Performance of PRP Associated with Porous Chitosan as a

Research ArticlePerformance of PRP Associated with Porous Chitosan asa Composite Scaffold for Regenerative Medicine

Andreacutea Arruda Martins Shimojo Amanda Gomes Marcelino PerezSofia Elisa Moraga Galdames Isabela Cambraia de Souza Brissacand Maria Helena Andrade SantanaDepartment of Engineering of Materials and of Bioprocesses School of Chemical EngineeringUNICAMP PO Box 6066 13083-970 Campinas SP Brazil

Correspondence should be addressed to Maria Helena Andrade Santana mariahelenasantanagmailcom

Received 30 November 2014 Accepted 8 January 2015

Academic Editor Faik Oktar

Copyright copy 2015 Andrea Arruda Martins Shimojo et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

This study aimed to evaluate the in vitro performance of activated platelet-rich plasma associated with porous sponges of chitosanas a composite scaffold for proliferation and osteogenic differentiation of human adipose tissue-derived mesenchymal stem cellsThe sponges were prepared by controlled freezing (minus20 minus80 or minus196∘C) and lyophilization of chitosan solutions (1 2 or 3 wv)The platelet-rich plasma was obtained from controlled centrifugation of whole blood and activated with calcium and autologousserumThe composite scaffoldswere prepared by embedding the spongeswith the activated platelet-rich plasmaThe results showedthe performance of the scaffolds was superior to that of activated platelet-rich plasma alone in terms of delaying the release ofgrowth factors and increased proliferation of the stem cells The best preparation conditions of chitosan composite scaffolds thatcoordinated the physicochemical and mechanical properties and cell proliferation were 3 (wv) chitosan and a minus20∘C freezingtemperature while minus196∘C favored osteogenic differentiation Although the composite scaffolds are promising for regenerativemedicine the structures require stabilization to prevent the collapse observed after five days

1 Introduction

In tissue engineering a scaffold is a three-dimensionalmatrixfor the stimulation of cell proliferation and the formationof new tissue To achieve this goal scaffolds must meetsome specific requirements such as mimicking the nativeextracellular matrix (ECM) of the target tissue allowing cellattachment migration proliferation and differentiation toand maintenance of the target phenotype promoting vascu-larization and nutrient delivery and having a biodegradationrate and mechanical properties that are adequate to supportthe formation of the new tissue [1 2]

According to Crane and Everts tissue regeneration isbased on a proliferation triangle composed of the threefundamental elements of cells growth factors (GFs) andscaffolds which are in a close relationship through theircapabilitiesThus scaffolds provide the conductivematrix forsupporting the genic capability of progenitor cells mediated

by the inductive capability of GFs [3] Optimal conditionsfor tissue regeneration must come from the interaction of theproperties of those elements

The cell responses to the surface chemistry of scaffoldsdepend on their hydrophobicity protein adsorption surfacecharge and roughness softness and stiffness Additionallythe porous architecture characterized by the pore size poros-ity connectivity and tortuosity plays important roles asdescribed and discussed by Chang and Wang [4] Thereforebringing together all these properties in solid scaffolds is anenormous technological challenge

The fibrin matrix is the natural scaffold formed from thecoagulation cascade in the healing process Under injurythrombin cleaves the soluble plasma protein fibrinogen intopeptide fragments yielding insoluble fibrin peptides thataggregate and form fibrils A fibrin network is then formedwhich entraps platelets and other blood components [5 6]

Hindawi Publishing Corporatione Scientific World JournalVolume 2015 Article ID 396131 12 pageshttpdxdoiorg1011552015396131

2 The Scientific World Journal

Calcium acts as a cofactor of thrombin modulates theelongation of fibers during polymerization by promoting lat-eral branching and functions in clot stability [7 8] Calciumand thrombin also activate platelets allowing the release ofGFs and cytokines Therefore the fibrin matrix provides anoptimized medium for cell proliferation and healing

Based on these features platelet-rich plasma (PRP) hasbeen widely used in regenerative medicine PRP is an autol-ogous product prepared from whole blood by separatingthe red blood cells and concentrating the platelets andother components of plasma However although viscoelasticthe fibrin matrix alone lacks stability to be efficient whenregenerative medicine is the goal

To mimic the natural healing process and improve thestability of the fibrin matrix we propose in this work theuse of the fibrin network from activated PRP with porouschitosan as a composite scaffold for tissue regeneration

Our hypothesis is that the chemical nature of the chi-tosan surface supports the fibrin network by electrostaticattachment thus prolonging its stability without changingthe paracrine affinity of mesenchymal cells to fibrin fibers Asa consequence the composite scaffoldmust improve cell pro-liferation and tissue differentiation compared to PRP alone

Related works showed the effects of chitosan on bloodcoagulation through the strong adhesion of platelets tothe surface of chitosan particles as well as chitosan (01ndash1mgmL) incorporated with PRP to enhance the releaseof PDGF-AB and TGF-1205731 from platelets [9 10] Shen etal demonstrated that chitosan could be an appropriatesubstitute for thrombin as an agonist in PRP preparation[11] Chang et al showed that growth factors could sustainrelease until 12 h at approximately 1 ngmL from chitosanCaSO

4PRP microspheres after activation with thrombin

[12] Kutlu et al showed that scaffolds prepared by the freeze-drying of PRP added to chitosan gel (2 wv) were moreeffective than chitosan sponges soaked with PRP on con-trolled GF release [13] Oktay et al applied PRP-embeddedchitosan sponges to defects and showed a histological ten-dency toward increased bone formation [14] Mathews etal demonstrated that chitosan enhanced mineralization byupregulating the genes associated with mineralization andcalcium-binding proteins [15]

Chitosan is a polysaccharide derived from chitin (copoly-mers 120573-(1rarr 4)-2-amino-2-deoxy-D-glucose and 120573-(1rarr 4)-2-acetamido 2-deoxy-D-glucose) found in the shells ofmarine crustaceans and insects and the cell walls of somefungi [16 17]

In the last ten years considerable attention has been givento chitosan-based materials in the field of tissue engineering[18 19] The beneficial properties of chitosan have beenproven such as biodegradability [20 21] biocompatibility[22 23] antibacterial activity [24 25] cell adhesion [26 27]and wound healing properties [28 29] Moreover the chem-ical nature of chitosan gives many possibilities for ionic andcovalent modifications that allow for the modulation of themechanical and biological properties of biomaterials [30 31]

Regarding the technological aspects chitosan can beeasily processed in diverse forms in the absence of toxicsolvents such as films [32] sponges [33 34] fibers [35]

beads [36] hydrogels [37] and microparticlesnanoparticles[38] Furthermore chitosan supports sterilization [39] isabundant in nature and requires only low-cost processingin nonaggressive ecological conditions before being used asa raw material [17]

Freeze-drying is the most common and simplest methodto produce porous chitosan scaffolds The freezing processprovides the nucleation of ice crystals from solution andfurther growth along the lines of thermal gradients Exclusionof the chitosan acetate salt from the ice crystal phase andsubsequent ice removal by lyophilization generate a porousmaterial [40 41]

Madihally and Matthew reported that the pore size ofchitosan scaffolds produced by lyophilization can be con-trolled within the range of 1ndash250120583m [33] A more uniformand interconnected pore structure can be obtained whenlower freezing temperatures are used Furthermore the poreorientation is related to the geometry of the moldings usedand can also be controlled by changing thermal gradientsduring freezing [42]

Themicrostructure crystallinity andmechanical strengthof the porous chitosan matrix also can be controlled by thechitosan concentration molecular weight and deacetylationdegree [33 43]

In this work the effects of PRP association were studiedregarding the porous structure of solid sponges which wereproduced by varying chitosan concentration and freezingconditions The biological performance of association wasevaluated in terms of growth factor release kinetics prolifera-tion and osteogenic differentiation of seeded human adiposetissue-derived mesenchymal stem cells (h-AdMSCs)

2 Experimental

21 Materials Chitosan (average molecular weight [Mw] =4 times 105Da degree of deacetylation = 83plusmn4) was purchasedfrom Polymar (Fortaleza CE Brazil) and purified accordingto the protocol described by Nasti et al [44] Other chemicalswere of reagent grade and were used without any furtherpurification All biological experiments were performed withhuman adipose tissue-derived stem mesenchymal cells (h-AdMSCs) and approved by the Ethics Committee of theMedical Sciences School of the University of Campinas(UNICAMP CAAE 09720146000-11) The donors werehealthy individuals aged between 30 and 40 years old whowere previously assessed through clinical examinations

22 Methods

221 Preparation of Porous Chitosan Scaffolds (PCHTs)PCHTs were prepared by freezing at a controlled temperatureof a chitosan solution previously poured in 24-well cultureplates (TPP polystyrene diameter = 154mm) to give thema cylindrical shape followed by lyophilizing in lyophilizerL101 (Liobras Sao Carlos SP Brazil) at a temperature ofapproximately minus30∘C for 48 hours

