Research Article Potentiated Osteoinductivity via ...downloads.hindawi.com/journals/bmri/2015/435253.pdfResearch Article Potentiated Osteoinductivity via Cotransfection with BMP-2

Research ArticlePotentiated Osteoinductivity via Cotransfection with BMP-2 andVEGF Genes in Microencapsulated C2C12 Cells

Yang Shen, Han Qiao, Qiming Fan, Shuhong Zhang, and Tingting Tang

Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopedic Surgery, Shanghai Ninth People’s Hospital,Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Road, Shanghai 20011, China

Correspondence should be addressed to Shuhong Zhang; [email protected] and Tingting Tang; [email protected]

Received 8 June 2015; Revised 18 August 2015; Accepted 26 August 2015

Academic Editor: David Markel

Copyright © 2015 Yang Shen et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Microcapsules with entrapped cells hold great promise for repairing bone defects. Unfortunately, the osteoinductivity ofmicrocapsules has been restricted by many factors, among which the deficiency of functional proteins is a significant priority.We potentiated the osteoinductivity of microencapsulated cells via cotransfection with BMP-2 and VEGF genes. Various tissue-derivedmesenchymal stem cells and cell lineswere compared for BMP-2 andVEGF cotransfection. Ethidiumbromide (EB)/CalceinAM staining revealed that all of the cell categories could survive for 4 weeks after microencapsulation. An ELISA assay indicatedthat all microencapsulated BMP-2 or VEGF transfected cells could secrete gene products constitutively for 1 month. Particularly,the recombinant microencapsulated C2C12 cells released the most desirable level of BMP-2 and VEGF. Further experimentsdemonstrated that microencapsulated BMP-2 and VEGF cotransfected C2C12 cells generated both BMP-2 and VEGF for 4weeks. Additionally, the cotransfection of BMP-2 and VEGF in microencapsulated C2C12 cells showed a stronger osteogenicinduction against BMSCs than individual BMP-2-transfected microencapsulated C2C12 cells. These results demonstrated that thecotransfection of BMP-2 andVEGF intomicroencapsulatedC2C12 cells is of potent utility for the potentiation of bone regeneration,which would provide a promising clinical strategy for cellular therapy in bone defects.

1. Introduction

One major obstacle encountered by clinical orthopedicpractice is the repair of bone defects caused by trauma,malignant disease, and prosthetic replacement [1]. The mostcommon approaches to repair bone defects, such as thetransplantation of autologous and allogeneic bone grafts orsubstitution of artificial bone, exhibit many disadvantages.These disadvantages include the scarcity of supply resources,the risk of disease dissemination, and deficient osteogenesis,which lead to the delayed union or the nonunion of the bone[2–4]. However, cell microencapsulation represents a noveland promising tissue engineering strategy that involves thecarrying of viable cells with biologically active molecules orgenes that promote bone regeneration [5, 6].

The microencapsulation technique involves the for-mation of a semipermeable membrane that is able toboth entrap functional and feasible cells and permit theflow of nutrients inwards and the waste of interior cells

outwards [7]. Historically, microencapsulation approacheswere applied to many medical conditions, such as anemia,delayed growth, and diabetes [8–10]. Furthermore, becauseit is an immune-tolerated biocompatible therapeutic vector,microencapsulation should assist the inner cell in avoidinghost immune exterminations [11].The alginate-poly-L-lysine-alginate (APA) microcapsules first reported by Lim andSun [12] appear to exert an immune-protective effect onentrapped cells and form a spherical shape with a smoothsurface and consistent uniformity. These were considered tobe suitable standards for the use ofmicroencapsulation in celltreatment research.

Bone morphogenetic protein-2 (BMP-2), which func-tions as amember of the transforming growth factor-𝛽 super-family, plays a vital role during osteogenic and endochondralregeneration [13–16]. Moreover, angiogenesis appears to bea prerequisite for bone rehabilitation, and vascular endothe-lial growth factor (VEGF) has been proposed as the mostpotent induction stimulus [17]. Additionally, VEGF is capable

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015, Article ID 435253, 10 pageshttp://dx.doi.org/10.1155/2015/435253

2 BioMed Research International

of enhancing osteoblast differentiation by interacting withBMP-2 in a series of sequential processes [18, 19]. BMP-2is able to enhance angiogenesis by stimulating an increasedexpression of VEGF on osteoblast-like cells. In turn, theaccelerated establishment of new blood vessels promotes thedifferentiation of osteoblast cells and potentiates BMP-2-mediated bone formation [20].

