TISSUE ENGINEERING: Part A Volume 20, Numbers 13 and 14, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/ten.tea.2013.0604

In Vitro Osteogenic Induction of Bone Marrow Stromal Cells with Encapsulated Gene-Modified Bone Marrow Stromal Cells and In Vivo Implantation for Orbital Bone Repair Yuan Deng,* Huifang Zhou,* Chenxi Yan, Yefei Wang, Caiwen Xiao, Ping Gu, PhD, and Xianqun Fan, MD, PhD

Osteogenic induction with either growth factors or genetic modification has limitations due to the short half-life and cost of the former, or safety concerns regarding the latter. The objective of this study was to employ a microcapsulation technique to separate genetically modified and nonmodified bone marrow stromal cells (BMSCs) to establish a cost-effective and biosafe osteogenic induction methodology with functional evaluation in vitro and in vivo in a canine model. Autologous BMSCs were isolated and transduced with adenoviral vectors containing either BMP-2 or vascular endothelial growth factor (VEGF) or were dual transduced followed by encapsulation in alginate microcapsules using an electrostatic bead generator. After cocultured with encapsulated cells, normal autologous BMSCs were analyzed for osteogenic differentiation and seeded onto tricalcium phosphate (TCP) scaffolds for in vivo implantation to repair orbital wall bone defects (12 mm in diameter) in a canine model. In vitro assays showed that the expression of the transduced genes was significantly upregulated, with significantly more transduced proteins released from the transduced cells compared with control cells. Importantly, examination of the BMSCs induced by soluble factors released from the encapsulated cells revealed a significant upregulation of expression of osteogenic markers Runx2, BSP, OPN, and OCN in dual-transduction or induction groups. In addition, dual transduction and induction resulted in the highest increase of alkaline phosphatase activity and mineralization compared with other experimental groups. In vivo assays using CT, micro-CT, and histology further supported the qPCR and western blot findings. In conclusion, encapsulation of genetically modified BMSCs was able to release a sufficient amount of BMP-2 and VEGF, which effectively induced osteogenic differentiation of normal-cultured BMSCs and demonstrated bone repair of the orbital wall defect after implantation with b-TCP in vivo.

Introduction

O

rbital defects are a common injury caused by trauma, infection, tumor excision, and congenital malformation. The anatomical feature of the thin orbital wall surrounded by paranasal sinuses results in poor blood supply to this anatomical region. Due to its complex anatomy, there is difficulty in obtaining exposure for surgery and an increased tendency to bleed. Moreover, repair of orbital wall defects remains a great challenge in reconstructive surgery.1 To address the need for enhanced repair of skeletal tissue, tissue engineering has become an attractive approach, with growing attention focused on the combinational use of growth factors and scaffolding materials.2–4 For example, vascular endothelial growth factor (VEGF) and bone mor-

phogenetic proteins (BMPs), including BMP-2, BMP-4, and BMP-7, have been investigated for their ability to induce MSC osteogenesis and regenerative repair of bone defects in various animal models.5–8 The main challenges of growth factors used for clinical treatment are the high cost, short half-life, and other issues.9,10 Adenoviruses as a high-efficiency approach of gene delivery have been widely used in gene therapy.8,11,12 We have previously reported bone repair via adenovirusmediated gene transfer of BMP-2 and VEGF in autologous bone marrow stromal cells (BMSCs), which revealed significantly enhanced new bone formation by dual-gene delivery of BMSCs compared with cells with single-gene delivery, indicating a potential synergistic bone induction effect between BMP-2 and VEGF.13 Despite the successful

Department of Ophthalmology, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China. *These authors contributed equally to this work.

2019

2020

report of this genetic modification approach in bone regeneration, the biosafety and secondary immune response to the transfected cells remain an obstacle toward its clinical application.14,15 Encapsulating cells or tiny pieces of tissues in alginatebased microcapsules was originally described by Lim and Sun and is the most commonly applied procedure for controlled material release and immune isolation.16 Microencapsulation technology can separate the gene-transfected cells from other cells and can simultaneously enable the encapsulated cells to release the proteins of transfected genes in a safe manner.17–20 Thus, we encapsulated the gene-modified BMSCs as a source of BMP-2- and VEGFreleasing cells and cocultured these cells with normalcultured BMSCs for osteogenic induction. On the basis of the synergistic effects of BMP-2 and VEGF on bone formation and the immune-isolation effect of microencapsulation, the goal of the current study was to explore the potential use of encapsulation as a distributive source of recombinant factors for BMSC osteogenic induction and to analyze their effect on bone regeneration in vivo in a canine orbital wall bone repair model.

