brane. In group 4, a first PLGA membrane was put intothe dura mater (Fig. 1, B). Then VEGF microsphereswere placed on the PLGA membrane (Fig. 1, C). Asecond PLGA membrane was placed to completelycover the VEGF microspheres and the marginal area ofthe bone defect (Fig. 1, D). After implantation of thematerials, the soft tissues were closed with 4-0 silksutures. Management of postoperative infection wascontrolled with the intraperitoneal injection of 1 mL(500 IU) penicillin G. After 3 months, rats werekilled with chloral hydrate and perfused transcardi-ally with 200 mL 5 mmol/L sodium phosphate (pH7.4)– buffered 0.9% (w/v) saline, followed by 200mL 4% (w/v) formaldehyde in 0.1 mol/L sodiumphosphate, pH 7.4. Subsequently, tissue sampleswere immediately fixed in 10% neutral buffered for-malin solution for 24 hours.

Assessment of bone regenerationBone regeneration at the site of bone defects was

assessed using Softex CSM-2 under 25 kV and 3 mA.The specimens’ positioning was standardized with aparalleling technique. The radiographic films usedwere industrial X-ray films (Fujifilm Co., Tokyo,Japan). All of the films were set under the conditionof X-ray exposure time 60 seconds, processing time20 minutes, and temperature of processing solution25°C.

We performed computer-assisted analysis usingWinRoof image processing software (Mitani Corp.,Tokyo Japan). Briefly, all radiopaque area includingexisting bone and regenerative bone was extractedautomatically using 2 binary-distinct macroinstruc-tions composed of algorithms for color identificationbased on grayscale. Radiolucent area in the calvariadefect 12 weeks after surgery was clearly extractedby reversing all radiopaque area. In advance, stan-dard 8 mm circle radiolucent area was also clearlyextracted in the same way. The extracted radiolucent

area was calculated by automated calibration on the

OOOOEVolume 109, Number 2 Yonamine et al. 227

software. Regenerative area ratio was determined asfollows:

Regenerative area ratio (%)

� (standard 8 mm circle radiolucent area

� radiolucent area in the calvaria defect

12 weeks after surgery)

� 100 ⁄ standard 8 mm circle radiolucent area

Bone specimens were demineralized with 0.5 mol/Lethylenediamine tetraacetic acid (pH 7.4) at 4°C for 21days. After dehydration using a graded series of ethanolsolutions, specimens were embedded in paraffin. Serialsections were prepared with a microtome. Thin sections(4 �m) were stained with hematoxylin and eosin.

Scanning electron microscopyThe morphology of PLGA microspheres was ob-

served by scanning electron microscopy (SEM; JSM-5510; Jeol, Tokyo, Japan). The PLGA microspheres

Fig. 1. Representative photographs of rat calvaria duringA, Critical-size bone defect (8 mm diameter) was created inC, VEGF microspheres were put over the PLGA membranmicrospheres and the marginal area of the bone defect.

were attached to sample stubs using conductive paint

and sputter-coated with an ultrathin (100 Å) layer ofgold in a Polaron E 5100 coating apparatus. The sam-ples were viewed under SEM at an accelerating voltageof 15 kV.

Statistical analysisAll data were statically analyzed using a 1-way

analysis of variance with post hoc Scheffé test formultiple comparisons. Statistical significance wasaccepted for P values of �.05. The data were ex-pressed as mean � SD.

RESULTS

Morphology of PLGA microspheres by SEMFigure 2 shows representative images of the surface

and morphologies of the microspheres. VEGF micro-spheres presented smooth and homogeneous surfacemorphology (Fig. 2, A). A porous structure was ob-served on the outer surface of VEGF microspheres

al treatment with VEGF microspheres/PLGA membrane.lvaria. B, A PLGA membrane was put into the dura mater.

A second PLGA membrane completely covered the VEGF

surgicthe ca

e. D,

(Fig. 2, B).

OOOOE228 Yonamine et al. February 2010

Kinetics of VEGF165 release from PLGAmicrospheres in vitro

The VEGF165 release from PLGA microspheres was

Fig. 2. Morphology of VEGF microspheres. A, Scanningelectron microscope (SEM) image of VEGF microspheres.B, Higher-magnification SEM picture of the VEGF micro-sphere surface.

