in vitro and in vivo evaluation of l -lactide/ε-caprolactone copolymer scaffold to support myoblast...

In Vitro and In Vivo Evaluation of L-Lactide/e-Caprolactone Copolymer Scaffold

to Support Myoblast Growth and Differentiation

Balaji Bandyopadhyay, Viru Shah, Memtombi Soram, Chandra Viswanathan, and Deepa GhoshTissue Engineering Group, Regenerative Medicine, Reliance Life Sciences Pvt. Ltd., 282-TTC Area of MIDC,Rabale, Navi-Mumbai, Maharashtra, India 400701

DOI 10.1002/btpr.1665Published online December 20, 2012 in Wiley Online Library (wileyonlinelibrary.com).

Skeletal muscle regeneration involves the activation of satellite cells to myoblasts, fol-lowed by their proliferation and fusion to form multinucleated myotubes and myofibers. Thepotential of in vitro proliferated myoblasts to treat various diseases and tissue defects canbe exploited using tissue-engineering principles. With an aim to develop a biocompatibleand biodegradable scaffold that supports myoblast growth and differentiation, we havedeveloped a porous sponge with 70/30 L-lactide/e-caprolactone copolymer (PLC) using aphase inversion combined with particulate leaching method. Degradation studies indicatedthat the sponge retained its structural integrity for 5 months in vitro and had undergonecomplete biodegradation within 9 months in vivo. The sponge supported human myoblastsattachment and its proliferation. Myoblasts seeded on the PLC sponge differentiated andfused in vitro to form myotubes expressing myosin heavy chain. Histological and molecularanalyses of the PLC scaffolds seeded with green fluorescent protein-labeled human myo-blasts and implanted ectopically under the skin in SCID mice demonstrated the presence ofmultinucleated myotubes expressing human muscle-specific markers. Our results suggest thatPLC sponges loaded with myoblasts can be used for skeletal muscle engineering or forinducing muscle repair. VVC 2012 American Institute of Chemical Engineers Biotechnol.Prog., 29: 197–205, 2013Keywords: biocompatibility, biodegradation, muscle, myoblasts, scaffold

Introduction

Skeletal muscle regenerates by activating the satellite cellsor muscle precursor cells that are present beneath the basallamina, to form myoblasts. The activated myoblasts prolifer-ate and undergo differentiation and fusion to form myotubesand myofibers.1 Although skeletal muscle can self-repair, itis unable to restore significant tissue loss because of trauma,congenital defects, etc.2 Success with conventional surgicalreconstruction procedures using muscle tissue obtained fromlocal or distant sites lead to significant donor site morbiditycausing functional loss and volume deficiency. Transplanta-tion of exogenous myogenic cells (satellite cells and myo-blasts) has been proposed to increase the regenerativecapacity of skeletal muscle.3 Regeneration of damagedmuscles has been predominantly attempted by injecting smallvolumes of in vitro expanded myoblasts suspension directlyinto the damaged site. Drawbacks of this method includecell loss during its implantation as well as inhomogeneouscell distribution at the site of damage. The limited success ofmyoblasts implantation to enhance skeletal muscle regenera-tion could thus be related to its poor survival and migrationin the transplanted tissue.4 In cell therapy, to induce repair,the cells are either directly injected into the damaged muscleor delivered to the site of damage on suitable scaffolds.5,6

The materials used in the scaffold preparation should be

biocompatible, biodegradable, and support cell attachment,

proliferation, and differentiation. Materials of biological ori-

gin such as collagen, fibrin, chitosan, and alginate have been

used for myoblasts delivery or muscle tissue engineering.7–9

Some of the drawbacks facing the use of materials of biolog-

ical origin include its batch-to-batch variation and induction

of immunological response. To overcome these issues,

synthetic biocompatible polyester materials such as poly(e-caprolactone) (PCL), poly(lactic acid) (PLA), poly(glycolic

acid) (PGA), and their copolymers have been tested.

