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Biomaterials 33 (2012) 7422e7434

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Biomaterials

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

Immune responses during healing of massive segmental femoral bone defectsmediated by hybrid baculovirus-engineered ASCs

Chin-Yu Lin a,1, Kun-Ju Lin b,c,1, Kuei-Chang Li a, Li-Yu Sung a, Swei Hsueh d, Chia-Hsin Lu a, Guan-Yu Chen a,Chiu-Ling Chen a, Shiu-Feng Huang e, Tzu-Chen Yen b,f, Yu-Han Chang f,g,**, Yu-Chen Hu a,h,*

aDepartment of Chemical Engineering, National Tsing Hua University, Hsinchu 300, TaiwanbDepartment of Nuclear Medicine and Molecular Imaging Center, Chang Gung Memorial Hospital, Taoyuan 333, TaiwancDepartment of Medical Imaging and Radiological Sciences, Chang Gung University, Taoyuan 333, TaiwandDepartment of Anatomic Pathology, Chang Gung Memorial Hospital, Taoyuan 333, Taiwane Institute of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli 350, TaiwanfCollege of Medicine, Chang Gung University, Taoyuan 333, TaiwangDepartment of Orthopaedic, Chang Gung Memorial Hospital, Taoyuan 333, Taiwanh Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan

a r t i c l e i n f o

Article history:Received 9 June 2012Accepted 27 June 2012Available online 13 July 2012

Keywords:BaculovirusAdipose-derived stem cellsInflammationImmune responseSegmental bone defectTissue engineering

* Corresponding author. Department of ChemicalHua University, Institute of Biomedical EngineerTel.: þ886 3 5718245.** Corresponding author. College of Medicine, Chan333, Taiwan. Tel.: þ886 3 328 1200x2420.

E-mail addresses: hardbone2002@yahoo.commx.nthu.edu.tw, yuchen@che.nthu.edu (Y.-C. Hu).

1 These two authors contribute equally to this wor

0142-9612/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.biomaterials.2012.06.083

a b s t r a c t

Baculovirus holds promise for genetic modification of adipose-derived stem cells (ASCs) and boneengineering. To explore the immune responses during bone healing and the cell fate, ASCs were mock-transduced (Mock group), transduced with the baculovirus transiently expressing growth factorspromoting osteogenesis (BMP2) or angiogenesis (VEGF) (S group), or transduced with hybrid baculovi-ruses persistently expressing BMP2/VEGF (L group). After allotransplantation into massive femoraldefects in rabbits, these 3 groups triggered similar degrees of transient inflammatory response (e.g.neutrophil proliferation and immune cell infiltration into the graft site), revealing that baculovirus andtransgene products did not exacerbate the inflammation. The cells in all 3 groups underwent apoptosisinitially, persisted for at least 4 weeks and were eradicated thereafter. The L group prolonged the in vivoBMP2/VEGF expression (up to 4 weeks), extended the antibody responses, and slightly enhanced the cell-mediated cytotoxicity. Nonetheless, the L group led to remarkably better bone healing and remodelingthan the Mock and S groups. These data confirmed that the ASCs engineered with the hybrid BVimparted prolonged expression of BMP2/VEGF which, although stimulated low levels of humoral andcell-mediated immune responses, essentially augmented the healing of massive segmental bone defects.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Massive segmental bony defects following trauma or tumorresection often end up as non-union were they not treated prop-erly. Currently autografting is the gold standard for clinical repair oflarge defects, but it is restricted by donor shortage and morbidity[1] and unsatisfactory healing occurs in up to 30% of cases [2]. Dueto the clinical demand, the marriage of gene therapy and stem cell

Engineering, National Tsinging, Hsinchu 300, Taiwan.

g Gung University, Taoyuan

(Y.-H. Chang), ychu@

k.

All rights reserved.

therapy has captured interests for the treatment of bone defects atvarious sites including femora [3,4], tibia [5], radii [6] and calvaria[7,8]. Among the cell sources, bone marrow-derived mesenchymalstem cells (BMSCs) and adipose-derived stem cells (ASCs) areparticularly promising thanks to their immunoprivilege propertiesand ability to differentiate into osteoblasts [9].

The gene delivery vectors often used in bone engineeringinclude lentivirus [7,10], adenovirus [11,12], adeno-associated virus[3,5] and plasmid [6,13,14]. Each of these vectors, however,possesses its respective drawbacks. For instance, plasmid trans-fection of stem cells suffers from low delivery efficiency whilelentivirus may result in insertional gene inactivation and evenmurine leukemia [15]. In contrast to these vectors, baculovirus (BV)is an insect virus in nature but can efficiently transduce variousmammalian cells without appreciable signs of cytotoxicity and viralreplication [16,17], thereby prompting researchers to exploit BV asa gene transfer vector for diverse applications ranging from vaccine

C.-Y. Lin et al. / Biomaterials 33 (2012) 7422e7434 7423

delivery, protein production to cancer therapy [18e25]. Moreover,BV transduces BMSCs and ASCs at efficiencies exceeding 95%[26e28]. Therefore, we have constructed 2 recombinant BV vectors,Bac-CB and Bac-CV, to transiently express the potent osteogenicfactor BMP2 (bone morphogenetic protein 2) and angiogenesisstimulator VEGF (vascular endothelial growth factor), respectively[27]. Transplantation of allogeneic Bac-CB-transduced and Bac-CV-transduced BMSCs into critical-size (10 mm) femoral defects inNew Zealand White (NZW) rabbits not only promoted the osteo-genesis and angiogenesis, but also augmented the segmental bonehealing [27].