DENG ET AL. Alginate encapsulation

Three groups of gene-transduced cells, including Trans-B (Cap-B), Trans-V (Cap-V), and Trans-B + V (Cap-B + V), and nontransduced BMSCs (Cap-C) were used for encapsulation in microcapsules. As previously described,24 transduced BMSCs were harvested and carefully homogenized with 1.75% alginate solution (MW: 100,000–200,000 g/mol; viscosity = 250 cps; G Content: 65–70%) (Sigma). Subsequently, the alginate–cell suspension was fed into a syringe pump, and beads were produced using an electrostatic bead generator (University of Shanghai for Science and Technology). The diameter of the generated beads was controlled by the flow rate of the alginate solution, nozzle gauge, electrostatic potential, and distance between the gelling bath and needle tip.25 Following the coating by 0.05% (w/v) poly-L-lysine (MW: 30,000–70,000 g/mol; Sigma) as the outer alginate layer,26 the calcium was replaced by 55 mM sodium citrate in the microsphere, and the cores of the microspheres were liquefied. Uniform alginate–poly-L-lysine–alginate (APA) microcapsules (100–200 mm in diameter) were then obtained.27 After two rinses in a-MEM, the microcapsules were cultured in 10cm dishes. These dishes were gently shaken for 5 min every 8 h, and the culture medium was changed every 7 days.

Materials and Methods Experimental animals

A total of 18 adult beagle dogs aged 1 year were used in this study. All of the animals were purchased from Shanghai Agriculture College, and an institutional review committee of the Shanghai Jiao Tong University School of Medicine approved all of the animal protocols used in this study. Canine BMSC culture

Bone marrow was harvested from the ilium of beagle dogs as previously described. The bone marrow (1 mL) was mixed with 10 mL of a-MEM (Gibco) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin and was then placed in a 10-cm dish. All of the cell cultures were incubated in a humidified environment with 5% CO2 at 37C, subcultured to passage 3, and then used for gene transduction. Flow cytometry was used to characterize BMSCs with CD29, CD31, CD34, CD44, CD45, and CD90 staining (BD Biosciences).21 Gene transduction

Adenoviruses containing human BMP-2 cDNA (advBMP-2), human VEGF165 cDNA (adv-VEGF), and GFP cDNA (adv-GFP) were constructed as previously described and used for gene transduction.13,22,23 Briefly, BMSCs were divided into four groups: Group 1 was transduced with advBMP-2 (Trans-B), Group 2 was transduced with advVEGF165 (Trans-V), Group 3 was dually transduced with both adv-BMP-2 and adv-VEGF165 at a 1:1 ratio (TransB + V), and Group 4 was transduced with adv-GFP (TransG). The viruses were applied at a dose of 150 multiplicity of infection (MOI pfu/cell). To determine the transduction efficiency, flow cytometry was used to quantify the GFP expression rate of the transduced cells. The expression of the transduced genes was examined using real-time (RT)-PCR.

Permeability, cell metabolic activity, and ELISA analysis of the APA capsules

The permeability of the APA microcapsules was tested with 100 mL of 0.05% w/v fluorescein-isothiocyanate (FITC)–labeled IgG (KPL) in a six-well plate. After 1 h of incubation, the permeability of the FITC-IgG was examined using confocal laser scanning microscopy (Leica Microsystems).24,28 The dark spheres indicated that the APA membranes inhibited antibody penetration into the interior of the microcapsules. Postencapsulated BMSCs were collected in a 0.1-mL suspension in a 96-well plate with 2 · 103 cells per well (n = 4). As a control, the nonencapsulated BMSCs were seeded at 2 · 103 cells per well (n = 4) and 20 mL of MTS solution (AQueous One Solution Cell Proliferation Assay; Promega) was added into each well at 7, 14, and 21 days. The microcapsules were incubated for 3 h at 37C, and the absorbance was measured at 490 nm with a reference to 630 nm in a microplate reader (Bio-Tec).29 The supernatant culture of the microcapsules was collected every week and centrifuged for 5 min at 300 g to remove any remaining cells. A BMP-2 ELISA Kit (R&D) and VEGF ELISA Kit (Invitrogen) were used to determine the released protein levels,24 and the protein concentration in the supernatant was normalized to ng per 106 BMSCs (n = 4). Coculture of the encapsulated and nonencapsulated BMSCs

Nonencapsulated BMSCs were seeded onto a six-well plate at 3 · 105 cells per well, and encapsulated BMSCs were separately added to these wells at 3 · 105 cells per well with 2 mL of a-MEM for coculture. Nonencapsulated BMSCs were induced by the encapsulated BMSCs of the Cap-B + V (B + V-induce), Cap-B (B-induce), Cap-V (V-induce), and Cap-C (control) groups, and the floating encapsulated BMSCs were removed by pipetting in the coculture at 7, 14, and 21 days postculture.

