Branched peptide-amphiphiles as self-assembling coatings for tissue engineering scaffolds

Branched peptide-amphiphiles as self-assembling coatingsfor tissue engineering scaffolds

Daniel A. Harrington,1,2,3 Earl Y. Cheng,2,3 Mustafa O. Guler,4 Leslie K. Lee,5 Jena L. Donovan,3

Randal C. Claussen,4 Samuel I. Stupp1,4,6

1Department of Materials Science and Engineering, Northwestern University, Evanston, Illinois 602082Department of Urology, Northwestern University, Chicago, Illinois 606113Division of Pediatric Urology, Children’s Memorial Hospital, Chicago, Illinois 606144Department of Chemistry, Northwestern University, Evanston, Illinois 602085Department of Biomedical Engineering, Northwestern University, Evanston, Illinois 602086Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611

Received 10 November 2005; revised 21 December 2005; accepted 12 January 2006Published online 17 April 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.30718

Abstract: An important challenge in regenerative medicineis the design of suitable bioactive scaffold materials that canact as artificial extracellular matrices. We reported previ-ously on a family of peptide-amphiphile (PA) molecules thatself-assemble into high-aspect ratio nanofibers under phys-iological conditions, and can display bioactive peptideepitopes along each nanofiber’s periphery. One type of PAdisplays its epitope at a branched site using a lysine den-dron, a molecular feature that improves epitope availabilityon the nanofiber surface. In this work, we describe theapplication of these branched PA (b-PA) systems as self-assembling coatings for fiber-bonded poly(glycolic acid)scaffolds. b-PAs bearing variations of the RGDS adhesionepitope from fibronectin were shown by elemental analysis

to coat repeatably onto fiber scaffolds. The retention of su-pramolecular organization after coating on the scaffold wasdemonstrated through spectroscopic identification of�-sheet structures and the close association of hydrophobictails in a model pyrene-containing PA system. Primary hu-man bladder smooth muscle cells demonstrated greater ini-tial adhesion to b-PA-functionalized scaffolds than to barescaffolds or to those coated with linear PAs. This strategy ofmolecular design and coating may have potential applica-tion in bladder tissue regeneration. © 2006 Wiley Periodi-cals, Inc. J Biomed Mater Res 78A: 157–167, 2006

Key words: regenerative medicine; bladder; tissue engineer-ing; supramolecular; self-assembly

INTRODUCTION

Research on the design of scaffolds for regenerativemedicine is shifting away from the use of inert syn-thetic materials towards an increasing emphasis oninteractive scaffolds, which can influence cell adhesionor phenotype expression through selective ligand pre-sentation or targeted degradation.1 One of the possiblestrategies to introduce bioactivity is to modify the

surface chemistry of well-established synthetic biode-gradable scaffold materials.

The biodegradable poly(�-ester)s, such as poly(gly-colic acid) (PGA), poly(l-lactic acid) (PLLA), and theircopolymers (PLGA), have found extensive application inboth research and commercial use. Despite their utility,these materials contain none of the natural signalingchemistries that can directly influence cell behavior, norare they well-suited to functionalization (although sur-face treatment with plasma, entrapment of surface pep-tidic groups,2 or copolymerization with peptidic func-tionality3 have been explored). Extensive work hasdemonstrated the benefits of spatial organization of pep-tide ligands in two dimensions, through application ofself-assembled monolayers,4 covalently coupled mole-cules,5 or Langmuir-Blodgett films.6 Our laboratory andothers have directed their efforts towards the develop-ment of molecules that are preprogrammed to self-as-semble on the nanoscale into a variety of liquid crystal-line and supramolecular structures.7–11 Through their

The Supplementary Materials referred to in this article canbe found online at http://www.interscience.wiley.com/jpages/1549-3296/suppmat/.

No benefit of any kind will be received either directly orindirectly by the author(s).

Correspondence to: S. I. Stupp; e-mail: [email protected]

Contract grant sponsor: National Institutes of Health; con-tract grant numbers: T32 DK062716–02, R01 EB003806–01,DE015920–01

© 2006 Wiley Periodicals, Inc.

self-organization, such systems can present specificchemical functionality with spatial selectivity.

We have reported previously on “peptide-amphi-philes” (PAs),11–13 which are designed to self-assembleinto cylindrical nanostructures. These molecules con-sist of a hydrophobic alkyl tail, covalently coupled toa charged peptide sequence containing a �-sheet-forming segment and a bioactive segment as the mol-ecule’s terminus. Self-assembly into cylindrical nano-structures can be triggered electrostatically, forexample, through pH changes or charge screening ofthe peptide segment. The amphiphilicity of the mole-cule drives the formation of the cylindrical aggre-gate,14 which is stabilized by a hydrogen-bonded sec-ondary structure and hydrophobic collapse of theinterior alkyl segments.15,16 These features promotethe formation of long, cylindrical nanostructures, withtypical aspect ratios exceeding 50:1. Previous work hasdemonstrated the utility of these nanostructures ininfluencing cell phenotype, most recently in inducingrapid and selective differentiation of neural progenitorcells into neurons.13