In comparisonwith BMSCs, ASCs are easy to isolate in abundancethrough liposuction, but ASCs are inferior in terms of osteogenesispotential [29]. As such, we surmised that sustained expression offactors promoting osteogenesis (BMP2) and angiogenesis (VEGF) isnecessary should ASCs be used as the cell source. Therefore, weemployed a hybrid BV system that constitutes two BV vectors [30]:one expressing the FLP recombinase (BacFLP) while the otherharboring the human BMP2 (Bac-FCBW) or VEGF (Bac-FCVW)expression cassette flanked by the flippase recognition target (Frt)sequences. Co-transduction of ASCs with BacFLP/Bac-FCBW orBacFLP/Bac-FCVW resulted in gene cassette excision off the BVgenome, re-circularization of the gene cassette and formation ofepisomal DNA circle at efficiencies approachingz45e50%, therebyenabling persistent transgene expression for >28 days in vitro [28].Allotransplantation of ASCs transduced with the hybrid BVexpressingBMP2 (BacFLP/Bac-FCBW)andVEGF (BacFLP/Bac-FCVW)into the critical-size femoral segmental defects in NZW rabbitsaccelerated thehealing, improved thebonequalityandangiogenesis[28]. The hybrid vector was also exploited to engineer BMSCs andameliorate thehealingof critical-size calvarial defects in rabbits [31].Furthermore, the hybrid BV vector transduction of BMSCs neitherinfluenced the differentiation potential nor altered the karyotype[32]. These data thus implicated the potential of the hybrid BVvectors for stem cell engineering and bone regeneration [17].

Despite the success, a number of questions remained unan-swered. In particular, whether the hybrid BV system indeed pro-longed the growth factor expression in vivo and whether theexpressed human factors triggered anti-transgene antibodies wereunknown. It also remained to be examined whether trans-plantation of ASCs transduced with the hybrid BV vector provokedexcessive immune responses against the engineered cells. Thus,this study primarily aimed to explore whether the genetic modi-fication and transgene expression induced immune responses andhow the immune responses correlatedwith the fate of transplantedcells and bone healing.

2. Materials and methods

2.1. Cell culture and recombinant BV

All animal experiments were approved by the National Tsing Hua UniversityInstitutional Animal Care and Use Committee. Male rabbit ASCs were harvestedsubcutaneously from the inguinal fat pads surrounding epididymis of NZW rabbitsas detailed in Supplementarymethods. Primary human foreskin fibroblast (HFF) waskindly provided by Dr. Mei-Chi Wang (Industrial Technology Research Institute,Hsinchu, Taiwan). ASCs and HFF were cultured in DMEM (Invitrogen) containing 10%fetal bovine serum (FBS, Hyclone) and 1% penicillin/streptomycin. Cells of passage 3through 5 were used for experiments.

All BV vectors were constructed previously [27,28,33] and were titered by end-point dilution method [34]. Bac-CB and Bac-CV were constructed to transientlyexpress human BMP2 and VEGF under the control of CMV-IE promoter, respectively(Fig. S1). BacFLP expressed FLP [30] while hybrid vectors Bac-FCBW and Bac-FCVWaccommodated the Frt-flanking human bmp2 and vegf165 genes, respectively (Fig. S1).

2.2. Preparation of implants and surgical procedures

Transduction with Bac-CB or Bac-CV at multiplicity of infection (MOI) 100 wasperformed in T-75 flasks for 4 h using NaHCO3-free DMEM as the surrounding

solution [30]. The ASCs transduced with Bac-CB or Bac-CV were mixed at a numberratio of 4 to 1, loaded to concentric cylindrical poly (L-lactide-co-glycolide) (PLGA)scaffolds (z7 mm in outer diameter, z2 mm in inner diameter, z5 mm in height;total cell number¼ 1.5�106) and designated as S group. In parallel, ASCs cultured inT-75 flasks were transduced with BacFLP (MOI 100) first for 4 h and then transducedwith Bac-FCBWor Bac-FCVW (MOI 100) for another 4 h [28]. Following transduction,the cells were cultured in complete medium containing 3 mM sodium butyrate(Sigma). After 12 h incubation, the medium was replaced by normal completemedium (without osteogenic supplements and butyrate) and the ASCs were tryp-sinized at 1 day post-transduction. The Bac-FCBW- and Bac-FCVW-transduced cellswere mixed at a ratio of 4 to 1, loaded to PLGA scaffolds and designated as L group.Likewise, mock-transduced ASCs were loaded to PLGA scaffolds and designated asMock group. Moreover, mock-transduced HFF were loaded to the PLGA scaffolds anddesignated as HFF group.

One day after the cell loading, 10 mm section at the mid-shaft of the left femurof 5-month-old female NZW rabbit was osteotomized and the defect was filledwith 2 implants (i.e. 3 � 106 cells/animal) as described in details in Supplementarymethods.

2.3. Blood count, ELISA and cell-mediated cytotoxicity

Before and after the surgery, blood samples were collected at different timepoints from the marginal ear vein of each rabbit under anesthesia. The bloodsamples were analyzed for the differential count of blood cells using blood counter(ADVIA 2120 Hematology System, Siemens). After centrifugation, the sera weremeasured for human BMP2/VEGF concentrations using respective ELISA kits(Quantikine� ELISA, R&D Systems).

