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Biomaterials 31 (2010) 2893–2902

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Biomaterials

journal homepage: www.elsevier .com/locate/biomateria ls

The ability of a collagen/calcium phosphate scaffold to act as its own vector forgene delivery and to promote bone formation via transfection with VEGF165

Michael Keeney a, Jeroen J.J.P. van den Beucken b, Peter M. van der Kraan c, John A. Jansen b,Abhay Pandit a,*

a Network of Excellence for Functional Biomaterials, National University of Ireland Galway, NFB Building, IDA Business Park, Newcastle Road, Dangan, Irelandb Department Periodontology & Biomaterials, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlandsc Experimental Rheumatology & Advanced Therapeutics, NCMLS, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

a r t i c l e i n f o

Article history:Received 4 December 2009Accepted 14 December 2009Available online 30 December 2009

Keywords:Bone tissue engineeringGene therapyCalcium phosphateCollagen

* Corresponding author. Tel.: þ353 91492758; fax:E-mail address: abhay.pandit@nuigalway.ie (A. Pan

0142-9612/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.biomaterials.2009.12.041

a b s t r a c t

Collagen/calcium phosphate scaffolds have been used for bone reconstruction due to their inherentsimilarities to the bone extracellular matrix. Calcium phosphate alone has also been used as a non-viralvector for gene delivery. The aim of this study was to determine the capability of a collagen/calciumphosphate scaffold to deliver naked plasmid DNA and mediate transfection in vivo. The second goal of thestudy was to deliver a plasmid encoding vascular endothelial growth factor165 (pVEGF165) to promoteangiogenesis, and hence bone formation, in a mouse intra-femoral model. The delivery of naked plasmidDNA resulted in a 7.6-fold increase in mRNA levels of b-Galactosidase compared to the delivery of plasmidDNA complexed with a partially degraded PAMAM dendrimer (dPAMAM) in a subcutaneous murinemodel. When implanted in a muirne intra-femoral model, the delivery of pVEGF165 resulted in a 2-foldincrease in bone volume at the defect site relative to control scaffolds without pVEGF165. It was concludedthat a collagen/calcium phosphate scaffold can mediate transfection without the use of additionaltransfection vectors and can promote bone formation in a mouse model via the delivery of pVEGF165.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Calcium phosphate has been commonly used as a transfectionagent in non-viral gene delivery. This process relies on the fact thatcationic Ca2þ acts in a similar manner to cationic divalent metalions such as Mg2þ, Ba2þ and Mn2þ, which form ionic complexeswith helical phosphates [1]. Calcium phosphate therefore formsa complex with the nucleic acid backbone of plasmid DNA [2,3].Complexes are typically formed by mixing negatively chargedplasmid DNA with positively charged CaCl2 followed by the addi-tion of a phosphate buffer. Addition of a phosphate buffer causesprecipitation of nanoparticles containing plasmid DNA, which arereadily taken up by cells [4]. The cationic bonding of plasmid DNAwith the calcium phosphate used in this study was demonstratedby gel electrophoresis. (For more information on this technique seesupplementary data) As a control transfection agent in this researcha commercially available partially degraded poly(amidoamine)dendrimer was used (dPAMAM, Superfect�, Qiagen, France). dPA-MAMs are among the most efficient non-viral gene transfection

þ353 91 563991.dit).

All rights reserved.

agents available [5–7] and previous research in our lab hasdemonstrated the versatility and efficiency of this polymer [8].

VEGF is critical in angiogenesis (the formation and differentia-tion of the vascular system) and is responsible for endothelial cellproliferation and migration [9–11]. VEGF165 is the most abundantform of VEGF present in the human body and remains cell and ECMassociated [12–15]. As angiogenesis is a requirement for boneformation, VEGF has been commonly used for the treatment ofbone defects [16–22].

It was hypothesised that the presence of calcium phosphatewithin the scaffold would act as a non-viral vector for plasmid DNA(Fig. 1), and that the delivery of pVEGF165 through this systemwould lead to increased bone formation via stimulated angiogenicdevelopment.

Two in-vivo models (within a single animal) were performedto address the hypothesis. First, a subcutaneous murine modelwas used to determine if a collagen/calcium phosphate scaffoldcan deliver naked plasmid DNA and mediate transfection usingb-Galactosidase (b-Gal) as a marker. Second, the collagen/calcium phosphate scaffold combined with pVEGF165 wasimplanted in a murine intra-femoral model to determine if thedelivery of pVEGF165 from this scaffold stimulates boneformation.