In the assembled nanofibers, the peptide headgroupis displayed at high density along the fiber periphery.Known bioactive sequences such as RGDS for celladhesion, IKVAV for neurite extension, and YIGSR forneuron adhesion have been employed in these sys-tems to act as recognizable epitopes for influencingcell behavior.17 Recently,18,19 we modified the archi-

tecture of PA molecules to incorporate a branchedl-lysine dendron at the terminus, with the followingexpectations: (1) the highly charged lysine dendroncould provide steric bulk, pushing individual PA mol-ecules further apart in the nanostructure, thus improv-ing accessibility of the RGDS epitope to integrin re-ceptors, and (2) the modified fibers would be wrappedin an envelope of positively charged lysines, which areoften used in vitro in the form of poly(d-lysine) toenhance cell adhesion through nonspecific electro-static attraction. The chemical structures of these“branched PA” (b-PA) molecules are shown in Figure1. Since the RGDS sequence is known to be recognizedby integrins as a part of an outward-facing loop onfibronectin, we envisioned that the RGDS sequence ona branched-PA nanofiber could have sufficient spaceand flexibility to “flop over,” thus improving accessi-bility, and potentially, recognition.

In this article, we describe our efforts to coat thesurface of porous PGA scaffolds with b-PA moleculesthat display a variety of terminal RGDS sequences.Our interest was to characterize the retention of thesemolecules on a PGA scaffold, and to determinewhether self-assembly of the bioactive nanostructureswould occur on their surfaces. To test these coatedscaffolds in vitro, we considered their application inbladder tissue regeneration, which has long been atarget in tissue engineering.20 Especially in pediatriccases, bladder function can be severely affected by

Figure 1. Chemical structures of branched and linear PAs and abbreviations.

158 HARRINGTON ET AL.

Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a

conditions that form during development. Bladder tis-sue is composed primarily of two cell layers: a thinlayer of urothelial cells facing the bladder interior,which is supported internally by a thicker layer ofsmooth muscle cells (SMCs). Previous in vivo modelshave used either naturally derived materials (e.g., por-cine-derived small intestinal submucosa21), or degrad-able polymeric biomaterials (e.g. PGA22) as scaffolds.For our application, it was clear that the mechanicalstrength of a polymeric scaffold would eventually benecessary to support the expansion and contraction ofa bladder cavity. We therefore tested bladder SMCadhesion onto b-PA coated PGA scaffolds to deter-mine whether the presence of a self-assemblednanoscale coating could improve upon existingseeded technology.

MATERIALS AND METHODS

Protected amino acids and reagents for automated pep-tide synthesis were obtained from Applied Biosystems, andRink resin was purchased from Nova Biochem. PLGA (50:50) and other reagents for peptide synthesis and histologywere obtained from Sigma-Aldrich. All cell culture mediawere from Gibco/Invitrogen. Cell culture plasticware wasfrom Corning and Falcon. Hoechst 33258 was purchasedfrom Molecular Probes. Supplies for electron microscopywere from Electron Microscopy Services. Buffer salts andorganic solvents were obtained from Fisher Scientific. Non-woven PGA fiber scaffolds, with 12-�m fiber diameter and3-mm fabric thickness, were purchased as a sheet fromAlbany International.

Peptide-amphiphile synthesis

b-PA molecules were synthesized manually on solid-phase resins, using standard fluorenylcarboxylmethyl(Fmoc) chemistry as previously described.19 Briefly, an or-thogonally protected N-�-Fmoc-N-�-4-Mtt-l-lysine aminoacid was coupled to a Rink resin, followed by removal of theMtt group with 5% trifluoroacetic acid (TFA) in methylenechloride for 5 min. Palmitic acid was then coupled to the free�-amine, followed by Fmoc removal from the � amine. Stan-dard solid-phase peptide synthesis techniques were used tocouple later residues. At branching points, (Fmoc)Lys(Mtt)OH was again used with removal of Mtt from selectedbranches to continue growth. Boc-protected residues wereemployed to end a branch. Ninhydrin (Kaiser) tests for freeamines were performed at each step to ensure completecoupling. An RGD PA with the sequence palmitic acid-AAAAGGGS�PO4�RGD was synthesized as described previ-ously.12

Completed PAs were cleaved from resin beads by 3 h ofshaking in a 95/2.5/2.5 mixture of TFA/deionized (DI) wa-ter/triisopropylsilane. The PA/TFA solution was rotaryevaporated at 60°C to remove excess TFA, leaving a viscous

yellow liquid. The product was triturated repeatedly withcold (�20°C) diethyl ether, precipitating as a white powder.The powder and solvent were slowly poured onto a fine fritfunnel, and the solids were recovered and dried under vac-uum.