Effects of Chitosan Concentration Chitosan solutions withconcentrations of 1 2 or 3 (wv) were prepared by

The Scientific World Journal 3

dissolution in 02mol Lminus1 acetic acid for 24 hours at roomtemperatureThe solutions were frozen at minus20∘C for 24 hoursand lyophilized

Effects of Freezing Conditions Chitosan solution (3wv) wasprepared as previously described The solution was frozen atminus20∘C in a freezer for 24 hoursminus80∘C in an ultrafreezer for 24hours or minus196∘C by immersion in liquid N

2 and lyophilized

222 Characterization of Porous Chitosan Scaffolds (PCHTs)

Morphology and Pore Size The morphology of PCHTs wasevaluated by scanning electron microscopy (SEM) using anLEO 440i Electron MicroscopyOxford (Cambridge Eng-land) operated at 5 kV accelerating voltageThe scaffolds weregold-coated using a sputter coater POLARON SC7620 VGMicrotech (Uckfield England) for 180 s at a current of 3mAPore size (119899 = 20) was measured using software Image J 147t

Mechanical Properties Mechanical compression tests ofPCHTs (119899 = 3) were performed using a Universal TestingMachineMTSmodel 810-Flex Test 40 (MTS Systems Corpo-ration Eden Prairie MN USA) up to 60 strain accordingto Correia et al [45] The testing machine was equipped witha 15 kN load cell and the loading rate was 5mmmin Youngrsquosmodulus was calculated in the initial linear section of thestress-strain curve when the strain was lower than 10

Degradation in PBS The degradation profile of the PCHTsin PBS at 37∘C was performed by the gravimetric methoddescribed by Tang and Spector through measurements ofremaining weight [46]

Water Sorption The water sorption was determined byswelling of PCHTs (previously weighted) in PBS (LB Labor-clin Pinhais PR Brazil) at pH 74 for 24 hours at 37∘C Theswollen PCHTs were weighed after the removal of excesswater by keeping the surfaces on a filter paper The swellingratio (SR) was also calculated using

SR = 119908119904119908119889

(1)

where 119908119904 and 119908119889 are the weights of the scaffolds in theswelled state and the dry state respectively

Porosity The porosity (120576) of the PCHTs was determinedaccording to the protocol used by Wang et al and calculatedby [47]

120576 () =119881119898 minus (119908119898120588)

119881119898

times 100

(2)

where119881119898 is the total volume of PCHTs (cm3) 120588 is the densityof nonporous CHT (1342 gcm3) and 119908119898 is the weight ofsponge (g) Values are expressed as the means plusmn standarddeviation (119899 = 3)

Cell Compatibility The compatibility was carried out byexposing PCHTs to h-AdMSCs followed by cultivationat 37∘C for 24 hours and evaluation by MTT assay

(3-[45-dimethyl-thiazol-2-yl]-25-diphenyltetrazolium bro-mide) (MTT Molecular Probes) according to a modifiedMosmann method [48] TheMTT assay is a colorimetric testbased on the reduction of yellow tetrazolium salt into a purpleformazan product in presence of cells [49]

223 PRP Preparations

PRP Concentration P-PRP (plasma rich in platelets andpoor in leukocytes) was prepared according to Perez et al[50] Briefly whole blood (WB) was collected into 35mLvacuum tubes (Vacuette Campinas SP Brazil) containingsodium citrate 32 (wv) as an anticoagulant WB wasinitially centrifuged in a Rotina 380R centrifuge (HettichZentrifugen Tuttlingen Germany) at 100timesg for 10 minutesat 25∘C After the formation of three layers (a bottom layercomposed mainly of red blood cells (RBCs) an upper layercomposed of plasma platelets and some WB cells andan intermediate layer or buffy coat composed mostly ofWB cells) only the upper layer was collected to obtain P-PRP The concentration of platelets WB cells and RBCsin WB and P-PRP were determined using the ABX MicrosES 60 hematology analyzer (HORIBA ABX DiagnosticsMontpellier France)

P-PRPActivation Activated P-PRP (119886P-PRP)was obtained byadding autologous serum (Ser) and 10 (wv) CaCl

2solution

as agonists using the following proportions agonistP-PRP =20 (vv) SerCaCl

2volumetric ratio = 9 Autologous serum

was prepared by collecting 5mL of WB in tubes withoutanticoagulant After 30 minutes to form clots WB wascentrifuged at 2000timesg for 10 minutes [51]

224 The Composite Scaffolds (119886P-PRPPCHTs)

Preparation 119886P-PRPPCHTs was prepared for embedding bydripping 119886P-PRP immediately after activation onto PCHTsThe preparation was carried out in 48-well microplates usinga ratio of 200120583L of 119886P-PRP to 10ndash20mg of PCHTs

Release of GFs The release of platelet-derived growth factorAB (PDGF-AB) and transforming growth factor 1205731 (TGF-1205731) was performed after 1 hour of gelation of 119886P-PRPassociated with PCHTs in the presence of the culturemedium(Dulbeccorsquos Modified Eaglersquos Medium (DMEM-LG) (GibcoGrand IslandNYUSA)with low glucose concentration)Theculturemedium (15mL) was added to 119886P-PRPPCHTs in 48-wellmicroplates whichweremaintained in an incubatorwith5 CO

2throughout the assays The total volume of culture

medium was withdrawn at 3 6 12 24 and 72 hours and thesame volume of fresh medium was replaced without remov-ing the hydrogels from the wells The samples were storedat minus80∘C for further characterization The concentrationsof the released GFs PDGF-AB and TGF-1205731 were measuredusing enzyme-linked immunosorbent assay (ELISA) kits(RampD Systems Minneapolis MN USA) according to themanufacturerrsquos instructions and specifications

h-AdMSCs Proliferation The cultivation of h-AdMSCs wascarried out in 24-well tissue culture plates by adding 1mL


DMEM to the seeded composite scaffolds (119899 = 4) Thecomposite scaffolds seeded were maintained at 37∘C for 10days Cell proliferation was quantified using the thiazolylblue tetrazolium bromide (MTT) assay At 3 5 7 and 10cultivation days the composite scaffolds were removed andtransferred to 24-well plates MTT (1mL of 1mgmL) wasthen added and the cultivation proceeded at 37∘C for 4hours The MTT solution was then discarded and 1mL ofDMSO was added to dissolve the purple formazan crystalsThe samples were shaken at 120 rpm for 30min to ensurehomogeneous dissolution of the formazan dye and then200120583L of each sample was transferred to a 96-well plateOptical density was measured at 595 nm using a microplatereader (FilterMax F5 Molecular Devices)

The isolation and precultivation of h-AdMSCs as well asthe cell seeding were performed as described below

h-AdMSCs Isolation and Precultivation Human subcuta-neous adipose tissue that was initially acquired from lipo-suction surgery was washed with sterile PBS separatedinto fractions of 10 g digested with 20mg of collagenasetype 1A and maintained in 20mL of DMEM-LG containing10 BSA (bovine serum albumin) and 10 120583L of gentamicinfor 30min in a 37∘C bath After complete digestion thereaction was quenched with 10mL fetal bovine serum (FBS)and immediately centrifuged for 15min at 1500 rpm Thesupernatant was discarded and the pellet was suspended in10mLDMEM-LGwith 10FBSAfter precultivation for 24 hthe culturemediumwas changed every 3 days after the fourthpassage the cells were characterized by immunophenotyping(data not shown) using flow cytometry and according to theiradipogenic osteogenic and chondrogenic differentiation andthen used in the subsequent experiments

h-AdMSCs-Seeding The precultured h-AdMSCs were trypsi-nized and resuspended in P-PRP to a final cell concentrationof 1 times 104 cellsmL P-PRP containing h-AdMSCs was acti-vated and immediately embedded to the PCHTs in a 24-welltissue culture plate using 200 120583L of h-AdMSCs + 119886P-PRP per10ndash20mg of PCHTsThe composite scaffolds with h-AdMSCswere kept at room temperature for 45 minutes for consolida-tion of the fibrin network Pure PRP was used as control

225 Images of the h-AdMSCs-Seeded Composite ScaffoldsThe images of the cell-seeded composite scaffolds wereobtained by scanning electron microscopy after 5 days ofh-AdMSCs proliferation The cell-seeded composite scaffoldswere fixed in a solution of 4 paraformaldehyde and 25glutaraldehyde in phosphate buffer pH 74 for 2 hours Thesamples were than dehydrated in ethanol for 15min intervalsin aqueous 50 70 95 and 100 ethanol solutions(2x) and dried at the critical point dryer BAL-TEC CPD030 (Schalksmuhle Germany) After gold coating (SputterCoater POLARON SC7620 VG Microtech) the cell-seededcomposite scaffolds were visualized with a SEM (Leo440iLEO) with an accelerating voltage of 20 kV

