Research ArticleUse of Self-Assembling Peptides to Enhance Stem CellFunction for Therapeutic Angiogenesis

Hyung Sub Park ,1,2 Geum Hee Choi,2 Daehwan Kim,1,2 Tae Woo Jung,2 In Mok Jung,1,3

Jung Kee Chung,1,3 and Taeseung Lee 1,2

1Department of Surgery, Seoul National University College of Medicine, Seoul, Republic of Korea2Department of Surgery, Seoul National University Bundang Hospital, Gyeonggi, Republic of Korea3Department of Surgery, Seoul Metropolitan Government Seoul National University Boramae Medical Center,Seoul, Republic of Korea

Correspondence should be addressed to Taeseung Lee; tslee@snubh.org

Received 5 February 2018; Accepted 21 May 2018; Published 13 June 2018

Academic Editor: Benedetta Bussolati

Copyright © 2018 Hyung Sub Park 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.

The use of nanomaterials for biomedical applications has become a promising field in regenerative medicine. Self-assemblingpeptides (SAPs) have been proposed as a good candidate because they are able to self-assemble into stable hydrogels andinteract with cells or molecules when combined together. This in turn can lead to the improved survival or action of cellsor molecules to obtain the desired effects. In this study, we investigated whether the combination of mesenchymal stemcells (MSCs) with SAPs could improve angiogenesis in ischemic hindlimbs of rats compared to MSC or SAP treatmentalone. The combination of MSCs and SAPs showed an overall higher expression of angiogenesis markers on fluorescentimmunohistochemical analysis and a lower degree of fibrosis and cell apoptosis, which in turn led to an overall tendencyfor improved perfusion of the ischemic hindlimbs. Finally, SAPs also showed the ability to recruit endogenous host MSCsinto the site of action, especially when modified to incorporate substance P as a functional motif, which when injected withexogenous MSCs, allowed for the dual presence of MSCs at the site of action. Overall, these results suggest that SAPs canbe applied with stem cells to potentiate angiogenesis, with potential therapeutic application in vascular diseases.

1. Introduction

Stem cell therapy for treatment of vascular diseases is apromising field of research. This is especially so for caseswhere ischemic insults and consequent end-organ perfusiondefects cannot be overcome. In the clinical setting, peripheralvascular diseases such as Buerger’s disease or ischemic dia-betic foot are related with poor wound healing outcomesand high amputation rates mainly due to the failed resolutionof the underlying ischemia using currently available revascu-larization methods. In such cases, an increase in angiogenesiscan overcome the situation by the formation and outgrowthof new vessels. The role of stem cells in therapeutic angiogen-esis has been widely investigated, and its possible applicationin multiple vascular diseases has also been studied previously[1, 2]. However, the problem with stem cell therapy lies in its

limited survival rate and durable effects, especially whenadministered by external injection [3]. Stem cells can beadministered locally or systematically, and depending onthe mode of administration, the target for stem cell potentia-tion may vary. In the case of systemic delivery, an improve-ment in stem cell homing can be a target for enhancedefficacy, while for local delivery, enhancing stem cell survivalcan be more important. In any case, new technologies arebeing studied to increase survival and prolong the effects ofexogenously administered stem cells. One of these includebiological scaffolds, which can provide a 3-dimensionalmicroenvironment in the form of extracellular matrices(ECMs) to protect the stem cells from apoptosis whileprolonging their effects in vivo.

Self-assembling peptides (SAPs) are one form of biologi-cal scaffold which typically consist of 8–16 amino acids with

HindawiStem Cells InternationalVolume 2018, Article ID 4162075, 12 pageshttps://doi.org/10.1155/2018/4162075