Prior to use on the PGA scaffolds, the PAs were addition-ally purified to remove TFA salts. Reports have indicatedthat excess TFA anion can be detrimental in primary cellculture,23 requiring an exchange with a more benign anion,such as chloride. As a final step in peptide synthesis, PAs arecleaved from solid-phase using TFA, which then also servesas a counter-ion to each charged amine. The high lysinecontent of the b-PA systems made this an especially relevantconsideration. PA molecules were therefore treated to re-duce TFA salts by mixing 10 mg/mL aqueous solutions ofeach PA 1:1 with 1M HCl for 30 min at room temperature.Under these conditions, displaced TFA, as well as excessHCl, should be easily removed under high vacuum duringlyophilization. The solutions were snap-frozen in liquid ni-trogen and lyophilized overnight. The lyophilized powderswere analyzed for TFA content by mixing 1:20 (w/w) withKBr and pressing the resulting powder into a solid film. KBrpellets were scanned by Fourier transform infrared spectros-copy (FTIR) on a Bio-Rad FTS-40 at 2 cm�1 resolution and 32scans/sample. The peaks at 1135 and 1202 cm�1 (COF) and1673 cm�1 (acetate) were monitored to gauge the removal ofTFA from the molecules (Supplemental Information, Fig.SI-1). Each treated PA was redissolved in filtered DI waterand adjusted to pH 6.5 with potassium hydroxide solution.PAs were then relyophilized and stored at –20°C prior to use.

Samples of each PA were characterized by electrosprayionization mass spectroscopy from water solution, and ma-trix-assisted laser-desorbed ionization time-of-flight massspectroscopy, using an �-cyano-4-hydroxycinnamic acidmatrix. Both methods yielded majority peaks correspondingto the expected molecular weights of each molecule (Sup-plemental Information, Fig. SI-2). Molecules were addition-ally characterized by nuclear magnetic resonance spectros-copy, which yielded expected peaks for the specificfunctionalities on each PA. Circular dichroism (CD) spectraof each PA at 3.33 � 10�6M in aqueous solution werecollected with a Jasco J-715 spectrometer.

PGA scaffold preparation and characterization

Nonwoven PGA fiber scaffolds were cut into 1 � 1 cm2

from the PGA fabric and dipped for 5 s in a 40 mg/mLsolution of 50:50 PLGA in methylene chloride. The wetscaffolds were tapped briefly onto a wire mesh to removeexcess liquid, and dried in glass Petri dishes under vacuum.The fiber-bonded scaffolds were then sterilized by ethyleneoxide exposure, and stored under vacuum with desiccantprior to use. Scaffold surface area was determined by mea-surement of adsorbed krypton gas using a Quantasorb dy-namic flow analyzer.

Scaffold coating and characterization

As a general coating procedure, sterilized, fiber-bondedscaffolds were rehydrated under aseptic conditions, with

BRANCHED PEPTIDE-AMPHIPHILE COATINGS 159


regular exchanges of 25, 50, 75, 95, and 100% sterile DI waterin ethanol. PA (25 �L of each) solution in water, at varyingconcentrations, was added dropwise across the large face ofeach damp scaffold and allowed to dry under vacuum.Dried scaffolds were flipped over, and another 25 �L of eachPA solution was added onto the reverse face and allowed todry.

To initially determine the dependence of coating level oninitial PA concentration, scaffolds were treated with a pre-viously reported12 RGD PA (palmitic acid-AAAAGGGGS�PO4�RGD) at 5, 10, 20, and 40 mg/mL. Two sections, 3 mm2

each, were cut from separate coated scaffolds and examinedby elemental analysis for carbon, hydrogen, and nitrogenlevels (CHN).

To determine any effect of the individual PA chemistrieson coating retention, fiber-bonded scaffolds were coatedwith 20 �L of a 15 mg/mL solution of each of the b-PAmolecules shown in Figure 1, using the coating proceduresdescribed earlier. At least three samples of each type of thedried coated scaffolds were examined by CHN analysis.

Characterization of PA supramolecular structureretention on scaffolds

Scaffolds coated with each of the PAs in Figure 1 wereexamined on a Thermo Nicolet Nexus 870, using a diffusereflectance infrared Fourier transform (DRIFT) attachment.Samples 5 � 10 mm2 in size were placed directly into thesample holder and scanned at 2 cm�1 resolution for 256scans. Data were converted to Kubelka-Munk units usingOmnic software.

To examine the association of hydrophobic tails in PAscoated onto scaffolds, a pyrene-functionalized PA was syn-thesized as a fluorescent analogue of the PAs in Figure 1,using previously described methods.24 Briefly, the moleculewas synthesized by standard Fmoc coupling of l-lysine andl-leucine to a Rink resin. Fmoc-protected 6-aminohexanoicacid was added after the peptidic segment to approximatethe length of the hydrophobic segment to that of the hydro-phobe (palmitic acid) used in other PAs. After deprotectionof this segment, 1-pyrenebutyric acid was coupled, and theentire molecule was cleaved, purified, and characterized asdescribed earlier.

Fluorescence measurements of pyr-PA, either in solutionor coated onto PGA scaffolds, were done on a MolecularDevices SpectraMax Gemini EM fluorescence plate reader inspectrum mode, with 2-nm step size, 7 repeats/sample inbottom-read mode. Pyr-PA in aqueous solution had a max-imum emission at an excitation wavelength of 343 nm,which was used for all subsequent pyr-PA experiments.Fiber-bonded PGA scaffolds were coated with a 10 mg/mLaqueous solution of pyr-PA, as described earlier. For reten-tion experiments, a pyr-PA-coated scaffold of 3 mm2 wastransferred consecutively among four wells containing 200�L/each of phenol red-free DMEM for 15 min each. Thescaffold was then soaked in 5 mL of phenol red-free DMEMfor an additional 45 min and scanned to determine coatingretention.