Scaffolds in two-dimensional (2D)10,11 and three-dimen-sional (3D)12,13 forms have been used for myoblast culture.Compared to cells grown in 2D, the cells grown in 3D areknown to differ considerably in their morphology as well ascell–cell and cell–matrix interactions with the latter provid-ing an in vivo-like environment.14 Although electrospun non-woven membrane scaffolds provide a 3D environment to themyoblasts, the membranes however are extremely thin (\1mm) thereby limiting their cell-loading capacity.15

To overcome some of the limitations, we have developeda scaffold in the form of a highly porous sponge. We hadprepared a biocompatible and biodegradable synthetic 3D-scaffold with 70/30 L-lactide/e-caprolactone copolymer(henceforth referred to as PLC) using a phase inversion com-bined with particulate leaching method.16 The large intercon-necting pores in the scaffold facilitated seeding of large

Correspondence concerning this article should be addressed toD. Ghosh at [email protected].

VVC 2012 American Institute of Chemical Engineers 197

number of cells. The sponge was biodegradable and biocom-patible and displayed adequate structural integrity. In vitro,the PLC scaffold supported human myoblasts attachment,proliferation, and differentiation. Histological and molecularanalyses of ectopically implanted sponges seeded with greenfluorescent protein (GFP)—expressing myoblasts demon-strated differentiation and fusion, indicating the suitability ofthe PLC sponge for use in skeletal muscle engineering aswell as for myoblasts delivery to encourage muscle repair.

Materials and Methods

Materials

PLC was purchased from Purac Biochem, Netherland.N,N-Dimethylformamide (DMF) was purchased from Merck,India. Myoblast culture media (SKGM-2 bullet kit) was pur-chased from Lonza. DMEM and all other cell culturereagents were purchased from Sigma. Plasticware for cellculture was obtained from NUNC. Calcium phosphate trans-fection kit was purchased from Promega, and antibodies(desmin and myosin 1A-heavy chain) were from Abcam.The cDNA of the GFP constructed into the lenti-viral vectorPrlSinDeco, was a kind gift from Dr. Wei Li (Department ofDermatology, University of Southern California).

Human primary myoblasts used in the study were isolatedfrom skeletal biopsies obtained from the biceps or triceps ofdonors undergoing elective surgery after receiving informedconsent, which has been approved by an independent institu-tional ethics committee.

Animals

SCID mice and Wistar rats were bred in-house. Allanimals were handled in accordance with the CPCSEAguidelines for the welfare of laboratory animal practices laiddown by the Government of India.

Preparation of PLC sponge

The sponge was prepared by using a phase inversioncombined with particulate leaching method to generate a po-rous sponge.17 A 6% w/v PLC (inherent viscosity 1.59 dL/gm) solution was prepared in dimethyl formamide (DMF).The solution (5 mL) was mixed thoroughly with 5G sodiumchloride (particle size 200–300 lm) and poured in 3.5-cmglass petri-dishes and frozen O/N at �86�C. The spongeswere then immersed in water to induce phase inversion andsalt leaching and sterilized by exposure to gamma irradiationat 25 MRad (AV Processors, India).

Scaffold characterization

The structural characteristics of the sponge were analyzedusing a FEI Inspect Scanning Electron Microscope, QuantaInspect (Oxford Instruments, Switzerland). The average poresize was calculated from 25 fields obtained from five differ-ent scanning electron microscopy (SEM) images using ImageJ software.

Degradation studies

In Vitro Studies. The in vitro degradation study was con-ducted as described by Holy et al.18 Briefly, 0.5-cm-diameterdiscs were cut out from the sponge and weighed in triplicate

and incubated in normal sterile saline at 37�C. The pH of thesolution was monitored regularly during the study. At definedintervals, triplicate samples were removed from the solution,washed with H2O, and dried under vacuum. The driedsamples were evaluated for changes in mass, its structural in-tegrity and molecular weight. While the structural integrity ofthe discs was evaluated by its intactness during handling, theaverage molecular weight was determined by gel-permeationchromatography in tetrahydrofuran at ambient temperature(Series 200, Perkin Elmer). Weight loss was calculated as theratio between difference in initial and final weight to initialweight. Each experiment was performed in triplicate, theresults of which are presented as the mean � SD.

