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Biomaterials 31 (2010) 1213–1218

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Biomaterials

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The effect of VEGF on the myogenic differentiation of adipose tissue derivedstem cells within thermosensitive hydrogel matrices

Min Hwan Kim b, Hea Nam Hong c, Joon Pio Hong d, Chan Jeoung Park a, Seog Woon Kwon a,Soon Hee Kim e, Gilson Kang e, MiJung Kim a,b,*

a Dept. of Laboratory Medicine, University of Ulsan College of Medicine, Asan Medical Center, 388-1 PoongNap-Dong, SongPa-Gu, Seoul 138-736, Republic of Koreab Cell & Molecular Biology Laboratory, Asan Institute for Life Sciences, Asan Medical Center, 388-1 PoongNap-Dong, SongPa-Gu, Seoul 138-736, Republic of Koreac Dept. of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul, Republic of Koread Dept. of Plastic Surgery, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Koreae Polymer BIN Fusion Research Team, Department of Polymer Science and Technology, Chonbuk National University, Jeonju, Republic of Korea

a r t i c l e i n f o

Article history:Received 1 October 2009Accepted 25 October 2009Available online 14 November 2009

Keywords:Adipose tissue derived stem cellThermosensitive hydrogelMPEG–PCLSoft-tissue engineeringVascular endothelial cell growth factorVascularization

* Corresponding author. Cell & Molecular Biology LLife Sciences, Dept. of Laboratory Medicine, UniversityAsan Medical Center, 388-1 PoongNap-Dong, SongPaof Korea. Tel.: þ82 2 3010 4147; fax: þ82 2 3010 418

E-mail address: mjkimhsc@korea.com (M. Kim).

0142-9612/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.biomaterials.2009.10.057

a b s t r a c t

We investigated the combination of human adipose tissue derived stem cells (ADSC) and in vivo gel-forming methoxy poly (ethyleneglycol)-poly (3-caprolactone) (MPEG–PCL) as a muscle regenerationmatrix, with and without inclusion of vascular endothelial cell growth factor (VEGF). VEGF165-treatedstem cell grafts showed significant proliferation and differentiation into muscle tissue in vivo. Impor-tantly, the inclusion of VEGF enhanced vascularization. This scaffold supported preconditioned ADSC, andallowed them to differentiate into mature muscle tissues in vivo, indicating that ADSC of human originand MPEG–PCL scaffolds provided an appropriate environment for cellular growth and expansion. Ourresults thus provide a potential solution to the major obstacle encountered in the engineering of thickcomplex tissues, which require an adequate blood supply to maintain cell viability during tissue growthand to induce appropriate structural organization. Therefore, the combination of ADSC and in vivo gel-forming MPEG–PCL with VEGF165 might serve as a suitable non-invasive biomaterial for clinical muscleregeneration applications.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Soft-tissue engineering solutions, for example those used torepair defective muscle, require a soft muscle conductive vehiclethat differs from that used for hard tissue (e.g., bone, cartilage).

The scaffolds that have been developed for in vivo delivery ofcells for muscle tissue engineering applications are implantable invitro cultured polymer-based scaffolds, such as poly (L-lactide-tri-methylene carbonate) (PLLA-TMC) copolymer, and injectable gel-type scaffolds, including alginate microcapsule [1,2].

Recently, the triblock copolymer, poly(ethylene glycol-b-[DL-lacticacid-co-glycolic acid]-b-ethylene glycol) (PEG–PLGA–PEG) [3], anddiblock copolymer, methoxy poly(ethylene glycol) poly(3-capro-lactone) (MPEG–PCL) [4], have been used as effective tools for

aboratory, Asan Institute forof Ulsan College of Medicine,

-Gu, Seoul 138-736, Republic2.

All rights reserved.

targeted cell delivery. These in vivo gel-forming, degradable scaffoldsexhibit temperature-dependent phase-change properties, convertingfrom a solution to a gel at approximately body temperature. Thus,when injected into the body, the scaffold forms a gel-type scaffold,providing a matrix for the proliferation and differentiation of deliv-ered cells without the need for additional surgery.