In vitro experiments with primary human bladdercells

Human bladder tissue was obtained from consenting pe-diatric patients at the University of Oklahoma Health Cen-ter, and SMCs were isolated from these tissues using previ-ously described procedures.25 All subjects enrolled in thisresearch responded to an Informed Consent that has beenapproved by the Institutional Committee on Human Re-search at the University of Oklahoma, Children’s MemorialHospital, and Northwestern University, and this protocolhas been found acceptable by them. Briefly, pediatric blad-der tissue was transferred in cold M199 media to the labo-ratory, where urothelium and stroma were separated. Mus-cle tissue was incubated in collagenase IV solution andmechanically disrupted to promote SMC outgrowth fromtissue explants. Frozen SMCs were transferred to North-western University and expanded in complete SMC culturemedia (M199 media (9.55 g M199 powder, 2.2 g NaHCO3,3.56 g HEPES, 1 g dextrose, 0.5 g bacteriologic peptone) 10% NCS 1% P/S 1% MEM vitamins 1% MEM aminoacids) at 37°C/5% CO2 by standard methods.

Spinner flasks were modified for use with coated PGAscaffolds (Supplemental Information, Fig. SI-3). Holes werecut in a Teflon seal at the top of a 250-mL spinner flask toaccommodate the Luer-lock fittings of eight 22-gauge nee-dles. The needles were cut to 15–20 cm length, and spacedevenly around the circumference of the flask spindle. Mod-ified flasks were sterilized prior to use. Fiber-bonded scaf-folds were coated with the PA molecules shown in Figure 1,using 20 �L/side of a 15 mg/mL solution of PA in DI water,using the coating procedures described earlier. Control baresamples were coated with DI water alone. Four coated scaf-folds of a given PA type were skewered onto each needle,separated by 2–3 mm sterilized 1/8� Tygon tubing betweenscaffolds, and supported by a 5 mm3 sterile silicone plug atthe end of each skewer.

Each spinner flask was soaked in M199 media (no serumor other additives) for 1 h prior to seeding. Expanded SMCswere trypsinized, pelleted, and resuspended in completeSMC media. Flasks were emptied of media, and 3.0 � 107

cells were added to each flask (1.7 � 105 cells/mL completemedia; 1.3 � 106 cells/scaffold). Spinner flasks were filled to180 mL of media to fully cover all scaffolds. Scaffolds werecultured under standard conditions at 60 rpm, with half-volume media replacements every other day. At 4, 10, 17,and 31 days, all samples from a given flask were removed,with one half of each scaffold fixed overnight at 4°C in 10%neutral buffered formalin (for SEM and histological evalua-tion), and the other half snap-frozen in liquid nitrogen andlyophilized (for biochemical assay).

Analysis of in vitro samples

Samples for SEM evaluation were postfixed 1 h in 1%OsO4, dehydrated, critical-point dried, sputter-coated with 3nm Au/Pd, and imaged at 3 kV (25 mm working distance)on a Hitachi 4500 FE-SEM. Samples for histological evalua-tion were dehydrated, cleared in xylenes, infiltrated at 60°C



in 1:1 (v/v) xylene/paraffin, embedded, sectioned at 2 �mthickness, and stained with Masson’s trichrome stain. Ly-ophilized scaffolds were digested with papain solutions andanalyzed for DNA content via the Hoechst 33258 assay asdescribed elsewhere.26,27 Five repeat measurements of eachof four digested samples for the Hoechst assay were con-ducted on a Molecular Devices SpectraMax Gemini EMfluorescence plate reader, with calf thymus DNA standards.DNA content was normalized to the dry weight of thedigested scaffold. ANOVA calculations for p values betweensamples were performed using Microsoft Excel software.

RESULTS

Branched PA synthesis

A variety of molecules were synthesized, representingmany alternatives for enhancing cell adhesion. Asshown in Figure 1, PA structure variations with RGDSsequences on one or both �-amines were created to testthe effect of epitope density. Another molecule was syn-thesized that included the PHSRN sequence, which isfound on fibronectin’s tenth type-III repeat, 30 Å fromthe RGDS loop,28 and is known to enhance integrin-mediated adhesion after the initial recognition ofRGDS.29 A fourth molecule was designed to test for thespecificity of epitope recognition by reversing theepitope stereochemistry with a D-RGDS. A version ofthe molecules with linear RGDS presentation was alsosynthesized to compare against the branched structures.

Scaffold coating and characterization

PGA fabrics, as received, are soft, pliable felts,which are frequently coated with a thin layer of 50:50

PLGA to selectively weld the individual fibers at theirintersections and increase the scaffolds’ mechanicalproperties (“fiber-bonding”).30 We tested this methodat varying PLGA concentrations, and found that 40mg/mL was an optimal level for forming strong in-tersections without blocking pores (Supplemental In-formation, Fig. SI-4). For all later experiments, scaf-folds were cut and coated at a standard size of 1 � 1cm2. Fragments of these scaffolds were analyzed bythe Brunauer–Emmett–Teller (BET) method using ad-sorbed krypton gas, and were found to have an aver-age normalized surface area of 0.17 m2/g, within theexpected range from other literature reports.31

To test the retention of PA molecules on a scaffoldsurface, fiber-bonded scaffolds were rehydrated fromethanol through to water. Scaffolds were coated withan aqueous solution of a previously reported RGD PA,varying in concentration from 5 to 40 mg/mL. Theamount of PA retained after coating was evaluated byCHN analysis, as shown in Figure 2. Although CHNdetermination does not allow for recovery of the sam-ples, it proved useful for analysis, as only the PAmolecules would be expected to contribute signifi-cantly to nitrogen levels in the coated scaffolds. Theresponse of retained PA was linear with respect tocoating solution concentration.