alternating hydrophilic and hydrophobic residues that assem-ble itself into a 3-dimensional configuration when adminis-tered into the site of action [4]. The hydrophilic portionconsists of alternatively repeating units of positively and neg-atively charged amino acids which form stable β-sheets inwater, which in turn combine to form flexible nanofiberhydrogels of 7–20nm in diameter under physiologic condi-tions [5, 6]. In this way, these hydrogels are able to support3-dimensional culture of not only different cell types, but alsosmaller proteins such as growth factors or chemokinesthrough noncovalent bonding, allowing for controlled deliv-ery and sustained release. These SAPs have the advantage ofbeing versatile, easy to manufacture, cheap, biocompatible,noncytotoxic, and biodegradable, making them an attractivesource for application in regenerative medicine [7]. SAPs canalso be modified in a wide variety of ways to provide uniquefeatures and perform specific functions, allowing for promis-ing applications in the field of drug delivery, tissue engineer-ing, and biosensor device applications [7–9]. SAPs are knownto have an ECM-resembling configuration for cell and pro-tein delivery, but they have also been shown to have intrinsicmesenchymal stem cell- (MSC-) recruiting ability, thus beingable to recruit circulating host MSCs into the site of action.One of these SAPs is known as RADA (16-I or 16-II), whichhas been demonstrated in previous animal studies to havethe ability to recruit host MSCs into ischemic limbs toimprove hindlimb perfusion [10]. Its potential for recruit-ment can be further enhanced if modified to combine afunctional motif in the sequence. Despite the numerousadvantages and potential applications of SAPs, there are alsodisadvantages, mainly related to limited availability of data.The kinetics and reproducibility of protein or cell deliveryin vivo has not been evaluated yet, and the extent of degrada-tion is not well known either. Further immunological testingis also needed.

In this study, we examined whether the combinedadministration of MSCs with SAPs into ischemic hindlimbsof rats could increase angiogenesis and decrease cell apopto-sis and fibrosis, probably by mechanisms related to anincrease in MSC survival, which in turn could lead toimprovements in perfusion. The role of SAPs in the recruit-ment of circulating host MSCs was also investigated, andthe possibility of a synergistic effect between exogenouslyadministered MSCs and endogenously recruited host MSCswas verified.

2. Materials and Methods

2.1. Isolation and Culture of Murine MSCs. MSCs were iso-lated from the femoral bones of 3-4wk-old Sprague-Dawleymale rats using the Ficoll-Hypaque medium (1.077 g/mL,GE Healthcare Life Sciences) and cultured in T-25 culturedishes using Dulbecco’s Modified Eagle’s Medium (DMEM,Sigma-Aldrich), 10% fetal bovine serum (FBS, Gibco BRL),100U/mL penicillin, 100μg/mL streptomycin (Gibco BRL),and 2mM L-glutamate (Gibco BRL) at 37°C and 5% CO2atmosphere. After 3–5 passages, cells were sorted using theMACS and CD105 MultiSort Kit (PE, Miltenyi Biotec Inc.).These cells were then characterized for CD105, CD73,

CD29, and CD45 (knownmarkers for rat bonemarrowMSCsin particular) [11, 12] with either mouse immunoglobulin-FITC or mouse immunoglobulin-PE using a BD FACSCali-bur™ Flow Cytometer (BD Biosciences). These cells werethen transfected with a lentivirus (Macrogen) tagged with agreen fluorescent protein (GFP) marker for use in in vitroand in vivo studies.

2.2. Self-Assembling Peptide Preparation. The self-assemblingpeptide used in this study was RADA 16-II (Peptron) havingthe configuration AcN-RARADADARARADADA-CONH2(hereinafter simplified as RADA), with its characteristicshaving been already described elsewhere [13]. RADA wasdissolved in sterile 295nM sucrose at 1% (wt/vol) and soni-cated for 30min.

2.3. In Vitro Culture and MTT Assay of MSCs and SAPs. Forcolony-forming unit fibroblast (CFU-F) identification,DMEM solution and 1.2% agar were mixed and placed oneach well in a 6-well plate. Once solidified, MSCs (2500 cells)or MSCs+RADA were mixed with 0.6% agar, dispensed ondifferent wells, and cultured at 37°C and 5%CO2 atmosphere.After 14 days of culture, the wells were stained with crystalviolet for 1 hr and observed under a microscope system(Olympus). Colonies were counted at 100x magnification in10 different random points.

For the MTT assay, a 96-well plate was used where MSCs(1× 104 cells) or MSCs+RADA were placed on differentwells with culture medium and cultured at 37°C and 5%CO2 atmosphere. A control group was also used where onlyculture medium was placed. Then, 10μL of theMTT solution(Cell Counting Kit-8, Dojindo Molecular Technologies, Inc.)was added into each well and placed in a CO2 incubatorwhere an ELISA reader (SpectraMax Plus 384, MolecularDevices) was used to measure the absorbance at 450nmwavelength at 0.5, 1, 1.5, and 2hr.

