We report the modified release of platelet-rich plasma from alginate platelet-rich plasma hydrogels altered by the presence of silica. These PRP–alginate–silica compositions can be used as injectable carriers for viable mesenchymal stem cells

Alini, M

Eglin, D

Gimeno-Fabra, M

Peroglio, M

Perry, CC

English

Nottingham Trent Institutional Repository (IRep)

Dynamic Article LinksC<Journal ofMaterials ChemistryCite this: DOI: 10.1039/c0jm04463fwww.rsc.org/materials COMMUNICATIONDownloaded by Nottingham Trent University on 21 February 2011Published on 21 February 2011 on http://pubs.rsc.org | doi:10.1039/C0JM04463FView OnlineCombined release of platelet-rich plasma and 3D-mesenchymal stem cellencapsulation in alginate hydrogels modified by the presence of silica†Miquel Gimeno-Fabra,ab Marianna Peroglio,a David Eglin,a Mauro Alinia and Carole C. Perry*bReceived 20th December 2010, Accepted 2nd February 2011DOI: 10.1039/c0jm04463fWe report the modified release of platelet-rich plasma from alginateplatelet-rich plasma hydrogels altered by the presence of silica.These PRP–alginate–silica compositions can be used as injectablecarriers for viable mesenchymal stem cells.Platelet-rich plasma (PRP) is an autologous source of growth factors(GF) derived from blood plasma. It holds great promise in variousclinical applications such as periodontal and maxillofacial surgery,spinal fusion, bone augmentation and treatment of skin and softtissue ulcers.1 In a study comprising 88 patients Marx et al. haveshown that bone autogenous bone grafts with PRP displayeda significantly higher bone trabecular density and maturity in thereconstruction of maxillofacial defects.2 In another study, Margoliset al. showed on a large set of patients that PRP treatment for dia-betic foot ulcers was more effective than the standard one.3 However,other studies have shown that PRP does not accelerate or improvethe healing process.4,5 These contradictory results have been attrib-uted to the low control of the spatial and temporal delivery of PRP6and the variability in the quantity of growth factors between differentPRP preparations.7In typical preparations, the release kinetics of pure PRP is very fastwith more than 95% released in the first hour.7 This can be sloweddown by combining PRP with biomaterials (e.g. ceramic,8 polymerscaffolds9 or gelatin gels10) which is found to increase PRPefficiency. In vitro, Lu et al. have shown using PRP–alginate beadsthat by controlling the spatial and temporal release of PRP growthfactors, the osteogenic differentiation of stem cells could bepromoted.6Mesenchymal stem cells (MSCs) are a cell type of choice for tissueengineering applications. As for PRP, MSCs are of autologous originand possess the ability to differentiate to different cell types underappropriate stimuli.11 They can be used in combination with a carrier(e.g. scaffold, hydrogel, etc.) for a variety of applications. ForaAO Research Institute Davos, Clavadelerstrasse 8, 7270 Davos,Switzerland; Fax: +41 (0)81 414 24 80. E-mail: david.eglin@aofoundation.org; +41 (0)81 414 22 88bBiomolecular and Materials Interface Research Laboratory, School ofScience and Technology, Nottingham Trent University, NG11 8NSNottingham, UK. E-mail: carole.perry@ntu.ac.uk; Fax: +44 (0)115 8486616; Tel: +44 (0)115 848 6695† Electronic supplementary information (ESI) available: Experimentaldetails. See DOI: 10.1039/c0jm04463fThis journal is ª The Royal Society of Chemistry 2011example several studies have investigated the combination of PRPwith mesenchymal stem cells (MSCs), showing positive results invarious in vivo models.12,13 However, there are other experimentalresults that showed no significant improvement in the presence ofPRP,14 emphasising the need for a better control of PRP release.Ideally, a PRP delivery system should be injectable, provide fine-tuning of PRP release and allow for cell encapsulation. Current two-component systems composed of alginate and PRP allow forinjectability and cell encapsulation,6,12 but the ability to finely tunePRP protein and growth factors release is limited.In our study, we introduced a third component, silica nano-particles, during alginate–PRP gel synthesis to generate novel parti-cles with the end goal being to modulate the release of TGF-b1 fromthe beads. It is known that the size or curvature of silica nano-particles influences