MICROCAPSULES USED IN ORBITAL BONE REPAIR Quantitative real-time PCR of encapsulated BMSCs

Quantitative real-time polymerase chain reaction (Q-PCR) was used to detect the mRNA expression of osteogenic transcriptional factors. Briefly, after 7, 14, and 21 days of coculture, supernatant samples of the encapsulated BMSCs were collected and incubated with EDTA (50 mM) for 5 min to dissolve the APA membranes.29 Cells were then washed in phosphate-buffered saline (PBS) after centrifugation, and the total RNA was extracted from the BMSCs of the Cap-B + V, Cap-B, Cap-V, Cap-C, and noncapsulated control BMSC groups using the RNeasy Mini Kit (Qiagen). The procedures of reverse transcription were the same as those previously described.30 Gene expression was analyzed using q-PCR with the primers listed in Supplementary Table S1. Data were expressed as the fold change relative to the nonencapsulated control BMSCs after normalization to GAPDH. q-PCR and western blotting analysis of nonencapsulated BMSCs

For detecting the induced BMSCs in coculture, microcapsules were removed with the supernatant, and then total RNA was extracted from adherent cells (B+V-induce, Binduce, V-induce). The groups of Trans-B+V and noninduced BMSCs were set as control. Next, Q-PCR was used to analyze the gene expression of runt-related transcription factor 2 (Runx2), bone sialoprotein (BSP), osteopontin (OPN), and osteocalcin (OCN) to determine the effect of osteogenic induction derived from the microencapsulatedtransduced cells after 3 weeks of coculture. The primers and parameters used for the real-time PCR analysis are listed in Supplementary Table S1 (Supplementary Data are available online at www.liebertpub.com/tea), and the relative mRNA levels were normalized to the untreated BMSCs. For western blotting analyses, the cells were washed three times with ice-cold PBS, solubilized in protein extraction reagent as previously described.31 The protein concentration was determined using a BCA Kit (Pierce) according to the manufacturer’s protocol. For each sample, 30 mg of protein was loaded onto a 10% SDS/PAGE gel. The gel-separated proteins were then transferred to a PVDF membrane (Millipore) at 80 V for 90 min and incubated with rabbit anti-human Runx2, BSP, OPN, or OCN polyclonal antibody (Abcam) or mouse anti-human b-actin monoclonal antibody (Invitrogen) at 37C for 2 h, followed by incubation with horseradish peroxidaseconjugated secondary antibodies (Sigma). Protein expression was visualized using Odyssey V 3.0 image scanning (LICOR). Semiquantification of the protein concentrations was determined on the basis of three independently performed experiments. The densitometric intensities of the protein bands were quantified using the Bandscan 5.0 software, and the values were normalized against b-actin for each sample. Alkaline phosphatase activity and alizarin red S staining

Once cocultured with encapsulated BMSCs on day 14, the induced cells were fixed in 4% paraformaldehyde, and alkaline phosphatase (ALP) staining was performed according to the manufacturer’s instructions (Rainbow). On day 21, after fixation in 95% ethanol for 20 min, the cells were incubated with 40 mM alizarin red S (ARS) staining solution

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(Sigma) for 20 min at room temperature. A semiquantitative analysis of ALP activity and ARS staining was performed as previously described.32 Briefly, the total protein was determined using a BCA Protein Assay kit. ALP activity was detected at 405 nm using p-nitrophenyl phosphate (p-NPP; Sigma) as the substrate, and ARS staining was determined at 590 nm using cetylpyridinium chloride (Sigma) by a spectrophotometer (Thermo). Finally, the ALP and ARS levels were normalized to the total protein content. Animal experiments