Therefore, we intend to determine whether the enhancedosteoinductivity created via the cotransfection of BMP-2 andVEGF can be realized in a certain type of microencapsulatedcells to provide an enhanced instrument for future cellulartherapeutic progress.

Furthermore, due to the limitations of the quantity orquality of entrapped engineered cells, it is critical to identifythe most satisfactory transfected cell type from amongthe remaining unsatisfactory cell categories to achieve thehighest level of secreted functional molecules. Herein, weinvestigated the viability of microcapsules in various tissue-derived mesenchymal stem cells, including rat bone marrowmesenchymal stem cells (BMSCs), adipose-derived stemcells (ADSCs), synovium-derived mesenchymal stem cells(SMSCs), and divergent mouse cell lines (mouse fibroblastcell line C3H10T1/2, mouse myoblast cell line C2C12, andmouse preosteoblast cell line NIH/3T3). This investigationwould significantly contribute to the development of a supe-rior platform for microencapsulated cell delivery systems.

2. Methods and Materials

2.1. Cell Preparations

2.1.1. Cell Culture of Rat Bone Marrow-Derived MesenchymalStem Cells (BMSCs). Male Sprague-Dawley rats weighing300 g were purchased from the Shanghai Laboratory AnimalCenter, Chinese Academy of Sciences. After cervical dislo-cation and 75% ethanol submergence for 10min, the rats’bilateral femurs were dissected and soft tissues were removedunder aseptic conditions. Metaphyses from both proximaland distant ends were removed via diaphysis into dishesat 37∘C in humidified conditions with 5% CO

2following

a flush of 10mL 𝛼MEM medium (HyClone, Logan, UT,USA) containing 10% fetal bovine serum (FBS, HyClone,Logan, UT, USA), penicillin 100U/mL, and streptomycin100 𝜇g/mL (Gibco, Invitrogen Ltd., Carlsbad, CA, USA).Single cell suspension was created via a syringe. The culturemediums were changed every other day until the appearanceof adherent BMSCs.

2.1.2. Cell Culture of Rat Adipose-Derived Stem Cells (ADSCs).Male Sprague-Dawley rats weighing 300 g were purchasedfrom the Shanghai Laboratory Animal Center, ChineseAcademy of Sciences. After cervical dislocation and 75%ethanol submergence for 10min, rat abdominal adipose tis-sueswere excised. After removal of red blood cells (RBC) via aphosphate-buffer saline (PBS)wash, adipose tissuewasmixedwith a 3x volume of 0.1% type I collagenase (Gibco, InvitrogenLtd., Carlsbad, CA, USA) and incubated at 37∘C for 50min.Subsequently, an equal volume of complete 𝛼MEM culture

medium was added and centrifuged. After removal of thesupernatant together with the undigested adipose tissue,cell pellets were resuspended in a 5mL complete 𝛼MEMculture medium and a red blood cell lysis buffer, respectively,following several filtrations through a nylon mesh screen.Single cell suspension was created via a syringe. ADSCs werecultured at 37∘C in humidified conditions with 5% CO

2, and

the culture medium was changed every other day.

2.1.3. Cell Culture of Rat Knee Synovium-Derived Mesenchy-mal StemCells (SMSCs). Male Sprague-Dawley ratsweighing300 g were purchased from the Shanghai Laboratory AnimalCenter, Chinese Academy of Sciences. After cervical disloca-tion and 75% ethanol submergence for 10min, knee synovialmembrane tissues were harvested under low temperatureand washed vigorously several times in a PBS wash. Smoothand shiny synovial tissues were exposed after the removal ofadipose and connective tissues. Following several washes ofPBS, the synovial tissues were divided into 1-2mm3 pieceswith sterile blades or scissors and mixed with 0.4% typeI collagenase and incubated at 37∘C for 4 hr. Cell pelletswere then resuspended in PBS, centrifuged several times,and cultivated at 37∘C in a humidified condition with 5%CO2. Single cell suspension was created via a syringe. Culture

medium was changed every three days.