For live cell screening analysis, 1× 104 cells of MSCs(MSC group) or MSCs with RADA (MSC+RADA group)were placed in a 6-well plate and monitored using the Incu-Cyte Live Cell Analysis System (Essen BioScience) at 37°Cand 5% CO2 atmosphere. The culture media was changedat 3 days and the screening of cell growth was recorded bythe IncuCyte system software for 7 days.

2.4. Animal Preparation. Hindlimb ischemia models werecreated in 3-4wk-old Sprague-Dawley rats by applying con-tinuous isoflurane general anesthesia. A longitudinal incisionwas made in the left hindlimb at the thigh level, and thefemoral artery was identified. The femoral artery was metic-ulously dissected along its entire course and was removedafter ligation of the proximal and distal ends. Care was takennot to injure the surrounding structures including the femo-ral vein and nerve. A total of 32 rats were divided into 4groups: MSC, RADA, combination of MSCs+RADA, andcontrol. For the MSC group, 1× 107 cells were injected intra-muscularly at 4-5 different locations in the thigh immedi-ately after femoral artery excision and at postoperative day3. In the same manner, RADA was mixed in 200μL ofphosphate-buffered saline (PBS) and injected for the RADA

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group, and the combination group included a mixture of1× 107 MSCs and RADA in 200μL of PBS, while for thecontrol group, the same amount of saline was injected. Thisstudy was approved by the Institutional Animal Care andUse Committee (IACUC) of Seoul National UniversityBundang Hospital.

2.5. Measurement of Hindlimb Perfusion with Laser Doppler.Measurements of perfusion were carried out using a laserDoppler perfusion monitoring system (Periflux System5000, Perimed AB) immediately after the surgery and at days7, 14, and 28. A standardized protocol was used for the mea-surement of perfusion in an attempt to decrease the variabil-ity inherent to this method, where the anesthetized rats wereplaced in the same position with the leg fixed in a slightlyextended position and under a warm environment using aheating system. The probe was then placed at the same placeevery time on the forefoot and fixed with adhesive tapesagainst the table. The probe was set at 37°C and the measure-ment was carried out for 2min once the set temperature wasachieved. The mean perfusion was obtained as perfusionunits (PU) for each rat at different time points for the differ-ent groups.

2.6. Immunohistochemical (IHC) Staining and ReverseTranscriptase Polymerase Chain Reaction (RT-PCR). Thestudy rats were harvested at 7, 14, and 28 days after the mea-surement of perfusion by removing their thigh muscles andwere fixed in 10% formalin for 24hr. The tissues were thenembedded in paraffin and sliced at 5–7μm thickness. Immu-nohistochemical analysis was performed for MSC markersCD105, CD90, and CD29 by using their respective antibod-ies: anti-CD105/Endoglin antibody (Abcam), anti-CD90/Thy1 antibody (Abcam), and anti-CD29/integrin beta-1antibody (Abcam). For measurement of angiogenesis, IHCstaining was performed for angiogenesis markers using pri-mary polyclonal antibodies von Willebrand factor (vWF1 : 100, Dako), CD31 (1 : 200, Abcam), and α-smooth muscleactin (α-SMA 1 : 200, Abcam) at 4°C overnight and thenwith goat anti-rabbit IgG (rhodamine, Abcam) as secondaryantibody at 4°C for 3 hr. Nuclei counterstaining was per-formed with 4′,6-diamidino-2-phenylindole (DAPI, VectorLaboratories). Capillary density was measured by countingthe positive stains at 6 different random fields for 3 timesunder 100x magnification microscopy.