Fig. 1. Concept diagram. It is hypothesised that a collagen/calcium phosphate scaffoldcarrying naked plasmid can mediate transfection without the presence of a non-viralvector. It is further hypothesised that up-regulation of vascular endothelial growthfactor (VEGF) would promote angiogenesis which is critical for bone formation.

M. Keeney et al. / Biomaterials 31 (2010) 2893–29022894

2. Materials and methods

2.1. Materials and reagents

All materials were purchased from Sigma–Aldrich unless otherwise stated. TheVEGF165 plasmid used in this research was purchased from Genecopeia�, USA. Theplasmid was inserted into a pReceiver-M02 vector. The b-Gal plasmid was kindlydonated by Dr. Udo Greiser (Regenerative Medicine Institute, Galway).

2.2. Scaffold fabrication

Scaffolds were produced as described previously [23]. Briefly, a dilute solution ofcollagen type I containing CaCl2 was mixed with a solution of KH2PO4 at neutral pHand 37 �C. The gel-like composite formed over a period of five hours. Followingincubation the gel-like composite was centrifuged to form a precipitate andsubsequently processed for subcutaneous or intra-femoral implant generation.Subcutaneous implants were generated via moulding the precipitate into a discshape (diameter 7 mm, height 1.5 mm), into which pore channels (380 mm diameterin the axial direction at a spacing of 1 mm) were introduced to encourage tissueinfiltration. For intra-femoral defects, scaffolds were produced without specificdimensions as they were shaped and fitted into the defect during surgery.

2.3. Experimental groups

The experimental groups used in this study are presented in Table 1. ComplexedDNA was prepared prior to the implantation procedure by mixing pVEGF or pb-Galwith a partially degraded PAMAM dendrimer (dPAMAM, Superfect�, Qiagen,France) at a ratio of 6:1 (wt:wt). Implants were loaded with 0.35 mg/mm3 solution ofplasmid DNA in either complexed or non-complexed form 10 min prior to implan-tation. Scaffolds in groups A and C were loaded with naked plasmid DNA whilegroups B and D received complexed plasmid DNA. Scaffolds used in Group E wereloaded with sterile filtered water.

2.4. In-vivo experiments

Forty C57BL/6 male mice (weight: 24.9–29.0 g; Elevage Janvier, Le Genest Saint-Ile, France) were used in this study and divided into treatment groups as describedin Table 1. Ethical approval for the procedures was obtained from the InstitutionalAnimal Ethics Committee of Radboud University, Nijmegen, Netherlands (DEC2008-164). Each animal received one subcutaneous and one intra-femoral implant,

Table 1Subcutaneous and femoral defect experimental groups. S indicates collagen/calciumphosphate scaffold. n¼ 8 for all implant groups. Group 1 was reduced to n¼ 6 due totwo deaths occurring during the implantation procedure.

Group A Group B Group C Group D Group E

pVEGF165 pb-Gal

Subcutaneous Sp Sc Sp Sc SIntra-femoral defect Sp Sc Sp Sc S

p indicates the scaffold was loaded with naked plasmid while c indicates the scaffoldwas loaded with plasmid complexed with dPAMAM.

resulting in 40 subcutaneous (n¼ 8) and 40 intra-femoral (n¼ 8) implants totally forthe five experimental groups.

Mice were anaesthetised with 4% isoflurane and hair was shaved from theimplant site. The site was washed with 70% ethanol. A longitudinal incision wasmade through the outer dermal layers in the mid back to expose the underlyingfascia. A subcutaneous pocket was created using blunt dissection with scissors. Thesubcutaneous implant was laid flat on the fascia and the wound was closed with twosurgical staples. Subsequently, the animal was placed on its back and a longitudinal,parapatellar incision was made medially from the knee of the left hind limb. Afterexposure of the distolateral femoral condyle, a sharp implement was used topuncture the outer cartilage surface. Rotary dental files of increasing size were thenused to create a 1 mm diameter defect with a depth of 8 mm. The scaffold wassubsequently placed into the defect, after which the wound site was closed with twosurgical staples (Agraves, InstruVet C.V., Cuijk, the Netherlands). To reduce pain aftersurgery, all the animals were injected with buprenorfine 0.15 mg/kg 3 times a day for2 days postoperatively.

