Improving the osteogenic potential of BMP-2 with hyaluronic acid hydrogelmodi!ed with integrin-speci!c !bronectin fragment

Marta Kisiel a, Mikaël M. Martino b, Manuela Ventura d, Jeffrey A. Hubbell b,c, Jöns Hilborn a,Dmitri A. Ossipov a,*

aDivision of Polymer Chemistry, Department of Chemistry-Ångström Laboratory, Science for Life Laboratory, Uppsala University, SE-751 21 Uppsala, Swedenb Institute of Bioengineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerlandc Institute of Chemical Sciences and Engineering, School of Basic Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, SwitzerlanddDepartment of Biomaterials, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands

a r t i c l e i n f o

Article history:Received 18 August 2012Accepted 5 October 2012Available online 24 October 2012

Keywords:rhBMP-2Hyaluronic acid hydrogelCell adhesionIntegrinsFibronectinBone regeneration

a b s t r a c t

While human bone morphogenetic protein-2 (rhBMP-2) is a promising growth factor for bone regen-eration, its clinical ef!cacy has recently shown to be below expectation. In order to improve the clinicaltranslation of rhBMP-2, there exists strong motivation to engineer better delivery systems. Hyaluronicacid (HA) hydrogel is a suitable carrier for the delivery of rhBMP-2, but a major limitation of this scaffoldis its low cell adhesive properties. In this study, we have determined whether covalent grafting of anintegrin-speci!c ligands into HA hydrogel could improve cell attachment and further enhance theosteogenic potential of rhBMP-2. A structurally stabilized !bronectin (FN) fragment containing the majorintegrin-binding domain of full-length FN (FN III9*-10) was engineered, in order to be incorporated intoHA hydrogel. Compared to non-functionalized HA hydrogel, HA-FN hydrogel remarkably improved thecapacity of the material to support mesenchymal stem cell attachment and spreading. In an ectopic boneformation model in the rat, delivery of rhBMP-2 with HA-FN hydrogel resulted in the formation of twiceas much bone with better organization of collagen !bers compared to delivering the growth factor innon-functionalized HA hydrogel. This engineered hydrogel carrier for rhBMP-2 can be relevant in clinicalbone repair.

! 2012 Elsevier Ltd. All rights reserved.

1. Introduction

The delivery of growth factors to injured sites is believed to beable to resolve many issues in regenerative medicine. However,clinical translation of growth factors has been very limited. In fact,a number of growth factors have been tested in phase II clinicaltrials and showed bene!ts to patients below expectations [1e3].Many growth factors showed poor ef!cacy and the ones that showsclinical bene!ts still present issues related to safety [4,5] and cost-effectiveness [1]. For example, the use of rhBMP-2 in spinal surgerydemonstrated an incidence of adverse events of 10e50% of thecases, depending on the approach [5]. Therefore, to improve theunsatisfactory clinical translation of growth factors, there exists

strong motivation to engineer better growth factor deliverysystems. Besides the fact that the extracellular matrix (ECM) bindsgrowth factors, a deeper understanding of the dynamic interactionsbetween ECMproteins, growth factors, cell-adhesion receptors, andgrowth factor receptors is rising [6e8]. Interestingly, because somemolecules involved in the signaling machinery of growth factorsand integrins are common [7,8], the formation of molecularcomplexes between growth factors and ECM proteins such as!bronectin (FN) [9,10] can greatly enhance the potency of growthfactors [11].

Hyaluronic acid (HA) is a non-sulfated glycosaminoglycanwidely distributed in the extracellular matrix (ECM). HA plays animportant role in regulation of cell adhesion, morphogenesis andmodulation of in"ammation [12,13], and in numerous importantphysiological processes such as wound healing [14,15]. HA hydrogelscaffolds have been one of the most promising materials for bonetissue repair. Furthermore, HA hydrogels are biodegradable andnon-immunogenic, which enables their use for various clinicalapplications such as ophthalmic surgery and arthritis treatment[16]. HA hydrogels have been used as a carrier for growth factors

* Corresponding author. Division of Polymer Chemistry, Department of Chem-istry, Ångstrom Laboratory, Uppsala University, Box 538, SE-751 21 Uppsala,Sweden. Tel.: !46 18 471 73 35; fax: !46 18 471 34 77.

E-mail addresses: dmitri.ossipov@mkem.uu.se, dmitri.ossipov@kemi.uu.se(D.A. Ossipov).

Contents lists available at SciVerse ScienceDirect

Biomaterials

journal homepage: www.elsevier .com/locate/biomateria ls

0142-9612/$ e see front matter ! 2012 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biomaterials.2012.10.015

Biomaterials 34 (2013) 704e712

such as bone morphogenetic protein-2 (BMP-2) [17], and the localdelivery of recombinant human BMP-2 (rhBMP-2) has been foundpromising for the assessment of non-healing bone fractures [18,19].For example, we have previously shown that the encapsulation ofrhBMP-2 in HA hydrogel provides its sustained release in vitrofor almost a month. This ef!cient encapsulation was corroboratedwith potent in vivo bone formation on ectopic [20] and orthotopicsites [21,22].

HA can interact with several cell surface receptors, called hya-ladherins, such as CD44 and RHAMM, which are involved in regu-lating growth factor signaling [23], and ICAM-1, a metabolicreceptor for HA [24]. However, HA is unable to bind key adhesionreceptors such as integrins. Because cells need to properly assimi-late within a biomaterial that is designed to promote tissue regen-eration, HA hydrogel should be able engage integrins. Therefore,great attention has been paid to modify native HA hydrogels withintegrin ligands derived from ECM [25,26]. The most ubiquitousintegrin-binding peptide used to functionalize hydrogels is based onthe arginineeglycineeaspartic acid (RGD) sequence, which ispresent within many ECM proteins, such as !brinogen and !bro-nectin (FN) [27,28]. Despite the speci!city differences that can beobtained by modifying the surrounding amino acid environment,RGD-based peptides are limited to signal through the so-called“RGD-binding integrins” [27]. However, many tissue morphoge-netic processes require integrins that cannot be engaged by RGDonly. For example, it has been shown that angiogenesis [29] andbone formation or regeneration [11,30] are dependent of theintegrin a5b1. Functionally, ECM interaction with a5b1 requiresboth the RGD sequence located in the 10th type III repeat of FN (FNIII10) as well as the “synergy sequence” (PHSRN) in the adjacent 9thtype III repeat (FN III9). In several reports, it has been shown thatbiomaterials designed to preferentially engage a5b1 could enhancetissue morphogenesis. For example, recombinantly expresseddomains of FN, FN III7-10, were shown to improve osseointegrationof implants in bone defects and enhance osteoblastic differentiationof mesenchymal stem cells (MSCs), when compared to surfacesfunctionalized with the linear RGD peptide [31]. Moreover, !brinmatrix functionalized with a stabilized variant of FN III9-10 (FNIII9*-10, where the leucine1408 is substituted by a proline) [32]signi!cantly enhanced osteogenic differentiation of MSCs andbone regeneration, even compared to full-length FN [11,33].