The pie-shaped porous b-tricalcium phosphate (b-TCP) scaffolds (Shanghai Bio-lu Biomaterials Co., Ltd) (Supplementary Fig. S2C) were specifically prefabricated and were 12 mm in diameter, 3 mm in height, and 500 – 100 mm in pore size; the interconnected pore was 150 – 50 mm in the diameter; the pore interconnectivity is around 90%; and the porosity rate is about 75%. Next, 1 · 107 cocultured induced BMSCs were concentrated and evenly seeded onto b-TCP scaffolds using negative pressure suctioning. The cell-seeded scaffolds were incubated at 37C with 5% CO2 for 8 h to allow for cell attachment followed by the subsequent immersion of the scaffolds in a-MEM with a medium change every 2 days. The composites were subjected to scanning electron microscopic (SEM; Philips) examination after in vitro incubation for 3 days. To establish a canine model of an innerside orbital bone defect of both sides, a trephine that was 12 mm in diameter was used to produce a circular defect of the orbital wall and the periosteum was removed (Supplementary Fig. S2D). A total of 18 beagle dogs were divided into six groups (n = 3) with six sides for every group. Composites containing Trans-B + V, B + V-induce, B-induce, V-induce, and control BMSCs were separately implanted into the five groups, and the blank control group received no implant. CT examination

A 64-channel CT scanner (Philips) was applied at 4 and 16 weeks postoperation to scan the canine head in a coronal view with an interval thickness of 0.625 mm. Three-dimensional reconstruction with a thickness of 1.25 mm per layer was performed on the basis of the CT data to observe the bone regenerative effect at 4 and 16 weeks postoperatively. Micro-CT imaging

After animals were euthanatized under general anesthesia at 16 weeks postoperatively, all of the specimens were harvested using a saw, and the calcification levels of the specimens were analyzed using micro-CT (m80; Scanco Medical) examination with a spatial resolution of 50 · 50 mm2 and a slice thickness of 50 mm for the specimens. The percentages of new bone volume to tissue volume (BV/ TV) and bone mineral density (BMD) were also determined using the micro-CT image analysis software to calculate the BV/TV ratio and BMD of the harvested specimens. Histological analysis

The harvested specimens were dehydrated in ethanol and embedded in methylmetacrylate. The sagittal sections of the central segment were cut, hand-ground, and polished to a final thickness of *40 mm. Six sections per sample were

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subjected to Van Gieson staining to quantify the boneforming area versus nonbone-forming area. To determine the percentage of the formation of new bone of the total area, an image analysis system was used (Image-Pro Plus; Media Cybernetic). Statistical analysis

All of the experiments were performed in triplicate unless otherwise specified. The differences between the experimental and control groups were determined using a group ttest. In addition, differences in the encapsulated BMSCs obtained from different weeks were analyzed using one-way ANOVA, where p < 0.05 was considered statistically significant. The B + V-induce group compared with the Binduce group (#p < 0.05), the B-induce group compared with V-induce group (**p < 0.05), and the V-induce group compared with control group (*p < 0.05) were used throughout this study. Results Gene transduction and osteogenic differentiation

The isolated canine BMSCs showed high expression of the mesenchymal stem cells markers CD29, CD44, and CD90, whereas low expression of the hematopoietic lineage markers CD31, CD34, and CD45 was detected using flow cytometry (Supplementary Fig. S1). The percentage of GFP-positive cells was 90.00% – 3.41% as revealed by fluorescent microscopy and flow cytometry analyses (Fig. 1A, B) and represents the adenovirus-mediated gene transduction rate. RT-PCR analysis verified the high gene expression of BMP-2 and VEGF after transduction within 24 h (Fig. 1C, D). Formation and permeability of microcapsules

In this study, control parameters, including a cell density of 3 · 106 cells/mL, 10 mL/h flow rate, 30 gauge nozzle size, and 10 kV/cm electrostatic potential with 3 cm length from the needle tip, and 1.75% alginate solution generated uniform-size microbeads with a diameter of *120 mm (Fig. 2B,

FIG. 1. Characterization of gene-transduced bone marrow stromal cells (BMSCs). (A, B) The percentage of GFP-positive cells was 90.00% – 3.41% as revealed by fluorescent microscopy and flow cytometry analyses. (C, D) Real-time-PCR of BMP-2 and VEGF mRNA expression in transduced BMSCs. The scale bar represents 100 mm in A. Color images available online at www.liebertpub.com/tea