For RT-PCR analysis, harvested tissues were preserved at4°C in RNAlater (Qiagen) and the total RNA was extractedusing an RNeasy Mini Kit (Qiagen) according to the manu-facturer’s instructions. Quantification of the extracted RNAwas done at 260 and 280 nm wavelengths using a NanoDropSpectrophotometer (Thermo Fisher Scientific). The forwardand reverse primers used for CD31, α-SMA, vWF, andGAPDH (control) were as follows: CD31 forward 5′-GTGGAACTGGGGACAAAGAA-3′, reverse 5′-TCCGGATGAATTCTGAGGTC-3′; α-SMA forward 5′-TGCTGTCCCTCTAGCCTCT-3′, reverse 5′-GAAGGAATAGCCACGCTCAG-3′; vWF forward 5′-TGCTGCCCAGAGTATGAGTG-3′, reverse 5′-GATGTTCCTCTGCTGCACAA-3′; GAPDH

forward 5′-TGCCACTCAGAAGACTGTGG-3′, reverse 5′-GCATGTCAGATCCACAATGG-3′. PCR was performedusing a PTC-200 PCR Thermal Cycler (MJ Research) underthe following cycling conditions: 94°C for 2min (initial step),94°C for 30 s (denaturation), 53–55°C for 45 s (annealing),and 72°C for 30 s (elongation) for a total of 35 cycles; 72°Cfor 10min was used for the final extension. Electrophoresisof PCR products in 1-2% agarose gel with ethidium bromidewas performed and images were analyzed using a bioimageanalyzer EXT-20MX (Vilber Lourmat). Band intensities werenormalized against GAPDH.

2.7. Apoptosis and Fibrosis. Apoptosis at the cellular levelwas measured using the terminal deoxynucleotidyl transfer-ase deoxyuridine triphosphate nick end labeling (TUNEL)method. A TUNEL assay kit (Millipore) was used for stain-ing of harvested tissues at days 7, 14, and 28, and the apo-ptosis ratio was calculated by counting the number ofTUNEL-positive nuclei and dividing it by the total numberof nuclei at 5 different random fields (200x magnification)using a Mantra Quantitative Pathology Imaging System(PerkinElmer).

Degree of fibrosis formation was analyzed by staining theparaffin-embedded sections with Masson’s trichrome stainand by calculating the area of fibrosis (per mm2) in 10 differ-ent random fields (100x magnification) using a MantraQuantitative Pathology Imaging System (PerkinElmer).

2.8. Modification of SAPs for Enhanced Recruitment ofIntrinsic MSCs. In order to enhance recruitment of intrinsicMSCs into the site of action, RADA was modified to incorpo-rate a specific motif required to achieve the desired action.For this purpose, substance P was used, which is well knownas a neurotransmitter at the peripheral terminals of sensorynerve fibers, but has also been demonstrated to have a rolein the mobilization and recruitment of endogenous MSCsinto the site of action [14]. The new peptide, namedRADA-SP (Peptron), had the configuration AcN-RARADA-DARARADADAGGRPKPQQFFGLM-CONH2. A subgroupanalysis was performed where RADA-SP (RSP) was added toMSCs with RADA and injected into ischemic hindlimbs. Theother 3 groups consisted of control, MSCs, and RSP, and thehindlimbs were harvested at 7, 14, and 28 days for IHC stain-ing of stem cell markers CD105, CD90, and CD29 (as previ-ously described).

2.9. Statistical Analysis. All continuous variables are pre-sented as mean± standard error of the mean (SEM). Statisti-cal analysis between multiple comparison groups was doneby one-way analysis of variance (ANOVA) with post hoctesting using the Tukey method, while 2 group comparisonswere performed using the t-test. IBM SPSS Statistics 20(IBM) was used for analysis, and differences were consideredstatistically significant when P < 0 05.

3. Results

3.1. In Vitro Characteristics of MSCs and SAPs. Isolated andcultured murine MSCs exhibited characteristic MSC markers

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(positive CD105, CD29, and CD73, and negative CD45markers) and showed multilineage differentiation to osteo-genic, neurogenic, endothelial, and smooth muscle cell differ-entiation in vitro, while features of cell senescence were notobserved after 15 passages (data not shown). When MSCswere cultured in vitro, they formed characteristic colonies,but when cocultured with RADA, their colony-forming abil-ity was decreased (Figures 1(a) and 1(b)). Live cell screeninganalysis also demonstrated that MSCs continued to growup to 160 hours but the combination of MSCs andRADA showed a decrease in growth after 100–120 hours(Figures 1(c) and 1(d)). The MTT assay also showed thatthe combination of MSCs and RADA showed significantlylower cell viability compared to MSCs alone (Figure 1(e)).These results demonstrate that, in vitro, the combination ofMSCs and RADA did not improve cell survival of MSCs.