2.5. Explantation

All animals were sacrificed 30 days postoperatively. Subcutaneous implants andsurrounding tissue were removed and cut in half at a random angle. One half wasfixed in 4% neutral buffered formalin while the other half was placed in RNALater�

(Applied Biosystems, Nieuwerkerk a/d IJssel, the Netherlands) for real time reverse-transcriptase polymerase chain reaction (RT-PCR). Mouse femurs were removed andthe bone was cut at the proximal end to enable formalin infiltration. The femurswere then placed in 4% neutral buffered formalin for a maximum of 24 h. Afterfixation, tissues were placed in 70% ethanol until histological processing wasperformed.

2.6. Evaluation of subcutaneously implanted scaffolds

2.6.1. Histological processingAll subcutaneous explants were dehydrated in a graded series of ethanol baths

and embedded in paraffin. Paraffin embedded samples were sectioned at 5 mmthickness and stained with Haematoxylin and Eosin.

2.6.2. Immunohistochemistry for blood vesselsImmunohistochemistry staining was performed to identify blood vessels in

subcutaneous explants using an antibody against von Willebrand factor (vWF).Antigen retrieval was performed at 37 �C using proteinase K (10mg/ml). Blocking wasperformed using hydrogen peroxide (DakoCytomation, Ireland) for 5 min and thenwith 10% donkey serum in tris buffered saline for 30 min. A rabbit anti-vWF primaryantibody (1:800, Abcam, United Kingdom, Ab6994) diluted in blocking buffer wasadded and incubated at room temperature in a humidity chamber for 1 h. A rabbitHRP labelled secondary antibody (DakoCytomation) was applied for 30 min fol-lowed by addition of DAB chromagen (DakoCytomation). Sections were counter-stained with Haematoxylin and Eosin.

2.6.3. StereologyThree sections per implant were used for all stereology measurements. For

inflammatory response, a minimum of six fields of view were chosen per sectionalong the reaction zone between scaffold and host tissue. The reaction zone wasidentified as the newly synthesised collagen around the perimeter of the scaffoldwhich stained dark pink in histological sections. Stereology was performed along theskin side of the implant. Details of the stereological analysis are provided below andin Table 2.

2.6.4. Volume fraction of inflammatory cellsVolume fraction measurements were performed on sections examined at

a magnification of 400� using image analysis software (ImageJ). A grid containing234 squares was placed over each field of view. Minimum number of fields of viewwere determined in a pilot study using a method reported by Garcia et al. [24]. Thecumulative volume fraction of inflammatory cells was plotted against the number offields of view which were examined. When the variance of cumulative volumefraction diminished to within 5% of the final cumulative volume fraction, theminimum number of fields of view was determined. The diminished varianceconfirmed the number of tissue sections which must be examined for sectionstereological analysis.

The area of interest was measured in each field of view and a grid overlaid onscreen. The intersections between grid corners and inflammatory cells were coun-ted. The identification of inflammatory cells was based on their morphology. Thevolume fraction of inflammatory cells (VV) was based on the ratio of grid pointintersections (PP) to total grid points within the area of interest (PT).

2.6.5. Surface density of blood vesselsThe surface density of blood vessels was measured within the reaction zone.

A cycloid grid was placed over histology sections stained with anti vWF and inter-sections between cycloids and blood vessels were counted. The formula stated inTable 2 was used to calculate the surface density of blood vessels.

Table 2Histomorphometry parameters used in the evaluation of subcutaneous and intra-femoral bone explants.

Measuring parameter Tissue type Stain Method Formula

Volume fraction inflammatory cells (VV) Subcutaneous Haematoxylin and eosin Intersection of test grid with inflammatorycells in reaction zone (PP). PT: Total number ofgrid points within the area of interest.

VV ¼ PP/PT

Surface density of blood vessels Subcutaneous Von Willebrand factor Intersection of blood vessels with a cycloidgrid within the reaction zone (I). Cycloid arc height,11 mm; 10 arcs/line; 6 lines/grid.

SV ¼ 2 � (I/LT)

Volume fraction of bone Bone Methylene Blue andBasic Fuchsin

Colour segmentation of trabecular bone area (BN).AT: total area within the outer compact bone sheath.

BV ¼ BN/AT

Area of remaining scaffold Bone Methylene Blue andBasic Fuchsin

Colour segmentation of scaffold area. AS

VV¼ Volume fraction, PP¼ Grid point intersections, PT¼ Total number of grid points, BV¼ Bone volume fraction, BN¼ Trabecular bone area within outer compact bone sheath,AT ¼ Total area within outer compact bone sheath, AS ¼ Area of scaffold.