protein adsorption and conformation15,16 suchthat a synthesis method for the particles was adopted whereby silicaparticles of 200–250 nm could be rapidly produced under mildconditions (pH 7) compatible with alginates, PRP and othercomponents such as cells.17 The synthesis was performed by additionof pentaethylenehexamine (PEHA), itself a mimic of molecules foundin many silicifying organisms.18,19 PEHA was chosen as variation ofthe reaction conditions including concentration and modest changesin pH could lead to a wide variety of particle sizes being formed. Theother main component, alginate is a well-known injectable carrier,often used for cell and growth factor encapsulation, due to its mildgelation and controlled release properties, respectively.20 It is a linearunbranched polysaccharide containing varying amounts of 1,40-linked b-D-mannuronic acid and a-L-guluronic acid that can becrosslinked by divalent ions (e.g. Ca2+, Mg2+). This gelation is suffi-ciently mild to preserve cells and growth factors viability.20–22 Thekinetics of diffusion of molecules from the alginate network can bemodified by changing the sizes of its pores via changes to the ionicstrength used during synthesis.23 The combination of silica nano-particles with alginate and PRP would offer thereby a powerful toolto tune the release of PRP growth factors.The aim of this study was to validate for the first time the use ofa novel PRP–alginate–silica composite gel for combined controllablePRP release and cell-based therapy. Following characterisation of thematerials formed, the effect of the presence of silica nanoparticleswithin the new composite materials on the release of total proteinsand the transforming growth factor b1 (TGF-b1, one of the mostabundant growth factors in PRP) was investigated. Finally,a successful protocol for MSCs encapsulation was established and theJ. Mater. Chem.Downloaded by Nottingham Trent University on 21 February 2011Published on 21 February 2011 on http://pubs.rsc.org | doi:10.1039/C0JM04463FView Onlineeffect of silica condensation in an alginate–PRP hydrogel on theviability of MSCs over three days was studied.PRP–alginate beads with or without silica were prepared. Adetailed description of the experimental procedure is available asESI†. Briefly, sodium alginate was dissolved in Iscoves ModifiedDulbecco’s medium (IMDM), followed by the addition of appro-priate amounts of sodium metasilicate nonahydrate(Na2SiO3$9H2O). This solution was then mixed with an equalvolume of a PRP/thrombin solution. Macro-beads with an averagediameter of 4 mm were prepared by adding the mixture dropwise intoa CaCl2 solution (pH 7) containing PEHA by use of an auto-injector.The presence of the amine promoted the rapid condensation andaggregation of silica.17,18 Gel beads were formed immediately and leftmaturating for 10 min in the CaCl2 solution at room temperature.The novel gel beads were lyophilised before being characterisedby infrared spectroscopy (Fig. 1A) and electron microscopy(Fig. 1B and C). Compared to the reactants and the PRP–alginatebead mid-IR profiles, the PRP–alginate–silica bead mid-IR spec-trum showed additional vibrational bands at 1000 cm1, 950 cm1and 450 cm1, attributable to Si–O stretching and bending vibra-tions in silica, indicating the polycondensation of silicate (orsilicon species) within the beads (Fig. 1A). This was confirmed bythe use of a colorimetric assay for silicon,24 with the total siliconcontent in the PRP–alginate–silica beads representing 22% of theinitial silicon content added.Fractured and calcined (700 C) PRP–alginate–silica beadsexamined by SEM showed aggregated silica particles of size around50–200 nm suggesting PEHA catalyzed condensation of siliconspecies within the PRP–alginate beads (Fig. 1B and C), similarly toprevious studies on silica condensation in the presence of PEHA andpolysaccharide/protein compositions.18,25,26Fig. 1 Mid FT–IR spectra of lyophilized PRP–alginate and PRP–algi-nate–silica beads (A). SEM images of lyophilized PRP–alginate–silicabead 100 mM sodium metasilicate (B) and calcined PRP–alginate–silicabead 100 mM sodium metasilicate concentration with a higher magnifi-cation image in the inset (C).J. Mater. Chem.To prove the first evidence that silica nano-particles modify proteinrelease from PRP–alginate gels, a high concentration of silicate(100 mM) was initially added to the