In addition to the need for biodegradable scaffolds of goodbiocompatibility, cell-based tissue engineering solutions can exploita variety of growth factors to create the appropriate stimulatoryenvironment [5]. Complex tissues, such as muscle and adiposetissues, are embedded with blood vessels, which are critical forsupplying necessary nutrients and removing toxic metabolites.Thus, angiogenic processes that establish and maintain blood vesselnetworks are important for preventing muscle cell death and tissuenecrosis. Enhanced angiogenesis may thus improve muscle functionin ischemic tissue [6–9]. Insulin-like growth factor (IGF) andvascular endothelial cell growth factor (VEGF) are potent mediatorsof angiogenesis [10]. VEGF is known to be a critical and specificgrowth factor for blood vessel formation [11,12]. In particular, theVEGF-A subtype, which includes VEGF165 used in the current study,is a dominant and potent member of the VEGF family.

Table 1Primers used for RT-PCR analysis.

Primer Sequence Annealingtemp.

Size

MyoD (GenBank accession no. X56677) Forward 50-AAGCGCCACTCTTGAGGTA-30

Reverse 50-GCGCCTTTATTTTGATCACC-3051 �C 500 bp

Myosin heavy chain (MHC) (GenBank accession no. NM_005963) Forward 50-TGTGAATGCCAAATGTGCTT-30

Reverse 50-GTGGAGCTGGGTATCCTTGA-3056 �C 750 bp

GAPDH (GenBank accession no. NM_002046) Forward 50-ACCACAGTCCATGCCATCAC-30

Reverse 50-TCCACCACCCTGTTGCTGTA-3056 �C 452 bp

M.H. Kim et al. / Biomaterials 31 (2010) 1213–12181214

In addition to scaffolding material and growth factors, thesource of cells is a major consideration in tissue engineering. Recentstudies have shown that stem cells can be isolated from a widevariety of sources, including bone marrow, muscle and adiposetissue. These stem cells have been widely utilized to repairdamaged tissues and organs [13]. However, no clearly ideal stemcell source has been established, and it is likely that clinical appli-cations of stem cells may vary depending on the target tissue.

Like bone marrow, adipose tissue is of mesodermal origin andincludes a population of various stromal cells, including osteo-blasts, pre-adipocytes and muscle precursor cells [14]. As a stemcell source, adipose tissue presents a number of potential benefits,both from the standpoint of acquisitiondtissue can be easilyobtained from liposuction procedures under local anesthesiadandregulation of use. Adipose tissue derived stem cells (ADSC) havebeen used mainly to repair hard tissue, such as bone and cartilage.However, to date, the potential applicability of these cells forregeneration of soft-tissue, like muscle, has remained largelyunexplored.

In a previous study, we suggested that the combination of ADSCobtained from liposuction and injectable poly (lactic-co-glycolicacid) (PLGA) spheres are promising materials for adipose andmuscle tissue engineering [15]. In the current study, we tested thehypothesis that the combination of ADSC and an in vivo gel-forming scaffold containing an angiogenesis-enhancing growthfactor is suitable for muscle tissue engineering. Specifically, weevaluated muscle regeneration and blood vessel formation in vivo

Fig. 1. (A) Expression of the myogenic differentiation markers, MyoD and MHC, in myogen(Cy3) protein expression in vitro at 4, 6, 11 days by immunofluorescence staining. Nuclei w

after implantation of a hydrogel matrix containing the diblockcopolymer, MPEG–PCL, and ADSC with or without VEGF165.

2. Materials and methods

2.1. Synthesis of MPEG–PCL diblock copolymer

MPEG–PCL diblock copolymer was synthesized as described previously [16].MPEG (1.5 g, 2 mmol) (Sigma Aldrich Inc., USA) and toluene (80 ml) were introducedinto a sterilized round flask. The MPEG solution was distillated by azeotropic distil-lation to remove water. Toluene was then distilled off completely. CH2Cl2 (25 ml) (JinChem. Pharm. Co., Ltd., Korea) was added to MPEG, followed by the addition of CL(4.8 g, 42 mmol) using a syringe. The polymerization was initiated by the addition of1.0 M solution of HCl in diethyl ether (4 ml, 4 mmol) at 25 �C. After 24 h, the reactionmixture was poured into n-hexane (Junsei Chemical Co., Ltd., Japan) to precipitatea polymer, which was separated from the supernatant by decantation. The obtainedpolymer was re-dissolved in CH2Cl2 (Jin Chem. Pharm. Co., Ltd., Korea) and thenfiltered. The polymer solution was concentrated by a rotary evaporator and dried ina vacuum to give a colorless polymer of quantitative yield [16].