In Vivo Studies. In vivo degradation of the sponge wasassessed in male Wistar rats. Briefly, the rats (12) were anes-thetized with a cocktail of 80 mg/kg ketamine, 40 mg/kg xyla-zine, and 0.05 mg/kg atropine by intraperitoneal injection.Hair on the dorsal side of the rats was shaved and skin cleanedwith 70% ethanol. Small subcutaneous incisions of about 1cm were made on the dorsal side of the animals and the indi-vidual sponges (0.5 cm diameter) were inserted into thepouches. The incisions were sutured according to standardpractice and the animals were housed in individual cages. Atregular intervals of 1, 3, 6, and 9 months after implantation,the rats (n ¼ 3) were sacrificed, and the sponges were excisedand microscopically evaluated after staining with hemotoxylinand eosin (H&E) using a standard procedure.

Myoblasts isolation, culture, and characterization

Skeletal muscle biopsies received from the hospitals wererinsed in Hanks buffered saline solution (HBSS) and theirsurface was decontaminated by immersing in Povidone-Io-dine (Win Medicare, India) for 1–2 min. The tissues werefurther incubated for 20 min serially in 10X, 5X, and 1Xconcentration of 100X ampicillin-amphotericin-streptomycin(AAS) solution (Gibco).19 The tissues were chopped intosmall pieces and digested in a solution containing a mixtureof 1.2 units of dispase and 4 mg/mL of collagenase IV (1:1),for 30 min at 37�C with intermittent shaking. The resultingtissue suspension was passed through 70-lm strainer (BectonDickinson) and centrifuged for 5 min at 1200 rpm. The cellpellet was then resuspended in myoblast growth media(SKGM-2 bullet kit from Lonza) supplemented with 10%fetal bovine serum (Lonza), plated in tissue culture dishes,and incubated at 37�C in a humidified atmosphere containing5% CO2. To obtain an enriched myoblast population, theunattached cells in the dishes were transferred after 48 h tocollagen-I (Sigma) coated plates (100 ng/mL). Media waschanged every third day in the coated dishes till the cellsreached 70–80% confluency. The cultured myoblasts werepurified by MACS

VR

separation (Millteny Biotec) using anti-human desmin antibody. Differentiation of the purified myo-blasts to myotubes was induced by culturing highly confluentmyoblasts ([80% confluency) in differentiation media con-taining DMEM and 2% horse serum (Lonza) for 2 weeks.

Myoblasts were identified by standard immunofluorescencemethod. Briefly, cells were fixed with cold acetone for 20 minfollowed by incubation with monoclonal anti-human desminantibody (1:50). Positive cells were identified by counterstain-ing with FITC-conjugated anti-mouse antibody (1:500) (BDBioscience). Differentiated myoblasts were identified afterincubation with monoclonal myosin heavy chain (MHC) anti-body (1:100), followed by Alexa-fluor-568 conjugated

198 Biotechnol. Prog., 2013, Vol. 29, No. 1

secondary antibody. Total cells in each field were identifiedby 40-6-diamidino-2-phenylindole (DAPI, Sigma) staining andvisualized using fluorescence microscope (Nikon, Japan).

Cell culture on sponge

Cultured myoblasts were seeded on the PLC sponge using adynamic cell seeding method. The sponges (0.5 cm diameter)were soaked in media for 1 h at 37�C. To each sponge, 8 �106 myoblasts suspended in culture media were added andincubated at 20 rpm on a rotary shaker (PMR–30, Grand-Bio,UK) at 37�C in a CO2 incubator for 24 h. The seeding effi-ciency after 24 h was 99–100% based on the formula

Cell seeding efficiencyð%Þ ¼NðT0Þ � NðT24Þ� �

NðT0Þ� 100

where, N(T0) is the number of cells in the culture media atthe time of seeding and N(T24) is the number of cells remain-ing in the media after 24 h.

The sponges were maintained on the shaker throughoutthe culture period to minimize hypoxic conditions within thesponge.