M. Keeney et al. / Biomaterials 31 (2010) 2893–2902 2895

2.6.6. RNA extractionRNA extraction was performed on subcutaneous implants to determine b-Gal

transfection. RNA extraction was performed on tissue sections after 4 weeks. One mLof TriReagent (Applera Ireland, Dublin, Ireland) was added to each construct andincubated for 5 min at room temperature. Scaffolds were mechanically disruptedusing a rotorstator homogeniser (Qiagen). The homogenate was then heated for15 min at 37 �C. Phase separation was performed by adding chloroform, and totalRNA was purified using an RNeasy kit (Qiagen), according to the supplier’s recom-mended procedure.

2.6.7. Reverse transcription polymerase chain reaction (RT-PCR)Total RNA quantity and purity were determined by UV spectrometry at 260 and

280 nm using a UV spectrometer (NanoDrop Technologies, NanoDrop� ND-1000Spectrophotometer, Wilmington, DE). Reverse transcription (RT) was performed bythe ImProm-II� reverse transcription system from 1 mg of RNA according to themanufacturer’s protocol. Gene transcription was examined by real time RT-PCR.Reactions were performed and monitored using a StepOnePlus� detection system(Applied Biosystems, Foster City, CA) using a real time gene expression mastermix(QuantiFast Sybr Green, Qiagen) and specific primer sequence for b-Gal, forwardprimer 50-AAAACAACTGCTGACGCC-30 , reverse primer 50-TCGCCATTTGACCACTACC-30 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward primer 50-ACTCCCACTCTTCCACCTTC-30 , reverse primer 50-TCTTGCTCAGTGTCCTTGC-30 . Genetranscription was inferred from calibration samples and normalised in relation totranscription of the housekeeping gene GAPDH rRNA. The 2�DDCt method was usedto calculate relative gene expression for the target gene.

Table 3Total number of implants placed, retrieved, and used for analysis.

GroupA

GroupB

GroupC

GroupD

GroupE

pVEGF165 pb-Gal

Subcutaneous Implants Placed 8 8 8 8 8

2.7. Evaluation of intra-femorally implanted scaffolds

2.7.1. Histological processingTwo explants from each group A–D were decalcified, dehydrated in a graded

series of ethanol baths and embedded in paraffin. The remaining intra-femoralexplants were embedded in methylmethacrylate (MMA) without decalcification.MMA embedded samples were stained with methylene blue and basic fuchsinbefore creating 10 mm sections using a diamond saw.

2.7.2. HistomorphometryThree sections per implant were used for all histomorphometrical measure-

ments. Absolute values were derived for bone formation and scaffold degradation byexamining the complete histological section. Details of the histomorphometricalanalysis are provided below and in Table 2.

2.7.3. Bone volume fractionBone volume measurements were performed on sections examined at

a magnification of 100� using image analysis software (QWin, Leica, Rijswijk,Netherlands). The trabecular bone area was measured on each section by coloursegmentation and expressed as a fraction of the total area within the outer compactbone sheath.

2.7.4. Area of remaining scaffoldArea of remaining scaffold was determined in each section by colour segmen-

tation and calculated as a percentage of the total area within the outer compact bonesheath.

Implants Retrieved 8 8 8 8 6StereologicalAnalysis

8 8 8 8 6

Real Time RT-PCR 8 8 8 8 6

Intra-Femoral Implants Placed 8 8 8 8 8Implants Retrieved 8 8 8 8 6HistomorphometryAnalysis

6 6 6 6 6

2.8. Statistical analysis

Minitab� (Minitab Inc., USA) software was used for statistical analysis. Theoutliers were previously calculated using Grubb’s test and eliminated from theresults. Normal distribution was determined using the Anderson–Darling test. One-way analysis of variance (ANOVA) with a Tukey’s post-hoc analysis was used todetermine statistical significance between groups. Data are presented as

mean � standard error mean, as an average of the mean data from each animal wasused. p values of <0.05 were considered statistically significant.

3. Results

3.1. General observations

Implantation of the subcutaneous and intra-femoral scaffoldswas an uneventful procedure, except for two animals that diedfrom a rapid decrease in body temperature due to insufficientisolation. Placement of the scaffolds did not result in any macro-scopic signs of inflammation or adverse tissue responses. The totalnumber of implants placed, retrieved, and used for analysis isdepicted in Table 3.