The serawere also inactivated at 56 �C for 30min and 2-fold serially dilutedwithPBS (pH 7.4) for the measurement of anti-transgene antibody titers by directcompetitive ELISA. Briefly, eachwell in the ELISA plateswas coatedwith 100 ml (2 mg/ml in PBS) recombinant human BMP2 or human VEGF (PeproTech) for 12 h at roomtemperature and blockedwith 200 ml blocking buffer (0.05% Tween 20 and 1% BSA inPBS) for 2 h at 37 �C. After wash with PBST buffer (0.05% Tween 20 in PBS), theserially diluted serum samples were added to the wells (100 ml/well) and incubatedfor 1 h at 37 �C. After wash, the HRP-conjugated goat anti-rabbit IgG (1:200 dilution,Kirkegaard & Perry Laboratories) was added to the wells (100 ml/well) and incubatedfor 1 h at 37 �C. After the final wash, the color development was initiated by adding100 ml TMB solution (3, 30 , 5, 50-tetramethyl benzidine, Sigma) to each well, andterminated by adding 50 ml sulfuric acid (2 M). The absorbance was measured by anELISA reader (Molecular Devices) and the titers of anti-BMP2 and anti-VEGF anti-bodies were determined as the highest dilutions at which the absorbance was 0.2unit higher than that of non-operated rabbits.

To quantify the cell-mediated cytotoxicity, the spleens harvested from therecipient animals in each group were crushed through the iron mesh and pressedthrough a 70 mm strainer (BD Biosciences) to yield suspended cells. The cells wereincubated in ammonium chloride lysis (ACK) buffer for 5 min on ice to lyse red bloodcells and washed with RPMI medium (without FBS, Invitrogen) twice. In parallel,3 � 106 ASCs were treated (e.g. transduced with BacFLP/Bac-FCVW) in the samemanner as the ASCs transplanted into the recipient animals, and then lysed by freezeand thaw for 5 times. Approximately 1�108 splenocytes were resuspended in T-150flasks in 15 ml complete RPMI medium containing the crude ASCs lysates forstimulation. After 3 day stimulation, the splenocytes were centrifuged and co-cultured for 4 h with the same ASCs that were transplanted into the animals forcytotoxicity assay, using the CytoTox 96� Non-Radioactive Cytotoxicity Assay(Promega). Alternatively, after 5 day stimulation, the medium from the splenocyteculture stimulated with ASCs lysates was collected for ELISA analyses of interferon g(IFN-g) (Quantikine� ELISA, R&D Systems) using recombinant rabbit IFN-g (King-fisher Biotech) as standard.

2.4. Single-photon emission computed tomography/computed tomography (SPECT/CT)

Before imaging, we constructed a lentiviral vector (LV-TK) encoding herpessimplex virus-derived thymidine kinase (TK) as described in Supplementarymethods. Primary (P0) rabbit ASCs were transduced with LV-TK at MOI 25 in thepresence of 8 mg/ml polybrene, incubated for 24 h, and cultured in fresh DMEMcontaining 10% FBS and 25 mg/ml Zeocin� (InvivoGen) for 7 days to removeuntransduced ASCs. After subculture to passage 3, these ASCs were transduced,seeded and transplanted in the same manner as the ASCs in the L group.

To track the fate of the transplanted ASCs, recipient animals were injectedintravenously with 5 mCi of radioiodine-labeled 5-tributylstannyl-1-(2-deoxy-2-fluoro-b-D-arabinofuranosyl) uracil (123I-FTAU, Amersham Biosciences) via themarginal ear vein. The rabbits were anesthetized 24 h later with 3% isoflurane inprone position and the SPECT/CT study was performed over the pelvic and femurregions with a hybrid system (Infinia� Hawkeye� 4, GE Healthcare) composed ofa dual-head gamma-camera with integrated low-dose, four 5-mm-slice-thicknessCT installed in the gantry. The CT had a fixed anode oil-cooled X-ray tube and wasoperated at 120 kV and up to 2.5 mA. The X-ray tube and detector array, located on

C.-Y. Lin et al. / Biomaterials 33 (2012) 7422e74347424

the slip ring gantry of the gamma-camera, rotated together in a fixed geometry at2.0 rpm for 90� L-mode scans.

Emission SPECT images were first acquired over 360� (180� per head) at the159 keV photopeak and �10% energy window to detect the 123I-FTAU signal. A128 � 128 matrix was used, with a 3� angular step, an acquisition time of 30 s perframe (30 min in total) and a zoom factor of 1.0. The body contouring system wasused to minimize the distance between the rabbit and the collimator. The CT scanswere acquired within 4.5 min after the SPECT study to provide anatomic maps forattenuation correction and image fusion. Multiple slices were obtained in the helicalmode; 4 slices were acquired simultaneously with a beam coverage of 2 cm in eachgantry rotation and reconstructed online to a 512 � 512 image matrix. Cross-sectional attenuation maps (128 � 128 image matrix) in which each pixel repre-sents the attenuation of the imaged tissue were generated in all cases. Attenuationcorrected SPECT images were reconstructed with the iterative method and fusedwith CT images using a PMOD software package (PMOD Technologies).