Cell growth assay

Myoblast growth on the PLC sponge was assessed in vitrousing the methyl thiazolyl tetrazolium (MTT, Sigma)assay.20 Human myoblasts (3 � 104) were seeded as men-tioned above in 0.5 cm discs of the sponge in triplicate.MTT (0.5 mg/mL) was added to the sponges at 24, 48, 72,and 96 h after seeding. Cell proliferation was indirectlydetermined by the ability of the viable cells to convert solu-ble MTT to insoluble formazan crystals. The crystals weredissolved in DMSO and the absorbance was determined at570 nm using UV–vis spectrophotometer (Shimadzu, Japan).

Cell attachment and differentiation assay

To study the attachment and differentiation of myoblasts inthe PLC sponge, myoblasts seeded sponges were transferredto the differentiation media at the end of 48 h after cell seed-ing. A part of the sponge was rinsed in phosphate bufferedsaline and fixed with 10% formalin for SEM studies. After 2weeks in differentiation media, the cell-seeded sponge wasfixed as described above. The fixed sponge was incubatedserially in 50, 75, 90, and 100% ethanol for 15 min each,vacuum dried and examined under an FEI Inspect ScanningElectron Microscope, Quanta Inspect (Oxford Instruments,Switzerland). The other part of the sponge was processed forimmunofluorescence studies. Briefly, the above sponges wereembedded in paraffin blocks using a standard protocol. Cellsin the deparaffinized sections were identified by staining withdesmin and myotubes were detected by staining with MHCantibodies as indicated above and detected using Alexa-fluor-568 secondary antibody (Molecular Probes, Invitrogen,Oregon) and visualized under a Apotome 2 Observer.Z1 DiskScan microscope (Carl Zeiss, Germany). Total cells in eachfield were identified by counterstaining with DAPI.

Transduction of GFP into myoblasts usingLenti-viral vector

The lentivirus-derived vector pRRLsinhCMV was insertedwith eGFP cDNA using EcoRV. This construct was used to

cotransfect 293T cells together with packaging vectorspCMVDR8.2 and VSVG. Typical viral titers were 1�7 �106 transduction units/mL as measured by previouslydescribed method.21 The cell infection efficiency was 66%(data not included) as monitored by the percentage of cellspositive for GFP expression using FACS analysis [FACSCalibur (E3851), (Beckton Dickenson)]. The above cellswere used in the following ectopic implantation study.

Ectopic implantation

SCID mice (6) were anesthetized and prepared as men-tioned under the degradation studies. In each animal, anempty PLC sponge that served as a control was implantedsubcutaneously under the skin in the left dorsal side of theanimals and 8 � 106 GFP-labeled myoblasts-seeded spongewas implanted in the right dorsal side of the animal. Theanimals (n ¼ 3) were sacrificed at the end of 2 and 4 weeksafter implantation, and the implants were excised and proc-essed for molecular and histological analysis.

Histological analysis

For histological analysis, the excised sponges were fixedin 10% buffered formalin, and embedded in paraffin blocks.The GFP-positive myoblasts in each section (4–5 lM) wereidentified by their green staining using a Apotome 2 Observ-er.Z1 disk scan microscope (Carl Zeiss, Germany). Humanmuscle-specific marker, MHC was detected in the sectionsusing standard immunofluorescence method as mentionedabove. While positive cells expressing the specified proteinswere detected using Alexa-fluor-568 secondary antibody(Molecular Probes, Invitrogen, Oregon), total cells werevisualized by staining with DAPI. The cells were analyzedusing Apotome 2 Observer.Z1 Disk Scan microscope (CarlZeiss, Germany).