reaction medium and totalprotein release from the beads and appropriate controls without silicawas monitored over 4 days (Fig. 2). For the PRP–alginate beads (nosilica), there was an initial fast release (95% of protein released withinthe first 2 h), followed by a plateau with no further material beingreleased over the remainder of the experiment. For the PRP–algi-nate–silica beads, the amount of protein initially released was lower(approximately 80% after 2 h) and slow protein release continuedover the next 78 h. Although the protein encapsulation efficiencieshave not been specifically addressed, a previous study of proteinrelease from alginate–PRP beads showed that protein loading in suchsystems is close to 100%.6 Interactions between polycondensedsilica, a protein (gelatin) and alginate25 are reported to be low betweenalginate and silica, and strong between the protein studied and silicaand we anticipate the yield from the alginate–silica–protein beadsdescribed in this contribution to be as high as from alginate–proteinbeads.Once evidence for the ability of the gel beads containing silicanano-particles to modify the release profile of ‘total protein’ wasobtained, the next step was to investigate the effect of silica nano-particles on PRP growth factors release and viability of encapsulatedcells in the alginate–PRP–silica hydrogel. Therefore, the maximumsilicate concentration explored was reduced to 50 mM to matchphysiological osmolarity (300–330 mOsm).To assess the ability of silica nano-particles to moderate the releaseof growth factors, the release of TGF-b1 from PRP–alginate beadsprepared with different concentrations of sodium metasilicate (0, 5,25 and 50 mM) was measured (ESI†). The variation of TGF-b1release by the PRP–alginate beads with different concentrations ofsodium metasilicate was difficult to interpret due to the large standarddeviation values associated with material inhomogeneities (e.g.aggregates) that are commonly found in this kind of preparation.Nonetheless, all the sodium metasilicate containing beads showeda slower and significantly higher release of TGF-b1 at 48 h than thePRP–alginate system alone.Finally, the potential of this novel injectable composite system forcell encapsulation was tested. Human MSCs (hMSCs) were isolatedFig. 2 Plots of total protein release from the PRP–alginate beads (B)and PRP–alginate–silica beads prepared with 100 mM silicon concen-tration (,) as a function of time in PBS solution at 37 C; average andstandard deviation are reported.This journal is ª The Royal Society of Chemistry 2011Downloaded by Nottingham Trent University on 21 February 2011Published on 21 February 2011 on http://pubs.rsc.org | doi:10.1039/C0JM04463FView Onlineaccording to a reported procedure.27 hMSCs were suspended inalginate, alginate–silica, PRP–alginate with thrombin, PRP–alginate–silica with thrombin (cell density of 3 106 cells.ml1). Three differentconcentrations of sodium metasilicate (5, 25 and 50 mM) were testedand the PRP–alginate volume ratio was set as 0.9. The pH and theionic strength of the solutions were adjusted respectively to 7.4 and330 mOsm or as close as possible in the case of the 50 mM silicateconcentration. Once the pH and ionic strength had been adjusted forthe different solutions, hMSCs could be encapsulated and were thencultured for 3 days. After dissolution of the beads,28 the proportion ofviable cells in the various silica-containing beads compared to theinitial number of cells seeded was measured by the trypan blueexclusion assay (Fig. 3).After one day of culture, the addition of silicate significantlyincreased the proportion of viable cells, both in alginate and PRP–alginate samples as compared to the controls (alginate and PRP–alginate without silicate) suggesting that the presence of the silicate inthe preparation reduces any negative effect of the alginate gelformation on cell viability. The highest proportion of viable cells wasachieved at a low silicate concentration (5 mM) which is probably dueto the ability of low molecular weight silica species being mosteffective in disrupting the alginate network. With increasing amountsof silicate in the preparation (5 to 50 mM), the proportion of viablehMSCs in alginate–silica–PRP/no PRP (Fig. 3A and B) after 1 day ofculture decreased in a linear fashion (Fig. 3B has R2 ¼ 0.999). It islikely that reactive silicon