226 Induction of Osteogenic Differentiation h-AdMSCs-seeded in the composite scaffolds were induced to

differentiate into the osteogenic lineage by providing theosteogenic medium containing DMEM-LG supplementedwith 10 FBS 1 120573-glycerol-phosphate (Sigma-Aldrich StLouis MO USA) 1 L-ascorbic acid (Sigma-Aldrich StLouis MO USA) 1 dexamethasone (Sigma-Aldrich StLouis MO USA) and 1 penicillinstreptomycin solution(Gibco Grand Island NY USA) The medium was changedevery 7 days

Evaluation of Differentiation Differentiationwas evaluated bymeasuring the alkaline phosphatase activity (ALP) on day14 The supernatant (200120583L) was collected and mixed withthe same volume of p-nitrophenyl phosphate (SIGMAFASTp-nitrophenyl phosphate tablets Sigma Saint Louis MOUSA) as substrate and incubated at room temperature for 30minutes Absorbance was read immediately at 405 nm

227 Statistical Analysis Each experiment was carried out intriplicate unless otherwise specified All results are presentedas themeanplusmn standard deviation (SD)The experimental datafrom all the studies were analyzed using Analysis of Variance(ANOVA) Statistical significance was set to 119875 value le 005

3 Results and Discussion

31 Experimental Design In this study we prepared novelcomposite scaffolds by association of PRPwith the 3D-porousstructure of chitosan First different 3D-porous structuresof chitosan (PCHTs) were prepared by freeze-drying byvarying the CHT concentration and freezing conditions(temperature and freezing rate) Second the microstructureand mechanical properties of the PCHTs were characterizedand evaluated for cell compatibility The surface chemistryhydrophilicity and positive charge of ndashNH

2groups in acidic

medium were maintained unalteredThe composite scaffolds (119886P-PRPPCHTs) were obtained

by embedding the PCHTs with immediately activated P-PRPin a 24-well microplate The release of the GFs (PDGF-ABand TGF-1205731) from the composite scaffolds was evaluated inDMEMculturemediumAfterwards precultured h-AdMSCswere added to PRP before activation in order to obtaincell-seeded composite scaffolds In vitro examination of h-AdMSCs proliferation kinetics was performed over 10 daysby using the cell-seeded composite scaffolds in DMEMAdditionally osteogenic differentiationwas evaluated byALPactivity measurements after 14 days

Thus we verified the correlation between the structureand function of PCHTs by controlling the concentration andfreezing conditions of chitosan solution

32 Effects of Freezing Conditions and ChitosanConcentration on PCHTs

321 Images of Porous Structure Figures 1(a)ndash1(c) showimages of the porous structure from controlled freezing atminus20∘C by varying the concentration of the chitosan solution(1 2 or 3) and subsequent lyophilization The imagesshow highly porous structures with rounded pores thatare uniformly distributed radially oriented and visually


(a) (b)

(c) (d) (e)

Figure 1 SEM micrographs of PCHTs Cross-sectional morphologies of (a) PCHTs 1 (minus20∘C) (b) PCHTs 2 (minus20∘C) (c) PCHTs 3(minus20∘C) (d) PCHTs 3 (minus80∘C) and (e) PCHTs 3 (minus196∘C) Original magnification is times100 and the scale bar represents 200 120583m

interconnected regardless of CHT concentration Differ-ences in chitosan molecular weight deacetylation degreeand purity make difficult comparisons even for similartreatment Madihally and Matthew showed a higher poreinterconnectivity degree and more open pores for 1 (ww)chitosan concentration for a similar deacetylation degree butother properties were not characterized [33]

Freezing conditions produced amore prominent effect onthemorphology of the pores (Figures 1(d)-1(e)) attributed thevariations in the freezing rate used to achieve the tempera-tures evaluated In contrast with rounded pores at minus20∘C (3CHT) (Figure 1(c)) we observed pores with leaf structure atminus80∘C (Figure 1(d)) while smaller and elongated open poreswere produced at minus196∘C (Figure 1(e)) In all cases the poreswere uniformly distributed and radially oriented and had ahigh degree of interconnectivity

The pore structures allow uniform cell spatial distributionthroughout the scaffold facilitating homogeneous tissueformation Moreover the differences in pore morphologiesobtained are suitable to several cell linage and type of tissueto be regenerated

322 Characterization of PCHTs

Physicochemical and Mechanical Properties Table 1 showsphysicochemical characterization of the PCHTs At minus20∘Cthe mean diameter of rounded pores could be controlledaround the 300ndash400 120583m regardless of the CHT concentra-tion although there is a large distribution

However the elongated pores from Figures 1(d)-1(e) weremarkedly decreased with freezing conditions from 2615120583m

(minus80∘C) to 280120583m (minus196∘C) because crystal growth andhence pore size are functions of both heat and mass transferrates determined by the temperature and freezing rate Atminus80∘C the observed differences in pore shape and size sug-gest parallel ice crystal growth which in turn was caused bythe strongly one-dimensional nature of the thermal gradientsestablished during freezing as discussed by Madihally andMatthew [33]

The diverse nature of tissue architectures requires scaf-folds with optimal pore sizes such as 5 120583m for neovascu-larization [52] 5ndash15120583m for fibroblast ingrowth [53] 20120583mfor the ingrowth of hepatocytes [54] 200ndash900120583m for osteo-conduction [55] and 20ndash125120583m for regeneration of adultmammalian skin [56]

According to these data the rounded pores obtainedat minus20∘C or elongated at minus196∘C are adequate for bonetissue although other properties could also influence thechoice Moreover because human adipose tissue-derivedmesenchymal stem cells (h-AdMSCs) exhibit a spindle-likeshape and are 80ndash100120583m in diameter and sim200120583m in length[57] the range of pore sizes in our PCHTs allows cells tomigrate freely into the scaffolds favoring the formation of anew tissue

The porosity is also an important aspect of cell migrationand proliferation guiding and promoting the formation ofnew tissue Porosity is defined as the percentage of voidspace in a solid and is a morphological property independentof material [58] A porosity higher than 90 and poreinterconnectivity are basic requirements for scaffolds in tissueengineering because they affect the diffusion of physiolog-ical nutrients and gases to and the removal of metabolic


Table 1 Physicochemical characterization of PCHTs

CHTconcentration()

Freezing temperature(∘C)

Swellingratio(119899 = 3)

Pore size(120583m)

(119899 = 20)

Porosity()

(119899 = 3)

Youngrsquos moduli(MPa)(119899 = 3)

1 minus20 65 plusmn 04 425 plusmn 102 971 plusmn 03 0037 plusmn 00022 minus20 55 plusmn 09 418 plusmn 113 947 plusmn 08 05 plusmn 013 minus20 47 plusmn 09 336 plusmn 138 931 plusmn 05 11 plusmn 013 minus80 51 plusmn 09 2615 plusmn 600lowast 917 plusmn 03 028 plusmn 0013 minus196 64 plusmn 06 280 plusmn 62lowast 925 plusmn 01 101 plusmn 005lowastMean longitudinal size

50

40

30

20

10

0

Wei

ght l

oss (

)

Time (days)0 3 6 9 12 15

Figure 2 The weight loss of scaffolds with time in PBS at 37∘C asa percentage of the original weight of the scaffold (119899 = 3) The dataare plotted with the means plusmn standard error (◼) PCHTs 1 (minus20∘C)(e) PCHTs 2 (minus20∘C) (998771) PCHTs 3 (minus20∘C) (◻) PCHTs 3(minus80∘C) and (O) PCHTs 3 (minus196∘C)

waste and byproducts from cells that have penetrated thescaffold [59 60] Moreover the porosity often compromisesthe mechanical and structural stability of the scaffolds andmust be evaluated in accordance with the application anddegradation rate of materials utilized [61]

The porosity values of PCHTs scaffolds ranged from 910to 970 which are adequate for TE independent of CHTconcentration or freezing conditions However the porositydecreased with increasing chitosan concentration The high-est porosity was provided from 1 chitosan solution butthere was not a direct relationship with freezing conditionsThe PCHTs prepared at minus80∘C showed a significantly lowerporosity even with higher pore size probably due to their leafstructure (Table 1)

To guide tissue regeneration scaffolds should also havesufficient mechanical strength during in vitro culturing tomaintain the spaces required for cell in-growth and matrixelaboration [59] Moreover their mechanical propertiesshould be similar to the properties of tissues generated toprovide an adequate structural support in the stage of healing[62]

Cel

l num

ber

wel

l

30

25

20

15

10

00

PCH

Ts1

(minus

20∘ C)

PCH

Ts2

(minus

20∘ C)

PCH

Ts3

(minus

20∘ C)

PCH

Ts3

(minus

80∘ C)

PCH

Ts3

(minus

196∘ C)