Reverse transcription-polymerase chain reaction analysis

Total RNA was extracted from the excised sponges usingthe RNeasy Mini Kit (Qiagen) according to the manufac-turer’s protocol. The cDNA was prepared using the Super-Script First-Strand System (Invitrogen). Gene amplificationwas carried out in 25-lL reaction volume each containing1.5 lL of cDNA in PCR Supermix (Invitrogen). PCR condi-tions used were 5 min at 95�C followed by 95�C for 45 s,55�C for 45 s, and 72�C for 45 s with a final extension of10 min at 72�C for a total of 35 cycles. Amplification wasperformed in a thermal cycler (Biometra, Germany) with thefollowing specific primers designed from sequences obtainedfrom Genbank. Human desmin, 303 bp, (F-CGAGCTGCTG-GACTTCTCAC, R-AGGTCGTCGAGCAGGTTGTC) andhuman myogenin, 638 bp (F-CCATGGAGCTGTATGAGACATC, R-ATCTTCCACTGTGATGCTGTCC), were used toconfirm the presence of undifferentiated and differentiatedhuman myoblasts. Human glyceraldehyde phosphate dehy-drogenase (GAPDH), 750 bp (F-GGGCTGCTTTTAACTCTGGT, R-TGGCAGGTTTTTCTAGACGG) and mouse betatubulin, 317 bp (F-GGAACATAGCCGTAAACTGC, R-TCACTGTGCCTGAACTTACC) were used to identify humanand murine cells respectively. Detection of the PCR productswas performed after separation on a 1.5 % agarose gel andthe PCR products were visualized on ImageMaster (Amer-sham Biosciences).

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Results

Scaffold characterization

Porosity. PLC sponges (3 cm diameter and 2–3 mmthickness) with an average pore size of 322.37 � 67.61 lmwere fabricated using phase inversion combined with aparticulate leaching method. The cross-sectional SEMimages revealed a homogeneous distribution of intercon-nected pore structures (Figure 1).

In Vitro Degradation. In vitro degradation of the spongewas evaluated following its incubation in sterile normalsaline solution at 37�C. The sponges maintained its structuralintegrity up to 5 months and subsequently disintegrated bythe sixth month. During the study period, a significant fall inmolecular weight was observed in the sponge material withno substantial weight loss (Figure 2).

In Vivo Biodegradation. The sponges implanted ectopi-cally in rats were excised at the end of 1, 3, 6, and 9 monthsafter implantation and stained with H&E (Figure 3). Crosssections of the excised sponges showed host cell migration.

One month after its implantation, the surface of the spongehad a smooth uniform appearance (Figure 3A). Progressivedegradation was seen in the sponges at 3 (Figure 3B) and 6(Figure 3C) months after implantation as evidenced by thedisruption of the smooth surface of the scaffold. Very fewgiant cells were observed at 3 and 6 months around the scaf-fold. At 9-month postimplantation, the PLC material as wellas the giant cells had mostly disappeared leaving behind atissue like structure (Figure 3D).

Cultured myoblasts characterization

Adult human myoblasts were isolated and culturedin vitro. Spindle shaped morphology of myoblasts in culturewas seen under phase-contrast microscope (Figure 4A). Thepurity of the cultures at passage 2 was assessed by their pos-itive staining with desmin antibody (Figure 4B). Immuno-fluoresence staining indicated that [99% cells weremyoblast. Differentiation of the cultured myoblasts in low-serum media resulted in multinucleated myotubes asobserved under phase-contrast microscope (Figure 4C) thatexpressed MHC (Figure 4D).

In vitro response of myoblasts seeded in the PLC sponge

PLC scaffold support myoblast proliferation as seen in theMTT assay. The increase in absorbance observed with timeindicated that the PLC scaffold supports myoblast prolifera-tion (Figure 5). Similar to the cells in 2D, myoblasts seededin the PLC scaffold attached to the sponge and when incu-bated in differentiation media for 2 weeks had undergonedifferentiation (Figure 6). SEM analysis of myoblasts seededin PLC scaffold at 48 h after seeding appeared round(Figures 6A,B) while after 2 weeks in differentiation media;the cells had differentiated and formed myotube like struc-tures (Figures 6C,D). Paraffin embedded sections of theabove scaffolds on fluorescence imagining demonstrated pos-itive staining of cells with desmin (Figure 7A) and MHC(Figure 7D) antibodies, respectively. Same sections werecounterstained with DAPI (Figures 7B,E).

In vivo response of myoblasts seeded in the PLC sponge

SCID mice were dorsally implanted on either sidewith plain PLC sponges (control) or sponges seeded with

Figure 1. Scanning electron micrographs of PLC sponge (A) at low magnification and (B) at higher magnification.

Figure 2. In vitro degradation of the PLC sponge. Average mo-lecular weight and average weight of 0.5-cm discs ofPLC sponge incubated in sterile saline and analyzedat different time points.