2.2. Cell Culture of Mouse C3H10T1/2, C2C12, and NIH/3T3Cell Lines. Briefly, mouse fibroblast cell line C3H10T1/2 andmouse preosteoblast cell line NIH/3T3 were incubated with𝛼MEM culture medium and mouse myoblast cell line C2C12was nurtured with DMEM (HyClone, Logan, UT, USA)culture medium. C3H10T1/2, C2C12, and NIH/3T3 cell lineswere purchased from the Cell Bank of the Chinese Academyof Sciences, Shanghai. Each culture medium contained 10%fetal bovine serum, penicillin 100U/mL, and streptomycin100 𝜇g/mL at 37∘C in humidified conditions with 5% CO

2.

2.3. EB/Calcein AM Staining. Following rigorous abstersionwith PBS, microcapsules with entrapped cells were incubatedin 100mg/L ethidium bromide (EB) solution (Sigma-Aldrich,St. Louis, MO, USA) as well as in 5 𝜇mol/L Calcein AM(Sigma-Aldrich, St. Louis, MO, USA) for 30min at 37∘C.Themorphology was observed via fluorescence microscope.

2.4. Transfection of BMP-2 and/or VEGF Genes. The recom-binant adenovirus with the human BMP-2 gene (Adv-BMP-2) transfection was kindly provided by Professor Lou [14,21]. The recombinant adenovirus with the human hVEGFl65gene (Adv-VEGF) transfection was previously constructedin our lab [19]. Upon reaching 90% confluence, variouscells were incubated in a transfection medium containingthe culture medium and the recombinant adenovirus at therequired multiple of infection (MOI) (MOI = 200 for Adv-BM-2 transfection;MOI = 150 for Adv-VEGF transfection) at37∘C overnight. Infected cells were cultivated in 𝛼MEM cul-ture medium supplemented with 10% FBS; the transfectionmedium was changed continuously.

BioMed Research International 3

2.5. Microencapsulation of Recombinant Transfected Cells.Procedures similar to those previously described were uti-lized [22, 23]. Briefly, after purification, a specially designedhigh-voltage electrostatic microcapsule generator was uti-lized to manufacture APA microcapsules [24]. Recombinanttransfected cell pellets were resuspended in a sterile sodiumalginate solution at a concentration of 3 × 106/mL via a 27-gauge needle with a syringe pump. Cell suspensions wereextruded as droplets and then mixed with a 1.1% Cacl

2

solution.The exterior layer of the microencapsulated sphereswas cross-linked with poly-L-lysine (PLL) (Sigma-Aldrich,St. Louis, MO, USA) for 10min following a soak in sodiumcitrate for 6min to dissolve the unpolymerized alginate usedfor producing microencapsulated cells. The entire procedurewas carried out at 4∘C. All resultant microcapsules werestored in culturing dishes under 37∘C for further research.

2.6.Measurement of Cytokine Expressions. An identical num-ber of entrapped cells (2.5× 106/group) inmicrocapsules withspecific transfection were seeded and incubated for 48 hr.Then, the supernatants were collected and the concentrationsof BMP-2 and VEGF were measured via enzyme-linkedimmunosorbent assay (ELISA) kits (BioSource, Camarillo,CA, USA) according to the manufacturer’s guidelines.

2.7. Establishment of Coculture System between Microencap-sulated Recombinant Cells with BMSCs. Six-well plates wereused to investigate the interplay between upper microcap-sules and lower undifferentiated bone marrow mesenchymalstem cells (BMSCs) (5.0 × 105 cells/well). The basementmembrane of the cell culture insert (BDFalcon, NJ, USA)wasan 8 𝜇m porous PET membrane, and 2mL DMEM culturingmedium was added into each well to allow the intimatecommunication of cytokines between the upper and lowerchambers but inhibit cell penetration.

2.8. Alkaline Phosphatase (ALP) Activity Assay and ALPStaining. After 3 and 7 days of coculture with recombinantmicrocapsules and BMSCs, the lower chamber basementswere first washed twice with PBS. The ALP activity wasmeasured in differentiated BMSCs that were lysed with 1%Triton-100 through four standard freeze and thaw cycles.Next, a 50 𝜇L sample was mixed with 50𝜇L of p-nitrophenylphosphate (pNPP) (Sigma-Aldrich, St. Louis, MO, USA) thatserved as the substrate (1mg/mL) in a 1M diethanolaminebuffer containing 0.5mMMgCl

2, pH 9.8, and was further

incubated at 37∘C for 15 min. The reaction was stoppedwith the addition of 25𝜇L of 3NNaOH for each 100 𝜇Lof reaction mixture. Enzyme activity was quantified viaabsorbance measurements at 405 nm. The intracellular totalprotein expression was investigated with a BCA protein assaykit (Bicinchoninic Acid Kit, Pierce Biotechnology, ThermoFisher Scientific, Waltham, MA, USA). ALP staining wasconducted with an ALP staining kit (Renbao, Shanghai,China) on day 3 and day 7. The lower stimulated cells werefixed with 4% buffered formalin for 30 s, washed twice withcold ddH

2O, and then stained with a staining reagent for

2 hr at 37∘C according to the manufacturer’s instructions.