2.2. Expansion of ADSC

ADSC were prepared and seeded in 100-mm culture dishes at a density of4�105 cells/dish, as previously described [15]. Cells were maintained in ModifiedEagle Medium alpha (MEM-a, Hyclone, USA) containing 10% FBS (Gibco, USA) and 1%antibiotic/antimycotic solution (Gibco, USA) at 37 �C in a humidified 5% CO2

atmosphere.

2.3. Myogenic induction of ADSC in vitro

Expanded ADSC were obtained by trypsinization using a 0.25% Trypsin/EDTAsolution (Gibco, USA). Cell pellets were collected by centrifugation at 150g for 5 min

ic-induced ADSC in vitro, examined by RT-PCR. (B) Detection of MyoD (FITC) and MHCere stained with the DNA-binding dye, DAPI. Magnification 200�.

Fig. 2. Macroscopic observation of newly formed tissue in MPEG–PCL–only graft, ADSC/MPEG–PCL grafts without VEGF, and ADSC/MPEG–PCL grafts with VEGF165.

M.H. Kim et al. / Biomaterials 31 (2010) 1213–1218 1215

at 4 �C, and resuspended in myogenic medium (MEM-a supplemented with 10% FBS,5% horse serum, 1% antibiotic/antimycotic solution, and 50 mM hydrocortisone[Sigma, USA]). Cells were cultured in vitro in myogenic medium for 11 days to inducemyogenesis.

2.4. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis

Total RNA was isolated from myogenic-induced ADSC in vitro on days 4, 6 and 11using Qiazol� lysis reagent (QIAGEN Sciences, USA). Total RNA (1 mg) was reversetranscribed using Superscript II reverse transcriptase (Invitrogen, USA) and oligo-dTprimers to generate complementary DNA (cDNA). Synthesized cDNA was used astemplate for amplification of human MyoD, human myosin heavy chain (MHC) andglyceraldehyde phosphate dehydrogenase (GAPDH; control) in PCR reactions con-taining Taq polymerase and the primers listed in Table 1.

2.5. Immunostaining of cultured cells

Immunofluorescence staining was performed using mouse anti-human MyoD(Santa Cruz Biotechnology Inc., USA), mouse anti-human MHC (Upstate Biotech-nology Inc., USA), mouse anti-human dystrophin (Santa Cruz Biotechnology Inc.,USA) and mouse anti-human smooth muscle actin-a (SMA-a) antibodies (R&DSystems, Inc., USA). Cells cultured in vitro on sterilized cover slides, or slide-mounted, 10-mm-thick frozen sections obtained from animal experiments werefixed in acetone for 15 min at room temperature (RT) and then rinsed with PBS.Following additional 15-min rinses with PBS/0.1% Tween-20, the slides were incu-bated for 1 h at RT in PBS/0.1%Tween-20/1% BSA containing 20% normal goat ordonkey serum. Specimens were then incubated with primary antibody for 1.5 h atRT. After rinsing with PBS/0.1% Tween-20, slides were incubated with FITC-conju-gated goat anti-mouse IgG antibody (Santa Cruz Biotechnology Inc., USA) orCy3-conjugated donkey anti-mouse IgG antibody (Jackson Immune Research, USA)for 2.5 h at RT. Slides were then rinsed with PBS/0.1% Tween-20 and incubated withthe nuclear stain, 40 ,6 diamidino-2 phenyl indole dihydrochloride (DAPI) (SigmaAldrich Inc., USA), for 5 min at RT. Finally, slides were rinsed with PBS/0.1% Tween-20, mounted with aqueous mounting medium and observed under a fluorescencemicroscope (Olympus 1X71, Olympus Corporation, Japan).