2.5. Histological and immunohistochemical staining (IHC)

Rabbits were sacrificed by overdose of 2% lidocaine HCl. Femora were harvested,formalin-fixed, cut into small pieces, decalcified in 0.5 M EDTA (pH 8.0) for 7e10days, paraffin-embedded and sectioned (5 mm thickness). Serial sections closest tothe defect center were collected, floated in a water bath at 40 �C, placed onorganosilane-coated silanized microscope slides and baked at 37 �C overnight. Thesections were de-paraffined in xylene and rehydrated in serial ethanol baths forhematoxylin and eosin (H&E) staining.

The sections were also de-paraffined, rehydrated, incubated with 0.025% trypsinfor 40 min at 37 �C for antigen retrieval and subjected to immunohistochemicalstaining (IHC) using mouse MAb specific for macrophage (1:25 dilution, AcrisAntibodies), for CD4þ T cells (1:50 dilution, Novus Biologicals) or for CD8þ T cells(1:30 dilution, Novus Biologicals) as primary antibodies. The secondary antibodywas goat anti-mouse Dylight 488 MAb (dilution 1:200, Jackson ImmunoResearchLaboratories). The sections were incubated with primary and secondary antibodiesfor 60 and 30 min, respectively, and blocking was performed for 1 h with PBScontaining 2.5% FBS and 10% normal goat serum. All incubations and washes wereperformed with PBS containing 2.5% FBS (Hyclone) plus 0.1% Triton X-100 (Sigma).The sections were observed under the confocal microscope.

The cell death was examined by TdT-mediated dUTP nick-end labeling (TUNEL)staining, using the DeadEnd� Fluorometric TUNEL System (Promega). To examinethe bone turnover, the sections were simultaneously stained for alkaline phosphatase

Fig. 1. Transgene expression and anti-transgene antibody titers in vivo. (A) ELISA analysis oand anti-VEGF antibody titers by direct competitive ELISA. The L (n ¼ 6), S (n ¼ 5) and Mocksize (10 mm) femoral segmental bone defects in NZW rabbits. Serum samples were collectedwith either Bac-CB or Bac-CV (1 � 109 pfu) twice to elicit the anti-BMP2/anti-VEGF antibod

(ALP) and tartrate-resistant acid phosphatase (TRAP) using the TRAP/ALP double stainkit (Cat. #MK300, Takara).

2.6. Fluorescence in situ hybridization (FISH)

For FISH that detected the SRY gene on the Y chromosome, a 200 bp SRY probewas designed as described [35] and then labeled by random priming with biotin-dUTP (Biotin-High Prime, Roche). Tissue sections were de-paraffined, dehydrated,dried, retrieved, digested with Digest All-III (Zymed Laboratories), re-fixed in 10%buffered formalin, dehydrated in an ethanol series and dried. Chromosomes weredenatured at 94 �C for 5min and hybridized with the labeled SRY DNA probe for 24 hat 37 �C in an automated hybridizer (HYBrite�, Abbott Molecular). Slides werewashed with 0.5 � SSC buffer at 72 �C for 5 min, blocked at room temperature for10 min (CAS-Block, Zymed Laboratories) and incubated with avidin-fluorescein(1:200 dilution, Roche) at room temperature for 1 h. Slides were mounted inmounting medium containing DAPI (Vectashield�, Vector Laboratories) and exam-ined by fluorescence microscopy.

2.7. Statistical analysis

All quantitative data were analyzed using student’s t-test and are expressed asmeans � standard deviations (SD). p < 0.05 was considered significant.

3. Results

3.1. In vivo growth factor expression and anti-transgene antibodytiters

For animal studies, 3 groups of constructs (Mock, S and L groups)consisting of PLGA scaffold and male ASCs were fabricated. ASCs inthe L group were transduced with BacFLP/Bac-FCBWor BacFLP/Bac-FCVW, mixed at a cell number ratio of 4:1 and seeded to the scaffoldfor persistent BMP2/VEGF expression. The ASCs in the S group weretransducedwith Bac-CB or Bac-CV, mixed and seeded to the scaffoldfor transient BMP2/VEGF expression. The Mock group encompassed

f human BMP2 and VEGF concentrations in the serum. (B) Measurement of anti-BMP2(n ¼ 4) groups were prepared as described in the text and transplanted into the critical-from the marginal ear vein. The positive control animals were injected intramuscularlyies and the sera were collected at week 8.

C.-Y. Lin et al. / Biomaterials 33 (2012) 7422e7434 7425

mock-transduced ASCs. All constructs were transplanted into thecritical-size femoral segmental bone defects in female NZW rabbits.

ELISA analysis of the sera depicted that the BMP2 concentration(Fig. 1A) was negligible (z0.2 ng/ml) in the non-operated (n ¼ 8)and theMock group (n¼ 4) animals but was elevated in the S group(n ¼ 5), which peaked at day 3 (z1.5 ng/ml) and became barelydetectable at 28 days post-transplantation (dpt). The L group(n ¼ 6) conferred significantly higher and sustained BMP2 levels,which reached the maximum (z5.7 ng/ml) at 7 dpt, remained>3.1 ng/ml for 28 days and descended to near baseline levels(z0.6 ng/ml) at 56 dpt. The VEGF levels (Fig. 1A) in the L groupculminated at 7 dpt (z69.9 pg/ml) and remained higher than thosein the S and Mock groups for 14 days before returning to back-ground levels at 28 dpt. These data attested that the hybrid BV (Lgroup), when compared with the conventional BV (S group),extended the in vivo BMP2/VEGF expression.