Fig. 3. In vitro VEGF release curve of VEGF microspheres.VEGF microspheres were incubated in PBS at 37°C for theindicated period. The amount of VEGF released from VEGFmicrospheres was determined using a human VEGF ELISAkit.

evaluated in vitro for 21 days. No release of VEGF165

during the initial 7 days was observed. After 10 days,the release increased linearly with time (Fig. 3). By day20 of the release study, the levels of VEGF165 reacheda plateau, indicating that the release of VEGF wascontrolled by polymer degradation.

Soft X-ray evaluationFig. 4 shows soft radiographic images of bone de-

fects 3 months after treatment. In group 1, there waslittle bone regeneration (Fig. 4, A). In group 2 andgroup 3, some bone regeneration was observed (Fig. 4,B and C). However, in group 4, VEGF microsphereswith a PLGA membrane resulted in remarkable boneregeneration in selected sites (Fig. 4, D).

Histologic evaluationFigure 5 shows histologic sections of bone defects 3

months after treatment. There were no inflammatorysigns in any of the groups. Interestingly, group 4showed mature thick bone regeneration compared withother groups (Fig. 5, D). In group 1, the defect wasalmost completely filled with connective tissue. Therewas a little newly formed bone at the peripheral area ofthe defect (Fig. 5, A). In group 2 and group 3, thedefects were filled with thin regenerated bone (Fig. 5, Band C).

Evaluation of bone regeneration in calvarialbone defects

In Fig. 6, radiopaque areas are compared among thegroups. The radiopacity of bone defects in group 4 wassiginificantly higher than that of bone defects in group2 and group 1 in selected sites of calvarial defect.Group 3 showed significantly greater radiopaque areascompared with group 1. However, there were no sig-nificant differences in radiopaque areas between group1 and group 2. Group 4 did not show any significantdifferences in radiopaque areas compared with group 3.

DISCUSSIONThe present study showed that the application of a

combination of VEGF165 encapsulated into PLGA anda PLGA membrane promoted bone maturation andgrowth at critical-size bone defects in rat calvaria. Theapplication of VEGF microspheres alone showed sig-nificantly less regenerative effect (Fig. 6). The applica-tion of PLGA microspheres and PLGA membranecaused bone formation, but the regenerated bone wasthin. From these results, we suggest that sustainedrelease from VEGF microspheres combined withPLGA membrane is helpful for induction of thick bone-on-bone regeneration at critical-size bone defects in rat

calvaria.

es. D,

staininPLGA

OOOOEVolume 109, Number 2 Yonamine et al. 229

When carrier-free VEGF is applied locally, rapiddiffusion occurs at the site. Moreover, the in vivo

Fig. 4. Representative soft radiographic images of surgicalmicrospheres. C, PLGA microspheres with PLGA membran

Fig. 5. Representative photographs of hematoxylin and eosinsurgery. B, VEGF microspheres. C, PLGA microspheres withOriginal magnification �2.5.

half-life of free VEGF is short because of denaturation

and enzymatic degradation.22 Therefore, the predict-ability of the biologic effects of carrier-free VEGF or

months after each treatment. A, Sham surgery. B, VEGFVEGF microspheres with PLGA membranes.

g on tissue sections 3 months after each treatment. A, Shammembranes. D, VEGF microspheres with PLGA membranes.

sites 3

other growth factors on tissue regeneration seems to be

OOOOE230 Yonamine et al. February 2010

poor. PLGA microspheres have been used to encapsu-late several growth factors in different systems.21 In thepresent study, we used PLGA microspheres as a carrierto develop a VEGF controlled release system, whichenables VEGF to be released over a long period tofacilitate bone regeneration in the critical-size defects.The initial VEGF release from microspheres from po-rous polymer matrices is very likely due to the disso-lution of lyophilized VEGF absorbed to the surface ofthe PLGA, or to the release of VEGF contained withinsurface-accessible pores.23 However, as shown in Fig.3, the initial release did not occur for up to 7 days,indicating that the present microspheres were con-trolled by the poor porous form, as shown in Fig. 2, B.In addition, VEGF microspheres showed a release ofVEGF165 for a long period from day seven (Fig. 3).Therefore, slower release of VEGF in vitro may becontrolled by degradation of the PLGA and by diffu-sion of the VEGF through the PLGA.