We have previously exploited hydrazone cross-linking reactionto obtain suitable injectable carriers for rhBMP-2 and subsequentlyuse the hydrogel to form bone in ectopic [20,34] and orthotopic[21,22,35] animal models. In the present work, we aim to func-tionalize HA hydrogel with the a5b1 integrin-speci!c FN fragment(FN III9*-10), in order to improve MSCs integration to the material.Moreover, we hypothesized that the delivery of rhBMP-2 throughan HA hydrogel functionalized with the FN fragment would furtherenhance the osteogenesis in vivo.

2. Materials and methods

2.1. Material synthesis

2.1.1. Production and puri!cation of the FN fragmentFN III9*-10 was recombinantly produced as described elsewhere [33]. In addi-

tion, the FN fragment was engineered with a single cysteine at its N-terminus forfurther functionalization with vinyl sulfone. After puri!cation, the protein wasstored in Tris-buffered saline (TBS; 150 mM NaCl, 20 mM TriseHCl, pH 7.4) andreduced with 10 M excess of dithiothreitol. Directly after, the solution was dialyzedagainst TBS for 2 h. Then,100 M excess (relative to the FN fragment) of divinyl sulfone(SigmaeAldrich) was added in the dialysis bag and the mix was further extensivelydialyzed against TBS. The protein was then repuri!ed by size exclusion (HiLoad 16/60 Superdex 75 prep grade, GE healthcare) to remove eventual FN fragment dimersand !nally stored in TBS. Functionalization of the FN fragment with a single vinylsulfone per protein was veri!ed by MALDI-TOF analysis. Moreover, endotoxin levelwas veri!ed as under 0.01 EU/g (HEK-Blue LPS Detection Kit, InvivoGen).

2.1.2. Coupling of the dually modi!ed HA-hydrazide-thiol to the FN fragmentHydrazide-thiol HA (HA-hy-SH) and aldehyde-derivatized HA (HA-al) were

prepared from 150 kDa hyaluronic acid (HA was purchased from Lifecore Biomed-ical) according to our previously established protocol [36,37]. We veri!ed theattachment of the FN fragment to the HA-hy-SH backbone by sodium denaturingdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. A GibcoMini-V 8/10 Vertical Gel Electrophoresis System and power supply were used. Thesamples were prepared from 5 mg of the lyophilized components (HA-hy-SH, HA-hy) by !rst dissolving them in 235 mL of phosphate buffered saline (PBS). 15 mL ofthe solution of vinyl sulfone terminated FN (4.3 mg/mL) was added to each HAcomponent solution (either HA-hy-SH or HA-hy) and the mixtures were incubatedovernight. The samples were mixed with the sample buffer (ClearPAGE, CBSScienti!c) and loaded onto 4e10% SDS polyacrylamide gels (CBS Scienti!c) in non-reducing conditions. Then gel was stained with Coomassie Blue (Fermentas) over-night at room temperature, destained in distilled water, and photographed by digitalcamera.

2.1.3. Hydrogel preparation and rheological evaluationHA-al and HA-hy-SH were dissolved at 2 wt.% in PBS and equal volumes of these

solutions were mixed in a cylindrical glass vials to make 0.4 mL hydrogel samples(HA hydrogel). To make hydrogels containing the FN fragment (HA-FN hydrogel),12 mL of the vinyl sulfone terminated FN solution (4.3 mg/mL) was !rst added to0.2 mL of 2 wt.% solution of HA-hy-SH in PBS. The reaction was allowed to proceedfor 15 min after which the mixture was combined with 0.2 mL of 2 wt.% solution ofHA-al in PBS. The hydrogels were left in the capped vials for another 24 h tocomplete cross-linking reactions. The cylindrically shaped hydrogels were thencarefully transferred from the vials and incubated in PBS for further 24 h at roomtemperature. The mechanical properties were characterized by rheology (AR200advanced Rheometer; TA Instruments). The shear storage modulus, G0 was deter-mined. A custom-made parallel plate aluminum geometry of 8 mm diameter ortitanium geometry of 19 mm diameter was used. The wet hydrogels were weighted(Mt) and then freeze-dried to get dry masses (M0). The swelling ratio (S) is de!ned asfollows: S " (Mt # M0)/M0.

2.2. In vitro studies

2.2.1. Cell maintenanceThe mouse MSC line W20 clone 17 (W20-17) was obtained from the American

Type Culture Collection (ATCC-LGC Standards, Sweden). Cells were expanded inDulbecco’s Modi!ed Eagle’s medium (DMEM) supplemented with 10% fetal bovineserum (FBS; PerBio Science) and 1% antibiotics (penicillin/streptomycin) in T75"asks (Nunc, VWR International). Cells were maintained at 37 $C/5% CO2 and the cellculture medium was changed every second day. When cells reached 80% ofcon"uence, they were detached by trypsin/ethylenediaminetetraacetic acid (Gibco,Invitrogen) and subjected to further in vitro experiments.

2.2.2. Cell viability in HA-al and HA-hy-SH solutionTo quantitatively evaluate the toxicity of the soluble hydrogel precursors, Cell-

Titer 96AQueous One Solution Cell Proliferation Assay (MTS, Promega) was per-formed. HA-al and HA-hy-SH were dissolved in cell culture media at concentrationsof 100 or 150 mg/mL and the solutions were sterilized by !ltration through a 0.45 mm!lter. The W20-17 cells were plated at a density of 5 % 104 cells per well in 200 mL ofculture media and incubated at 37 $C/5% CO2. After 24 h the culture medium wasreplacedwith the dissolved hydrogel components. Hyaluronic acid (HA) dissolved inthe cell culture medium at a concentration of 150 mg/mL was used as a positivecontrol. After 48 h of incubation, cells were treated with MTS reagent for 4 h and theabsorbance was measured at 450 nm. The absorbance of the cells alone treated withMTS was related to 100% of viability and the number of viable cells treated with theHA components was calculated as percent of the number of viable cells in the controlexperiment.

2.2.3. Cells spreading on gels surfaceIn order to analyze cell spreading on gels surface, MSCs were seeded on top

of the HA hydrogels. 200 mL of hydrogel samples were prepared in wells of a 24-well plate by placing HA-hy-SH (with or without FN fragment) !rst and thenadding HA-al solution to the same wells. The contents of the wells were mixedwith the help of a sampler tip and allowed to form hydrogels for 2 h. Serum-freecell culture medium was added to the hydrogels for 15 min and then aspirated.This washing procedure was repeated three times to wash out any unbound FNfragment. The cells in 500 mL culture medium were placed on top of thehydrogel samples at a concentration of 20 % 104 cells per well and incubated at37 $C/5% CO2. The cell morphology and spreading were evaluated at differenttime points (0 h, 24 h, and 48 h) by phase contrast microscopy (Eclipse TE2000U, Nikon).