2.6. Animal experiments

Twelve male nude mice (BALB/c-nu, 6 weeks old, Orient Bio Inc., Korea) weredivided into the three experimental groups (four mice/group): (1) MPEG–PCL alone,(2) MPEG–PCL/myogenic-committed ADSC mixture with 200 ng VEGF165 (R&DSystems, Inc., USA), and (3) MPEG–PCL/myogenic-committed ADSC mixture withoutVEGF. The indicated solutions (300 ml) were injected subcutaneously into the necksof mice using a 23-gauge needle. Mice were sacrificed by CO2 inhalation after 2, 4and 6 weeks. Newly formed muscle tissues obtained from the neck cavity area wereanalyzed by Masson trichrome (MT) staining and immunofluorescence staining.

3. Results

3.1. Confirmation of gel–sol transition of the MPEG–PCL solution

The temperature-dependent phase-change behavior of theMPEG–PCL solution (20 wt%) was confirmed by warming thesolution to 37 �C in a water bath. At 37 �C, the MPEG–PCL solutionwas transformed into gel state, and returned to a solution state atroom temperature.

3.2. Myogenic differentiation of ADSC in vitro

ADSC collected and expanded in basal medium were switched tomyogenic differentiation medium containing FBS, horse serum andhydrocortisone. Myogenic induction was maintained for 11 days invitro. On days 4, 6 and 11, myogenic-induced ADSC were collected andanalyzed for the myogenic markers, MyoD and MHC, by RT-PCR(Fig. 1A) and immunofluorescence staining (Fig. 1B). MyoD geneexpression was detected as early as day 4 by RT-PCR analysis, then

Fig. 3. (A) MHC and dystrophin expression were detected in ADSC/MPEG–PCL (a–h) and ADSC/MPEG–PCL treated with VEGF165 (i–p) at 4 and 6 weeks, and in native muscle (q–t) byimmunofluorescence staining. Expression of MHC and dystrophin was detected in newly formed muscle tissue of nude mouse injected with ADSC/MPEG–PCL containing VEGF165 asearly as 4 weeks (i–l) and was maintained for 6 weeks (m–p). ADSC/MPEG–PCL without VEGF expressed MHC and dystrophin at 6 weeks (e–h). (B) MT staining of ADSC/MPEG–PCLwithout VEGF (u, v), and ADSC/MPEG–PCL with VEGF165 (w, x) at 4 and 6 weeks; (y) MT staining of native muscle. Magnification 400�.

M.H. Kim et al. / Biomaterials 31 (2010) 1213–12181216

gradually decreased; in contrast, MHC gene expression was detectedon day 6 and continued throughout the 11-day in vitro culture period(Fig. 1A). MyoD protein, detected by immunofluorescence staining,was steadily expressed from day 4 and was maintained to day 11, butMHC was only weakly expressed during this in vitro culture period(Fig. 1B).

3.3. Effect of VEGF165 on muscle regeneration

After 2, 4 and 6 weeks, MPEG–PCL/ADSC grafts were macroscopi-cally examined (Fig. 2), and newly formed muscle tissues wereanalyzed by MT staining (Fig. 3). Mice injected with MPEG–PCL aloneexhibited an outer fibroblastic wall formed by cells of mouse originand showed no muscle tissue-like structures. However, in miceinjected with MPEG–PCL/ADSC with or without VEGF165, newlyformed muscle tissue was observed over a broad region (Fig. 3B).Expression of the mature muscle markers, MHC and dystrophin, wasdetected in both ADSC-containing groups (Fig. 3A). This was especiallymarked in the VEGF165-treated MPEG–PCL/ADSC graft, which showedmuch earlier and wider development of new muscle tissue comparedto the MPEG–PCL/ADSC matrix lacking VEGF (Fig. 3A and B).

3.4. Increased angiogenesis in newly formed muscle tissue withVEGF165 treatment

New blood vessel formation was observed by immunofluores-cence staining of tissue sections using an antibody against SMA-a.

In the VEGF165-containing cell graft, vessel formation graduallyincreased with in vivo cultivation. At 4 weeks, a number of newlyformed blood vessels were evident in the area adjacent to muscleregeneration. Compared to the MPEG–PCL/ADSC–only graft, theVEGF165-treated graft showed much more abundant SMA-a–posi-tive sites at 4 weeks, and a better vessel formation pattern at6 weeks (Fig. 4). Thus, treatment of stem cell grafts with VEGF165

induced enhanced muscle regeneration and blood vessel formationin vivo.