NTC PT

C

times104

05

Figure 3 Proliferation of h-AdMSCs exposed to the PCHTsscaffolds after 24 hours of cultivation Negative control (NTC) =DMEMwith 10 FBS positive control (PTC) =DMEMwith phenol05 Mean plusmn standard deviation 119899 = 3 The population means aresignificantly different from positive control at lowast

119875

lt 005

Scaffolds for the regeneration of hard tissue must exhibita mechanical modulus in the range of 10ndash1500MPa whilescaffolds for soft tissues must exhibit a mechanical modulusin the range of 04ndash350MPa [63]

In this work we observed a drastic decrease in Youngrsquosmodulus (Table 1) with a decrease in CHT concentrationBetter mechanical properties were found for a CHT concen-tration of 3

The PCHTs 3 (wv) prepared at minus80∘C showed lowermechanical strength than those prepared at minus20∘C andminus196∘C with the same CHT concentration which could beattributed to its leaf structure and pore size There was nosignificant difference (119875 lt 005) in mechanical properties forPCHTs 3 (wv) frozen at minus20∘C or minus196∘C Thus the rangeof Youngrsquosmoduli found for PCHTs suggests their applicationfor soft tissue engineering For hard tissues these scaffoldsmust be crosslinked andor reinforced by the addition offillers


(a) (b) (c)

(d) (e) (f)

Figure 4 Scanning electron microscopic images of 119886P-PRPPCHTs after 5 days of cultivation of h-AdMSCs (a) 119886-PRP (b) 119886P-PRPPCHTs1 (minus20∘C) (c) 119886P-PRPPCHTs 2 (minus20∘C) (d) 119886P-PRPPCHTs 3 (minus20∘C) (e) 119886P-PRPPCHTs 3 (minus80∘C) and (f) 119886P-PRPPCHTs 3(minus196∘C)

The water absorption capacity (swelling property) of thescaffolds affects cell growth indirectly [64] The hydration ofthe scaffolds is a necessary step for cell incorporation andproliferation

PCHTs scaffolds showed a high swelling capacity in PBS(pH 72) regardless of chitosan concentration and freez-ing conditions allowing for rapid hydration when culturemedium was added

Degradation in PBS Scaffold degradation is also an importantparameter for the formation of new tissue The scaffolddegradation ratemust be tuned appropriately with the growthrate of the new tissue in such a way that by the time the injurysite is completely regenerated the scaffold is completelydegraded [55] Degradation can occur through mechanismsthat involve physical or chemical processes andor biologicalprocesses that are mediated by biological agents such asenzymes [1]

Here we evaluated in vitro the profile degradation ofPCHTs in PBS (pH 72) at 37∘Cwe supposed that degradationoccurred by solubilization due to the presence of residualacetate molecules

Figure 2 shows the degradation profile of PCHTsWe observed the highest weight loss approximately 45

for PCHT 1 (minus20∘C) after 7 days while for PCHTs 2(minus20∘C) 3 (minus20∘C) and 3 (minus196∘C) weight loss wasapproximately 35 after 3 days The PCHTs 3 (minus80∘C)showed the lowest weight loss probably due to their leafstructure retaining lower concentration of acetate molecules

Our results suggest that the PCHTs need further stabiliza-tion to ensure the balance between tissue regeneration anddegradation rate of the scaffold

323 Cell Compatibility Figure 3 shows the cell compati-bility of h-AdMSCs cultured in the presence of PCHTs asassayed by MTT The results revealed no potential cytotox-icity in 24 hours for the scaffolds according to the standardvalues (PTC)

However we observed lower proliferation of h-AdMSCson PCHTs prepared with chitosan concentration of 3 (wv)compared to the negative control

Nevertheless the PCHTs are potentially useful for in vivoapplications regardless of CHT concentration and freezingconditions

33 Characterization of the Composite Scaffolds

331 Images of the Cell-Seeded 119886P-PRPPCHTs SEM char-acterization (Figure 4) of the 119886P-PRPPCHTs on the 5thday of culture showed fibrin networks covering the poresand surface of PCHTs as a consequence of the interactionof fibrin fibers and CHT This interaction supported cellproliferation by improving the mechanical strength of thefibrin network in addition to providing additional surfacearea to cell adhesion proliferation and differentiation

332 Growth Factor Release Figure 5 shows PDGF-AB andTGF-1205731 release kinetics from 119886P-PCHTs determined byELISA


25000

20000

15000

10000

5000

0

Cum

ulat

ive T

GF-120573

(pg

mL

PRP)

Time (hours)0 10 20 30 40 50 60 70 80

(a)

Time (hours)0 10 20 30 40 50 60 70 80

12000

10000

8000

6000

4000

2000

0

Cum

ulat

ive P

DG

F-A

B (p

gm

L PR

P)

(b)

25000

20000

15000

10000

5000

0

Cum

ulat

ive T

GF-120573

1 (p

gm

L PR

P)

Time (hours)0 10 20 30 40 50 60 70 80

(c)

Time (hours)0 10 20 30 40 50 60 70 80

15000

12000

9000

6000

3000

0

Cum

ulat

ive P

DG

F-A

B (p

gm

L PR

P)

(d)

Figure 5 Release profiles of growth factors from 119886P-PRP in porous chitosan scaffolds as a function of ((a) (b)) chitosan concentrations and((c) (d)) freezing conditions (◼) PCHTs 1 (minus20∘C) (e) PCHTs 2 (minus20∘C) (998771) PCHTs 3 (minus20∘C) (◻) PCHTs 3 (minus80∘C) (O) PCHTs3 (minus196∘C) and (998779) P-PRP activated with Ca+2thrombin (used as control) TGF-1205731 ((a) (c)) and PDGF-AB ((b) (d)) The concentrationof platelets in P-PRP was 472250 pqmm3 Activated P-PRP alone was used as control

The curves show diffusion profiles indicating no collapseof the porous structure of chitosan scaffolds during thecourse of the assays regardless of the CHT concentration andfreezing conditions

PDGF-AB released from PCHTs ended within 24 hoursof incubation whereas TGF-1205731 tended to continue after 72hours

A controlled release of TGF-1205731 and PDGF-AB wasachieved for all CHT concentrations related to the scaffoldsof activated P-PRP onlyThe slowest release was observed fora chitosan concentration of 3

In contrast for the PCHTs 3 freezing at minus80∘C andminus196∘C we observed a controlled release of TGF-1205731 but astrong burst release of PDGF-AB

Thus PCHTs prepared with 3 CHT at minus20∘C provideda controlled release of PDGF-AB and TGF-1205731 and can be anefficient vehicle for release of GFs from PRP

333 h-AdMSCs-Seeded Proliferation Figure 6 shows theproliferation profile of h-AdMSCs cultured in 119886P-PRPPCHTs

The cell number per well determined after 3 daysexceeded the number of seeded cells (14 times 104 cellswell)in all the 119886P-PRPPCHTs meaning that the cells kept inthe matrices retained their viability regardless of the CHTconcentration and freezing conditions

The cell number per well of 119886P-PRPPCHTs preparedwith 1 2 and 3 (wv) of chitosan (Figure 6(a)) showed


Cel

l num

ber

wel

l

5

6

4

3

2

1

0

times105

Time (days)2 3 4 5 6 7 8 9 10 11

(a)

Cel

l num

ber

wel

l

times105

Time (days)

25

20

15

10

50

002 4 6 8 10

(b)

Figure 6 Proliferation kinetic profiles of h-AdMSCs seeded in 119886P-PRPPCHTs (a) CHT concentration and (b) freezing conditions (◼)PCHTs 1 (minus20∘C) (e) PCHTs 2 (minus20∘C) (998771) PCHTs 3 (minus20∘C) (◻) PCHTs 3 (minus80∘C) (O) PCHTs 3 (minus196∘C) and (998779) P-PRPactivated with Ca+2thrombin (control) The concentration of platelets in P-PRP was 374000 pqmm3 Activated P-PRP alone was used ascontrol

significant differences (119875 lt 005) compared to control(119886P-PRP) Therefore PCHTs should have stabilized fibrinnetworks as we initially hypothesized

However regarding freezing condition no significantvariations for 119886P-PRPPCHTs prepared at minus80∘C or minus196∘Cand 119886P-PRP were observed (Figure 6(b)) In all cases cellviability decreased after 7 days probably due to collapse ofthe structure

We also observed that the exponential phase of thecultures (Figure 6(a)) started after the maximum release ofGFs (Figure 5) and the decline phase matched the largestweight loss of the PCHTs (Figure 2)

334 Induction of Osteogenic Differentiation Osteogenic dif-ferentiation of h-AdMSCs was investigated 14 days after cellseeding ALP an early marker of osteogenic differentiationwas determined and the results are shown in Figure 7 Theexpression of ALP activity showed no significant difference(119875 lt 005) with CHT concentration However higherosteogenic differentiation was obtained with 119886P-PRPPCHTs3 (minus196∘C)