3.2. In Vivo Cell Characteristics and Angiogenesis. Harvestedtissues at different time points (7, 14, and 28 days) werestained for MSC markers CD105, CD90, and CD29 to detectfor the presence of MSCs (Figure 2). The control group didnot show any positive stain for all MSC markers, while MSCsshowed a positive stain for all three markers at different timepoints. Since injected MSCs were also tagged with GFP, apositive GFP stain was observed (second column). RADAalso demonstrated a positive stain for MSC markers whichwas not observed in the control group, demonstrating thatSAPs have the potential to recruit MSCs to the site of action.For the MSC+RADA combination group, the tissuesstained positive for GFP and for MSC markers, but whenmerged together, only the yellow stain was visible. Thismeant that only the injected MSCs were present in the tis-sues and no MSCs were recruited by RADA, which wouldhave stained red after merging (since GFP is not present).In terms of stem cell presence or survival, there was a defi-nite difference between control and the 3 groups, but therewas no visible correlation between the 3 groups or betweendifferent time points.

IHC staining for angiogenesis markers CD31, vWF, andα-SMA was performed from harvested tissues at differenttime points (Figure 3). The results show that compared tocontrol, all the other groups showed a significant increasein angiogenic markers at all time points. MSCs showed anoverall higher (or at least similar) angiogenic potential thanRADA, demonstrating that exogenously injected MSCs werestronger than MSCs recruited by RADA in forming capil-laries in ischemic tissues. Finally, the combination groupshowed an overall higher angiogenic potential than bothMSCs and RADA alone (although statistically not significantat some time points), suggesting that an increase in survivalof MSCs when combined with RADA led to a better abilityto form capillaries in ischemic tissues.

RT-PCR to determine the induction of angiogenesis atthe genetic level was performed for CD31, vWF, and α-SMA from harvested tissues, and there was a significantlyhigher expression of angiogenic genes for all 3 groups com-pared to control, but when the 3 groups were compared,there was no overall correlation between the groups (datanot shown).

3.3. Hindlimb Perfusion Measurements. The perfusion of hin-dlimbs as measured by laser Doppler was expressed as theratio of the measured perfusion postoperatively divided bythe preoperative baseline perfusion (Figure 4). As expected,at day 0 (immediate postoperation) there was no differencebetween the 4 groups. Furthermore, there was no statisticallysignificant difference between the 4 groups at the other timepoints, yet there was tendency for the combination groupto show a higher perfusion compared to the other groups at7, 14, and 28 days.

3.4. Apoptosis and Fibrosis. Apoptosis was determined usingTUNEL staining to demonstrate the degree of cell survivalfrom harvested tissues (Figures 5(a) and 5(b)). The controlgroup showed the highest rate of apoptosis compared to allother groups at all time points, with statistical significance.The combination group showed a significantly lower rate ofapoptosis, followed by the MSC group and the RADA group.This difference was most definite at 14 and 28 days, suggest-ing that the combination group had the strongest cell regen-erative effect after ischemic injury to the hindlimbs.

The degree of fibrosis formation was also determinedfrom harvested tissues using Masson’s trichrome staining(Figures 5(c) and 5(d)). Similar to cell death, the controlgroup showed the highest degree of fibrosis compared to allother groups at 7, 14, and 28 days with statistical significance.Overall, the combination group showed the lowest degree offibrosis, followed by the MSC group and the RADA group.These differences were mostly significant at 14 and 28 days.

3.5. RSP-Mediated Recruitment of Intrinsic MSCs. A sub-group analysis was performed where RSP was added to theprevious combination group and fluorescent IHC was per-formed for stem cell markers. Similar to the previous results,MSCs and RSP alone showed positive staining for stem cellmarkers CD105, CD90, and CD29, and injected MSCs alsoshowed positive stain for GFP, thus showing a yellow stainwhen merged, while the control group did not show any stainfor stem cell markers or GFP, as expected (data not shown).In the combined MSC+RADA+RSP group, injected MSCswere stained green on GFP stain, while all MSCs (bothinjected and intrinsic) were stained red for the respectivestem cell markers (CD105, CD90, and CD29). As a result,injected MSCs showed a yellow stain, while endogenouslyrecruited MSCs showed only a red stain on merged images.This was apparent in this new, RSP added, combinationgroup where the merged stains showed both yellow andred stains on the same slide for all stem cell markers atdifferent time points (Figure 6). These results demonstratethat by adding RSP, intrinsic host MSCs were recruited tothe site of action in combination with injected MSCs,which was not so apparent when MSCs and RADA werecombined alone.