Afterwards, the plates were washed rigorously with ddH2O

and staining images were collected under a scanner (Hewlett-Packard Scanjet 2400;Hewlett-Packard, PaloAlto, CA,USA).

2.9. Alizarin Red Staining (ARS). After 21 days of coin-cubation with microencapsulated cells and BMSCs, thelower chamber basements were first washed twice with PBSand then fixed in 4% buffered formaldehyde for 30min.Subsequently, the fixed cells were incubated with 40mMAlizarin Red (Sigma-Aldrich, St. Louis,MO,USA) for 15min,followed by a thorough wash with distilled water until theabstergent water was transparent. The final staining imageswere obtained with a scanner (Hewlett-Packard Scanjet 2400;Hewlett-Packard, Palo Alto, CA, USA).

2.10. Statistical Analysis. An analysis of variance (ANOVA)and post hoc analysis were performed to assess the statisticalsignificance of the data collected. A 𝑃 value of less than 0.05was indicative of a statistically significant difference. All ofthe data were analyzed with SPSS 13.0 software (StatisticalPackage for the Social Sciences, Chicago, IL, USA) and arepresented as the means ± SD.

3. Results

3.1. Observations of Cultured Cells. Inverted optical micro-scopic observations indicated that all of the nontransfectedcell types demonstrated a specific morphology (Figure 1).BMSCs exhibited a rod-like or spindle-shaped formation.After 7 days of incubation, the initial cell colony increasedto merge with the other clones, bringing along the adherentspindle- and rod-shaped cells (Figure 1(a)-(A)). Regardingthe ADSCs, after the first inoculation of 5–7 hr, somecells began to adhere and exhibited elongated pseudopodia,thereby contributing to a spindle-like appearance. 24 hr later,the cells completely adhered to the dishes, exhibiting afibroblast-like cell growthwith a proliferative peak at 3–5 days(Figure 1(a)-(B)). The presence of SMSCs indicated apparentadherent cells with elongated or polygonal shapes. After72 hr of cultivation, a multitude of spindle-shaped cells withsmall round cells scattered inside were present. Cell colonyformations were detectable at around day 5 (Figure 1(a)-(C)).Optical microscopic observations of C3H10T1/2, C2C12, andNIH/3T3 cell lines (Figure 1(b)) showed an elongated cellappearance with a rapidly proliferative bioactivity.

3.2. Characteristics of Microcapsules and MicroencapsulatedCells. The microcapsules with or without entrapped non-transfected cells generally exhibited a smooth and sphericalshape with diameters of approximately 250𝜇m (Figure 2(a)).This uniform structure enabled the collection of results thatwere sufficiently reliable. Further EB/Calcein AM stainingrevealed that, after the microencapsulation of various cellcategories, the entrapped cells were capable of survivingat least 4 weeks with no evident cell death (Figures 2(b)and 2(c)), meaning that a 4-week span of observation wasable to comprehensively evaluate the transition of cytokinesexpressions.


BMSCs ADSCs SMSCs

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C3H10T1/2 C2C12 NIH/3T3

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Figure 1: The morphology of different tissue-derived mesenchymal stem cells and cell lines. (a) The morphology of different tissue-derivedmesenchymal stem cells with the appearance of a colony-forming unit. (A) indicates the bone marrow-derived mesenchymal stem cells(BMSCs), (B) indicates the adipose-derived stem cells (ADSCs), and (C) indicates the synovium-derived mesenchymal stem cells (SMSCs).(b) The morphology of different cells lines. (A) represents the C3H10T1/2 cells, (B) indicates the C2C12 cells, and (C) indicates the NIH/3T3cells. Scale: 20𝜇m.