4 Conclusions

Composite scaffolds were prepared with porous chitosanand 119886-PRP The performance of the composite scaffoldswas superior to 119886P-PRP alone indicating that the porouschitosan stabilized the fibrin network supporting our initialhypothesis

Chitosan concentration and freezing conditions influ-enced the physicochemical and biological properties of thescaffolds On the average physicochemical mechanical and

ALP

activ

ity in

405

nm (A

U)

16

14

12

10

08

06

04

02

00

Blan

k

aP-P

RPP

CHTs

1

(minus20

∘ C)

aP-P

RPP

CHTs

2

(minus20

∘ C)

aP-P

RPP

CHTs

3

(minus20

∘ C)

aP-P

RPP

CHTs

3

(minus80

∘ C)

aP-P

RPP

CHTs

3

(minus196∘ C)

Figure 7 ALP activities of cells cultured on 119886P-PRPPCHTsscaffolds prepared with different CHT concentrations and freezingconditions (statistically significant differences from blank 119899 =3 lowast119875

lt 005) Blank = the reagents used in the assay onlyThe concentration of platelets in whole blood donors (averageof 2 donors) was 163500 pqmm3 After preparation of the PRPthe platelets were concentrated approximately 174 times with anaverage final concentration of 303000 pqmm3


h-AdMSCs proliferation improved by the use of 3 (wv)chitosan and minus20∘C freezing temperature while minus196∘Cfavored osteogenic differentiation Additional stabilization ofthe porous structure is needed for applications in regenerativemedicine

Conflict of Interests

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

Acknowledgments

The authors acknowledge the financial support from theNational Council of Technological and Scientific Develop-ment (CNPq Brazil) They also thank Dr William DiasBelangero and Dr Ana Amelia Rodrigues of the Facultyof Medical Sciences (University of Campinas) for the MTTresults andDr AngelaCristinaMalheiros Luzo of theHaema-tology and Hemotherapy Center (University of Campinas)for the donation of h-AdMSCs

References

[1] R Langer and J P Vacanti ldquoTissue engineeringrdquo Science vol260 no 5110 pp 920ndash926 1993

[2] G Chen T Ushida and T Tateishi ldquoScaffold design for tissueengineeringrdquoMacromolecular Bioscience vol 2 no 2 pp 67ndash772002

[3] D Crane and P AM Everts ldquoPlatelet rich plasma (PRP)matrixgraftsrdquo Practical Pain Management vol 8 pp 12ndash27 2008

[4] H-I Chang and Y Wang ldquoCell responses to surface andarchitecture of tissue engineering scaffoldsrdquo in RegenerativeMedicine and Tissue EngineeringmdashCells and Biomaterials DEberli Ed chapter 27 pp 569ndash588 InTech Rijeka Croatia2011

[5] W Bensaıd J T Triffitt C Blanchat K Oudina L Sedel andHPetite ldquoA biodegradable fibrin scaffold for mesenchymal stemcell transplantationrdquoBiomaterials vol 24 no 14 pp 2497ndash25022003

[6] J W Weisel and C Nagaswami ldquoComputer modeling of fibrinpolymerization kinetics correlated with electron microscopeand turbidity observations clot structure and assembly arekinetically controlledrdquo Biophysical Journal vol 63 no 1 pp 111ndash128 1992

[7] M R Falvo O V Gorkun and S T Lord ldquoThe molecularorigins of the mechanical properties of fibrinrdquo BiophysicalChemistry vol 152 no 1ndash3 pp 15ndash20 2010

[8] E A Ryan L F Mockros J W Weisel and L LorandldquoStructural origins of fibrin clot rheologyrdquo Biophysical Journalvol 77 no 5 pp 2813ndash2826 1999

[9] K Kojima Y Okamoto K Miyatake H Fujise Y Shigemasaand S Minami ldquoEffects of chitin and chitosan on collagensynthesis in wound healingrdquo Journal of Veterinary MedicalScience vol 66 no 12 pp 1595ndash1598 2004

[10] Y Okamoto R Yano K Miyatake I Tomohiro Y Shigemasaand S Minami ldquoEffects of chitin and chitosan on bloodcoagulationrdquo Carbohydrate Polymers vol 53 no 3 pp 337ndash3422003

[11] E-C Shen T-C Chou C-H Gau H-P Tu Y-T Chen andE Fu ldquoReleasing growth factors from activated human plateletsafter chitosan stimulation a possible bio-material for platelet-rich plasma preparationrdquo Clinical Oral Implants Research vol17 no 5 pp 572ndash578 2006

[12] S J Chang S M Kuo C-W Lan I Manousakas and PH Tsai ldquoEvaluation of chitosanCaSO

4

platelet-rich plasmamicrosphere composites as alveolus osteogenesis materialrdquoBiomedical Engineering Applications Basis and Communica-tions vol 21 no 2 pp 115ndash122 2009

[13] B Kutlu R S T Aydin A C AkmanM Gumusderelioglu andRM Nohutcu ldquoPlatelet-rich plasma-loaded chitosan scaffoldspreparation and growth factor release kineticsrdquo Journal ofBiomedical Materials Research Part B Applied Biomaterials vol101 no 1 pp 28ndash35 2013

[14] E O Oktay B Demiralp S Senel A Cevdet Akman KEratalay and H Akincibay ldquoEffects of platelet-rich plasma andchitosan combination on bone regeneration in experimentalrabbit cranial defectsrdquoThe Journal of Oral Implantology vol 36no 3 pp 175ndash184 2010

[15] S Mathews R Bhonde P K Gupta and S Totey ldquoA noveltripolymer coating demonstrating the synergistic effect ofchitosan collagen type 1 and hyaluronic acid on osteogenic dif-ferentiation of human bonemarrow derivedmesenchymal stemcellsrdquo Biochemical and Biophysical Research Communicationsvol 414 no 1 pp 270ndash276 2011

[16] R Muzzarelli ldquoChitosanrdquo in Natural Chelating Polymers RMuzzarelli Ed pp 144ndash176 Pergamon Press Oxford UK 1973

[17] J Berger M Reist J M Mayer O Felt N A Peppas and RGurny ldquoStructure and interactions in covalently and ionicallycrosslinked chitosan hydrogels for biomedical applicationsrdquoEuropean Journal of Pharmaceutics and Biopharmaceutics vol57 no 1 pp 19ndash34 2004

[18] N Nwe T Furuike and H Tamura ldquoThe mechanical andbiological properties of chitosan scaffolds for tissue regener-ation templates are significantly enhanced by chitosan fromGongronella butlerirdquoMaterials vol 2 no 2 pp 374ndash398 2009

[19] AMMartins G Eng S G Caridade J FMano R L Reis andGVunjak-Novakovic ldquoElectrically conductive chitosancarbonscaffolds for cardiac tissue engineeringrdquo Biomacromoleculesvol 15 no 2 pp 635ndash643 2014

[20] R J Nordtveit KM Varum andO Smidsroslashd ldquoDegradation ofpartiallyN-acetylated chitosans with hen egg white and humanlysozymerdquo Carbohydrate Polymers vol 29 no 2 pp 163ndash1671996

[21] D Koga ldquoChitin enzymologymdashchitinaserdquo inAdvances in ChitinScience R Chen and H C Chen Eds vol 3 pp 16ndash23 1998

[22] GMolinaro J-C Leroux J Damas andA Adam ldquoBiocompat-ibility of thermosensitive chitosan-based hydrogels an in vivoexperimental approach to injectable biomaterialsrdquoBiomaterialsvol 23 no 13 pp 2717ndash2722 2002

[23] M Rucker M W Laschke D Junker et al ldquoAngiogenic andinflammatory response to biodegradable scaffolds in dorsalskinfold chambers of micerdquo Biomaterials vol 27 no 29 pp5027ndash5038 2006

[24] R Muzzarelli R Tarsi O Filippini E Giovanetti G Biaginiand P E Varaldo ldquoAntimicrobial properties of N-carboxybutylchitosanrdquo Antimicrobial Agents and Chemotherapy vol 34 no10 pp 2019ndash2023 1990

[25] X Li M Min N Du et al ldquoChitin chitosan and glycatedchitosan regulate immune responses the novel adjuvants for


cancer vaccinerdquo Clinical and Developmental Immunology vol2013 Article ID 387023 8 pages 2013

[26] P He S S Davis and L Illum ldquoIn vitro evaluation of themucoadhesive properties of chitosan microspheresrdquo Interna-tional Journal of Pharmaceutics vol 166 no 1 pp 75ndash88 1998

[27] U Bertram and R Bodmeier ldquoIn situ gelling bioadhesive nasalinserts for extended drug delivery in vitro characterization ofa new nasal dosage formrdquo European Journal of PharmaceuticalSciences vol 27 no 1 pp 62ndash71 2006

[28] S F Antonov E V Kryzhanovskaya Y I Filippov S MShinkarev and M A Frolova ldquoStudy of wound-healing prop-erties of chitosanrdquo Russian Agricultural Sciences vol 34 no 6pp 426ndash427 2008