4. Discussion

In this study, we evaluated whether the combination of MSCsand SAPs improves angiogenesis in ischemic hindlimbs ofrats. SAPs have been demonstrated to provide a niche for

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Figure 2: Fluorescent immunohistochemical staining of stem cell markers (a) CD105, (b) CD90, and (c) CD29 for MSCs, RADA, MSCs+RADA, and control (10x magnification). Injected MSCs previously tagged with GFP stained green in the presence of MSCs (secondcolumn for each time point). The different stem cell markers were stained for (third column for each time point) and showed the presenceof stem cells, not only in the MSC and MSC+RADA group, but also in the RADA-alone group, suggesting the presence of stem cellsprobably from the recruitment of endogenous MSCs by RADA. The merged images showed a characteristic yellow color from doublestaining of GFP and stem cell markers (second and fourth row for the different stem cell markers at different time points), yet there wasno presence of endogenously recruited MSCs in the combination group which would have shown a red stain only in the merged images(fourth rows).

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improvement in stem cell survival, which in turn leads toprolonged effects. RADA in particular has been demon-strated to improve myocardial infarction when combinedwith either platelet-derived growth factor-BB (PDGF-BB)[15] or with a combination of PDGF-BB and fibroblastgrowth factor-2 (FGF-2) [13]. This was achieved by con-trolled local delivery of the respective growth factors whencombined with RADA, while injection of growth factors orRADA alone did not achieve the desired cardioprotectiveeffects. The combination of stem cells with SAPs has also

been studied widely in a variety of different animal models,including ischemic hearts [16], osteoarthritis [17], and carti-lage defects [18]. In these studies, the role of SAPs was to actas a carrier to increase engraftment of injected stem cells,thereby improving survival and retention of administeredcells and thus improving their desired therapeutic effects.

Our study results demonstrated that the combination ofMSCs and RADA in vitro did not cause any improvementin cell survival. In fact there was a statistically significantdecrease in colony formation and cell viability when MSCs

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were combined with RADA compared to MSCs alone. Livecell screening analysis also demonstrated that after a certainpoint in cell growth, the combination group showed a suddendecrease in cell proliferation. Similar results were observed ina study by Liu et al. where in vitro 3-dimensional culture ofhuman adipose stem cells with RADA 16-I showed a lowerproliferation than 2-dimensional culture of adipose stemcells alone, and only when combined with a functionallymodified RADA 16-I to include stem cell homing motifswas cell proliferation significantly improved [19]. Stevensonet al. also showed that the incorporation of specific sequencesas binding sites into SAPs was associated with increasedmicrovascular network formation, while SAPs without thesebinding sites showed no network formation [20]. The reasonfor the lower proliferation of MSCs with RADA in ourin vitro results is not well understood, but the suddendecrease in cell proliferation after a certain point in time sug-gests that there may be competitive inhibition of stem cellgrowth, possibly due to overcrowding. Another possibleexplanation is that despite the stable formation of a hydrogelnetwork under a physiologic aqueous environment, the self-assembly process of SAPs is known to be affected or evenreversed depending on external environmental factors suchas pH, temperature, ionic strength, and mechanical forces[21]. It may be possible that the physiologic environmentrequired for stable hydrogel formation and proliferation ofstem cells in a 3-dimensional manner may not have beenachieved in our in vitro cultures.

WhenMSCs were injected into in vivo ischemic hindlimbanimal models in combination with RADA, the results weremore promising. Fluorescent IHC staining for stem cellmarkers (CD105, CD90, and CD29) demonstrated the pres-ence of stem cell survival in the different groups at 7, 14,and 28 days. The presence of stem cell markers in the RADA