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C). The membrane of 99% microcapsules was intact and showed no FITC-IgG penetration into the inferior of the microcapsules (Fig. 2A). The cell viability of the encapsulated BMSCs (Cap-B + V, Cap-B, Cap-V, and Cap-C groups) showed profiles that were similar to that of nonencapsulated cells (control) according to optical density (OD) values obtained from an MTS test. Although there were no significant differences in the statistics among the five groups at 7 days ( p > 0.05), the OD values of the nonencapsulated BMSCs were higher compared with the encapsulated BMSCs at 14 and 21 days ( p < 0.05). There was no difference in cell viability among the four encapsulated BMSC groups ( p ‡ 0.05, Fig. 2D). Gene expression and protein secretion of encapsulated BMSCs

The adenoviral vector increases the mRNA expression levels of the transduced cells. The mRNA expression of BMP-2 in the Cap-B + V group increased 1000-fold compared with the control group within 2 weeks and then decreased to 300-fold more than the control group at 3 weeks ( p < 0.05). However, the BMP-2 mRNA levels at 3 weeks in the Cap-B + V group were similar to the Cap-B group, and the Cap-V group was similar to the control group ( p ‡ 0.05, Fig. 2E). Further, the mRNA levels of VEGF in the CapB + V group were 1000-fold higher compared with the control group. In addition, the VEGF expression levels in the Cap-B + V group were similar to the Cap-V group. No significant differences in the VEGF mRNA expression were found in the Cap-B and Cap-C groups ( p ‡ 0.05, Fig. 2F). Similar to the increase observed in mRNA expression, protein secretion was also increased. ELISA quantitative analyses of secreted proteins in the Cap-B + V group revealed a significant overexpression relative to the control group in the production of BMP-2 and VEGF ( p < 0.05). Secretions of BMP-2 and VEGF proteins in the Cap-B + V group were highly upregulated at day 7 and then significantly decreased ( p < 0.05) at days 14 and 21. The BMP-2 protein levels in the Cap-B group and VEGF protein levels in the Cap-V group were similar to those of the Cap-B + V

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FIG. 2. Characterization of microencapsulated gene-transduced BMSCs. (A) Confocal microscopy shows the intact membrane of APA microcapsules, which prevented the penetration of FITC-labeled antibody. (B, C) The microcapsule structures and encapsulated BMSCs observed using light microscopy. (D) MTS demonstrated optical density (OD) values of various groups of encapsulated BMSCs and nonencapsulated control BMSCs. (E, F) BMP-2 and VEGF mRNA expression levels in various groups of encapsulated and nonencapsulated cells (*p < 0.05, the Cap-B + V, Cap-B, or Cap-V group was compared with control group). (G, H) BMP-2 and VEGF proteins released from various groups of encapsulated and nonencapsulated cells. The scale bars represent 100 mm in A–C. Color images available online at www.liebertpub.com/tea group ( p ‡ 0.05). The BMP-2 protein level in the Cap-V group and the VEGF protein level in the Cap-B group were also similar to the Cap-C group ( p ‡ 0.05, Fig. 2G, H). Osteogenic differentiation of nonencapsulated BMSCs in coculture

Nonencapsulated cells were harvested at various time points and were subjected to qPCR analysis. These results showed a significant difference in the gene expression levels among the five groups for Runx2, BSP, OPN, and OCN ( p < 0.05, Fig. 3). For gene expression (Fig. 3A–D), the Trans-B + V and B + V-induce groups demonstrated the highest levels compared with the other groups ( p < 0.05), and both of them showed higher than B-induce group from day 14 ( p < 0.05); however, no significant differences between the groups were observed ( p > 0.05) at all the time points. In addition, four gene expression in the B-induce BMSCs was higher compared with V-induce group at days 7–21 ( p < 0.05); also, the V-induce BMSCs were higher than control BMSCs at days 14 and 21. The protein levels at various time points were determined using western blotting analyses. Runx2, BSP, OPN, and OCN protein levels exhibited an expression profile similar to that of their respective mRNAs (Fig. 3E). Quantitative analysis showed that the Trans-B + V and B + V-induce BMSCs were the most upregulated among the five groups within 3 weeks (p < 0.05). At day 14, the protein levels show a significant difference between B + V-induce and B-induce groups, the difference also show among B-induce, V-induce, and control groups. The induced BMSCs were also examined using ALP at day 14 and ARS at day 21. With no difference between

Trans-B + V and B + V-induce groups, ALP staining was enhanced at different degrees in the B + V-induce, B-induce, and V-induce groups. Further, ARS staining at day 21 revealed different degrees of increase in calcium deposition in the B + V-induce, B-induce, and V-induce groups (Fig. 4A). Semiquantitative analysis shows that the ALP activity in the V-induce and B + V-induce groups was lower and higher, respectively, than in the B-induce group on day 14 ( p < 0.05), but with no significant difference between TransB + V and B + V-induce groups ( p > 0.05). In addition, the semiquantitative analysis of ARS staining was in agreement with the results for the ALP activity (Fig. 4B). In vivo bone regeneration study