The molecules shown in Figure 1 were then coatedonto fiber-bonded scaffolds to determine whether differ-ences in PA chemistry would affect coating retention orreliability. Results from CHN analysis are shown in Fig-ure 3. The grey bar indicates the overall average coatinglevel for the five PAs (9.5 � 10�7 moles PA per gram ofscaffold) and a 95% confidence interval (1.5 � 10�7) forthat average. ANOVA comparison of all of the samplesyielded p � 0.05, except for the b-RGDS/PHSRN andb-D-RGDS for which p 0.046.

Figure 2. CHN analysis of PGA fiber scaffolds, coated withvarying concentrations of RGD PA. The detected levels ofthe PA indicate predictable, repeatable coating on the scaf-folds. Linear trend line on graph has R2 0.986. n 2samples tested per condition.

Figure 3. CHN analysis of PGA fiber scaffolds, coated withvarious branched and linear PAs. Error bars are � a 95%confidence interval for each PA type, with n 3 samplestested. The shaded bar represents an overall average for thefive sample types, � a 95% confidence interval.



Characterization of supramolecular structureretention for PAs on scaffolds

After determining that PA molecules were beingretained on PGA scaffolds, we assessed whether theyhad adopted any supramolecular organization. Previ-ous work has shown that a signature of PA self-as-sembly into nanofibers (including lysine branchedversions) is the formation of �-sheets that are observ-able by CD and FTIR. To assess assembly in our sys-tems, coated scaffolds were analyzed directly byDRIFT spectroscopy, showing absorption near 1627cm�1, which corresponds to the amide I CAO stretch

(Fig. 4). The observed absorption was above the base-line value observed for a bare PGA scaffold. Thisstrongly suggests that the b-PA molecules on the scaf-folds are adopting a self-assembled structure. Thespectrum for a scaffold coated with the linear-RGDanalogue appeared similar to the spectrum for barePGA (not shown). This is not unexpected, as the lin-ear-RGD has also been shown by CD to have a re-duced �-sheet character (Supplemental Information,Fig. SI-5).

As another determination of supramolecular assem-bly, we used a model PA molecule to assess the inter-action of the molecules’ hydrophobic tails. A fluores-cent tag was incorporated in the molecule shown inFigure 5(a) (pyr-PA) to probe the hydrophobic core ofthe assembled structure. It also included several ter-minal lysine residues to mimic the structure ofbranched systems bearing a terminal lysine dendron.Pyrene was selected as a fluorescent tag, as its emis-sion shifts when two molecules are in close proximity(within 4 Å), because of excimer formation.32 Asshown in Figure 5(c), a well-solubilized pyrene mole-cule (i.e. largely unassembled) demonstrated mono-mer emission maxima at 380 and 398 nm. Upon addi-tion of concentrated KOH to neutralize charges on themolecule and induce self-assembly, excimer formationat 460 nm was immediately observed, with substantialreduction in the lower-wavelength peaks. As pyr-PAhas been observed by TEM to form nanofibers at basicpH, excimer formation can be considered as anothersignature of a self-assembled state.

Scaffolds coated with pyr-PA were scanned directlyon a fluorescence plate reader, and demonstrated asimilar excimer signature [Fig. 5(d)], indicating su-

Figure 4. DRIFT scans of PGA scaffolds coated with vari-ous PAs. Peaks at 1627 cm�1 correlate with the formation of�-sheets, which are characteristic of assembled b- PAs. The�-sheet character of linear-RGDS molecules (not shown) isknown to be less intense, and therefore no peak is expectedin this range. Intensity is given as a Kubelka-Munk trans-form.

Figure 5. Fluorescence emission measurements. (a) Chemical structure of pyr-PA. (b) Schematic for the measurementsdepicted in (e) and (f). (c) Aqueous solutions of pyr-PA at 3.0 � 10�3M, at pH 6 (mostly unassembled) and pH � 14 (mostlyassembled), both normalized to their emission maxima. Excimer emission at 460 nm is only observed in the assembled state.(d) PGA scaffold coated with pyr-PA and dried. Excimer formation on the coated scaffold indicates that the PA molecules arein an assembled state. No fluorescence above background is observed for the bare scaffold. (e) Four DMEM solutions aftera pyr-PA coated scaffold were sequentially soaked in each. While some PA does dissolve into the DMEM, this amountdecreases significantly with each fresh soak. (f) Scan of a pyr-PA coated scaffold after five consecutive soaks in DMEM. Thepresence of the excimer peak indicates that pyr-PA is still present on the scaffold, and retains its assembled state.



pramolecular assembly on the PGA scaffold. This re-sult is consistent with previous observations that mostPA molecules self-assemble into nanofibers whendried onto surfaces.12