Since the growth factor genes were of human origin for futureclinical application, we assessed whether the expressed humanBMP2/VEGF elicited anti-transgene antibodies by ELISA. Fig. 1Bdelineates that the S group provoked low but detectable anti-BMP2 and anti-VEGF antibody titers (<24), which peaked at 8weeks post-transplantation (wpt) and declined to background

Fig. 2. Evaluation of systemic immune responses by blood cell counts. (A) Total white bloBasophils. The peripheral blood samples were collected from the marginal ear vein beforecounts of WBC. n > 11 for each group.

levels at 12 wpt. The anti-BMP2 and anti-VEGF antibody titers inthe L group were statistically similar to those in the S group atweeks 4 and 8, but remained higher than the background levelsat week 12. Nonetheless, the antibody titers were significantlylower than those of positive control animals which were intra-muscularly injected with either Bac-CB or Bac-CV to elicitantibodies.

3.2. Systemic responses elicited by the BV-engineered ASCs

To evaluate the systemic responses, the peripheral bloodsamples collected before (blank) and after transplantation wereanalyzed for the differential count (DC) of white blood cells (WBC).Fig. 2 depicts that the Mock, S and L groups resulted in statisticallysimilar (p > 0.05) responses in terms of total WBC (Fig. 2A),neutrophils (Fig. 2B), lymphocytes (Fig. 2C), monocytes (Fig. 2D),eosinophils (Fig. 2E) and basophils (Fig. 2F) from 1 to 84 dpt, sug-gesting that the ASCs transduced with either conventional orhybrid BV vectors did not trigger stronger systemic immuneresponses than the mock-transduced ASCs.

At 1 dpt, the total WBC counts (�103 cells/ml) of the Mock, S andL groups (z9.3 � 0.2) sharply increased when compared with the

od cells (WBC). (B) Neutrophils. (C) Lymphocytes. (D) Monocytes. (E) Eosinophils. (F)surgery (blank) and at different time points post-transplantation for the differential

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blank (z5.0 � 1.1). The WBC primarily contributing to this rise wasneutrophil, a first-line responder to injury and hallmark ofinflammation, whose counts rose sharply from 1.5 � 0.5 (blank) toz3.5� 0.7 (for the L group, Fig. 1B). The lymphocyte and monocytecounts also slightly rose, but the eosinophil and basophil countswere barely disturbed. After 7 dpt, the counts of all 5 WBC typesreturned to the levels prior to surgery. These data indicated that thesurgery provoked transient inflammation and temporary neutro-phil proliferation, but did not markedly trigger the proliferation ofother WBCs modulating innate/adaptive immunity (lymphocyteand monocyte) and inflammation/asthma/allergy (eosinophil andbasophil).

Fig. 3. Infiltration of inflammatory cells into the graft. The NZW rabbits were transplanted wconstructs consisting of xenogenic HFF and scaffolds (HFF group, n ¼ 3) as positive control. Adecalcified, paraffin-imbedded and sectioned at 5 mm thickness. The sections at the mid-shaArrows indicate the WBC while arrowheads indicate the osteoblasts.

3.3. Local immune responses elicited by the BV-engineered ASCs

The local immune responses were first monitored by H&Estaining of sections from the mid-shaft of femora. As positivecontrols, rabbits (n ¼ 3) were transplanted with constructs con-sisting of scaffolds and human foreskin fibroblast (HFF). Fig. 3illustrates severe and lasting inflammatory responses against thexenogenic HFF as evidenced by the pronounced WBC infiltrationinto the grafts from weeks 1e4. At 1 wpt, evident, but lessprominent, WBC infiltration was also observed in the Mock, S andL groups. The local inflammatory responses subsided in the Mock,S and L groups after 2 wpt as the number of WBC sharply

ith the Mock (n ¼ 2), S (n ¼ 2) or L (n ¼ 4) group constructs, or additionally with thet different time points post-transplantation, the femora were harvested, formalin-fixed,ft were de-paraffined in xylene and rehydrated in serial ethanol baths for H&E staining.

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decreased. Notably, at 4 wpt the Mock and S groups were primarilyfilled with fibrous tissues with little new bone formation. In starkcontrast, trabecular bone formation and osteoblasts lining thebony tissues appeared in the L group, thereby confirming thesuperior bone regeneration in the L group and echoing ourfindings that sustained BMP2/VEGF expression was essential toheal massive segmental bone defects (Fig. S2 and reference [28]).

The IHC staining (Fig. 4) illustrated conspicuous macrophageinfiltration into the grafts of the Mock, S and L groups at 1 wpt,and extents of infiltration were comparable among these 3groups and the HFF group. Such serious macrophage infiltration

Fig. 4. Infiltration of macrophage into the graft. The femoral sections of recipient rabbits hstaining specific for macrophage. n ¼ 2, 2, 4 and 3 for the Mock, S, L and HFF groups, resp

for the Mock, S and L groups might arise from the coagulationthat accompanied surgery, which recruited macrophages toeliminate the blood clots [36]. Nevertheless, the numbers ofmacrophages in the Mock, S and L groups precipitously decre-ased at 2 wpt.