group but not in the control group also demonstrates thatRADA has the potential to recruit stem cells, most probablyendogenous host stem cells through homing mechanismsinto the site of action. In the combination group however,there was the presence of injected MSCs only, whichappeared as a yellow stain in the merged image (from thedouble staining of GFP and stem cell markers), and no pres-ence of endogenously recruited MSCs, which would havestained red in the merged image. Only when RSP, whichincorporates a functional motif to achieve a prolongedrelease of substance P, was added to the combination groupcould the presence of both injected MSCs and recruited hostMSCs be visualized. The reason for the absence of endoge-nous MSCs in the first combination group (MSCs+RADA)is not well understood, but it can be postulated that by com-bined injection, the MSCs immediately adhere to the assem-bled hydrogels, thereby attenuating the recruiting potentialof RADA. Another possibility is that the recruiting abilityof RADA itself is weak, and therefore cannot overcome thecompetition against already injected MSCs at the site ofaction. This is in accordance with a previous study wheredespite the ability of RADA to recruit endogenous MSCs,the in vivo effects were enhanced only when a second RADAwith substance P bound as a functional motif into thesequence was added [10]. SAPs are known to be modifiableto perform specific bioactive functions when functional pep-tide motifs are incorporated into the C termini of the peptidesequence separated by a GG spacer between the motif andthe ionic-complimentary sequence [22]. Substance P, in par-ticular, has been shown to enhance the recruitment ofendogenous stem cells into implanted scaffolds to increaseangiogenesis [23], mainly from circulating cells inside theblood circulation around the local site of action [24]. Theaddition of RSP to the combination group may have pro-vided a stronger drive for recruitment of circulating hostMSCs, which in turn may improve angiogenesis further,although further studies are needed.

The combination of MSCs and RADA resulted in anoverall increase in angiogenesis, as shown by the increase inangiogenesis markers CD31, vWF, and α-SMA. As statedpreviously, RADA may have potentiated the effects of MSCsby providing a stem cell niche in the form of a 3-dimensionalmicroenvironment in which MSCs can reside more stably byallowing for cell attachment, migration, and differentiation.In this microenvironment, stem cells were able to increasetheir angiogenic effect under the ischemic conditions inwhich they were exposed to by differentiating into endothe-lial and vascular smooth muscle lineage cells required fornew vessel formation. Whether the survival rate of MSCswas actually increased in vivo by the addition of RADA wasnot quantified in this study. We found that their angiogeniceffects were improved up to a certain point and the tendencywas clear although statistical significance was not achieved atall time points. The lack of statistical significance at 28 days isa limitation, since the improvement in the durability ofeffects by the combination of MSCs with RADA was adesired effect, yet it may also be possible that by 28 days,the ischemic insult caused by the ligation of the femoralartery may have been restored up to a certain point that the

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drive for angiogenesis may have attenuated. Although we didnot quantify stem cell survival in vivo, may reports havealready shown that stem cell survival may not be a crucialissue for prolonged effects, since one of the most widelyaccepted mechanism of action of stem cells is throughparacrine effects, releasing potent trophic factors that can

modulate the molecular composition of the environment toevoke responses from resident cells [25, 26].

Hindlimb necrosis or color changes were not visible inour experiment, and therefore the improvement in ischemiacould not be grossly detected. The use of laser Doppler to ver-ify the improvement in perfusion as a result of angiogenesis

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Figure 5: (a) Fluorescent TUNEL staining and (b) quantification of TUNEL-positive stains for the evaluation of apoptosis for MSCs, RADA,MSCs +RADA, and control. There is a statistically significant lower rate of apoptosis in the MSC+RADA group compared to MSCs orRADA alone. (c) Representative Masson’s trichrome staining showing fibrosis between muscle fibers (10x magnification) and (d)quantification of degree of fibrosis from immunohistochemical stains for the evaluation of fibrosis for MSCs, RADA, MSCs +RADA, andcontrol. There is an overall tendency for lower fibrosis formation in the MSC+RADA group compared to MSCs or RADA alone. Lowerdegree of fibrosis and apoptosis correlates with an improvement in limb perfusion from increased angiogenesis. †p < 0 05 for controlversus all other groups. ∗p < 0 05.

9Stem Cells International

showed that there was an overall increase in hindlimb perfu-sion in the combination group, as compared to the othergroups including control. However, laser Doppler measure-ments are known to have great variability depending on thesurrounding measuring conditions, site of measurement, oroperator dependency [27, 28]. We tried to strictly adhere toa standardized protocol with all measurements being per-formed by one operator under a controlled environment,yet our measurements showed a wide range as a result ofthe intrinsic variability inherent to the laser Doppler perfu-sion measuring system. The lack of statistical significance inour perfusion measurements may probably be due to thisvariability, as shown by the large error bars in our graph.