3.2. Subcutaneous implantation study

Collagen/calcium phosphate scaffolds were implanted subcu-taneously in mice. Scaffolds either contained naked plasmid DNA orplasmid DNA complexed with a degraded PAMAM dendrimer(dPAMAM). Both a marker (b-Gal) and therapeutic plasmid(VEGF165) were used for this study.

3.2.1. Reporter gene expressionReal time RT-PCR was used to determine the level of trans-

fection after 4 weeks in vivo. It was found that the scaffold con-taining naked plasmid DNA had a statistically higher mRNAexpression of b-Gal than scaffolds containing complexed plasmidDNA. The scaffold containing plasmid alone had a mean mRNAvalue 7.6-fold higher than that of the scaffold containing com-plexed plasmid DNA. No significant levels of b-Gal mRNA werefound in other groups. This result confirms that the collagen/calcium phosphate containing naked plasmid DNA was moreefficient at mediating transfection than that containing com-plexed plasmid DNA.

M. Keeney et al. / Biomaterials 31 (2010) 2893–29022896

3.2.2. Host tissue responseFig. 2 shows representative histological sections for each treat-

ment group. The reaction zone (R) was defined as the dark pinktissue surrounding the scaffold (S). Gross observation indicated anincreased size of the reaction zone and number of inflammatorycells surrounding scaffolds containing dPAMAM. A stereologicalanalysis was performed on the reaction area surrounding theimplant to quantify volume fraction of inflammatory cells and thepresence of blood vessels.

3.2.3. Inflammatory responseThe stereological results of the inflammatory response are

presented in Fig. 3. The addition of only naked plasmid DNA (eitherVEGF165 or b-Gal) to the scaffold did not induce an increase in thevolume fraction of inflammatory cells compared to the scaffold

Fig. 2. Representative histology sections from each treatment group of implants placed scaptured at a magnification of 400�. S indicates scaffold while R indicates reaction zone. (AScaffold þ pb-Gal þ dPAMAM and (E) Scaffold.

alone. In contrast, a substantial increase in the volume fraction ofinflammatory cells was observed for the groups containing dPA-MAM. However, this increase was statistically significant only forthe group containing pb-Gal þ dPAMAM, which showed a twofoldincrease in the volume fraction of inflammatory cells compared tothe control scaffold (Fig. 3).

3.2.4. AngiogenesisThe surface area density of blood vessels measures the total

area occupied by blood vessels within the reaction zone.(Fig. 4) After 4 weeks of implantation there was no statisticaldifference between any groups. Fig. 4 (inset) shows a repre-sentative immunohistochemical image of blood vessel staining.The majority of blood vessels resided outside the reactionzone.

ubcutaneously for 4 weeks. Sections were stained with Haematoxylin and Eosin and) Scaffold þ pVEGF165, (B) Scaffold þ pVEGF165 þ dPAMAM, (C) Scaffold þ pb-Gal, (D)

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Fig. 3. Volume fraction of inflammatory cells within the reaction zone of subcutaneously placed implants after 4 weeks. A statistical difference was observed between groupslabelled A0 and A (p < 0.005).

M. Keeney et al. / Biomaterials 31 (2010) 2893–2902 2897

3.3. Intra-femoral implantation study

To evaluate the effect of pVEGF165 delivery from the collagen/calcium phosphate scaffold on bone formation, implants wereplaced intra-femorally in a 1 mm diameter defect in mouse femurs.(Fig. 5) For these implants, analysis consisted of bone response andimplant degradation.

3.3.1. Bone formationRepresentative histological images for each group are shown in

Fig. 6. Gross observation showed a clearly distinguishable scaffold(or its remains) in the femoral cavity surrounded by bonetrabeculae.

Bone volume was measured as the volume of bone within thearea surrounded by the compact bone sheath, i.e. only the trabec-ular bone volume, into which the scaffold was inserted.

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Fig. 4. Surface density of blood vessels within the reaction zone. Inset, immunohistochemisbeen counterstained with Haematoxylin and captured at a magnification of 400�. S indicavessels.

Histomorphometry analysis showed that the group containingscaffold þ pVEGF165 had a statistically higher bone volume relativeto scaffold alone. (Fig. 7) Bone occupied w24% of the area of interestin the scaffold þ pVEGF165 group in comparison to w10% incontrols. Additionally, the scaffold þ pVEGF165 group showeda statistically significant higher bone volume than scaffold þ pb-Gal þ dPAMAM.