Besides macrophages, transplanting xenogenic HFF cells evokedprominent influx of CD4þ (Fig. 5A) and CD8þ (Fig. 5B) T cells atweeks 2 and 4. In comparisonwith the HFF group, theMock, S and Lgroups recruited noticeable, but considerably fewer, CD4þ andCD8þ T cells at week 2 and virtually all CD4þ and CD8þ T cellsvanished at week 4. The degrees of immune cell infiltration

arvested at 1 and 2 wpt were de-paraffined and rehydrated for immunohistochemicalectively.

Fig. 5. Infiltration of T cells into the graft. (A) CD4þ T cells. (B) CD8þ T cells. The femoral sections of recipient rabbits harvested at 2 and 4 wpt were subjected to immunohisto-chemical staining specific for CD4þ or CD8þ T cells. n ¼ 2, 2, 4 and 3 for the Mock, S, L and HFF groups, respectively.

C.-Y. Lin et al. / Biomaterials 33 (2012) 7422e74347428

(Figs. 3e5) were similar among the Mock, S and L groups, sug-gesting that the inflammation arose from the surgery (trans-plantation of ASCs) and BV transduction did not exacerbate theinflammatory responses.

Fig. 6. Cell-mediated cytotoxicity and IFN-g expression. (A) Cytotoxicity. (B) IFN-g expressiASCs lysates of the corresponding group for stimulation. After 3 day stimulation, the splecytotoxicity assay. After 5 day stimulation, the medium was collected for ELISA analyses of

3.4. Elicitation of cell-mediated cytotoxicity

To examine whether the transplanted BV-engineered cells eli-cited cell-mediated cytotoxicity, the splenocytes were harvested at

on. The splenocytess harvested at 12 wpt from each group were co-cultured with thenocytes were subcultured and co-cultured with ASCs of the same group for 4 h forIFN-g. n ¼ 6, 11 and 11 for the Mock, S and L groups, respectively.

Fig. 7. Fate of transplanted cells as tracked by SPECT/CT. The ASCs stably expressing TK were transduced and transplanted in the same manner as the ASCs in the L group. Atdifferent weeks post-transplantation, animals (n ¼ 7) were injected with 123I-FTAU and the TK expression was imaged with SPECT/CT. For each time point, the SPECT images wereoverlaid with CT scans in 3 axes to provide image slices precisely pinpointing the cells in the graft site (as indicated by the arrows).

Fig. 8. Cell fate tracked by FISH. The femora were sectioned and subjected to FISH analysis which specifically detected the SRY gene on the Y chromosome of male donor cells. Tissuesections were hybridized with the biotin-labeled SRY DNA probe, incubated with avidin-fluorescein, counter stained with DAPI and examined by fluorescence microscopy. n ¼ 3 foreach group. Arrows indicate the transplanted cells with the SRY gene.

C.-Y. Lin et al. / Biomaterials 33 (2012) 7422e74347430

12 wpt from the recipient animals in each group and co-culturedwith the ASCs lysates of the corresponding group for stimulation.For instance, the splenocytes from the L group animals werestimulated with lysates of Bac-FCBW- or Bac-FCVW-transducedASCs. After 3 day stimulation, the splenocytes were co-culturedwith ASCs of the same group for 4 h for cell killing assay.

Fig. 6A delineates that the Bac-CB-transduced ASCs lysatesprovoked the S group splenocytes to kill z4.0 � 2.5% of the Bac-CB-transduced ASCs. This cytotoxicity was statistically indistin-guishable (p > 0.05) from that imparted by the Mock-transducedASCs, indicating that short-term BMP2 expression by the Bac-CB-engineered cells did not enhance the cell-mediated cytotoxicityover the Mock-transduced cells. The Bac-FCBW-transduced ASCslysates, conversely, stimulated the L group lymphocytes (n ¼ 11)to kill z7.0 � 3.4% of Bac-FCBW-transduced ASCs, which washigher (p < 0.05) than the cytotoxicity endowed by the mock-and Bac-CB-transduced ASCs and suggested that prolongedBMP2 expression slightly elevated the cell-mediated cytotoxicity.Conversely, the cytotoxicity elicited by the Bac-FCVW-transducedASCs was statistically similar (p > 0.05) to that activated by theMock- and Bac-CV-transduced ASCs. The negligible effect ofprolonged VEGF expression on the cell-mediated cytotoxicitywas probably due to the relatively low VEGF expression level(Fig. 1A).

The concurrent measurement of IFN-g in the medium (Fig. 6B)showed that the splenocytes stimulated with Bac-CB- or Bac-CV-transduced ASCs secreted statistically comparable (p > 0.05) IFN-g when compared with the splenocytes stimulated with mock-transduced ASCs, but the splenocytes activated by Bac-FCBW-transduced ASCs secreted more IFN-g (p < 0.05) than those stim-ulated with Mock- and Bac-CB-transduced ASCs.

3.5. Cell fate tracked by SPECT/CT imaging and FISH

To track the fate of transplanted cells during bone healing, ASCswere engineered to stably express thymidine kinase (TK), trans-duced, and transplanted in the same manner as the ASCs in the Lgroup. The animals transplanted with the ASCs expressing TK andBMP2/VEGF were designated as TKL group (n ¼ 7) and the TKexpression was monitored by SPECT/CT.

Without TK expression, there were no signals in the Mock groupanimals (without TK and BMP2/VEGF expression) throughout theexperiment (Fig. S3), proving the absence of false positive back-ground in the SPECT/CT images. In contrast, apparent signals in thefemoral defects were detected in the TKL group (expressing TK) at0, 2 and 4 wpt (Fig. 7), thus revealing the persistence of BV-transduced, TK-expressing cells in the first 4 weeks. However, thesignals in the TKL group animals extinguished at weeks 8 and 12.