VEGF-containing PLGA microspheres have beenused to stimulate angiogenesis.24 When these micro-spheres are applied to bone defects, an additional pos-itive effect on new bone tissue formation can be ex-pected, because VEGF may recruit progenitor cells andosteoblasts and support their differentiation.25,26 Theseeffects can be achieved by preservation of VEGF mi-crospheres in the bone defects. However, poor bonewalls in the defects generally easily disperse releasedgrowth factors and PLGA microspheres. Accordingly,we developed a way to accelerate bone regeneration by

Fig. 6. Evaluation of bone regeneration areas on soft X-rayimages of surgical sites 3 months after each treatment. Group1: sham surgery; group 2: VEGF microspheres; group 3:PLGA microspheres with PLGA membranes; group 4: VEGFmicrospheres with PLGA membranes. *P � .05; **P � .01.

stimulating bone cell proliferation and differentiation

using VEGF microspheres in combination with aPLGA membrane. As shown in Fig. 5, D, the maturedand thick bone formation in group 4 indicates thatsustained release of VEGF and microspheres preservedby PLGA membranes at bone defects in calvaria maystimulate chemotactic migration of osteoblasts andmesenchymal progenitor cells. Recent studies haveshown that the combination of angiogenic and osteo-genic factors can stimulate bone healing and regenera-tion.11,27-29 Kaigler et al.27 have demonstrated increasedbone regeneration when VEGF-releasing PLGA scaffoldsare implanted into preirradiated noncritical-size (3.5mm diameter) defects. However, Patel et al.28 havereported that VEGF delivery from gelatin micropar-ticles alone have no impact on bone regeneration incritical-size (8 mm diameter) defects.

Those reports are consistent with our results thatVEGF microspheres alone (group 2) did not showsignificantly increased bone regeneration comparedwith the control group (group 1) at critical-size bonedefects in calvaria (Fig. 6). However, the combinationof VEGF microspheres and PLGA membrane (group 4)showed a significant increase in bone regeneration inselected sites of critical-size bone defects comparedwith that in rats receiving sham surgery treatment(group 1). Similarly, the combination of PLGA micro-spheres and PLGA membrane also led to significantbone regeneration (group 3). A PLGA membrane alonein calvaria defects did not show any increased boneformation (data not shown). It seems likely that aPLGA membrane combined with microspheres preventedmicrosphere resorption and diffusion. Consequently, thecontrolled release of VEGF from a combination of PLGAmicrospheres and a barrier membrane might have a su-perior effect on bone regeneration compared with thatof VEGF microspheres alone. The primary role of thespace making by a GBR membrane is the creation of amechanical barrier as it blocks fibrous tissue invasion atthe recipient site. The secondary role is the biologicinduction of osteoblasts from the remaining tissuearound the defect into the defect. The present resultssuggest that the combination of VEGF microspheresand a PLGA membrane is effective for bone maturationand growth in selected sites of the bone defects.

CONCLUSIONWe suggest that the combination of VEGF micro-

spheres and a PLGA membrane might be effective anduseful to increase the generation of matured and thickbone in guided bone regeneration. However, it shouldbe taken into consideration that growth factors may beeffective at specific time intervals over the course of

bone regeneration.

OOOOEVolume 109, Number 2 Yonamine et al. 231

The authors greatly thank to Drs. Motoharu Miyamotoand Daisuke Yamashita for their technical assistance inscanning electron microscopy and appreciate Prof. IchiroSemba’s help with the soft radiographic analysis.

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Reprint requests:Takashi Matsuyama, DDS, PhDDepartment of PeriodontologyKagoshima University Graduate School of Medical and Dental Sciences8-35-1, SakuragaokaKagoshima, 890-8544Japan

takashi@dentb.hal.kagoshima-u.ac.jp