A statistically significant decrease in molecular weight (repre-sented as bars) is observed with time (P ¼ 0.0028) calculatedusing linear regression analysis. During the same period, noconsiderable change in weight is observed (represented as aline graph).


GFP-labeled myoblasts respectively. The sponges at the endof the study were excised and part of it was processed forimmunohistological analysis. The deparaffinized sectionswere stained with MHC antibody and DAPI before visualiz-ing under a fluorescence microscope. Control sponges did

not show the presence of GFP positive cells. Representativepictures of only myoblasts seeded scaffolds are shown in Fig-ure 8. Sponges seeded with GFP-labeled myoblasts at 2 and 4weeks after implantation indicated the presence of GFP posi-tive cells. At 2-weeks post implantation GFP-positive cells

Figure 3. Histological observation of H&E-stained PLC sponge.

The structure of the PLC material (indicated with black arrows) changes with time. (A) At 1 month after implantation, the PLC material has asmooth appearance. Degrading surface of the PLC sponge seen at (B) 3 months and (C) 6 months after implantation. Giant cells are seen near thePLC polymer (white arrow) (D) At 9 months, the polymer has almost completely disappeared. Magnification bar represents 100 lM.

Figure 4. Characterization of myoblasts.

(A) Phase-contrast micrograph of cultured myoblasts in growth media. (B) Myoblasts are identified by their positive staining with desmin antibody.(C) Phase-contrast micrograph of multinucleated myotubes. (D) Myotubes-stained positive with myosin 1A—Heavy chain antibody. Nuclei in B andD are counterstained with DAPI. Magnification bar represents 50 lM.


had fused to form small myotube-like structures (Figure 8A),which at 4 weeks after implantation appeared more disctinctand contained several nuclei suggesting further fusion (Figure8C). Differentiation of the implanted cells was further con-firmed by immunohistological staining of the above sectionswith MHC antibody (Figure 8B,D).Total cells in the field wasdetected with DAPI staining.

A part of the implanted scaffolds that were excised fromthe animals was analyzed for the presence of human-specificmuscle markers. As indicated in Figure 9, while onlymyoblast-seeded scaffolds express human-specific desminand myogenin genes at 2 and 4 weeks after implantation,

expression of these genes were absent in the control sponges.A similar expression pattern was seen with human GAPDH,a housekeeping gene. Beta-tubulin, a murine-specific house-keeping gene, was used as internal control for equal loadingof RNA obtained from control and myoblast seeded sponges.

Discussion

Myoblast delivery to degenerated/damaged muscle on anappropriate scaffold that supports its growth and proliferationmight overcome some of the limitations of direct cell appli-cation. Although attempts to deliver myoblast have beenmade using natural polymers like collagen22 and fibrin,8

issues of batch to batch variability, immunogenicity anduncontrolled rate of degradation do exist. Synthetic polymerssuch as PGA, PLA, PCL, and their copolymers have been ofspecial interest to many researchers because of their biocom-patibility, mechanical, and biodegradation properties.Besides, these have the advantages of batch-to-batch consis-tency and their rate of degradation can be controlled.23,24

In an attempt to prepare a suitable scaffold that supportsmyoblast attachment, growth, and differentiation, we havedeveloped a sponge with PLC, a biodegradable syntheticaliphatic polyester copolymer. Scaffolds prepared with PLAare known to be hard, stiff, and nonflexible. Addition ofe-caprolactone to PLA during the scaffold preparationreduces the stiffness of the resulting scaffold. Our selectionof 70:30 L-lactide/e-caprolactone copolymer compositionresulted in a soft and highly flexible sponge.

A PCL sponge prepared by extrusion-particulate leachingmethod has been used for culturing smooth muscle cells.25 Car-tilage tissue was engineered using a nanofibrous PCL-PLLA

Figure 5. PLC sponge support myoblasts proliferation. Growthof myoblasts in the PLC sponge was assayed usingthe MTT assay.

Proliferation of myoblasts in the PLC sponge increases withtime (P\ 0.03). Data is represented as mean � SD, N ¼ 3.

Figure 6. PLC sponge supports myoblast differentiation.