2.2.4. Cell attachment on gels surfaceTo determine cell adhesion to hydrogel surfaces, MSCs were seeded on top of the

hydrogel samples as described in the previous section. Qualitative evaluation byLive/Dead Cell Viability assays (Invitrogen) and quantitative analysis by crystal violet

M. Kisiel et al. / Biomaterials 34 (2013) 704e712 705

assay were performed. Qualitative analysis was performed with 200 mL hydrogelsamples. The cells in 500 mL culture medium were seeded at a concentration of20% 104 cells per well and incubated at 37 $C/5% CO2 for 24 h. The cells werewashedtwice with PBS and once with 200 mL of the assay solution (the assay solution wasprepared by dissolving calcein and ethidium bromide in PBS according to themanufacturer protocol followed by dilution with cell culture medium). The assaysolution was added to the washed cells and the cells were incubated at 37 $C for45 min. The cells were washed !nally with PBS and imaged using inverted "uo-rescent microscopy. For the quantitative analysis, 100 mL of hydrogel samples wereformed in 96-well plate (n " 5). The cells in 100 mL culture mediumwere seeded ontop of the hydrogels at a concentration of 5 % 104 per well and incubated at 37 $C ina humidi!ed atmosphere of 5% CO2. After 4 h of incubation, the cells were washedtwice with PBS to avoid staining of non-adherent cells. The cells were subsequently!xated with 4% paraformaldehyde in PBS at room temperature for 30 min. 100 mLsolution of crystal violet dye in of 10% acetic acid was added to each well followed byincubation for 60 min. The absorbance was measured at 570 nm, using microplatereader (Kinetic Microplate Reader; Molecular Devices).

2.3. In vivo studies

2.3.1. Hydrogel preparation for in vivo studiesrhBMP-2 (InductOs" P!zer, delivered as a lyophilized powder in a formulation

buffer containing 2.5% glycine, 0.5% sucrose, 0.01% Polysorbate 80, 5 mM sodiumchloride and 5 mM L-glutamic acid) was reconstituted by the addition of deionizedwater at a concentration of 0.1 mg/mL and the obtained stock solution was stored at4 $C. The solutions of HA-al and HA-hy-SH in PBS were prepared at 2 wt.%concentration and !ltered through a 0.45 mm !lter for sterilization. For the prepa-ration of HA-FN hydrogels, the solution of vinyl sulfone terminated FN (4.3 mg/mL)was preliminary added to the !ltered 2 wt.% solution of HA-hy-SH in PBS at 3:50volume ratio and the resulting mixture was agitated at room temperature for15 min. The equal aliquots of the rhBMP-2 stock solution were added to the equalvolumes of the hydrogel precursor solutions (HA-al and HA-hy-SH for HA hydrogels,and HA-al and HA-hy-SH ! FN for HA-FN hydrogels). The resulting 1.6% w/v HAsolutions containing 20 mg/mL of BMP-2 were placed in two separate 1 mL syringes,and the tips of each syringe were subsequently interconnected by means of a luer-lock adapter. The solutions were mixed by passing the contents of the syringes fromone to the other. After 20 passages, the hydrogels were formed. The hydrogels wereallowed to set in the syringes for approximately 3 h before injections into animals.

2.3.2. Ectopic bone formation in ratsSix male SpragueeDawley rats (Taconic M&B, Lille Skensved, Denmark) of

approximately 250e300 g of weight were used for this study strictly following thelaws on animal experimentation. All protocols were approved by the Local AnimalCommittee of Uppsala University (246/8). Two animals per cage were maintainedwith a 12 h day/night cycle at room temperaturewith ad libitum access towater anda standard rat food. Hydrogels of 200 mL were injected under the skin of 6 rats by a 21G needle. The !nal concentration of rhBMP-2 was 20 mg/mL of hydrogel (4 mg persample). The concentration of the FN fragment in the HA-FN hydrogels was 136 mg/mL of hydrogel. Before the hydrogel injections, the rats were anesthetised with a 5%iso"urane/oxygen mixture that was reduced to 2% during the implantation of thehydrogels. After shaving the dorsal area of the animal and disinfecting it with 70%ethanol, two injections (HA-FN hydrogel and the control HA hydrogel) per animalwere performed. The animals weremonitored during the time of experiment and nosign of infection was noticed. Seven weeks after injections, the animals wereeuthanatized via CO2 asphyxiation. The harvested ectopic bone samples were !xedin 4% of paraformaldehyde in PBS for a minimum of 24 h.

2.3.3. Quanti!cation of ectopic boneEctopic bone samples were subjected to micro-CT analysis using Skyscan 1072

(Kontich, Belgium) with X-ray Source 100 kV/98 mA for bone volume/tissue volume(BV/TV) quanti!cation. All samples were placed vertically onto the sample holderand analyzed at a 14.16 mm % voxel resolution (magni!cation 20%, exposure time3.9 s, 1 mm !lter applied). The creator software (3D-DOCTOR 4.0, Able SoftwareCorp.) was used for 3-dimensional coronal section reconstructions.

2.3.4. Histological evaluationEctopic bone samples were decalci!ed with formic acid using electrophoresis

system Tissue-TekMiles scienti!c (Histolab Göteborg), dehydrated in a graded seriesof ethanol and embedded in paraf!n. Five mm sections were cut with microtomethrough the middle part of the specimen. For morphological observation, thespecimens were stained with Hematoxylin and Eosin (H&E, Merck) and Masson’sTrichrome (Merck). In order to detect possible remaining hydrogel in the ectopicbones, the sections were stained with Alcian Blue (SigmaeAldrich) at pH 2.5 [34].Sections were incubated in a phosphate buffer (pH 6.0) or in the phosphate buffercontaining 0.5 mg/mL of ovine testicular hyaluronidase (SigmaeAldrich) asa control. Additionally, the sections were stained with primary antibodies speci!cfor osteocalcin (OC, Santa Cruz Biotechnology). The epitopes were unmasked withantigen retrieval (0.05% trypsin solution, SigmaeAldrich). The primary antibodiesfor OC were diluted 1:50 in PBS containing 1% bovine serum albumin. After

overnight incubation at 4 $C, the sections were rinsed two times with TRIS buffer for10 min and labeled using Rabbit/Mouse EnVision# Peroxidase System (Dako).Antibody complexes were visualized with 3,30-diaminobenzidine (DAB kit).Sections of cranial bone and sections not treated with the primary antibody wereused for controls.

The sections were photographed with an optical microscope, using imageanalysis software (Eclipse TE 2000U, Nikon). For observation of collagen orientation,Sirius Red (Fluka) staining was used. Sirius red was dissolved at 0.1% in a saturatedaqueous solution of picric acid (Fluka). The images were made using polarized lightmicroscope with a slides inclined at 45$ to the incident light.