[29] T Dai M Tanaka Y-Y Huang and M R Hamblin ldquoChitosanpreparations for wounds and burns antimicrobial and wound-healing effectsrdquo Expert Review of Anti-Infective Therapy vol 9no 7 pp 857ndash879 2011

[30] S A Agnihotri N N Mallikarjuna and T M AminabhavildquoRecent advances on chitosan-based micro- and nanoparticlesin drug deliveryrdquo Journal of Controlled Release vol 100 no 1pp 5ndash28 2004

[31] I Adekogbe and A Ghanem ldquoFabrication and characterizationofDTBP-crosslinked chitosan scaffolds for skin tissue engineer-ingrdquo Biomaterials vol 26 no 35 pp 7241ndash7250 2005

[32] M Mucha I Michalak J Balcerzak andM Tylman ldquoChitosanscaffolds films and microgranules for medical applicationmdashpreparation and drug release studiesrdquo Polimery vol 57 no 10pp 714ndash721 2012

[33] S V Madihally and H W T Matthew ldquoPorous chitosanscaffolds for tissue engineeringrdquoBiomaterials vol 20 no 12 pp1133ndash1142 1999

[34] F Amaral P Sampaio and M A Barbosa ldquoThree-dimensionalculture of human osteoblastic cells in chitosan sponges theeffect of the degree of acetylationrdquo Journal of BiomedicalMaterials Research A vol 76 no 2 pp 335ndash346 2006

[35] TMajima T Irie N Sawaguchi et al ldquoChitosan-based hyaluro-nan hybrid polymer fibre scaffold for ligament and tendontissue engineeringrdquo Proceedings of the Institution of MechanicalEngineers Part H Journal of Engineering in Medicine vol 221no 5 pp 537ndash546 2007

[36] X Z Shu and K J Zhu ldquoControlled drug release propertiesof ionically cross-linked chitosan beads the influence of anionstructurerdquo International Journal of Pharmaceutics vol 233 no1-2 pp 217ndash225 2002

[37] H P Tan C R Chu K A Payne and K G Marra ldquoInjectablein situ forming biodegradable chitosan-hyaluronic acid basedhydrogels for cartilage tissue engineeringrdquo Biomaterials vol 30no 13 pp 2499ndash2506 2009

[38] M Lavertu S Methot N Tran-Khanh and M D BuschmannldquoHigh efficiency gene transfer using chitosanDNA nanoparti-cles with specific combinations of molecular weight and degreeof deacetylationrdquo Biomaterials vol 27 no 27 pp 4815ndash48242006

[39] S B Rao and C P Sharma ldquoSterilization of chitosan impli-cationsrdquo Journal of Biomaterials Applications vol 10 no 2 pp136ndash143 1995

[40] S Cardea P Pisanti and E Reverchon ldquoGeneration of chitosannanoporous structures for tissue engineering applications usinga supercritical fluid assisted processrdquoThe Journal of SupercriticalFluids vol 54 no 3 pp 290ndash295 2010

[41] A R Costa-Pinto R L Reis and N M Neves ldquoScaffolds basedbone tissue engineering the role of chitosanrdquoTissue EngineeringPart B Reviews vol 17 no 5 pp 331ndash347 2011

[42] F Shen Y L Cui L F Yang et al ldquoA study on the fabricationof porous chitosangelatin network scaffold for tissue engineer-ingrdquo Polymer International vol 49 no 12 pp 1596ndash1599 2000

[43] D L Nettles S H Elder and J A Gilbert ldquoPotential use of chi-tosan as a cell scaffold material for cartilage tissue engineeringrdquoTissue Engineering vol 8 no 6 pp 1009ndash1016 2002

[44] A Nasti N M Zaki P de Leonardis et al ldquoChitosanTPP andchitosanTPP-hyaluronic acid nanoparticles systematic opti-misation of the preparative process and preliminary biologicalevaluationrdquo Pharmaceutical Research vol 26 no 8 pp 1918ndash1930 2009

[45] C R Correia L S Moreira-Teixeira L Moroni et al ldquoChitosanscaffolds containing hyaluronic acid for cartilage tissue engi-neeringrdquo Tissue EngineeringmdashPart C Methods vol 17 no 7 pp717ndash730 2011

[46] S Tang and M Spector ldquoIncorporation of hyaluronic acidinto collagen scaffolds for the control of chondrocyte-mediatedcontraction and chondrogenesisrdquo Biomedical Materials vol 2no 3 pp S135ndashS141 2007

[47] Y-C Wang M-C Lin D-M Wang and H-J Hsieh ldquoFabri-cation of a novel porous PGA-chitosan hybrid matrix for tissueengineeringrdquo Biomaterials vol 24 no 6 pp 1047ndash1057 2003

[48] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[49] M Gumudereliolu and S Aday ldquoHeparin-functionalized chi-tosan scaffolds for bone tissue engineeringrdquo CarbohydrateResearch vol 346 no 5 pp 606ndash613 2011

[50] A G M Perez R Lichy J F S D Lana et al ldquoPrediction andmodulation of platelet recovery by discontinuous centrifuga-tion of whole blood for the preparation of pure platelet-richplasmardquo BioResearch Open Access vol 2 no 4 pp 307ndash3142013

[51] A G M Perez A A Rodrigues A C M Luzo J F S DLana W D Belangero and M H A Santana ldquoFibrin networkarchitectures in pure platelet-rich plasma as characterized byfiber radius and correlated with clotting timerdquo Journal ofMaterials Science Materials inMedicine vol 25 no 8 pp 1967ndash1977 2014

[52] J H Brauker V E Carr-Brendel L A Martinson J CrudeleW D Johnston and R C Johnson ldquoNeovascularization of syn-thetic membranes directed by membrane microarchitecturerdquoJournal of BiomedicalMaterials Research vol 29 no 12 pp 1517ndash1524 1995

[53] J J Klawitter and S F Hulbert ldquoApplication of porous ceramicsfor the attachment of load-bearing internal orthopedic applica-tionsrdquo Journal of Biomedical Materials Research A Symposiumvol 5 no 6 pp 161ndash229 1971

[54] S Yang K-F Leong Z Du and C-K Chua ldquoThe designof scaffolds for use in tissue engineering Part I Traditionalfactorsrdquo Tissue Engineering vol 7 no 6 pp 679ndash689 2001

[55] A J Salgado O P Coutinho and R L Reis ldquoBone tissueengineering state of the art and future trendsrdquoMacromolecularBioscience vol 4 no 8 pp 743ndash765 2004

[56] I V Yannas E Lee D POrgill EM Skrabut andG FMurphyldquoSynthesis and characterization of a model extracellular matrixthat induces partial regeneration of adult mammalian skinrdquo


Proceedings of the National Academy of Sciences of the UnitedStates of America vol 86 no 3 pp 933ndash937 1989

[57] C Chavez-Munoz K T Nguyen W Xu S-J Hong T AMustoe and R D Galiano ldquoTransdifferentiation of adipose-derived stem cells into keratinocyte-like cells engineering astratified epidermisrdquo PLoSONE vol 8 no 12 Article ID e805872013

[58] C A Leon y Leon ldquoNew perspectives in mercury porosimetryrdquoAdvances inColloid and Interface Science vol 76-77 pp 341ndash3721998

[59] K F Leong C M Cheah and C K Chua ldquoSolid freeform fab-rication of three-dimensional scaffolds for engineering replace-ment tissues and organsrdquo Biomaterials vol 24 no 13 pp 2363ndash2378 2003

[60] X Liu and P X Ma ldquoPolymeric scaffolds for bone tissueengineeringrdquo Annals of Biomedical Engineering vol 32 no 3pp 477ndash486 2004

[61] Q L Loh and C Choong ldquoThree-dimensional scaffolds fortissue engineering applications role of porosity and pore sizerdquoTissue Engineering Part B Reviews vol 19 no 6 pp 485ndash5032013

[62] H-Y Cheung Q-T Lau T-P Lu and D Hui ldquoA criticalreview on polymer-based bio-engineered materials for scaffolddevelopmentrdquo Composites Part B Engineering vol 38 no 3 pp291ndash300 2007

[63] S J Hollister ldquoPorous scaffold design for tissue engineeringrdquoNature Materials vol 4 no 7 pp 518ndash524 2005

[64] J Enrione F Osorio D Lopez et al ldquoCharacterizationof a Gelatinchitosanhyaluronan scaffold-polymerrdquo ElectronicJournal of Biotechnology vol 13 no 5 pp 1ndash11 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Calcium acts as a cofactor of thrombin modulates theelongation of fibers during polymerization by promoting lat-eral branching and functions in clot stability [7 8] Calciumand thrombin also activate platelets allowing the release ofGFs and cytokines Therefore the fibrin matrix provides anoptimized medium for cell proliferation and healing