Human myoblasts in the PLC sponge 48 h after seeding at low magnification (A) and higher magnification (B). The rounded cells are indicated withwhite arrows. The above constructs observed after 2 weeks in differentiation media at low magnification (C) and higher magnification (D). The myo-tubes are indicated with black arrows. Magnification bar in A and C is 50 lM and in B and D is 100 lM.


scaffold that was fabricated using a thermally induced phaseseparation method.26 To the best of our knowledge, we have,for the first time, prepared a PLC sponge using a phase inver-sion-combined with a particulate leaching method. The result-ing porous scaffold had largely open and interconnected poreswith an average pore size of 322.37 � 67.61 lm. The high cellseeding efficiency seen with the sponge could be due to thelarge pores seen in the PLC sponge.

The degradation of aliphatic polyesters such as PCL, pol-y(D,L-lactide), and its copolymers mainly occurs by nonenzy-matic random hydrolytic cleavage of ester linkages.27 Thein vitro studies conducted in sterile saline solution demon-strated a drop in molecular weight of the polymer with timesuggesting that the PLC sponge might have undergonehydrolytic degradation. The integrity of the sponge wasretained for 5 months after which it completely disintegrated.

Figure 7. Myoblasts seeded in PLC sponge express muscle-specific markers.

Immunofluorescence images of myoblasts in growth media expressing desmin (A) and in differentiation media expressing MHC (D). Cells in thesame section counterstained with DAPI (B and E). Costaining of cells with specific antibodies and DAPI are shown in (C and F). Scale bar repre-sents 50 lM.

Figure 8. Myoblasts seeded in PLC sponges and implanted in vivo display myogenesis.

Fluorescence micrographs of myoblasts at 2- and 4-week postimplantation. The implanted GFP positive cells (A and C) have differentiated andformed myotube-like structures. Cell differentiation is confirmed by MHC staining (B and D). Total cells in the field are identified by DAPI. Scalebar represents 20 lM.


The degradation process of lactides and caprolactonesnormally occurs in two stages. The first stage involves thediffusion of water into the amorphous region of the polymercausing simple hydrolytic chain scission of the ester groupsfollowed by the second stage wherein degradation of thecrystalline areas of the polymer occurs.28 In the PLC sponge,the first stage might have predominated during the initialmonths reflected by the fall in molecular weight followed bythe degradation of the crystalline regions resulting in itscomplete loss of mechanical properties.

As the rate of degradation is determined by factors suchas configurational structure, copolymer ratio, crystallinity,molecular weight, morphology, stresses, amount of residualmonomer, porosity, and site of implantation, we had con-ducted an ectopic study in rats to determine the performanceof the PLC sponge in vivo. Following its subcutaneous im-plantation, the scaffold retained its structure and could bephysically excised at various time points. During its exci-sion, the scaffold was observed to be attached to the dermisand supported by small capillaries. Bolgen et al. hadimplanted electrospun PCL patches subcutaneously in rats,and evaluated the degradation of material with time. Theyhad reported that at the later stages of in vivo degradation,after the fragmentation of PCL chain into low-molecular-weight fragments, intracellular degradation can take place byphagocytosis.16 Histological analysis of the ectopicallyimplanted PLC sponge showed continuous degradation ofthe sponge and by 9 months most of the polymer hadcompletely degraded. The scaffold supported host cell migra-tion and tissue formation. The absence of inflammatory cellsin the scaffold indicates its biocompatible property.

Besides biodegradability and biocompatibility of thebiomaterial, properties such as cell attachment, proliferation,and differentiation of the muscle cells are critical for muscletissue engineering. Bramfeldt et al. had reported that theproliferation rate of rat myoblasts in PLGA scaffolds wasnot affected by varying the pore size.29 The high seeding

efficiency as well as the consistent increase in OD seen withtime in the MTT study indicates that the porous PLC spongesupported myoblasts proliferation. The differentiation ofmyoblasts to myotubes was demonstrated on collagen I-fibrinnanofibers by Beier et al.30 Myoblasts attachment and myo-tube alignment on electrospun scaffolds was attributed to thealignment of the fibers by providing the necessary directionalcues along with architectural and mechanical support.31 SEMstudies of myoblasts seeded in PCL sponge showed cellattachment and myotube-like aligned cells. While some suchmyotubes were in parallel orientation, many of them dis-played random arrangement. This could be attributed to theabsence of directional cues and lack of alignment structuresin the sponge. This drawback may be overcome by usingappropriate mechanical31 or electrical stimulation.32 Expres-sion of MHC, a muscle differentiation marker by the cellsseeded in the sponge confirms cell differentiation.