To address the question of PA retention on the scaf-fold after immersion in culture media, a pyr-PA coatedscaffold (3 mm2) was soaked in four consecutive ex-changes of phenol red-free DMEM for 15 min each.Fluorescence scans of these liquids after soaking indi-cated some initial dissolution of pyr-PA into the media[Fig. 5(e)]. However, the excimer fluorescence observedin the first soak solution dropped substantially in subse-quent solutions. The scaffold used in these experimentswas then soaked a fifth time in a larger volume ofDMEM for 45 min, and finally the damp scaffold itselfwas scanned [Fig. 5(f)]. An excimer signature was stillretained from the scaffold, indicating that pyr-PA wasstill substantially present, and still largely maintained itssupramolecular structure. This strongly suggested thateven under immersion in cell culture media, PA nano-structures are retained on the scaffold surface, and arepresent in a self-assembled form.

Primary human bladder SMC culture on PA-coatedscaffolds

Modified spinner flasks were used for dynamic scaf-fold seeding and culture (Supplemental Information,Fig. SI-2). Based on preliminary experiments and lit-erature precedent,33 this approach was expected tooffer better initial cell attachment and later growththan a static (i.e. mutiwell plate) system. Coated scaf-folds were skewered into the flasks, seeded with pri-mary human bladder SMCs, and cultured with regu-lar media changes.

At 4, 10, 17, and 31 days, all scaffolds from an entirespinner flask were removed and processed as de-scribed in the Materials and Methods section. SEMimages of the six scaffold types are shown in Figures6 and 7. At day 4 (not shown), cells and matrix ap-peared sporadically along the scaffold fibers for PA-coated samples. Such features were significantly re-duced for linear-RGD coated scaffolds, and wererarely seen on bare PGA scaffolds. At day 10 (Fig. 6),SMCs were visible in greater number throughout all ofthe b-PA coated scaffolds, with increased matrix dep-osition, especially at fiber intersections. At this timepoint, some activity was visible on the linear-RGDSsample, while very few cells were visible on the barescaffolds. By day 17 (Fig. 7), cells on all of the b-PAscaffolds spread beneath the outer scaffold surface toform a near monolayer. Their spindled morphology ischaracteristic for SMCs both in culture and in vivo. Atthe same time point, bare and linear RGDS coatedscaffolds appeared to lag behind in cell coverage. Af-ter 1 month of culture (not shown), all of the scaffoldswere covered by cells and matrix, with no discernibledifference between the samples.

We examined cell penetration into the core of thescaffolds using both Masson’s trichrome staining ofparaffin sections, and SEM imaging of a critical point-dried scaffold in cross section. Figure 8 compares im-ages of scaffolds at day 10 coated with single b-RGDSversus bare PGA scaffolds. For all of the b-PA samples,good cell penetration was observed into the center ofthe scaffolds. Histological staining also demonstratedclear deposition of extracellular matrix in a mannerreminiscent of the same structures seen at fiber inter-sections by SEM. Conversely, bare scaffolds containedvery little biological material at this early time point,correlating with the results from Figure 6. Notably,

Figure 6. Representative SEM images of SMCs on coated scaffolds after 10 days in culture. Images are taken from one of thetop faces of the scaffold. Substantially more cells and cellular material are visible on the scaffolds coated with b-PA molecules:(a) single b-RGDS (b) double b-RGDS � 2 (c) b-RGDS/PHSRN (d) b-d-RGDS (e) linear-RGDS and (f) bare PGA.



cells on both scaffolds stained positive by immunohis-tochemistry for �-smooth muscle actin (SMA), aknown marker of SMCs (not shown).

In an effort to quantify cellular adhesion, the lyoph-ilized scaffolds from each time point were digestedand analyzed via a Hoechst 33258 assay for total DNAcontent (Fig. 9). As observed qualitatively by SEM andhistological cross section, the number of cells on scaf-folds coated with b-PAs was significantly higher thanon bare or linear RGDS-coated scaffolds at early timepoints. Surprisingly, there was little additional cellproliferation observed on the b-PA substrates. Analy-ses of b-RGDS samples by histology and SEM agree

with this data—cell number in each image did notincrease markedly across time points. Rather, cells onthese scaffolds adopted a stretched morphology overtime. Histologically, the ratio of nucleus:cytoplasmcross-sectional area appeared to be quite large at earlytime points, and decreased over time as cells spread.

DISCUSSION

The versatile self-assembly and gelation propertiesof PA molecules make them useful for a variety ofsoft-tissue applications. Combining these self-assem-bled nanofiber networks with a polymeric scaffoldcould create systems for regenerative medicine thatretain the bioactivity of the PA while meeting themore stringent mechanical requirements of a scaffoldto be used for regeneration of connective and muscu-loskeletal tissues.