4. Discussion

A reliable cell source for regenerative medicine and tissue engi-neering applications is very important for generating a tissue-engineered structure. Because their properties satisfy basic tissueengineering criteria, ADSC could be an ideal source of stem cells [14].

In our previous work, we showed that ADSC embedded in aninjectable PLGA matrix differentiated into muscle tissue in vivo [15].In the current study, we sought to identify an ideal scaffold systemfor muscle tissue engineering and improve vascularization for thickcomplex tissue grafts. We used the diblock polymer, MPEG–PCL, asinjectable scaffold in these experiments. This biomaterial, unlikea number of other injectable scaffolds, is a thermosensitive hydrogel.Once subcutaneously injected under the skin, MPEG–PCL forms a gelat body temperature [4].

VEGF is a potent mediator of angiogenesis, both during develop-ment and in the adult. Among the various VEGF subtypes, VEGF165 is

Fig. 4. SMA-a (Cy3) expression and nuclei (DAPI) were detected by immunofluorescence staining. (a–c) MPEG–PCL alone, (d–f) ADSC/MPEG–PCL without VEGF and (g–i) ADSC/MPEG–PCL with VEGF165 after 2, 4 and 6 weeks. VEGF165 treatment enhanced blood vessel formation. Arrow indicates blood vessel. Magnification 400�.

M.H. Kim et al. / Biomaterials 31 (2010) 1213–1218 1217

particularly dominant and potent [11]. Human VEGF165-inducedmyoblasts have been shown to produce myogenesis with concomi-tant angiogenesis in the regenerative heart [7]. Because increasedangiogenesis induced by VEGF165 may improve muscle function inischemic tissues [6,8,9], we investigated whether VEGF165, whencombined with ADSC and a new thermosensitive scaffold, could besuccessfully employed as a vascularizing tool for tissue engineering ofthick complex muscle tissue.

Our scaffold successfully supported preconditioned ADSC, andallowed them to differentiate into mature muscle tissues in vivo(Fig. 4). Newly formed tissue in MPEG–PCL – alone grafts was mostlycomprised of an outer layer of fibroblastic tissue that arose from cellsthat most likely originated from nude mouse skin. By contrast,MPEG–PCL/ADSC grafts with or without VEGF showed evidence ofmuscle differentiation. This was much more pronounced in VEGF165-treated stem cell grafts, which showed much earlier muscle tissueformation and significantly enhanced blood vessel formation (Figs. 3and 4). In the VEGF165-treated stem cells graft, blood vessel densitygradually increased and muscle tissues were generated within theMPEG–PCL/ADSC matrices at 4 weeks. At 6 weeks, MT stainingshowed that VEGF165-treated stem cells grafts formed maturemuscle tissue that was similar to native muscle tissue.

5. Conclusions

Here we show that ADSC of human origin and in vivo gel-forming MPEG–PCL scaffolds provided an appropriate environmentfor cellular growth and expansion. We further showed that VEGF165

increased angiogenesis function, generating newly formed bloodvessels necessary for the delivery of nutrients required for muscleregeneration. The limiting factor in the engineering of thickcomplex tissues such as skeletal muscle is vascularization, which isnecessary to maintain cell viability during tissue growth and for the

induction of structural organization. Our results suggest a means toovercome this obstacle, showing that the combination of ADSC andin vivo gel-forming MPEG–PCL with VEGF165 might serve asa suitable non-invasive biomaterial for clinical muscle regenerationapplications. Although elucidation of the detailed mechanismsunderlying muscle regeneration will require further research, webelieve that the muscle tissue engineering studies described heremay contribute to the field of muscle regeneration and cell therapy.

Acknowledgements

This study was supported by grants from the Asan Foundation(#2008-052), Seoul, Korea and from Korea Science and EngineeringFoundation (KOSEF), Korea (# R11-2002-097-06004-0).

Appendix

Figures with essential color discrimination. All figures of thisarticle are difficult to interpret in black and white. The full colorimages can be found in the online version, at doi:10.1016/j.biomaterials.2009.10.057.

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