To study the in vivo response, PLC sponges seeded withGFP-labeled human myoblasts were ectopically implanted inSCID mice. The presence of GFP-labeled cells in cut sec-tions of myoblasts seeded sponges indicates viability of thecells in the sponge. As expected, no GFP positive cells weredetected in control sponges. Although the viability of thecells post its implantation was not analyzed in this study, thefunctional capacity of these implanted cells to differentiateand fuse has been demonstrated at 2 and 4 weeks by positivestaining with anti-human MHC antibody. At 2-weeks smallmyotube-like structures containing few nuclei were observed,whereas at 4 weeks large multinucleated myotube like struc-tures were seen, indicating the possibility of more cell fusionresulting in the formation of thicker tubes. Morphology ofthese tubes although distinct was not typical to thoseobserved in the skeletal muscle. This difference could be theresult of lack of appropriate signals provided to the cells atits ectopic site if implantation.

While human muscle-specific desmin and myogenin geneexpression was observed only in the human myoblast-seededsponges, control sponges did not express these genes con-firming the presence of the implanted cells in the sponge.Expression level of these genes at 4 weeks as compared to2 weeks was lower. The weaker signal could be attributed tothe increased migration of the host cells into the implantedscaffold, which would result in decrease in the proportion ofthe implanted cells as compared to the total cell number.This is confirmed by the strong expression of mouse b-tubu-lin gene as well as the presence of non-GFP-labeled cells inthe scaffold sections.

Conclusions

A major challenge facing successful myoblast transplanta-tion is maintaining the long-term survival and functionalityof the implanted cells. Different scaffolds have been tried toenhance the survival and regenerative capacity of variouscells in vivo. To be clinically useful, such scaffolds need tobe biocompatible and made with clinical grade components.As a new approach, we have evaluated a PLC sponge pre-pared using a defined GMP grade material as a potentialscaffold for myoblast growth and differentiation.

A material that can be used as a scaffold in tissue engi-neering must satisfy a number of requirements. Theseinclude biocompatibility, biodegradation to nontoxic productswithin the time frame required for the application, appropri-ate porosity to support cell growth and differentiation, and

Figure 9. Reverse transcription-polymerase chain reactionanalysis of cell-specific markers in PLC controlsponges and in myoblasts seeded sponges followingits ectopic implantation at the end of 2 (A) and 4 (B)weeks, respectively.

Ethidium bromide-stained 1.5% agarose gel showing cDNAamplified with human-specific desmin, myogenin, and GAPDHprimers. Murine-specific b-tubulin serves as internal control forequal loading of RNA. Lanes 1 and 3 shows the gene expres-sion in control sponges (without any human myoblasts),whereas 2 and 4 reflect gene expression in sponges seeded withhuman myoblasts.


suitable mechanical properties. The potential of the PLCscaffold to meet the above requirement is reflected in ourstudies. While we have demonstrated the ability of the cellsto survive and maintain its functionality for 4 weeks in vivo,long-term studies in an appropriate muscle damage modelwould give a better understanding of the tissue formation.

Acknowledgment

The authors acknowledge the encouragement and support ofReliance Life Sciences Pvt. Ltd. in carrying out the researchwork (www.rellife.com). The authors thank Dr. HarinarayanRao, Dr. Anirban Thakur, and members of LARS and TissueEngineering Group for their support in carrying out the researchwork. They are thankful to Dr. V. K. Gupta and his team mem-bers, Reliance Technology Group-Hazira for their support inSEM imaging. They are grateful to Dr. Wei Li, Department ofDermatology, University of Southern California, USA, for pro-viding GFP constructs.

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Manuscript received May 25, 2012, and revision received Oct. 26,2012.


in vitro and in vivo evaluation of l -lactide/ε-caprolactone copolymer scaffold to support myoblast...

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