Previous work has demonstrated that self-assemblyof PA molecules into nanofibers can occur from dryingonto a substrate, as observed by TEM, SEM, andAFM.12,18,19 We anticipated similar behavior on PGAscaffolds, assuming the PA molecules could adhere tothe polymer surface and adopt the nanofiber structure.Several approaches were used to probe the assemblyand characterize the presence of the b-PA coating oneach scaffold. Qualitatively, we observed improvedwetting after coating one face of the scaffolds, whichprovided initial evidence of successful PA retention onthe fiber surface. While direct imaging of the nanofi-bers on the scaffolds proved difficult, CHN elementalanalysis provided quantifiable information on thepresence of PA molecules and the reliability of thecoating procedure. Notably, there appeared to be min-

Figure 7. Representative SEM images of SMCs on coated scaffolds after 17 days in culture. Images are taken from one of thetop faces of the scaffold: (a) single b-RGDS (b) double b-RGDS � 2 (c) b-RGDS/PHSRN (d) b-d-RGDS (e) linear-RGDS and(f) bare PGA. Cells appear to be forming a near-monolayer across the surface of the b-PA-coated scaffolds. Cellular activityis visible on the linear-RGDS and bare scaffolds, although it lags behind that of the b-PA samples.

Figure 8. Cross sections of single b-RGDS (a, c) and bare (b,d) scaffolds at day 10, as observed by trichrome staining ofparaffin sections (top) and SEM of critical point-dried sam-ples (bottom). More cells (red) and collagen matrix (blue) areseen in histology of the b-PA-coated scaffolds. Cellular ac-tivity was also observed to penetrate through to the center ofthe fiber scaffold, confirmed by SEM evaluation.



imal statistical difference in the coating efficiencyamong the five PAs, as shown in Figure 3.

On the basis of the normalized surface area (0.17m2/g) of a fiber-bonded scaffold measured by the BETmethod with adsorbed krypton gas, we can estimatethe surface coating efficiency. The average PA coatingfor a scaffold as determined by CHN (Fig. 3) was 0.95(�0.15) �mol PA/g scaffold. Estimating the spatialdensity of assembled PAs yields a range of 3–4 PAmolecules/nm2 on the scaffold surface. With the ex-pected increase in steric repulsion from lysine den-drons at the periphery of b-PAs, this indicates that atleast one layer of assembled fibers should be possibleacross the surface of the fiber scaffold. This is certainlyan average estimate for the entire surface area of agiven scaffold, and localized areas of multilayers oruncoated surfaces are possible. If thicker coverage isneeded, based on the results in Figure 2, it should bepossible to tune the amount of PA deposited (and thusthe coating thickness) by varying the initial concentra-tion of the PA solution.

In addition to determining the presence of the b-PAmolecules on the scaffolds, it was of interest to probetheir assembly state, since supramolecular structurecould influence their utility in cell signaling. AFM andSEM proved unsuccessful in directly imaging the PAfibers on the PGA scaffold, and so we attempted tostudy their assembled state using spectroscopic signa-tures. The most notable features of PA self-assemblyare (1) presentation of epitopes on the nanofiber pe-riphery,18 (2) extended association of the central Leu/Ala units in the form of a �-sheet structure,34 and (3)close interaction of the hydrophobic tails,35 buriedwithin the nanofiber core. In the system presentedhere, the presentation of charged epitopes on the sur-face of the fibers was inferred from a substantial im-

provement in scaffold wetting after coating with PAs.The highly charged lysine dendron, if present on theperiphery of an assembled structure, is likely respon-sible for this effect.

Further evidence for assembly was observed byDRIFT analysis of coated scaffolds. The presence ofthe amide I absorption correlated with �-sheet signa-tures observed in neat b-PAs. Although FTIR does notprovide definitive evidence for peptide secondarystructure,36 the presence of this peak does stronglysupport the proposed formation of nanofibers. FTIRsignatures for other secondary structures typically ap-pear at higher wavenumbers (1650–1700 cm�1), whichwould overlap with carbonyl absorptions from thePGA and PLGA polymer.

A third indication of PA assembly on the scaffoldsurface was the excimer fluorescence observed in thepyr-PA model system. This result supports the pro-posed model of closely aggregated hydrophobic tailsin an assembled state on the scaffold’s surfaces. Initialexcimer retention in the hydrated state (not shown),and after repeated soakings in cell culture media [Fig.5(d)], confirmed both that the pyr-PA molecule wassubstantially retained on the scaffold surface, and thatit maintained its supramolecular assembly underthese conditions. Both of these findings were vital forestablishing the utility of a coated scaffold for in vitrostudy. It should be noted that the data from thesepyr-PA experiments could not be compared quantita-tively, because of variations in retained sample vol-ume from the soaking experiments, and the inherentvariations in scanning a solid inhomogeneous objectsuch as a scaffold. The absolute ratios of intensitybetween the primary emission peaks (380 and 398 nm)and the excimer peak (460 nm) also could not becompared for similar reasons.

Figure 9. Quantitation of cell number on scaffolds by Hoechst 33258 fluorescence. Cell attachment at early time points wassignificantly higher on b-PA scaffolds than on linear-RGDS or bare PGA. This difference matches qualitative observationsfrom SEM and histology. At later time points, this observed difference decreased. n 4 scaffolds measured for each sampletype and time point. Error bars are � a 95% confidence interval. * p � 0.05 compared to linear RGD; † p � 0.05 compared tobare PGA.