C.-Y. Lin et al. / Biomaterials 33 (2012) 7422e7434 7431

To verify the finding, the femora were sectioned and subjectedto FISH analysis, which specifically detected the rabbit Y chromo-some and enabled us to discriminate the male donor cells from thefemale host cells. Fig. 8 demonstrates that the cells with Y chro-mosomes remained detectable in theMock, S and L groups at 4 wpt,but vanished at 12 wpt. Therefore, the SPECT/CT and FISH datacollectively confirmed that the transplanted ASCs, either mock-transduced or BV-transduced, persisted in the graft site for atleast 4 weeks, but were completely eradicated thereafter.

To examine how the transplanted cells were cleared, thesections were subjected to TUNEL assays for apoptosis assessment.Fig. 9 reveals abundant TUNEL-positive cells in all 3 groups at 1wpt,indicating that many cells were actively undergoing apoptosis inthe first week. After week 1, however, apoptotic cells were barelydetectable in all 3 groups.

3.6. Bone turnover in the femoral defects

Complete bone repair necessitates remodeling, which reshapesand reorganizes the repair tissue at the late stage of healing processand involves orchestrated activities of osteoblasts (for boneformation) and osteoclasts (for bone resorption). Whether therepair advanced to the remodeling stage was evaluated by histo-chemical staining for ALP (marker for osteoblasts) and TRAP(marker for osteoclasts). As shown in Fig. 10, the barely detectableTRAP (arrows) and ALP (arrowheads) signals in the Mock group atweeks 4 and 12 indicated very limited osteoblast and osteoclastactivities and suggested poor bone turnover without BMP2/VEGFstimulation.

In the S group, TRAP and ALP activities were prominent at week4 but became faint at week 12, revealing that short-term BMP2/

Fig. 9. Cell apoptosis as assessed by TUNEL assays. The femoral sections at 1, 2 and 4 wpt wecounterstain appeared red. n ¼ 3 for each group. (For interpretation of the references to co

VEGF expression initiated osteogenesis and bone turnover at week4, but was insufficient to sustain active bone remodeling at week12. In the L group, TRAP and ALP activities were enormous at week4 and remained pronounced at week 12, implicating the active andongoing bone resorption and osteogenesis. The data thusconfirmed that L group accelerated and prolonged bone regenera-tion, in which the bone remodeling commenced by week 4 andpersisted for at least 12 weeks.

4. Discussion

Bone graft healing is a sequential process involving inflamma-tion, revascularization, osteogenesis, remodeling and incorporationinto the host skeleton [37]. Although healing of bone defects bygenetically modified BMSCs or ASCs has beenwell documented, thehost immune responses against the genetically modified grafts andtransgene products remain elusive. Using a massive segmentalbone defect model treated with the hybrid BV-engineered ASCs, weconfirmed that the hybrid BV vector prolonged the expression ofhuman BMP2/VEGF both in vitro [28] and in vivo (Fig. 1A). Theprolonged expression also elicited more persistent anti-BMP2 andanti-VEGF antibody responses when compared with transientexpression (Fig. 1B), probably because the sustained human BMP2/VEGF expression provided antigens continuously to stimulate theantibody induction. Since pre-existing anti-BMP2/VEGF antibodiesmay potentiate immune responses against the graft, the prolongedanti-transgene antibody responses call for extra caution if trans-plantation of identical construct that expresses BMP2/VEGF isneeded in the future.

The cellular response of inflammation is manifested by themigration of granulocytes (e.g. neutrophil) into the graft site

re subjected to TUNEL assays. TUNEL-positive cells appeared green while DAPI nuclearlor in this figure legend, the reader is referred to the web version of this article.)

C.-Y. Lin et al. / Biomaterials 33 (2012) 7422e74347432

immediately after transplantation, followed by the influx of mono-cytes and lymphocytes [37]. The invaded neutrophils, macrophages,natural killer (NK) cells and CD8þ T lymphocytes provide an earlyresponse to injury in attempts to eliminate harmful stimuli [38].Here we observed that transplantation of mock-transduced ASCsinto the femoral defects triggered acute inflammation, as evidencedby the rapid neutrophil proliferation (Fig. 2B) and infiltration intothe graft (Fig. 3), modest macrophage and lymphocyte proliferation(Fig. 2CeD) and macrophage infiltration (Fig. 4). The inflammatoryresponses were pronounced in the first week but waned rapidlythereafter. Transplantation of mock-transduced ASCs also recruitedCD4þ andCD8þTcells into thegraft site,whichwereevident at 2wptbut virtually disappeared at 4 wpt (Fig. 5). These inflammatoryresponses were consistent with the inflammation occurring inallotransplantations [38,39] and in bone defects transplanted withBMSCs [40]. In general, inflammation contributes to tissue healingby facilitating the removal of necrotic tissues andby initiating repair.However, a recent study uncovered that transplantation of BMSCsintomiceprovokedTcell responses,which inhibitedbone repair andinduced BMSCs apoptosis via the secretion of IFN-g and TNF-a [41].Since surgical trauma results in secretion of IFN-g and TNF-a [42],one can envision that the transplantationprocess led to recruitmentof T cells, which impeded the bone repair via IFN-g/TNF-a andcontributed to the poor bone healing in the Mock group (Fig. S2).