3.3. BMP-2 Productions via Microencapsulated RecombinantCells. The productions of BMP-2 cytokines were contin-uously collected from the supernatants of the microcap-sules. Figure 3(a) shows the relatively low amount of BMP-2 expression in all six microencapsulated cells prior togene transfection. After transfection of BMP-2 (Figure 3(b)),C2C12 produced a higher level than any of the other celltypes from the first week to the fourth week. Also, after thepeak of BMP-2 generation at week 2 in microencapsulatedBMP-2-transfected C2C12 cells, a statistically significant dif-ference from the othermesenchymal stem cells waswitnessedthrough 4 weeks consistently.

3.4. VEGF Productions via Microencapsulated RecombinantCells. The productions of VEGF cytokines were consis-tently harvested from the supernatants of microcapsules.Figure 3(c) demonstrates a relatively reduced expression ofVEGFprior to targeted gene transfections. After themicroen-capsulation of the recombinant Adv-VEGF-transfected cells,the greatest generation of VEGFwas exhibited in BMSCs andADSCs during the initial three weeks (Figure 3(d)). Addi-tionally, C2C12 expressed the third level of VEGF expression(higher than the other three cell types).

3.5. BMP-2 and VEGF Productions via MicroencapsulatedCotransfected C2C12 Cells. Because C2C12 cells could releasethe most desirable level of BMP-2 and VEGF after a single

transfection, the further cotransfection of BMP-2 and VEGFat a ratio of 4 : 1 was performed in C2C12 cells. Figure 4(a)shows a significant increase in BMP-2 release after cotrans-fection during the first two weeks, followed by a dramaticdecline from week 2 to week 4. The generation of VEGF incotransfectedmicroencapsulatedC2C12 cells remained stableduring 4 weeks and demonstrated no significant differencefrom the first week. Altogether, the above data revealed thatthe cotransfection of BMP-2 andVEGF inmicroencapsulatedC2C12 cells could release both BMP-2 and VEGF for 4 weeks.This method could thus be utilized for further osteoinductiv-ity assessments.

3.6. Potentiated Osteoinductivity via Coculture of BMSCswith Microencapsulated BMP-2/VEGF Cotransfected Cells.The above results indicated an optimal expression of BMP-2in recombinant C2C12 microcapsules. This method was alsoable to generate a satisfactory level of VEGF proteins. There-fore, the advantages of the C2C12 cell line were utilized tofurther investigate the osteoinductivity of BMSCs via cotrans-fection with BMP-2/VEGF genes. An ALP activity assay, onequantitative determination method of early osteogenic dif-ferentiation, was used to evaluate the osteoinductivity of thecocultured cells. ALP revealed that recombinant cells cotrans-fected with BMP-2/VEGF exhibited more significant ALPactivity than those cocultured with the BMP-2-transfectedmicrocapsules group on both day 3 and day 7 (Figure 4(b)).


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(a)

BMSCs ADSCs SMSCs

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4w

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Figure 2: The morphology of APA microcapsules and the viability of various microencapsulated cells at 1 week or 4 weeks aftermicroencapsulation (EB/Calcein AM staining). (a) The morphology of APA microcapsules. (A) indicates the microcapsules without cellsand (B) indicates the microcapsules containing the BMSCs. Scale: 100 𝜇m. (b) The viability of different microencapsulated tissue-derivedmesenchymal stem cells at 1 week and 4 weeks (EB/Calcein AM staining).The green fluorescence indicates live cells, and the red fluorescenceindicates dead cells. Left: BMSCs, middle: ADSCs, and right: SMSCs. Scale: 20 𝜇m. (c)The viability of different microencapsulated cells linesat 1 week and 4 weeks (EB/Calcein AM staining). The green fluorescence indicates live cells, and the red fluorescence indicates dead cells.Left: C3H10T1/2, middle: C2C12, and right: NIH/3T3. Scale: 20 𝜇m.


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Figure 3: Measurement of cytokine expressions. (a) The basic BMP-2 secretion from the microencapsulated cells without gene transfection.(b) All encapsulated BMP-2 gene-transfected stem cells were capable of the constitutive secretion of BMP-2 proteins. Among the six cell types,C2C12 could secrete most BMP-2 protein, which was significantly higher than the other cell groups through four weeks after BMP-2 genetransfection and cells microencapsulation (#𝑃 < 0.05 compared with the C2C12 group). Among the three tissue-derivedMSCs, BMSCs couldsecrete most BMP-2 protein (significantly more than the other stem cells groups through four weeks after BMP-2 gene transfection and cellsmicroencapsulation) (#𝑃 < 0.05 compared with the C2C12 group). (c) The basic VEGF secretion from the microencapsulated cells withoutgene transfection (#𝑃 < 0.05 compared with the C2C12 group). (d) All microencapsulated VEGF gene-transfected stem cells were capable ofthe constitutive secretion of VEGF proteins. BMSCs or ADSCs could secrete most VEGF proteins. C2C12 cells expressed the third level ofVEGF (#𝑃 < 0.05 compared with the C2C12 group).