2.3.5. Angiogenesis evaluated by proximity ligation assayAngiogenesis in ectopic bone was detected with a mouse monoclonal antibody

to rat endothelial cell cytoplasmic antigen (RECA-1; Abcam) using in situ proximityligation assay (PLA) [27]. The 5 mm thick cross-sections were incubated in antigenretrieval solution (Dako) at 98 $C for 15 min, washed with PBS and incubated in Tris-Buffered Saline (TBS at pH 7.4) for 2 min. The sections were incubated overnightwith anti-RECA-1 (1:200) at 4 $C. The next day, sections were rinsed in PBS andincubated in a secondary probe ligation and ampli!cation, in accordance withmanufactures protocol (Duolink II Kit). The IHC stained sections were mounted withVectashield (Vector Laboratory) containing DAPI (100 ng/mL). The sections wereobserved and photographed using "uorescent microscopy (Eclipse TE 2000U,Nikon).

2.3.6. Statistical analysisThe statistical analysis was performed using SPSS for Windows (17.0, standard

version, SPSS"). For all data, mean values and standard deviations (SD) werecalculated. Comparison between groups was made by two-tailed, unpaired t tests. Ap-value <0.05 was considered signi!cant.

3. Results and discussion

3.1. Synthesis of HA-hydrazide-thiol and its conjugation to FNfragment

The limited capacity of HA hydrogels to bind integrins has beenpreviously addressed by Prestwich and co-workers, who coupledRGD peptides or FN fragments to the hydrogels [25,26]. By stimu-lating dermal !broblast responses, HA hydrogels functionalizedwith FN fragments improved wound healing, when compared toHA hydrogel only or to the HA hydrogel functionalized with RGDpeptide. The synthesis of the HA hydrogels was based on Michaeladdition of the thiolated HA and the cysteine-terminated integrin-ligands to the PEG-diacrylate cross-linker. In this work, wecombined our previously exploited hydrazone cross-linking reac-tion with the conjugation by Michael addition. Both Michaeladdition and hydrazone coupling reaction are “click” type reactionsthat can be performed in aqueous media under physiologicalconditions. More importantly, these reactions are chemicallyorthogonal, i.e. can undergo independently without interferingwith each other (Scheme 1). We therefore hypothesized that byusing an HA derivative that is dually functionalized with two typesof groups, hydrazide and thiol (HA-hy-SH) [36,38] we could achievebio-conjugation of a suitably functionalized FN fragment to HA-hy-SH as well as the subsequent hydrazone network formation ina modular manner. By combination of orthogonal “click” reactionswe aimed to achieve better control over the hydrogel assembly.

To improve the poor cell adhesive propriety of HA hydrogels, wehave chosen to incorporate FN III9*-10 into HA hydrogel. The a5b1integrin-speci!c fragment was engineered with a single cysteine atits N-terminus, in order to be further functionalized with vinylsulfone (VS-FN III9*-10). Next, we have tethered VS-FN III9*-10 toHA-hy-SH in the course of Michael type addition of the thiol groups(Fig. 1a). To verify whether the vinyl sulfone group speci!callyreacted with the thiol group or if a side reaction between the vinylsulfone and the hydrazide group took place, VS-FN III9*-10 was alsoincubated with the HA derivative containing only hydrazide groups(HA-hy). The reactions were examined by denaturing SDS-PAGEanalysis and compared with the free VS-FN III9*-10 (Fig. 1c). Theappearance of a retarded band was observed when VS-FN III9*-10

M. Kisiel et al. / Biomaterials 34 (2013) 704e712706

was mixed with the dually modi!ed HA-hy-SH (left lane in Fig. 1c).This band should correspond to the FN fragment covalently linkedto HA, due to considerably higher molecular weight and thenegative charge of the resulting adduct. In contrast, mixing of theHA-hy derivative with VS-FN III9*-10 resulted in only minoramount of the addition product (middle lane in Fig. 1c). This indi-cates that the addition of thiol groups to vinyl sulfone with theformation of thioester bond is much more ef!cient than the

addition of hydrazide groups. The side reaction of hydrazides,however, cannot be completely excluded due to very high reactivityof the vinyl sulfones. It should be noted that the Michael typeaddition to acryloyl derivatives might be more speci!c for thisparticular case.

The main problem with covalent conjugation of proteins is thepreservation of their initial structure and biological activity. The useof chemoselective reactions is particularly advantageous in suchconjugation approaches, especially when considering !nal thera-peutic applications [39,40]. In this respect, modi!cation of polymerprecursor molecules with orthogonal thiol and hydrazide groups istherefore suitable for the modular construction of complexpolymer-protein hydrogel materials without compromising thestructure of the respective protein. It also requires site-speci!cfunctionalization of the intended protein with a complementaryreactive “click”-type group. In the majority of cases, it is performedthrough a laborious genetic engineering and expression of therecombinant proteins in biological systems. However, only terminalsite-speci!c covalent immobilization of FN III9*-10 intoHAhydrogelcan ensure biological activity of the FN fragment after conjugation.Moreover, chemical linking of biologically active molecules issuperior to their physical encapsulation since it prevents their rapidclearance from the material by diffusion [41,42].

3.2. Mechanical properties of hydrogels

The HA-hy-FN adduct formed a hydrogel material (HA-FNhydrogel) upon in situ mixing with the aqueous solution of HA-al(Fig. 1b). The gel point was achieved in about 1 min after mixingthe two components. Table 1 reveals the mechanical properties ofthe HA-FN hydrogel in comparison with the control that has beenformed from the same HA precursors without FN fragment (HAhydrogel). As expected, theminor amount linked FN fragment (0.7%by mass per solid content of the hydrogel) did not in"uence toappreciable extent the mechanical properties of the parent HAhydrogel, albeit in the swollen state, the HA-FN hydrogel hadslightly lower elastic modulus. It can be explained by a differentchemical equilibrium that is likely achieved in the swollen networkcontaining a protein component. Amino groups of the protein can

Fig. 1. Conjugation of FN III9*-10 to HA. (a) Conjugation of VS-FN III9*-10 to HA-hy-SH derivative with the formation of HA-hy-FN adduct. (b) In situ cross-linking of the HA-hy-FN toform HA-FN hydrogel. (c) SDS-PAGE analysis of the reaction between VS-FN III9*-10 with HA-hy-SH (left lane) and HA-hy (middle lane) derivatives.