Fibrosis and cell apoptosis were measured to assess theeffects of angiogenesis at the histological level. The end resultof ischemia is known to be cellular death and tissue remodel-ing by fibrotic scarring. Our results demonstrate that thecombination group showed the lowest rate of cell death andfibrosis on TUNEL and Masson’s trichrome stain, respec-tively, especially during the later phases (14 and 28 days).This demonstrates that the combination of MSCs withRADA increases angiogenesis, which leads to decreased celldeath by improvement of ischemia and consequent lower tis-sue remodeling.

The use of SAPs to enhance the function of stem cells isvery promising since the application of stem cells alone hasmajor drawbacks in terms of survival and durability.Repeated injections and dosage modifications can overcomesome of these drawbacks, but recent trends in treatment

involve combinations of different treatment modalities toachieve the desired effects [29]. RADA in particular has beenpreferred for situations requiring angiogenesis due to itssuperiority in infiltrating endothelial and smooth musclecells, inhibiting endothelial cell apoptosis and prolonginglong-term survival [30, 31]. RADA provides a suitable 3-dimensional microenvironment where cells can adhere toand perform its normal cellular functions, which includeendothelial and smooth muscle cell attachment, migration,and differentiation required for capillary-like structure for-mation [32]. The potential translational application of SAPsto clinical practice is also promising since these peptides areknown to have the advantage of being nonimmunogenicand noninflammatory in nature [5, 33]. SAPs have beenwidely studied for the delivery of growth factors and drugs,but the use of stem cells is also very promising since the mul-tipotency and self-renewing characteristics of stem cells allowfor unlimited applications in the field of tissue engineeringthat cannot be easily achieved by single drugs or growth fac-tors. However, studies involving the combination of stemcells and nanomaterials need to be performed in greaterdepth since it has been shown that phenotypic expressionand function of stem cells are largely dependent on themicroenvironmental cues in which the cells are exposed to,and therefore the fate of stem cells can vary depending onthe type of nanomaterials used [34, 35].

Our study has several limitations, including the lack ofstatistical significance in some of the results despite showinga good tendency. The measurement of perfusion could have

DAPI CD105 MergeDAPI CD105 MergeDAPI CD105 MergeMSC-GFP MSC-GFP MSC-GFP

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DAPI CD29 MergeDAPI CD29 MergeDAPI CD29 Merge

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RADA +

RSP

Figure 6: Fluorescent immunohistochemical staining for stem cell markers CD105, CD90, and CD29 from ischemic hindlimbs injected witha combination of MSC, RADA, and RSP (10x magnification). RSP is proposed as being stronger in recruiting endogenousMSCs and thereforethe combination of MSCs with RADA and RSP showed the presence of both injected MSCs (yellow stain) and endogenously recruited MSCs(red stain) in the merged images for the different markers at different time points.

10 Stem Cells International

been improved in terms of variability by the use of laserDoppler imaging systems, which are known to be less sensi-tive to extrinsic factors. The effects of adding RSP to the com-bination group in improving angiogenesis also needs to befurther delineated in future studies.

5. Conclusions

In this study, we demonstrated that MSCs combined withSAPs were able to enhance angiogenesis in ischemic hin-dlimbs of rats by promoting endothelial and smooth musclecell proliferation, which led to a decrease in cell death and tis-sue remodeling in the form of fibrosis. In turn, the perfusionof the hindlimbs tended to improve as a result of increasedangiogenesis. RADA also showed the ability to recruit endog-enous MSCs into the site of action, especially so when a sec-ond SAP bound with substance P was added to allow for thedual presence of injected and intrinsically recruited MSCs atthe site of action. Although preliminary in nature, the combi-nation of MSCs and RADA can be an option for clinicalapplication in ischemic disease conditions such as peripheralvascular diseases.

Data Availability

Data is available upon request, please contact the corre-sponding author.

Conflicts of Interest

The authors declare that there is no conflict of interestregarding the publication of this paper.

Acknowledgments

This work was supported by Grant no. 14-2014-028 from theSeoul National University Bundang Hospital Research Fund.

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