3.3.2. Scaffold degradationScaffold þ pVEGF165 demonstrated the highest absolute values

of scaffold degradation compared with all other treatment groups,although not statistically significant. (Fig. 8) Remaining scaffoldarea within the area surrounded by the compact bone sheath after4 weeks was approximately 34%. Scaffold þ pb-Gal underwent thesecond highest degradation with 53% scaffold remaining and,interestingly, also resulted in the second largest bone volume.

25µm

S

SkinFascia

R

affold +GF165 +AMAM

Scaffold +pβ-Gal

Scaffold +pβ-Gal +dPAMAM

try image of subcutaneous tissue stained with anti-von Willebrand factor. Section hastes scaffold, R indicates reaction zone while the arrows indicate the presence of blood

Fig. 5. Defect created in intra-femoral model. (A) Photograph of defect created through the femoral condyle. (B) Micro-CT image of defect on day of implantation. A 1.0 mmdiameter defect can be seen protruding through the femoral condyle.

M. Keeney et al. / Biomaterials 31 (2010) 2893–29022898

4. Discussion

The present study was aimed at evaluating the potential ofa collagen/calcium phosphate scaffold as a delivery system fornaked plasmid DNA. It was hypothesised that transfection wouldoccur in vivo due to the presence of calcium phosphate within thescaffold. Using subcutaneous implantation in mice of scaffoldscontaining either dendrimer-complexed plasmid DNA or nakedplasmid DNA, it was demonstrated by real time RT-PCR thattransfection occurs, and that the level of transfection using nakedplasmid DNA succeeded that of dendrimer/plasmid DNAcomplexes. Furthermore, delivery of naked plasmid DNA inducedno increased inflammatory response, whereas the delivery ofdendrimer/plasmid DNA complexes was associated with a 2-foldincrease in inflammatory cells compared to empty control scaf-folds. Using an intra-femoral implantation model in mice, theeffects of collagen/calcium phosphate scaffold mediated deliveryof naked pVEGF165 DNA on bone formation were assessed. It wasobserved that the delivery of naked pVEGF165 significantlyincreased bone formation compared to control scaffolds withoutplasmid DNA. Together, the results demonstrate that collagen/calcium phosphate scaffolds (i) are powerful tools for the deliveryof naked plasmid DNA, (ii) do not induce an inflammatoryresponses compared to transfection vector associated equivalents,and (iii) increase bone formation when associated with nakedplasmid VEGF165 DNA.

Roy et al. have directly compared the dendrimer dPAMAM againstcalcium phosphate nano-particles and co-precipitates in-vitro fortheir transfection efficacy, but their results show that the calciumphosphate nano-particles only achieve about 80% of the transfectionlevels obtained by dPAMAM [25]. In view of eventual clinical appli-cation of plasmid delivery, however, it is more important to evaluatein vivo studies on plasmid delivery. Several in vivo studies have beenperformed using PAMAM dendrimers [26–36] that indicatemoderate levels of transfection and therapeutic benefits. However,a large variation in these results exists, e.g. Rudolph et al. found PEI toout perform dPAMAM in terms of transfection efficiency in a mousemodel [31] while Turunen et al. found the opposite effect in a rabbitmodel [33]. Despite these variations, the overall consensus was thatPAMAM dendrimers are relatively efficient in vivo plasmid com-plexing agents. In the current study, dPAMAM was also found totransfect cells in vivo. However, the level of transfection was

significantly lower (7.6-fold) than that of naked plasmid DNA teth-ered to the collagen/calcium phosphate scaffold.