Scheme 1. Modular “click” assembly of the essential ECM components and otherarti!cial ligands into a 3D hydrogel network. The stepwise modular “click” assembly ofan ECM mimicking multicomponent scaffold with a de!ned connectivity between thecomponents becomes readily accessible when utilizing multiple chemically orthogonalfunctionalities. Accordingly, different polymeric components (A and B) are !rst multi-functionalized with orthogonally reactive groups presented by different colors. Bio-logically relevant epitopes (X and Y), exhibiting their functionality in a cell-inductive orcell-responsive manner, are then installed on the macromolecular components usingtheir speci!c groups (blue and green colored). Finally, the obtained epitope-graftedcomponents are transformed into a hydrogel material in the course of a cross-linking reaction between the remaining free functionalities (red colored). (For inter-pretation of the references to color in this !gure legend, the reader is referred to theweb version of this article.)

M. Kisiel et al. / Biomaterials 34 (2013) 704e712 707

compete with the HA hydrazide groups for the aldehyde groups,reversibly forming imine linkages. The possible existence of anadditional imine bonding between the HA-al and the FN fragmentmay lower the cross-linking density of the resulting network. Thiswas also re"ected in higher swelling ratio of the HA-FN hydrogelcompared to the HA hydrogel (Table 1).

3.3. Cytotoxicity of the hydrogel individual components

As a natural-derived polymer, HA is known to be biologicallyvery safe. However, chemical modi!cation of HA and incorporationof reactive side groups onto HA hydrogel might be toxic for cells.We therefore performed a cytotoxicity test for the soluble HA-aland HA-hy-SH gel precursors, using an MTS assay. MSCs wereincubated with HA-al and HA-hy-SH derivatives dissolved at !nalconcentrations of 100 and 150 mg/mL. For comparison, native HA atconcentration of 150 mg/mL was used as a positive control (Fig. 2).We chose these concentrations because, when using HA hydrogelsin vivo, we inject the mixture of the hydrogel-forming componentsalready after their complete cross-linking (3 h after mixing thesoluble components). Thus, we do not expect to have free reactiveHA polymers at concentrations higher than 0.1 g/mL. After 48 h ofincubation of the cells with the hydrogel-forming HA components,the cell survival was more than 80% at concentration of 150 mg/mLand more than 90% at concentration of 100 mg/mL. Additionally, weobserved proliferative properties of HA-al and HA-hy-SH, when theviability exceeded 100%. These results show that the solublehydrogel precursors are not toxic at a concentration of 0.1 g/mL.

3.4. MSC attachment and spreading on hydrogel surfaces

The ability of HA-FN hydrogel to support cell attachment andspreading was determined by three different assays. As a cell

model, we have chosen MSCs, since they play a crucial role in bonerepair. Indeed, after bone injury, MSCs are recruited from thesurrounding tissues and greatly contribute to bone healing bymultiple mechanisms [43,44]. In all the following experiments, wehave analyzed the attachment, spreading and the morphology ofMSCs that have been seeded on the top of HA-FN hydrogel or HAhydrogel control. We found that MSCs could attach and spreadmuch faster on the hydrogels functionalized with the FN fragmentcompared to the control HA hydrogel (Fig. 3a). After 24 h of incu-bation, MSCs on HA-FN hydrogels presented a "at and spindleshape with a !broblastic morphology. After 48 h they formedbranches and covered the hydrogel with multicellular networks. Incontrast, MSCs on HA hydrogels were round shaped at 24 h timepoint and presented a stationary !broblastic morphology only after48 h. We have also evaluated cell attachment in a qualitative andquantitative manner. We !rst observed MSC attachment by a Live/Dead staining assay after 48 h of culture. We found that cellspreferably adhered to the surface of HA-FN hydrogel than to thesurface of the control HA hydrogel, where a fraction of the cellsdyed (Fig. 3b). Secondly, we quanti!ed the number of cells thatcould adhere to the hydrogels, by performing a crystal violet assay.After 4 h of culture, 79% of MSCs attached to the HA-FN hydrogel,while only 56% of the cells attached on the surface of the control HAhydrogel (Fig. 3c).

It has been demonstrated that MSCs can attach and spread veryrapidly on FN III9*-10 [33], due to their high expression of the a5b1integrin [45]. Here, we show that the incorporation of FN III9*-10into HA hydrogels can greatly enhance the capacity of the materialto support adhesion, spreading, and thus further proliferation ofMSCs. Moreover, we found that HA-FN hydrogel provides rapidintracellular branching of MSCs that may further improve osteo-genic differentiation due to ef!cient cellecell communication [42].We can exclude that the effect observed was due to mechanicalsignaling, since our rheology studies show similar substrate stiff-ness for both types of hydrogels.

3.5. Ectopic bone formation

In our previous studies, we have demonstrated that rhBMP-2 issustainably released for almost amonth, when encapsulatedwithinan injectable HA hydrogels. This remarkable capacity of HAhydrogel to retain rhBMP-2 translated into the formation of bone inectopic [20,34] and orthotopic [22,35] animal models. Here, wehypothesized that the delivery of rhBMP-2 through an HA hydrogelfunctionalized with the integrin-speci!c FN fragment wouldfurther enhance the osteogenic potential of the growth factor. Forthis purpose, we have selected the formation of ectopic bone in rat,as a model. This is a suitable and reproducible model used toevaluate the potential of biomaterials and growth factors for boneformation. HA-FN and HA hydrogels (0.2 mL) with or without 4 mgof rhBMP-2 per sample were prepared and injected under the skinof 7 week old rats. To maintain high reliability we randomlyinjected one sample from each group on the back of each animal.Seven weeks after injection, the animals were sacri!ced and theharvested specimens were studied. For the HA-FN and HA gelsinjected without BMP-2 we could not visually observe any residualgel or any bone formation. This complete HA hydrogel degradationcorroborated with results obtained from our previous subcuta-neous animal studies [20]. Contrary, at all sites where the hydrogelswere injected with entrapped rhBMP-2, we found ectopic bone!rmly attached to the subcutaneous soft tissue. Moreover, noin"ammatory response or formation of !brotic capsule around thehydrogel samples could be observed. All bone specimens showedrounded like shape that was visualized by 3D reconstructions frommicro-CT analysis (Fig. 4a and b). Quantitative measurement of the

Table 1Mechanical properties of HA hydrogels.

Gel composition G0formed

a (Pa) G0swollen

b (Pa) Sswollenc

HA-hy-SH ! HA-al 2520 & 30 405 & 78 59.9 & 1.0HA-hy-SH ! FN ! HA-al 2600 & 30 325 & 31 61.6 & 1.5

Data are mean & SD for n " 3.a G0

formed e elastic modulus measured for the hydrogels after setting for 24 h.b G0

swollen e elastic modulus measured for the hydrogels after setting for 24 h andsubsequent swelling in PBS for another 24 h.

c Swelling ratio of the swollen hydrogels.

Fig. 2. Toxicity of HA hydrogel precursors. Viability of MSCs after 48 h of incubation inthe presence of 100 and 150 mg/mL of HA-al, HA-hy-SH derivatives, or non-modi!edHA. Error bars indicate SD for n " 5.