After the cells were cultured in vitro for 3 days, the cellseeded scaffolds were subjected to scanning electron microscopic (SEM) examination, and the results showed b-TCP scaffold alone (Supplementary Fig. S2A) and induced BMSCs seeded on scaffold (Supplementary Fig. S2B). Three-dimensional reconstructed CT images revealed that the implants filled in the orbital wall defects and maintained their original form with clear joint lines between the implant and host at 4 weeks postimplantation (Fig. 5A1–5). At 16 weeks, the orbital walls were well repaired using the TransB + V and B + V-induce implants because the implants healed well with the host bone and the joint line became less evident with good osteointegration (Fig. 5B1–2), indicating complete bone regeneration. In contrast, the size of the implants decreased with time in the control groups, which showed only partial synostosis between the host bone and implanted graft with an uneven surface of the implants (Fig. 5B5). In addition, the orbital walls were only partially

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FIG. 3. The expression of osteogenic markers in nonencapsulated BMSCs induced with cocultured gene-transduced encapsulated cells. (A–D) The osteogenic gene expression of various groups of cells for Runx2, BSP, OPN, and OCN with statistical analysis at days 0, 7, 14, and 21 (#p < 0.05, the B + V-induce group compared with the Binduce group; **p < 0.05, the B-induce group compared with V-induce group; *p < 0.05, the V-induce group was compared with control group). (E) The protein expression of osteogenic markers in various groups using western blotting. Color images available online at www.liebertpub .com/tea

FIG. 4. Analysis of alkaline phosphatase (ALP) and ARS staining. (A) ALP expression on day 14, and the results of ARS staining on day 21. (B) Semiquantitative analysis of ALP and ARS staining (#p < 0.05, the B + V-induce compared with the B-induce group; **p < 0.05, the B-induce compared with V-induce group; *p < 0.05, the Vinduce was compared with control group). Color images available online at www .liebertpub.com/tea

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FIG. 5. CT image analysis of the repaired orbital bone defects in the various groups. (A1-5) Black arrow shows that the implants filled in the defects at week 4. At week 16, (B1-2) the bony union in the Trans-B + V and B + Vinduce groups; (B3-4) the bony union, but not well formed in the B-induce and V-induce groups; and (B5) partial bony union in the control group. (C1-5) White arrows show coronal views of implants in various groups at week 16. Color images available online at www .liebertpub.com/tea regenerated in both the B-induce and V-induce groups (Fig. 5B3–4). When examined in the coronal view, full ossification and good remolding of regenerated bone were observed in the Trans-B + V and B + V-induce groups (Fig. 5C1–2). In B-induce group, good ossification but relatively more re-

sidual b-TCP materials were found compared with the B + V-induce group (Fig. 5C3). Poor ossification and relatively poor scaffold degradation were observed in the Vinduce group (Fig. 5C4). No obvious ossifications and poor b-TCP degradation were found in the control groups

FIG. 6. Micro-CT imaging analysis. (A) The top and bottom panels represent the images scanned from the face to the eye ball and the images from the face to the ethmoid sinus in various groups at week 16 postsurgery. (B) The percentage of new bone volume to tissue volume (BV/TV). (C) The bone mineral density (BMD). (#p < 0.05, the B + V-induce compared with the B-induce group; **p < 0.05, the B-induce compared with V-induce group; *p < 0.05, the V-induce was compared with control group). Color images available online at www.liebertpub.com/tea

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(Fig. 5C5). A defect remained in the blank control group at 16 weeks (data not shown). The microstructure of the newly formed bone was also evaluated using micro-CT imaging, and representative images from each group are shown in Figure 6A. The majority of the orbital wall defects were filled with a substantial amount of newly formed bone tissue in the TransB + V and B + V-induce groups. There was partial repair of the bone defects in the B-induce and V-induce groups. A small amount of new bone in the peripheral area of the repaired defect, a large amount of material residue, and a honeycomb scaffold structure were observed in the control group. Quantitative analysis using the micro-CT analysis system showed that the percentages of new bone volume to tissue volume (BV/TV) in the Trans-B + V, B + V-induce, B-induce, V-induce, and control groups were (in %) 57.1 – 9.3, 57.9 – 7.1, 41.2 – 4.2, 25.8 – 6.4, and 15.5 – 5.2, respectively (Fig. 6B). The BV/TV of the TransB + V and B + V-induce groups was significantly higher than the B-induce, V-induce, and control groups ( p < 0.05). The bone densities of the Tran-B + V, B + V-induce, Binduce, V-induce, and control groups were 0.721 – 0.073, 0.717 – 0.062, 0.592 – 0.057, 0.434 – 0.052, and 0.331 – 0.043 g/cm3, respectively (Fig. 6C). The BMD of the B + Vinduce was close to Trans-B + V group ( p > 0.05) but higher