Based on these features platelet-rich plasma (PRP) hasbeen widely used in regenerative medicine PRP is an autol-ogous product prepared from whole blood by separatingthe red blood cells and concentrating the platelets andother components of plasma However although viscoelasticthe fibrin matrix alone lacks stability to be efficient whenregenerative medicine is the goal

To mimic the natural healing process and improve thestability of the fibrin matrix we propose in this work theuse of the fibrin network from activated PRP with porouschitosan as a composite scaffold for tissue regeneration

Our hypothesis is that the chemical nature of the chi-tosan surface supports the fibrin network by electrostaticattachment thus prolonging its stability without changingthe paracrine affinity of mesenchymal cells to fibrin fibers Asa consequence the composite scaffoldmust improve cell pro-liferation and tissue differentiation compared to PRP alone

Related works showed the effects of chitosan on bloodcoagulation through the strong adhesion of platelets tothe surface of chitosan particles as well as chitosan (01ndash1mgmL) incorporated with PRP to enhance the releaseof PDGF-AB and TGF-1205731 from platelets [9 10] Shen etal demonstrated that chitosan could be an appropriatesubstitute for thrombin as an agonist in PRP preparation[11] Chang et al showed that growth factors could sustainrelease until 12 h at approximately 1 ngmL from chitosanCaSO

4PRP microspheres after activation with thrombin

[12] Kutlu et al showed that scaffolds prepared by the freeze-drying of PRP added to chitosan gel (2 wv) were moreeffective than chitosan sponges soaked with PRP on con-trolled GF release [13] Oktay et al applied PRP-embeddedchitosan sponges to defects and showed a histological ten-dency toward increased bone formation [14] Mathews etal demonstrated that chitosan enhanced mineralization byupregulating the genes associated with mineralization andcalcium-binding proteins [15]

Chitosan is a polysaccharide derived from chitin (copoly-mers 120573-(1rarr 4)-2-amino-2-deoxy-D-glucose and 120573-(1rarr 4)-2-acetamido 2-deoxy-D-glucose) found in the shells ofmarine crustaceans and insects and the cell walls of somefungi [16 17]

In the last ten years considerable attention has been givento chitosan-based materials in the field of tissue engineering[18 19] The beneficial properties of chitosan have beenproven such as biodegradability [20 21] biocompatibility[22 23] antibacterial activity [24 25] cell adhesion [26 27]and wound healing properties [28 29] Moreover the chem-ical nature of chitosan gives many possibilities for ionic andcovalent modifications that allow for the modulation of themechanical and biological properties of biomaterials [30 31]

Regarding the technological aspects chitosan can beeasily processed in diverse forms in the absence of toxicsolvents such as films [32] sponges [33 34] fibers [35]

beads [36] hydrogels [37] and microparticlesnanoparticles[38] Furthermore chitosan supports sterilization [39] isabundant in nature and requires only low-cost processingin nonaggressive ecological conditions before being used asa raw material [17]

Freeze-drying is the most common and simplest methodto produce porous chitosan scaffolds The freezing processprovides the nucleation of ice crystals from solution andfurther growth along the lines of thermal gradients Exclusionof the chitosan acetate salt from the ice crystal phase andsubsequent ice removal by lyophilization generate a porousmaterial [40 41]

Madihally and Matthew reported that the pore size ofchitosan scaffolds produced by lyophilization can be con-trolled within the range of 1ndash250120583m [33] A more uniformand interconnected pore structure can be obtained whenlower freezing temperatures are used Furthermore the poreorientation is related to the geometry of the moldings usedand can also be controlled by changing thermal gradientsduring freezing [42]

Themicrostructure crystallinity andmechanical strengthof the porous chitosan matrix also can be controlled by thechitosan concentration molecular weight and deacetylationdegree [33 43]

In this work the effects of PRP association were studiedregarding the porous structure of solid sponges which wereproduced by varying chitosan concentration and freezingconditions The biological performance of association wasevaluated in terms of growth factor release kinetics prolifera-tion and osteogenic differentiation of seeded human adiposetissue-derived mesenchymal stem cells (h-AdMSCs)

2 Experimental

21 Materials Chitosan (average molecular weight [Mw] =4 times 105Da degree of deacetylation = 83plusmn4) was purchasedfrom Polymar (Fortaleza CE Brazil) and purified accordingto the protocol described by Nasti et al [44] Other chemicalswere of reagent grade and were used without any furtherpurification All biological experiments were performed withhuman adipose tissue-derived stem mesenchymal cells (h-AdMSCs) and approved by the Ethics Committee of theMedical Sciences School of the University of Campinas(UNICAMP CAAE 09720146000-11) The donors werehealthy individuals aged between 30 and 40 years old whowere previously assessed through clinical examinations

22 Methods

221 Preparation of Porous Chitosan Scaffolds (PCHTs)PCHTs were prepared by freezing at a controlled temperatureof a chitosan solution previously poured in 24-well cultureplates (TPP polystyrene diameter = 154mm) to give thema cylindrical shape followed by lyophilizing in lyophilizerL101 (Liobras Sao Carlos SP Brazil) at a temperature ofapproximately minus30∘C for 48 hours

Effects of Chitosan Concentration Chitosan solutions withconcentrations of 1 2 or 3 (wv) were prepared by




2 and lyophilized






SR = 119908119904119908119889

(1)



120576 () =119881119898 minus (119908119898120588)

119881119898

times 100

(2)







2solution






















2groups in acidic







(a) (b)

(c) (d) (e)














CHTconcentration()



Pore size(120583m)

(119899 = 20)

Porosity()

(119899 = 3)



50

40

30

20

10

0

Wei

ght l

oss (

)

Time (days)0 3 6 9 12 15





Cel

l num

ber

wel

l

30

25

20

15

10

00

PCH

Ts1

(minus

20∘ C)

PCH

Ts2

(minus

20∘ C)

PCH

Ts3

(minus

20∘ C)

PCH

Ts3

(minus

80∘ C)

PCH

Ts3

(minus

196∘ C)

NTC PT

C

times104

05


119875

lt 005





(a) (b) (c)

(d) (e) (f)
















25000

20000

15000

10000

5000

0

Cum

ulat

ive T

GF-120573

(pg

mL

PRP)

Time (hours)0 10 20 30 40 50 60 70 80

(a)

Time (hours)0 10 20 30 40 50 60 70 80

12000

10000

8000

6000

4000

2000

0

Cum

ulat

ive P

DG

F-A

B (p

gm

L PR

P)

(b)

25000

20000

15000

10000

5000

0

Cum

ulat

ive T

GF-120573

1 (p

gm

L PR

P)

Time (hours)0 10 20 30 40 50 60 70 80

(c)

Time (hours)0 10 20 30 40 50 60 70 80

15000

12000

9000

6000

3000

0

Cum

ulat

ive P

DG

F-A

B (p

gm

L PR

P)

(d)











Cel

l num

ber

wel

l

5

6

4

3

2

1

0

times105

Time (days)2 3 4 5 6 7 8 9 10 11

(a)

Cel

l num

ber

wel

l

times105

Time (days)

25

20

15

10

50

002 4 6 8 10

(b)






4 Conclusions



ALP

activ

ity in

405

nm (A

U)

16

14

12

10

08

06

04

02

00

Blan

k

aP-P

RPP

CHTs

1

(minus20

∘ C)

aP-P

RPP

CHTs

2

(minus20

∘ C)

aP-P

RPP

CHTs

3

(minus20

∘ C)

aP-P

RPP

CHTs

3

(minus80

∘ C)

aP-P

RPP

CHTs

3

(minus196∘ C)







Acknowledgments


References













4































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















2 and lyophilized






SR = 119908119904119908119889

(1)



120576 () =119881119898 minus (119908119898120588)

119881119898

times 100

(2)







2solution






















2groups in acidic







(a) (b)

(c) (d) (e)














CHTconcentration()



Pore size(120583m)

(119899 = 20)

Porosity()

(119899 = 3)



50

40

30

20

10

0

Wei

ght l

oss (

)

Time (days)0 3 6 9 12 15





Cel

l num

ber

wel

l

30

25

20

15

10

00

PCH

Ts1

(minus

20∘ C)

PCH

Ts2

(minus

20∘ C)

PCH

Ts3

(minus

20∘ C)

PCH

Ts3

(minus

80∘ C)

PCH

Ts3

(minus

196∘ C)

NTC PT

C

times104

05


119875

lt 005





(a) (b) (c)

(d) (e) (f)
















25000

20000

15000

10000

5000

0

Cum

ulat

ive T

GF-120573

(pg

mL

PRP)

Time (hours)0 10 20 30 40 50 60 70 80

(a)

Time (hours)0 10 20 30 40 50 60 70 80

12000

10000

8000

6000

4000

2000

0

Cum

ulat

ive P

DG

F-A

B (p

gm

L PR

P)

(b)

25000

20000

15000

10000

5000

0

Cum

ulat

ive T

GF-120573

1 (p

gm

L PR

P)