Notably, exposure to DMEM, M199, and most othertraditional culture media would be expected to directb-PA molecules to form supramolecular structures.37

High salt concentration is known to encourage self-assembly; in addition, for these particular PA systems,a pH � 6.5 also drives aggregation. Thus, exposure tocell culture media (pH 7.4) strongly encourages reten-tion of a supramolecular state.

The details of this assembly were initially difficult todetermine, as direct imaging of PA nanofibers on PGAfibers by SEM was not possible at nanoscale resolutionbecause of excess sample charging. DRIFT experi-ments could only identify the existence of an assembledstate, but not conclusively as cylindrical objects. How-ever, we have previously demonstrated that excimerformation in pyr-PA is only associated with the for-mation of assembled nanofibers; any other aggregatestate does not produce the same spectroscopic signa-ture.24 Thus, the detection of this signature for coatedPGA scaffolds, as shown in Figure 5, indicates that notonly was pyr-PA retained on these surfaces but alsothat it was assembled into cylindrical nanofibers.

The application of coated scaffolds to the in vitroculture of primary human bladder SMCs demon-strated a marked influence of the b-PAs on cellularadhesion. SMCs adhered in significantly higher num-ber at early time points to b-PA coated scaffolds thanto linear-RGDS or bare scaffolds. Also notable was thepenetration of cells to the center of the b-PA scaffolds,which is preferable to capsule formation on the outersurface. Cells initiated matrix production and gradu-ally adopted an aligned, spindled morphology. Thisobservation is not exclusive to SMCs, and would alsobe expected for other subpopulations (e.g. myofibro-blasts). However, the combined observations of aspindled morphology and the observed SMA expres-sion represent the expected behaviors for a populationof primary cells isolated from bladder smooth muscletissue.

The possible causes for this improvement could be thevarious presentations of the RGDS epitopes, or the ex-tended surface of amines along each PA assembly. Poly-(lysine) has long been used as a surface treatment toencourage adhesion in mammalian cell culture, and theselection of lysine dendrons for the b-PA systems wasdone deliberately with this history in mind. Other workby Guler et al. (submitted) has identified a strong influ-ence of these dendrons on b-PA assembly as measuredby pH titration. We have also previously demonstratedclear improvements in accessibility of the RGDS epitopein these systems, as compared to linear presentations, forthe interaction of a small molecule such as biotin.18 Otherwork has demonstrated enhanced cell attachment toPLLA fibers coated with self-organizing molecules bear-ing similar lysine dendrons.38

Interestingly, we did not identify a substantial dif-ference between the various RGDS presentations, even

for the b-D-RGDS PA, which presented the epitopewith abiotic stereochemistry (i.e. d-enantiomers).Given the nature of these experiments (i.e., culturewith serum proteins), we could not clearly distinguishbetween adhesion because of direct epitope recogni-tion or that because of electrostatic effects. Molecularbiology studies, involving focal adhesion formation ordeliberate blocking of integrin function, would pro-vide the necessary resolution to address these ques-tions. In the experiments reported here, the electro-static effects of a one-dimensional polyamine surfacemight dominate adhesion, through initial interactionwith cell membranes, preferential adsorption of solu-ble proteins, or other means. Conversely, the resultsshown in Figure 9 may be a combined influence ofboth the lysine surfaces and direct recognition of theextended RGDS epitopes. Our laboratory is currentlyconducting more detailed studies of integrin recogni-tion of b-PA surfaces, as well as the required condi-tions to obtain direct recognition of these and otherterminal epitopes.

While the mechanism of adhesion remains uncer-tain, it is apparent that cell attachment is greatly en-hanced for b-PA coated scaffolds. Notably, after initialattachment, SMCs did not demonstrate substantialproliferation, but instead were directed towards mor-phological changes and matrix production. Con-versely, cell numbers on bare PGA scaffolds did in-crease over time, which may have been due todetachment of cells from other scaffolds, or morelikely, from proliferation. Experiments could not beextended beyond 31 days because of degradation ofthe PGA scaffolds, preventing an assessment ofwhether this trend would continue. Future targets ofthis work will investigate more detailed markers, suchas protein production, gene expression, and rates ofproliferation/apoptosis, as we attempt to more con-cretely identify the influence of PA molecules on cellphenotype. We are additionally interested in seedingthese scaffolds at higher cell concentrations to createmore tissue-like structures.

In summary, we have demonstrated and character-ized the coating of branched and linear PA moleculesonto traditional PGA scaffolds in an effort to influencecell attachment and phenotype expression. PA mole-cules were shown to coat reproducibly onto PGA,with clear surface retention under cell culture condi-tions, and self-organization into nanofibers. BladderSMCs were influenced to attach preferentially to scaf-folds coated with b-PA at early time points. We plan tofurther investigate the utility of these scaffold compos-ites for bladder tissue engineering and for other phys-iologic systems.

The authors thank Yuan Yuan Zhang and Rick Cowan fortheir assistance in SMC culture, and Mona Cornwell for herassistance in histology. This work made use of instruments



in the following Northwestern University facilities: ElectronProbe Instrumentation Center (EPIC), Keck InterdisciplinarySurface Science Center (Keck II), Keck Biophysics Facility,the Analytical Services Laboratory, and the Biological Imag-ing Facility.

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Branched peptide-amphiphiles as self-assembling coatings for tissue engineering scaffolds

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