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than the B-induce ( p < 0.05), V-induce ( p < 0.05), and control ( p < 0.05) groups. Histological analysis

Histological analysis using Van Gieson staining of nondecalcified sections further supported the micro-CT findings (Fig. 7A). Calcified bone sections showed bright red with variations in the intensity that was dependent on the maturity of the bone and b-TCP stained black by Van Gieson staining. The percentages of the new bone area and b-TCP residue in the Trans-B + V group (48.2% – 7.84%, new bone area; 21.2% – 2.89%, b-TCP residue) showed similar to the B + V-induce group (47.3% – 8.12%, new bone area; 20.4% – 3.3%, b-TCP residue) ( p > 0.05). Less bone formation and less material degradation were observed in the B-induce (34.3% – 4.9%, new bone area; 38.7 – 4.32, b-TCP residue) and V-induce groups (13.3% – 3.7%, new bone area; 48.8% – 5.72%, bTCP residue) compared with the B + V-induce group, which showed a significant difference ( p < 0.05). The percentages of new bone area (7.7% – 1.23%) and b-TCP residue (58.4% – 6.83%) in the control group showed less newly formed bone and b-TCP degradation compared with the B-induce (p < 0.05), V-induce ( p < 0.05), and B + V-induce groups ( p < 0.05) with a remarkable difference (Fig. 7B, C).

FIG. 7. Histological analysis of regenerated bone and b-tricalcium phosphate (b-TCP) residue using van Gieson’s picrofuchsin staining. (A) From top to bottom: Trans-B + V BMSCs/b-TCP, B + V-induce BMSCs/b-TCP, B-induce BMSCs/b-TCP, V-induce BMSCs/b-TCP, and BMSCs/b-TCP (original magnification 4 · and 40 · ). New bone appears red, and b-TCP appears black. (B, C) There were significant differences between the B + V-induce, B-induce, V-induce, and control groups in the new bone area and b-TCP residue (#p < 0.05, the B + V-induce compared with the B-induce group; **p < 0.05, the B-induce compared with V-induce group; *p < 0.05, the V-induce was compared with control group). Color images available online at www.liebertpub.com/tea

MICROCAPSULES USED IN ORBITAL BONE REPAIR Discussion

Tissue engineering plays an indispensable role in tissue repair, and regeneration is one of the most promising strategies for bone repair.33 Although clinical application of tissue-engineered bone has been reported, some key technical issues for the successful clinical application of engineered bone regeneration remain unresolved.34,35 For example, enhanced angiogenesis of engineered bone is particularly important for promoting the survival of an implanted scaffold.36,37 In addition, improving the efficiency of BMSC osteogenic induction is also an important issue for successful clinical application.38,39 Thus, the application of BMP-2 and VEGF in bone engineering has been employed in bone tissue engineering.7,40,41 In general, growth factors can be directly applied for the lineage-specific induction of stem cell differentiation in vitro or can be integrated into a scaffold as a drug delivery system for the induced differentiation of seeded stem cells. However, the main challenges in growth factor clinical application are the high cost, short half-life, and drug overdose, which usually exceeds the physiological levels of a natural environment.9,10 The use of gene transfer technology to induce osteogenic differentiation of stem cells has been widely reported.11,42,43 Its advantages include the long-term expression of a transgene and low cost. Adenovirus is a commonly used vector for engineered bone defect repair due to its high efficiency of gene transfection.11,14 Our previous study showed that BMP-2- and VEGF-gene-transfected BMSCs by an adenoviral vector could construct tissueengineered bone and could successfully repair orbital defects in an animal model. We also showed that dual-gene transfection of both BMP-2 and VEGF could enhance the effects of bone repair compared with single-gene transfer.13 Nevertheless, inflammatory attack to the viral vector by the immune system after in vivo implantation is a major obstacle for this clinical application. Microencapsulation is a newly developed technology for immune separation, which allows for the exemption of genetically modified cells from immune attack, while encapsulated cells remain able to release transgene products, and may thus become an available system for growth factor delivery.17,44 Due to this feature, we have adopted this technology to encapsulate genetically modified BMSCs that serve as a source of recombinant factors. By coculturing with BMSCs, the released factors could induce osteogenic differentiation of BMSCs. Our results showed that microencapsulated adenovirus-transduced BMSCs were able to express high transgene levels and to release higher concentrations of recombinant proteins. Further, BMSCs were sufficiently induced for osteogenic differentiation via coculture with microencapsulated cells as demonstrated by the expression of osteogenic-specific markers, which were significantly increased in the induced cells at both the gene and protein expression levels. According to previous studies, dual delivery of both BMP-2 and VEGF shows superiority to BMP-2 alone in osteoinductive differentiation and bone formation.7,45 This was consistent with our study; the effects of dual transfection of BMP-2 and VEGF in the osteogenic differentiation and bone repair were significantly higher than that of transfection of BMP-2 or VEGF alone. But the synergistic