The precise reasons for the difference in transfection efficacyremain unclear. However, the delivery of plasmid DNA with orwithout dendrimer complexation is basically different. Withoutdendrimer complexation, calcium phosphate within the scaffold islikely to complex with the plasmid DNA, after which plasmiddelivery follows degradation of the collagen/calcium phosphatescaffold (the binding of calcium phosphate with plasmid DNA isshown in supplementary data). On the other hand, dPAMAM/plasmid DNA complexes are observed to elute from a collagen/calcium phosphate scaffold in an almost linear fashion over a 14 dayperiod in-vitro (see supplementary data) and likely elute overa shorter time period in vivo. Consequently, the relatively slowdegradation of the collagen/calcium phosphate scaffold will resultin slow delivery of calcium phosphate complexed plasmid DNA,whereas dendrimer/plasmid DNA complexes are relatively fastdelivered from the scaffold. This difference in release from thescaffold hence is a likely cause for the observed differences intransfection levels. Moreover, the fact that transfection can still bedetected at the 4 week period indicates that the naked plasmidDNA has not been degraded in vivo, probably due to the protectiverole of the collagen/calcium phosphate scaffold. To the authors’knowledge, a collagen/calcium phosphate scaffold has never beenused for non-viral gene delivery. Endo et al. have used a collagenscaffold to deliver plasmid DNA that was complexed with calciumphosphate to treat rat bone defects. In their study, a 5 mm tibiabone defect was bridged after 4 weeks with the delivery of 12 mgpBMP-2 complexed with calcium phosphate, indicating successfultransfection and a positive therapeutic result. It is speculated thata similar event has occurred in this study leading to increasedtransfection levels.

An increased inflammatory response was observed within thereaction zone of scaffolds containing dPAMAM. However, thevolume fraction of inflammatory cells only increased marginallyfrom a basal level of 3.1%–4.4% for pVEGF165þ dPAMAM and to 5.7%for pb-Gal þ dPAMAM.

There was no statistical difference observed in surface density ofblood vessels at the four week time point. An increased number ofblood vessels were expected in the groups containing pVEGF165,and more specifically in the group containing naked plasmid DNAin light of transfection results. There reason for no observed

Fig. 6. Representative histology sections from each treatment group. Sections were stained with methylene blue and basic fuchsin and captured at a magnification of 100�. Sindicates scaffold and arrows indicate bone forming around the perimeter of scaffolds. (A) Scaffold þ pVEGF165, (B) Scaffold þ pVEGF165 þ dPAMAM, (C) Scaffold þ pb-Gal, (D)Scaffold þ pb-Gal þ dPAMAM and (E) Scaffold.

M. Keeney et al. / Biomaterials 31 (2010) 2893–2902 2899

increase in blood vessels may be due to time point at which analysiswas performed. The therapeutic effect of pVEGF165 may haveresolved at this time point and tissue remodelling occurred. Normalwould healing involves the initial infiltration and sprouting of newblood vessels with a subsequent decrease as vessels mature. Breenet al. have used a rabbit ear ulcer model for the delivery of nitricoxide synthase via an adenoviral vector in a fibrin based scaffoldand demonstrated this phenomenon [37]. The surface density ofblood vessels was observed to increase at seven days postimplantation, however 14 days after implantation the surfacedensity decreased as remodelling occurred. A similar phenomenonis likely to have occurred in the current study. A second reason forno observed increase in blood vessels may be due to the immu-nohistochemical antibodies used. Rabbit produced antibodies areknown to be un-specific when used in mice and may haveproduced an under-estimation of blood vessel density.

The results of the intra-femoral study however clearly show thepotential of VEGF165 gene delivery in bone regeneration strategies,as mice receiving collagen/calcium phosphate scaffolds þ pVEGF165

demonstrated increased bone formation relative to control scaf-folds without plasmid DNA. Additionally, these scaffolds showedthe most rapid degradation. Geiger et al. also observed rapiddegradation when pVEGF165 was delivered via a calcium carbonatescaffold to bone defects in New Zealand white rabbits [18]. Ina separate study by the same research group, pVEGF165 was deliv-ered via collagen type I scaffolds to treat bone defects in NewZealand white rabbits. Bridging of the defect gap only occurred inanimals receiving pVEGF165 [17]. In view of the stimulatory effect ofVEGF on bone formation, Gerber at al. studied the inactivation ofVEGF and its effect on endochondral ossification in 24 day old mice[38]. Inactivation of the gene resulted in the suppression of bloodvessel formation in the growth plate, bone formation was

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Fig. 7. Volume fraction of bone within the compact bone outer sheath after 4 weeks implantation in an intra-femoral model. A statistical difference was observed between groupslabelled A0 and A (p < 0.005).

M. Keeney et al. / Biomaterials 31 (2010) 2893–29022900

disrupted, and the hypertrophic cartilage region began to expand.Cessation of the anti-VEGF treatment resulted in the invasion ofblood vessels and the subsequent regeneration of bone viaresorption of the excess hypertrophic cartilage. Consequently, theyconcluded that VEGF is an essential component in bone formationvia the remodelling of cartilage. Its critical role in this process is thereason why extensive research has been performed on this growthfactor for bone tissue engineering.