M. Kisiel et al. / Biomaterials 34 (2013) 704e712708

average bone volume formed revealed that HA-FN hydrogel formedtwice more bone tissue (p < 0.05), compared to non-functionalizedHA hydrogels (Fig. 4c).

3.6. Histology analysis of ectopic bone

In order to determine the morphology and quality of the boneformed ectopically, the samples were stained with Masson’s Tri-chrome (Fig. 5a and c) and H&E (Fig. 5b and d). In both HA-FN andHA groups, we could observe the presence of bone marrow.However, the bone formed in place of the HA-FN hydrogel hada more dense distribution of trabecular-like structures which grewdeeper toward the middle core of the samples, while, in the HAgroup, the remodeling process was limited to the edges. In the HAhydrogel group, the bone trabecula were observed only on theperipheral sides of the ectopic bone and the core part was !lled

with a bone marrow fat tissue. In contrast, analysis of the bonemarrow at higher magni!cation revealed that the HA-FN hydrogelgroup (Fig. 5b and f) had more homogenous distribution of osteo-blasts with less adipose tissue compared to the HA hydrogel group(Fig. 5d and h).

In addition, the obtained bone samples from both groups werepositive for osteocalcin (Fig. 6a and b). The staining was similar tothe control specimen of a rat autologous cranial bone (Fig. 6c).Furthermore, as an indication of the quality of the bone tissueformed, we examined the assembly of collagen !bers. We foundthicker collagen !bers of higher organization, in the HA-FNhydrogel group (Fig. 6e), compared to the HA hydrogel controlgroup (Fig. 6f). The dense trabecula distribution, with the betterorganization of collagen !bers in the HA-FN hydrogel group,suggests that the bone formed is mechanically stronger than in theHA-hydrogel group [46]. Finally, no hydrogel remnants were

Fig. 4. Ectopic bone formation. Reconstructed 3D structure of ectopic bone formed after 7 weeks for (a) HA-FN hydrogel and (b) HA hydrogel groups. Scale bars, 1 mm. (c)Quantitative analysis of the bone volume normalized to the total tissue volume (BV/TV). Error bars indicate SD for n " 6 (*p < 0.01).

Fig. 3. Attachment and spreading of MSCs on HA-FN and HA hydrogel surfaces. (a) Bright-!eld photomicrographs after 0 h, 24 h and 48 h of cell culture. (b) Calcein staining (ingreen, staining viable cells) and ethidium bromide staining (in red, staining dead cells) after 48 h of cell culture. As a control, MSCs were plated on standard cell-culture plate. Scalebars, 50 mm. (c) After 4 h of incubation, the number of MSCs that attached to the different HA hydrogel surfaces was quanti!ed using crystal violet. Data are expressed as percentadhesion relative to a 100% cell-adhesion control (cell plated on standard cell-culture plate). Error bars indicate SD for n " 5 (*p < 0.05). (For interpretation of the references to colorin this !gure legend, the reader is referred to the web version of this article.)

M. Kisiel et al. / Biomaterials 34 (2013) 704e712 709

detected within the ectopic bone formed as was judged fromglycosaminoglycan staining with Alician Blue (Fig. 6gej).

3.7. Evaluation of angiogenesis within the ectopic bone

Because blood supply is a critical factor for bone regeneration[44], we addressed whether there was a difference in angiogenesisbetween the two types of HA hydrogel. Because conventionalimmunohistology was not suf!cient to objectively evaluateangiogenesis we have performed in situ proximity-ligation assay(PLA) [47,48] with the rat endothelial cell antibody-1 (RECA-1)that is speci!c to the proteins presented in the rat endothelial cellsmembrane [49]. However, RECA-1 staining was not differentbetween the two HA hydrogel groups. In both groups, weobserved several single RECA-1 positive endothelial cells andsome of them formed lumen structures (inserts in Fig. 7). Thisresults indicated that angiogenesis was similar in both groups(Fig. 7), suggesting that enhanced vascularization was not thedriver behind improved ectopic bone formation in the HA-FNhydrogels laden with rhBMP-2.

We have shown that HA-FN hydrogels promote a betterattachment of bone precursor cells such as MSCs (Fig. 3). Thus, thebetter cell-adhesive capacity of HA-FN most likely supported therecruitment of cells that participate to the formation of new bone,in vivo. On the other hand, while FN III9*-10 alone does not have thecapacity to induce osteogenesis [33], it has been demonstrated thatthe fragment greatly enhances the osteoblastic differentiation ofMSCs when the cells are cultured in osteogenic conditions [33].Moreover, it has been shown that rhBMP-2 and FN III9*-10 syner-gistically induce the osteoblastic differentiation of MSCs, althoughthis effect depends on the spacing between the integrin and thegrowth factor receptor [11]. Indeed, the ability of the FN fragmentto potentiate osteoblastic differentiation is principally due to itsspeci!city to ligate the integrin a5b1 [11,33]. The improved osteo-genic properties of the HA-FN hydrogel could in principle arisefrom the alteration of BMP-2 release due to non-speci!c bindingbetween BMP-2 and the incorporated FN fragment. The FN III9*-10fragment used in the present study does, however, not have anyspeci!c af!nity for BMP-2. In previous studies we have in factshown that the neighboring FN III12e14 domain has signi!cant

Fig. 5. Ectopic bone morphology. Representative cross-sections of the ectopic bone formed in place of (aeb and eef) HA-FN hydrogel and (ced and gef) HA hydrogel. The sectionswere stained with (aed) Masson’s Trichrome and (eeh) H&E. Scale bars are 500 mm in a,c,e,g and 100 mm in b,d,f,h. T indicates trabecular bone and BM indicates bone marrow likestructures.

Fig. 6. Osteocalcin staining of representative cross-sections from the ectopic bone formed in place of (a) HA-FN hydrogel and (b) HA hydrogel. (c) Osteocalcin staining of the sectionof the rat cranial bone and (d) is a control where primary antibody was omitted. Scale bars are 50 mm. Collagen staining of the ectopic bone formed in place of (e) HA-FN hydrogeland (f) HA hydrogel. The specimens were stained with Sirius Red and imaged by polarized light microscope. Green-yellowish color represents thick and organized collagen !bers.Scale bars are 500 mm. Alician blue staining of the sections from the ectopic bone formed in place of HA-FN hydrogel (g, h) and HA hydrogel (i, j). Staining was performed either ina phosphate buffer (g, i) or in the buffer containing hyaluronidase (h, j). Scale bars are 100 mm. (For interpretation of the references to color in this !gure legend, the reader isreferred to the web version of this article.)