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role of VEGF in BMP-4-induced ectopic bone formation is dose- and cell-type dependent.46 Additionally, our data showed that VEGF also increased the expression of osteogenic markers of BMSCs and enhanced the bone regeneration; this finding agreed with previously published data that show VEGF increasing bone healing capacity by promoting osteogenic differentiation of adipose-derived stem cells and enhancing osteogenesis in large-segmental bone defects.47,48 More importantly, in vivo bone regenerative results also demonstrated that bone defects were best repaired by dualgene transfer groups as revealed by CT scanning, micro-CT evaluation, and histological analyses, as well as quantitative analysis of the bone volume and bone density when compared with other groups. This finding also supports our hypothesis that dual transfection of BMP-2 and VEGF is optimal for engineered bone regeneration, which may be potentially due to enhanced osteogenic differentiation by BMP-2, enhanced angiogenesis by VEGF, and the synergistic effect of both factors.3,7,49 These results also suggest that the simultaneous use of BMP-2 and VEGF is particularly useful in the engineered repair of bone defects located at the ischemic anatomical regions, such as the orbital wall. While biosafety remains a concern for the clinical application of adenovirus-mediated gene transfer,14 the use of cocultured encapsulated and nonencapsulated BMSCs is relatively safe. As observed in our studies, the encapsulated cells were usually suspended in culture medium, whereas the nonencapsulated cells were adherent to the culture dishes, and thus, it was fairly easy to identify the two cell types using this coculture system. Moreover, the genetically modified cells were not directly transplanted into the animal’s body. On the basis of this advantage, allogeneic cells or cell lines may be used as a cell carrier using this methodology with less concern for biosafety. In the future, longterm studies are required to investigate the fate of these induced BMSCs using this coculture system to determine whether the implanted cells are normal and that no carcinogenesis will occur. Conclusions

This study established a highly efficient BMP-2/VEGF gene transduction and recombinant-protein-releasing system by employing adenoviral vectors and microencapsulation. In cocultures of BMSCs and encapsulated cells, BMSCs could be sufficiently induced to osteogenic differentiation by BMP-2 and VEGF released from encapsulated cells. In addition, dual-gene transduction achieved enhanced osteogenic differentiation of BMSCs in vitro and enhanced bone regeneration in vivo. More importantly, encapsulation and the coculture system not only efficiently induced osteogenic induction of BMSCs but also provided a biosafety approach for its potential application. Acknowledgments

This work was supported by the National Natural Science Foundation of China (31271029, 81170876, 81200720, and 81320108010), Shanghai Science and Technology Innovation Project (13JC1403800), Research Fund for the Doctoral Program of Higher Education of China (20130073110015), the Outstanding Young Talents Training Plan of the Shanghai Health System (XYQ2011053), and Shanghai Jiao

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Tong University School of Medicine Doctor Innovation Fund (BXJ201228). 15. Disclosure Statement

No competing financial interests exist. References

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Address correspondence to: Ping Gu, PhD Department of Ophthalmology Ninth People’s Hospital Shanghai Jiao Tong University School of Medicine Shanghai 200011 People’s Republic of China E-mail: [email protected] Xianqun Fan, MD, PhD Department of Ophthalmology Ninth People’s Hospital Shanghai Jiao Tong University School of Medicine Shanghai 200011 People’s Republic of China E-mail: [email protected] Received: September 25, 2013 Accepted: January 29, 2014 Online Publication Date: April 8, 2014