In the current study, collagen type I and calcium phosphate havebeen combined to create a scaffold which closely mimics boneextracellular matrix. Calcium phosphate is an osteoconductivematerial that encourages osteoblast differentiation [39] and whencombined with collagen, enhances matrix production [40]. Geigeret al. have shown that both collagen and calcium phosphate arecapable of delivering plasmid DNA in vivo and the current researchstudy proves that the presence of a transfection agent is notnecessary to increase transfection and that decreased inflammationcan be obtained via the delivery of naked plasmid DNA. The aim of

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Fig. 8. Percent scaffold remaining after 4 week

the intra-femoral model was not to repair a bone defect (accord-ingly, the defect was not of critical size) but rather to evaluate theeffect of pVEGF delivery on bone formation. Bone was observedto form around the perimeter of the scaffold in this model, andto a larger extent in the treatment group containing scaffoldþ pVEGF165. As increased bone formation has been demonstratedin this model, it is recommended to use a critical size defect toexamine the regenerative capabilities of this non-viral system forpVEGF165.

As with in vivo experimental work, also the animal model usedin the present studies is associated with limitations. Due to animalexperimental work with plasmid DNA-enriched scaffolds, onlysmall rodent models were feasible within the laboratory animalhousing facility. Consequently, bone defect dimensions werelimited, resulting in the difference in scaffold dimensions andproperties for subcutaneous versus intra-femoral implantation: thesubcutaneously placed scaffolds were larger, loaded with higherabsolute amounts of plasmid DNA, and contained pore channels.

caffold +EGF165 +PAMAM

Scaffold +pβ-Gal

Scaffold +pβ-Gal +dPAMAM

s implantation in an intra-femoral model.

M. Keeney et al. / Biomaterials 31 (2010) 2893–2902 2901

These differences mean that delivery of plasmid DNA from thescaffolds at both implantation sites is incomparable, and hence theeffects this delivery would have on the biological surrounding.Furthermore, the intra-femoral implantation in mice only providesinformation on the stimulation of bone formation, not on theclinical potential of plasmid DNA-enriched scaffolds for the healingof critical sized defects. In view of this, it needs to be emphasizedthat the present study was aimed at only (i) evaluating the capacityof collagen/calcium phosphate scaffolds for plasmid DNA deliveryand (ii) determining the effect of pVEGF delivery on bone forma-tion. Additional experimental work is necessary to determine theefficacy of collagen/calcium phosphate scaffolds enriched withpVEGF for bone regeneration using critical sized bone defects.

5. Conclusion

The delivery of naked plasmid DNA encoding b-Gal viaa collagen/calcium phosphate scaffold resulted in over 7-foldhigher transfection efficiency than those obtained usingdendrimer-complexed DNA. Furthermore, the delivery of den-drimer-complexed plasmid DNA resulted in elevated levels ofinflammation in the subcutaneous model, whereas the delivery ofnaked plasmid DNA did not. The delivery of a naked therapeuticplasmid encoding VEGF165 from the collagen/calcium phosphatescaffold in a bone defect resulted in increased bone formation. Thepresent study demonstrates that a collagen/calcium phosphatescaffold can deliver naked plasmid DNA without the use of a genevector and achieve elevated levels of transfection without inflam-matory responses upon subcutaneous implantation, whereas theuse of this delivery system for pVEGF165 DNA results in increasedbone formation upon implantation at a bony site.

Acknowledgements

The authors would like to extend their thanks to Dr. Aoife Duffy(Regenerative Medicine Institute, Galway, Ireland) for her help withimmunohistochemical staining and Ms. Natasja van Dijk (Dept.Periodontology & Biomaterials, Radboud University NijmegenMedical Centre, the Netherlands) for her help with histologicalprocedures. The authors would also like to acknowledge the IrishCouncil for Science, Engineering and Technology, funded by theNational Development Plan, and the European Molecular BiologyOrganisation for providing research funding.

Appendix

Figures with essential colour discrimination. Most of the figuresin this article are difficult to interpret in black and white. The fullcolour images can be found in the online version, at doi:10.1016/j.biomaterials.2009.12.041.

Appendix. Supplementary data

Supplementary data associated with this article can be found inthe online version, at doi:10.1016/j.biomaterials.2009.12.041.

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