Additionally, the ALP activity of the BMP-2 recombinantmicrocapsules exhibited more significant strength than theregular culture medium-treated BMSCs group for up to 7days. Furthermore, ALP staining results demonstrated thatthe cotransfected microcapsules group showed strong posi-tive staining against BMSCs differentiation, with purple blue

within the cell cytoplasm and the stronger dyeing of localizedcells. In contrast, weak staining was found among the BMP-2-transfectedmicrocapsules groupwith barely stained signalson days 3 and 7 in the control group, which was coculturedwith undifferentiated BMSCs (Figure 4(c)). As one of theangiogenic factors, VEGF alone cannot induce the osteogenic


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Figure 4: Assessment of potentiated osteoinductivity. (a) Measurement of cytokine expressions after the cotransfection of BMP-2 and VEGFin microencapsulated C2C12 cells. After the cotransfection of BMP-2 and VEGF in C2C12 cells, the microencapsulated C2C12 cells couldrelease both BMP-2 and VEGF over a span of four weeks (#𝑃 < 0.05 compared with the 1-week observation time point). (b)The ALP activityof BMSCs cocultured with the microencapsulated C2C12 cells. BMP2 + VEGF: cocultured with the microencapsulated BMP-2 and VEGFgene-transfected C2C12 cells. BMP2: cocultured with the microencapsulated BMP-2 gene-transfected C2C12 cells. Control: coculture withthe microencapsulated C2C12 lacking gene transfection (#𝑃 < 0.05). (c) The ALP staining of BMSCs cocultured with microencapsulatedC2C12 cells. (A) showed the staining results after 3 days of coculture. (B) showed the staining results after 7 days of coculture. (1) Coculturedwith the microencapsulated BMP-2 and VEGF gene-transfected C2C12 cells. (2) Cocultured with the microencapsulated BMP-2 gene-transfected C2C12 cells. (3) Cocultured with the microencapsulated none gene-transfected C2C12 cells. (d) The Alizarin Red Staining ofBMSCs cocultured with the microencapsulated C2C12 cells. (A) Cocultured with the microencapsulated BMP-2 and VEGF gene-transfectedC2C12 cells. (B) Cocultured with the microencapsulated BMP-2 gene-transfected C2C12 cells. (C) Cocultured with the microencapsulatedC2C12 cells lacking gene transfection.

differentiation of BMSCs cells; because of this, we did not testthe efficacy ofC2C12 cells transfectedwithVEGF alone in thisstudy.

Extracellular matrix mineralization was evaluated usingAlizarin Red Staining. Figure 4(d) shows that the stain-ing on BMSCs cocultivated with BMP-2/VEGF-transfectedmicroencapsulated cells displayed a multitude of red nodulesafter a 3-week inducement. Similar staining was observedfor the BMSCs that went through BMP-2 recombinantmicrocapsules treatment and had a slightly weak expression.Nevertheless, few positive signals were found in BMSCsmineralization in the control microcapsules intervention.This finding was consistent with a previous study thatindicated that nontransfection microcapsules explicated anegligible osteoinductivity against BMSCs after a 3-weekdifferentiation.

4. Discussion

In this study, we explored the cultivation of microencap-sulated BMSCs, ADSCs, SMSCs, C3H10T1/2, C2C12, andNIH/3T3 cells, revealing that the microcapsules were ableto provide sufficient living conditions for the inner cells. Toinvestigate the osteoinduced capability via the cotransfectionof BMP-2 and VEGF, the C2C12 cell line was chosen forfurther in vitro coculturing with BMSCs due to its opti-mal expressions of both BMP-2 and VEGF. After coculturewith cotransfected microencapsulated C2C12 cells, the ALPactivity and ARS intensity of BMSCs were stronger thanthe single BMP-2-transfected group, signifying that more

desirable effects were achieved via the cotransfection of BMP-2 and VEGF, which promote the expressions of osteogenicmarkers at both the early and the late stages.