species are trapped in the alginate gel thatrapidly forms following Ca2+ diffusion with silica particles continuingto form over time within the gel structure. The resulting physicalchanges in the gel matrix could possibly damage the immobilized cellsin the hydrogel which could explain the reduced numbers of viablecells at higher silicate concentrations.After three days of culture, the proportion of viable hMSC in thePRP–alginate–silica samples was high (0.91–0.93), independent of thesilicate concentration. This is possibly due to the sustained release ofgrowth factors (Fig. 2) in the presence of silicate compared to algi-nate–PRP samples without silicate (Fig. 3B) as hMSCs express themembrane receptors to the growth factors present in PRP and PRP isknown to stimulate hMSCs proliferation.2,29 This effect was maximalwith the highest silicate concentration. It is known that gelatin–algi-nate interactions are weak while gelatin–silica are strong25 and thatalginate modulates the yield and release kinetics of the differentFig. 3 Plots of the proportion of viable hMSCs relative to the initialviability of the seeded hMSC at 1 and 3 days of culture in alginate–silicabeads with 0, 5, 25 and 50 mM silicate concentration (A) and in PRP–alginate–silica beads with 0, 5, 25 and 50 mM silicate concentration (B).(Data: average and standard deviation, lines p < 0.05 silicate concen-trations, *p < 0.05 day 1 versus day 3.)This journal is ª The Royal Society of Chemistry 2011growth factors contained in the PRP.6 Therefore, we would expectthat the yields and kinetics of the different proteins to be affected bythe presence of silica in the PRP–alginate composition.Without silica (see Fig. 3A), the proportion of viable hMSCs inalginate beads after 3 days of culture was lower (0.63). The additionof PRP to alginate was beneficial at 24 h, although, after 3 days ofculture, the proportion of viable cells in both alginate and PRP–alginate beads was still relatively low (0.68) suggesting that thepresence of PRP could only partially overcome the hMSCs initialadverse reaction to the alginate environment (lack of binding sites).The weak effect of PRP addition of later time points may beexplained by rapid protein release (4 h) in the absence of silicaadded to the preparation (Fig. 2). The exact mechanism of interactionbetween the composite materials and cells has not yet been elucidated.However, our hypothesis is that the small molecular weight oligomersand higher molecular weight nano-sized silica particles influence thephysical and chemical characteristics of the polymer network as wellas making possible the retention of proteins within the beads enablingan extended effect of PRP on encapsulated hMSCs.In summary, we have reported a novel method for combinedcontrolled PRP release and mesenchymal stem cell encapsulation. Inrecent years, it has been shown that combined growth factor deliveryis more effective than delivery of a single factor30 as growth factorseach have a different role to play in a time dependent manner.Although delivery systems for sequential release of recombinantgrowth factors have already been proposed30 using composites wheredifferent components are separately loaded with individual growthfactors they are not suitable for an autologous source of growthfactors such as PRP as it is itself a mixture of proteins and growthfactors. The synthetic approach described in this contributioncombining silica with alginate could offer for the first time theopportunity to tune PRP growth factor release. Furthermore, thiscomposite system is injectable and preserves the viability of hMSCs,thereby opening the way for combined tissue engineering and drugdelivery applications.Notes and references1 B. L. Eppley, W. S. Pietrzak and M. Blanton, Plast. Reconstr. Surg.,2006, 118, 147e–159e.2 R. E. 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Clin. Oral Implants Res.,

Drug Delivery Rev.,

Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod.,

This journal is ª The Royal Society of Chemistry

Tissue Eng.,

Combined release of platelet-rich plasma and 3D-mesenchymal stem cell encapsulation in alginate hydrogels modified by the presence of silica

Combined release of platelet-rich plasma and 3D-mesenchymal stem cell encapsulation in alginate hydrogels modified by the presence of silica

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