M. Kisiel et al. / Biomaterials 34 (2013) 704e712710

binding af!nity for growth factors of different families includingBMP-2 while FN III9*-10 domain has a negligible af!nity for growthfactors [50]. Therefore, the increased bone formation observed inHA-FN hydrogels is attributed to better cell recruitment/adhesion,enhanced assimilation of MSCs within the hydrogel, as well as dueto potential synergistic osteoinductive signaling between theintegrin a5b1 and BMP-2 receptor.

4. Conclusion

We have successfully elaborated a modular approach for theconstruction of an HA-FN hydrogel based on consecutive “clicks”:protein bioconjugation by Michael addition and HA hydrazonecross-linking reactions. To accomplish this, we have utilized an HAderivative that is dually functionalized with orthogonal thiol andhydrazide groups, each specialized for the particular step of themodular assembly. The advantage of this strategy is that cross-linking process does not affect biological activity of the integratedprotein. In addition, covalent binding of the protein to the in situforming network prevents the FN fragment from its rapid releasefrom the material. We have demonstrated that MSC assimilationinto HA hydrogel could be signi!cantly enhanced by incorporatingthe a5b1 integrin-speci!c FN fragment into the material. Takingadvantage of the improved cell-adhesive capacity of HA-FNhydrogel and the potential synergistic signaling between FN III9*-10 and rhBMP-2, we could greatly enhance the capacity of rhBMP-2to induce the formation of ectopic bone. The delivery of rhBMP-2through HA hydrogel functionalized with FN III9*-10 resulted inthe formation of twice as much bone compared to delivering thegrowth factor in HA hydrogel. Moreover, the presence of the FNfragment in the material resulted in morphologically morehomogenous bone tissue with a better organization collagen !bers.This work provides evidence that the modi!cation of HA hydrogelwith an engineered integrin-speci!c ligand can ef!ciently enhancethe osteogenic potential of rhBMP-2. Furthermore, this approachcan be relevant in clinical bone repair.

Con!ict of interest

The authors declare no con"ict of interest.

Acknowledgments

This work was supported by the European Community’sSeventh Framework Programme in the project Angioscaff NMP-LA-2008-214402 and the Multiterm Marie Curie INT Project. We arealso thankful to Dr. Elana Ossipova for help with SDS-PAGE analysis.

References

[1] Garrison KR, Donell S, Ryder J, Shemilt I, Mugford M, Harvey I, et al. Clinicaleffectiveness and cost-effectiveness of bone morphogenetic proteins in thenon-healing of fractures and spinal fusion: a systematic review. HealthTechnol Assess 2007;11:1e150. iiieiv.

[2] Axelrad TW, Einhorn TA. Bone morphogenetic proteins in orthopaedicsurgery. Cytokine Growth Factor Rev 2009;20:481e8.

[3] Kitamura M, Nakashima K, Kowashi Y, Fujii T, Shimauchi H, Sasano T, et al.Periodontal tissue regeneration using !broblast growth factor-2: randomizedcontrolled phase II clinical trial. PLoS ONE 2008;3.

[4] Frieden IJ. Addendum: commentary on becaplermin gel (Regranex) forhemangiomas. Pediatr Dermatol 2008;25:590.

[5] Carragee EJ, Hurwitz EL, Weiner BK. A critical review of recombinant humanbone morphogenetic protein-2 trials in spinal surgery: emerging safetyconcerns and lessons learned. Spine J 2011;11:471e91.

[6] Hynes RO. The extracellular matrix: not just pretty !brils. Science 2009;326:1216e9.

[7] Yamada KM, Even-Ram S. Integrin regulation of growth factor receptors. NatCell Biol 2002;4:E75e6.

[8] Giancotti FG, Tarone G. Positional control of cell fate through joint integ-rin/receptor protein kinase signaling. Annu Rev Cell Dev Biol 2003;19:173e206.

[9] Lin F, Ren XD, Pan Z, Macri L, Zong WX, Tonnesen MG, et al. Fibronectingrowth factor-binding domains are required for !broblast survival. J InvestDermatol 2011;131:84e98.

[10] Wijelath ES, Rahman S, Namekata M, Murray J, Nishimura T, Mostafavi-Pour Z, et al. Heparin-II domain of !bronectin is a vascular endothelialgrowth factor-binding domain: enhancement of VEGF biological activityby a singular growth factor/matrix protein synergism. Circ Res 2006;99:853e60.

[11] Martino MM, Tortelli F, Mochizuki M, Traub S, Ben-David D, Kuhn GA, et al.Engineering the growth factor microenvironment with !bronectin domainsto promote wound and bone tissue healing. Sci Transl Med 2011;3:100ra89.

[12] Menzel EJ, Farr C. Hyaluronidase and its substrate hyaluronan: biochemistry,biological activities and therapeutic uses. Cancer Lett 1998;131:3e11.

[13] Collis L, Hall C, Lange L, Ziebell M, Prestwich R, Turley EA. Rapid hyaluronanuptake is associated with enhanced motility: implications for an intracellularmode of action. FEBS Lett 1998;440:444e9.

[14] Toole BP. Hyaluronan: from extracellular glue to pericellular cue. Nat RevCancer 2004;4:528e39.

[15] Fraser JR, Laurent TC, Laurent UB. Hyaluronan: its nature, distribution, func-tions and turnover. J Intern Med 1997;242:27e33.

[16] Prestwich GD. Hyaluronic acid-based clinical biomaterials derived for celland molecule delivery in regenerative medicine. J Control Release 2011;155:193e9.

[17] Burdick JA, Prestwich GD. Hyaluronic acid hydrogels for biomedical applica-tions. Adv Mater 2011;23:H41e56.

[18] Aro HT, Govender S, Patel AD, Hernigou P, Perera de Gregorio A, Popescu GI,et al. Recombinant human bone morphogenetic protein-2: a randomized trialin open tibial fractures treated with reamed nail !xation. J Bone Jt Surg Am2011;93:801e8.

[19] Swiontkowski MF, Aro HT, Donell S, Esterhai JL, Goulet J, Jones A, et al.Recombinant human bone morphogenetic protein-2 in open tibial fractures. Asubgroup analysis of data combined from two prospective randomizedstudies. J Bone Jt Surg Am 2006;88:1258e65.

[20] Bergman K, Engstrand T, Hilborn J, Ossipov D, Piskounova S, Bowden T.Injectable cell-free template for bone-tissue formation. J Biomed Mater Res A2009;91:1111e8.

[21] Martinez-Sanz E, Ossipov DA, Hilborn J, Larsson S, Jonsson KB, Varghese OP.Bone reservoir: injectable hyaluronic acid hydrogel for minimal invasive boneaugmentation. J Control Release 2011;152:232e40.

[22] Docherty-Skogh AC, Bergman K, Waern MJ, Ekman S, Hultenby K, Ossipov D,et al. Bone morphogenetic protein-2 delivered by hyaluronan-based hydrogelinduces massive bone formation and healing of cranial defects in minipigs.Plast Reconstr Surg 2010;125:1383e92.