Fig. 10. Confirmation of bone turnover by TRAP/ALP staining. The femoral sections at 4 andwere indicated by arrowheads and arrows, respectively. n ¼ 3 for each group.

Very importantly, the inflammatory responses were similar amongthe Mock, S and L groups (Figs. 2e4), indicating that the inflam-mation stemmed from the transplantation procedures, and BVtransduction of ASCs did not exacerbate the inflammatoryresponses.

T cell-mediated immune responses are the major cause of allo-transplantation failure [43]. The processing of donor cells’ alloge-neic MHC molecules by host antigen presenting cells (APCs) maylead to allorecognition by CD4þ T cells, while MHC class I-mediatedpresentation of transgene products activates the host CD8þ T cells[38]. Compared with the Mock group, the S group gave rise tosimilar degrees of CD4þ/CD8þ T cells infiltration (Fig. 5) and cell-mediated cytotoxicity (Fig. 6), suggesting that BV transductionand short-term BMP2/VEGF expression did not boost apparentcellular immune responses. Conversely, the L group provokedslightly higher cell-mediated cytotoxicity and IFN-g production(Fig. 6) upon stimulation with the same antigens (ASCs of the Lgroup). Such elevated immunogenicity was attributed to the pro-longed expression of BMP2/VEGF, which not only were continu-ously secreted out of cells to stimulate antibody production (Fig. 1),but also were processed intracellularly and recognized by the hostAPCs, resulting in T cell activation (Fig. 6).

Regardless of Mock, S or L groups, the transplanted cells per-sisted for at least 4 weeks and were eradicated from the graft site

12 wpt were subjected to simultaneous histochemical staining for ALP and TRAP, which

C.-Y. Lin et al. / Biomaterials 33 (2012) 7422e7434 7433

thereafter, as confirmed by SPECT/CT (Fig. 7) and FISH (Fig. 8). Suchcell clearance was consistent with the cease of growth factorexpression after 4 wpt (Fig. 1A) and agreed with the notion that inmany situations the transplanted stem cells repair tissues through“touch and go” effects without significant engraftment or differ-entiation [44]. That is, the transplanted donor cells facilitated therepair primarily by secreting growth factors (e.g. BMP2/VEGF) andwere eliminated. The cell eradication can be ascribed to severalreasons. First, immediately after the surgery, phagocytes (e.g.neutrophil and macrophage) invading into the graft could phago-cytose and eliminate a large number of transplanted cells [38].Second, NK cells in the innate immunity arm might directlyrecognize stressed cells and impart cell-mediated cytotoxicity,resulting in cell killing by apoptosis [39]. Third, the transplantationgave rise to infiltration of CD4þ and CD8þ T cells (Fig. 5), whichcould secrete IFN-g and TNF-a that synergistically lead to cellapoptosis [41,45]. The second and third hypotheses were supportedby the pronounced apoptosis at week 1 (Fig. 9).

In the clinical setting, the clearance of transplanted ASCs isdesired because the ASCs would lose the immunoprivileged prop-erties upon differentiation into the osteogenic lineage [46,47],which could render the grafted cells more promiscuous targets ofhost immune surveillance and cause rejection. Furthermore, theextended presence of grafted cells might lead to excessively longBMP2/VEGF expression, hence triggering more potent antibodyresponse and undesired side effects such as heterotopic boneformation or dysregulated angiogenesis.

Despite the initial inflammation, enhanced immunogenicity andclearance of transplanted cells, the L group gave rise to remarkablysuperior bone healing and angiogenesis when compared with theMock and S groups, as confirmed by radiography, biomechanicalanalysis [28], gross appearance, mCT and histology (Fig. S2). Inparticular, after the clearance of transplanted cells the L group graftcontinued to undergo active bone remodeling whereas the Mockand S groups did not (Fig. 10). Normally, bony repair results fromthe activities of undifferentiated mesenchymal cells migrating tothe injury site during inflammation, which proliferate andsynthesize new matrix and later differentiate into osteoblasts.Given the clearance of transplanted cells, the later stage of boneformation and remodeling was ascribed to the host mesenchymalcells migrating into the graft site.

5. Conclusions

This study sheds light on the immune responses directed againstthe transplanted BV-engineered ASCs. The transplantation proce-dures resulted in inflammationandcell death, thereby leading to cellclearance after 4 wpt. The ASCs conferring short-term BMP2/VEGFexpression (S group) triggeredhumoral immune responses and onlyconferred suboptimal bonehealing. In contrast, theASCs engineeredwith the hybrid BV vectors conferred prolonged BMP2/VEGFexpression which, although elicited low levels of humoral and cell-mediated immune responses, was essential to heal the massivebone defects at the femora of NZW rabbits.

Acknowledgments

Theauthors acknowledge thefinancial support fromtheNationalTsing Hua University (Toward World-Class University Project100N2050E1), CGMH-NTHU Joint Research Program (99N2419E1,100N7753E1, 101N2753E1, CMRPG3B0431, CMRPG390141, CMRPG300161, CMRPG300131, CMRPG380101) and CGMH IntramuralProject (CMRPG300161, CMRPG391721, CMRPG381001).

Appendix A. Supplementary material

Supplementary data related to this article can be found online athttp://dx.doi.org/10.1016/j.biomaterials.2012.06.083.

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