Undoubtedly, the viable cells in microcapsules should bepreserved to promote functionality. Obviously, such condi-tions require the formation of semipermeable membranes,which on one hand shields the invasive effects exerted on theinner sensitive and functional cells and on the other handenables both intimate communication between the entrappedcells and the movement of exterior nutritional materialsthat support the stable survival of the cells entrapped inthe microcapsules. With such concerns in mind, our pre-vious study found that potentiated membrane strength andbiocompatibility can be accomplished via purified sodiumalginate- (PSA-) based microcapsules, indicating that thebiological activity of microcapsules can be enhanced throughalginate modification [25]. In this study, we also entrappedvarious potential osteodifferentiated cell types within APAmicrocapsules, showing that all of the cell categories couldmanage to survive within the APAmicrocapsules for 4 weeks.

In addition, we utilized APAmicrocapsules with rat bonemarrow-derivedmesenchymal stem cells transfectedwith theBMP-2 gene and demonstrated both the effective immune-isolation capability of microcapsules and the valid biologicalresponse induced via the consistent and efficient release ofBMP-2 from recombinant microcapsules [22]. Herein, theVEGF that served as a potent angiogenesis stimulus wasutilized to enhance BMP-2-induced bone formation. Initially,BMP-2 and VEGF cytokine expressions were measured viaELISA examinations after corresponding transfection intoeach cell type. The 4-week duration not only demonstrated a


consistent release due to the successfully microencapsulatedBMP-2 and VEGF transfections but also provided us withthe useful information that C2C12, which released the highestlevel of BMP-2 and a desirable level of VEGF, can be exploitedfor further cotransfection study. The C2C12 cell line acts asa premyoblast cell line [26] and could be shifted towardsosteoblast differentiation from myoblast differentiation withinterference of the BMP-2 protein [27–29]. Moreover, Liet al. [30] proposed that C2C12 cells were able to secretecytokines and survive within incapacious microcapsules inthe long term. Therefore, further research was carried out tocompare the osteoinductivity of C2C12 cells with a single genetransfection with those with a double gene cotransfection.

VEGF, as previously reported, is capable of enhancingosteoblast differentiation with the costimulus of BMP-2through a series of sequential processes [18, 20]. Our resultsshowed that recombinant BMP-2 and VEGF cotransfectedC2C12 cells in microcapsules could release both BMP-2and VEGF for 4 weeks. Additionally, the ALP activity,ALP staining intensity, and ARS staining of C2C12 cellscotransfected with BMP-2 and VEGF (when examined afterthe 3-day and 7-day coincubation periods) demonstrateda stronger tendency than BMSCs cocultured with merelyBMP-2-transfected cells, which indicated the facilitation ofosteodifferentiation via the coculture of BMP-2 and VEGFcotransfected microcapsules in both the early and the latestages.

Nonetheless, despite the comprehensive observation ofBMSC differentiation via secreting cytokines from recom-binant microcapsules, the impact on the entrapped cellsafter microencapsulation is not yet fully understood. SOX-9 and TGF-𝛽3-transfected MSCs after microencapsulationdifferentiated towards cartilage, indicating that a three-dimensional culturing systemofmicrocapsules could provideentrapped cells with an adequate microenvironment for pro-liferation and differentiation [31, 32]. Additionally, anotherstudy found that polysaccharide microcapsules, when usedas a scaffold-based culture platform, were able to reinforcethe adhesion and growth of hBMSCs [33]. Furthermore, thenew bone regeneration could be unraveled after microen-capsulation cultivation both in vitro and in vivo [33]. It isthus no surprise that the cellular events occurring insidemicrocapsules play an important role in microcapsules as aneffective drug delivery vector.

In conclusion, we successfully attempted the cultivationof microencapsulated BMSCs, ADSCs, SMSCs, C3H10T1/2,C2C12, and NIH/3T3 cells with long-term lifespans. More-over, the transfection of BMP-2 and VEGF into targetedmicroencapsulated cells can effectively facilitate the releaseof functional proteins from microcapsules. More impor-tantly, the BMSCs’ ability to differentiate can be significantlypotentiated through the cotransfection of BMP-2/VEGFinto C2C12 cells. This provides promising opportunities forcellular therapy in future clinical treatments of bone defects.

Conflict of Interests

There is no conflict of interests.

Acknowledgments

This research was funded by the National Natural ScienceFoundation of China (30772183, 81172549) and a grant fromthe Shanghai Science and Technology Development Com-mission (13JC1403900, 13DZ2294000).

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