[23] Turley EA, Noble PW, Bourguignon LY. Signaling properties of hyaluronanreceptors. J Biol Chem 2002;277:4589e92.

Fig. 7. Angiogenesis within the ectopic bone. In situ PLA detection of anti-RECA-1 (reddots) in sections of samples derived from (a) HA-FN hydrogel and (b) HA hydrogel.Nucleus was stained with DAPI (blue dots). Scale bars, 25 mm. (For interpretation of thereferences to color in this !gure legend, the reader is referred to the web version of thisarticle.)

M. Kisiel et al. / Biomaterials 34 (2013) 704e712 711

[24] McCourt PA, Ek B, Forsberg N, Gustafson S. Intercellular adhesion molecule-1is a cell surface receptor for hyaluronan. J Biol Chem 1994;269:30081e4.

[25] Ghosh K, Ren XD, Shu XZ, Prestwich GD, Clark RA. Fibronectin functionaldomains coupled to hyaluronan stimulate adult human dermal !broblastresponses critical for wound healing. Tissue Eng 2006;12:601e13.

[26] Shu XZ, Ghosh K, Liu Y, Palumbo FS, Luo Y, Clark RA, et al. Attachment andspreading of !broblasts on an RGD peptide-modi!ed injectable hyaluronanhydrogel. J Biomed Mater Res A 2004;68:365e75.

[27] Humphries JD, Byron A, Humphries MJ. Integrin ligands at a glance. J Cell Sci2006;119:3901e3.

[28] Pfaff M, Tangemann K, Muller B, Gurrath M, Muller G, Kessler H, et al.Selective recognition of cyclic RGD peptides of NMR de!ned conformation byalpha IIb beta 3, alpha V beta 3, and alpha 5 beta 1 integrins. J Biol Chem 1994;269:20233e8.

[29] Serini G, Valdembri D, Bussolino F. Integrins and angiogenesis: a stickybusiness. Exp Cell Res 2006;312:651e8.

[30] Hamidouche Z, Fromigue O, Ringe J, Haupl T, Vaudin P, Srouji S, et al. Primingintegrin alpha5 promotes human mesenchymal stromal cell osteoblastdifferentiation and osteogenesis. Bone 2009;44:S218e9.

[31] Petrie TA, Raynor JE, Reyes CD, Burns KL, Collard DM, Garcia AJ. The effect ofintegrin-speci!c bioactive coatings on tissue healing and implant osseointe-gration. Biomaterials 2008;29:2849e57.

[32] van der Walle CF, Altroff H, Mardon HJ. Novel mutant human !bronectin FIII9-10 domain pair with increased conformational stability and biological activity.Protein Eng 2002;15:1021e4.

[33] Martino MM, Mochizuki M, Rothen"uh DA, Rempel SA, Hubbell JA, Barker TH.Controlling integrin speci!city and stem cell differentiation in 2D and 3Denvironments through regulation of !bronectin domain stability. Biomaterials2009;30:1089e97.

[34] Kisiel M, Ventura M, Oommen OP, George A, Walboomers XF, Hilborn J, et al.Critical assessment of rhBMP-2 mediated bone induction: an in vitro andin vivo evaluation. J Control Release 2012;162:646e53.

[35] Martinez-Sanz E, Varghese OP, Kisiel M, Engstrand T, Reich KM, Bohner M,et al. Minimally invasive mandibular bone augmentation using injectablehydrogels. J Tissue Eng Regen Med 2012.

[36] Ossipov DA, Yang X, Varghese O, Kootala S, Hilborn J. Modular approach tofunctional hyaluronic acid hydrogels using orthogonal chemical reactions.Chem Commun 2010;46:8368e70.

[37] Ossipov DA, Piskounova S, Varghese OP, Hilborn J. Functionalization of hya-luronic acid with chemoselective groups via a disul!de-based protection

strategy for in situ formation of mechanically stable hydrogels. Bio-macromolecules 2010;11:2247e54.

[38] Yang X, Kootala S, Hilborn J, Ossipov DA. Preparation of hyaluronic acidnanoparticles via hydrophobic association assisted chemical cross-linking ean orthogonal modular approach. Soft Matter 2011;7:7517e25.

[39] Mantovani G, Lecolley F, Tao L, Haddleton DM, Clerx J, Cornelissen JJ, et al.Design and synthesis of N-maleimido-functionalized hydrophilic polymers viacopper-mediated living radical polymerization: a suitable alternative toPEGylation chemistry. J Am Chem Soc 2005;127:2966e73.

[40] Wong L, Boyer C, Jia Z, Zareie HM, Davis TP, Bulmus V. Synthesis of versatilethiol-reactive polymer scaffolds via RAFT polymerization. Biomacromolecules2008;9:1934e44.

[41] Keselowsky BG, Garcia AJ. Quantitative methods for analysis of integrinbinding and focal adhesion formation on biomaterial surfaces. Biomaterials2005;26:413e8.

[42] Kowalczynska HM, Nowak-Wyrzykowska M, Dobkowski J, Kolos R,Kaminski J, Makowska-Cynka A, et al. Adsorption characteristics of humanplasma !bronectin in relationship to cell adhesion. J Biomed Mater Res 2002;61:260e9.

[43] Nombela-Arrieta C, Ritz J, Silberstein LE. The elusive nature and function ofmesenchymal stem cells. Nat Rev Mol Cell Biol 2011;12:126e31.

[44] Marsell R, Einhorn TA. The biology of fracture healing. Injury 2011;42:551e5.[45] Docheva D, Popov C, Mutschler W, Schieker M. Human mesenchymal stem

cells in contact with their environment: surface characteristics and theintegrin system. J Cell Mol Med 2007;11:21e38.

[46] Shapiro F. Bone development and its relation to fracture repair. The role ofmesenchymal osteoblasts and surface osteoblasts. Eur Cell Mater 2008;15:53e76.

[47] Fredriksson S, Gullberg M, Jarvius J, Olsson C, Pietras K, Gustafsdottir SM, et al.Protein detection using proximity-dependent DNA ligation assays. Nat Bio-technol 2002;20:473e7.

[48] Soderberg O, Leuchowius KJ, Gullberg M, Jarvius M, Weibrecht I, Larsson LG,et al. Characterizing proteins and their interactions in cells and tissues usingthe in situ proximity ligation assay. Methods 2008;45:227e32.

[49] Loy DN, Crawford CH, Darnall JB, Burke DA, Onifer SM, Whittemore SR.Temporal progression of angiogenesis and basal lamina deposition aftercontusive spinal cord injury in the adult rat. J Comp Neurol 2002;445:308e24.

[50] Martino MM, Hubbell JA. The 12the14th type III repeats of !bronectin func-tion as a highly promiscuous growth factor-binding domain. FASEB J 2010;24:4711e21.

M. Kisiel et al. / Biomaterials 34 (2013) 704e712712