Time (hours)0 10 20 30 40 50 60 70 80

(c)

Time (hours)0 10 20 30 40 50 60 70 80

15000

12000

9000

6000

3000

0

Cum

ulat

ive P

DG

F-A

B (p

gm

L PR

P)

(d)











Cel

l num

ber

wel

l

5

6

4

3

2

1

0

times105

Time (days)2 3 4 5 6 7 8 9 10 11

(a)

Cel

l num

ber

wel

l

times105

Time (days)

25

20

15

10

50

002 4 6 8 10

(b)






4 Conclusions



ALP

activ

ity in

405

nm (A

U)

16

14

12

10

08

06

04

02

00

Blan

k

aP-P

RPP

CHTs

1

(minus20

∘ C)

aP-P

RPP

CHTs

2

(minus20

∘ C)

aP-P

RPP

CHTs

3

(minus20

∘ C)

aP-P

RPP

CHTs

3

(minus80

∘ C)

aP-P

RPP

CHTs

3

(minus196∘ C)







Acknowledgments


References













4































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























2groups in acidic







(a) (b)

(c) (d) (e)














CHTconcentration()



Pore size(120583m)

(119899 = 20)

Porosity()

(119899 = 3)



50

40

30

20

10

0

Wei

ght l

oss (

)

Time (days)0 3 6 9 12 15





Cel

l num

ber

wel

l

30

25

20

15

10

00

PCH

Ts1

(minus

20∘ C)

PCH

Ts2

(minus

20∘ C)

PCH

Ts3

(minus

20∘ C)

PCH

Ts3

(minus

80∘ C)

PCH

Ts3

(minus

196∘ C)

NTC PT

C

times104

05


119875

lt 005





(a) (b) (c)

(d) (e) (f)
















25000

20000

15000

10000

5000

0

Cum

ulat

ive T

GF-120573

(pg

mL

PRP)

Time (hours)0 10 20 30 40 50 60 70 80

(a)

Time (hours)0 10 20 30 40 50 60 70 80

12000

10000

8000

6000

4000

2000

0

Cum

ulat

ive P

DG

F-A

B (p

gm

L PR

P)

(b)

25000

20000

15000

10000

5000

0

Cum

ulat

ive T

GF-120573

1 (p

gm

L PR

P)

Time (hours)0 10 20 30 40 50 60 70 80

(c)

Time (hours)0 10 20 30 40 50 60 70 80

15000

12000

9000

6000

3000

0

Cum

ulat

ive P

DG

F-A

B (p

gm

L PR

P)

(d)











Cel

l num

ber

wel

l

5

6

4

3

2

1

0

times105

Time (days)2 3 4 5 6 7 8 9 10 11

(a)

Cel

l num

ber

wel

l

times105

Time (days)

25

20

15

10

50

002 4 6 8 10

(b)






4 Conclusions



ALP

activ

ity in

405

nm (A

U)

16

14

12

10

08

06

04

02

00

Blan

k

aP-P

RPP

CHTs

1

(minus20

∘ C)

aP-P

RPP

CHTs

2

(minus20

∘ C)

aP-P

RPP

CHTs

3

(minus20

∘ C)

aP-P

RPP

CHTs

3

(minus80

∘ C)

aP-P

RPP

CHTs

3

(minus196∘ C)







Acknowledgments


References













4































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d) (e)














CHTconcentration()



Pore size(120583m)

(119899 = 20)

Porosity()

(119899 = 3)



50

40

30

20

10

0

Wei

ght l

oss (

)

Time (days)0 3 6 9 12 15





Cel

l num

ber

wel

l

30

25

20

15

10

00

PCH

Ts1

(minus

20∘ C)

PCH

Ts2

(minus

20∘ C)

PCH

Ts3

(minus

20∘ C)

PCH

Ts3

(minus

80∘ C)

PCH

Ts3

(minus

196∘ C)

NTC PT

C

times104

05


119875

lt 005





(a) (b) (c)

(d) (e) (f)
















25000

20000

15000

10000

5000

0

Cum

ulat

ive T

GF-120573

(pg

mL

PRP)

Time (hours)0 10 20 30 40 50 60 70 80

(a)

Time (hours)0 10 20 30 40 50 60 70 80

12000

10000

8000

6000

4000

2000

0

Cum

ulat

ive P

DG

F-A

B (p

gm

L PR

P)

(b)

25000

20000

15000

10000

5000

0

Cum

ulat

ive T

GF-120573

1 (p

gm

L PR

P)

Time (hours)0 10 20 30 40 50 60 70 80

(c)

Time (hours)0 10 20 30 40 50 60 70 80

15000

12000

9000

6000

3000

0

Cum

ulat

ive P

DG

F-A

B (p

gm

L PR

P)

(d)











Cel

l num

ber

wel

l

5

6

4

3

2

1

0

times105

Time (days)2 3 4 5 6 7 8 9 10 11

(a)

Cel

l num

ber

wel

l

times105

Time (days)

25

20

15

10

50

002 4 6 8 10

(b)






4 Conclusions



ALP

activ

ity in

405

nm (A

U)

16

14

12

10

08

06

04

02

00

Blan

k

aP-P

RPP

CHTs

1

(minus20

∘ C)

aP-P

RPP

CHTs

2

(minus20

∘ C)

aP-P

RPP

CHTs

3

(minus20

∘ C)

aP-P

RPP

CHTs

3

(minus80

∘ C)

aP-P

RPP

CHTs

3

(minus196∘ C)







Acknowledgments


References













4































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















CHTconcentration()



Pore size(120583m)

(119899 = 20)

Porosity()

(119899 = 3)



50

40

30

20

10

0

Wei

ght l

oss (

)

Time (days)0 3 6 9 12 15





Cel

l num

ber

wel

l

30

25

20

15

10

00

PCH

Ts1

(minus

20∘ C)

PCH

Ts2

(minus

20∘ C)

PCH

Ts3

(minus

20∘ C)

PCH

Ts3

(minus

80∘ C)

PCH

Ts3

(minus

196∘ C)

NTC PT

C

times104

05


119875

lt 005





(a) (b) (c)

(d) (e) (f)
















25000

20000

15000

10000

5000

0

Cum

ulat

ive T

GF-120573

(pg

mL

PRP)

Time (hours)0 10 20 30 40 50 60 70 80

(a)

Time (hours)0 10 20 30 40 50 60 70 80

12000

10000

8000

6000

4000

2000

0

Cum

ulat

ive P

DG

F-A

B (p

gm

L PR

P)

(b)

25000

20000

15000

10000

5000

0

Cum

ulat

ive T

GF-120573

1 (p

gm

L PR

P)

Time (hours)0 10 20 30 40 50 60 70 80

(c)

Time (hours)0 10 20 30 40 50 60 70 80

15000

12000

9000

6000

3000

0

Cum

ulat

ive P

DG

F-A

B (p

gm

L PR

P)

(d)











Cel

l num

ber

wel

l

5

6

4

3

2

1

0

times105

Time (days)2 3 4 5 6 7 8 9 10 11

(a)

Cel

l num

ber

wel

l

times105

Time (days)

25

20

15

10

50

002 4 6 8 10

(b)






4 Conclusions



ALP

activ

ity in

405

nm (A

U)

16

14

12

10

08

06

04

02

00

Blan

k

aP-P

RPP

CHTs

1

(minus20

∘ C)

aP-P

RPP

CHTs

2

(minus20

∘ C)

aP-P

RPP

CHTs

3

(minus20

∘ C)

aP-P

RPP

CHTs

3

(minus80

∘ C)

aP-P

RPP

CHTs

3

(minus196∘ C)







Acknowledgments


References













4































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b) (c)

(d) (e) (f)
















25000

20000

15000

10000

5000

0

Cum

ulat

ive T

GF-120573

(pg

mL

PRP)

Time (hours)0 10 20 30 40 50 60 70 80

(a)

Time (hours)0 10 20 30 40 50 60 70 80

12000

10000

8000

6000

4000

2000

0

Cum

ulat

ive P

DG

F-A

B (p

gm

L PR

P)

(b)

25000

20000

15000

10000

5000

0

Cum

ulat

ive T

GF-120573

1 (p

gm

L PR

P)

Time (hours)0 10 20 30 40 50 60 70 80

(c)

Time (hours)0 10 20 30 40 50 60 70 80

15000

12000

9000

6000

3000

0

Cum

ulat

ive P

DG

F-A

B (p

gm

L PR

P)

(d)











Cel

l num

ber

wel

l

5

6

4

3

2

1

0

times105

Time (days)2 3 4 5 6 7 8 9 10 11

(a)

Cel

l num

ber

wel

l

times105

Time (days)

25

20

15

10

50

002 4 6 8 10

(b)






4 Conclusions



ALP

activ

ity in

405

nm (A

U)

16

14

12

10

08

06

04

02

00

Blan

k

aP-P

RPP

CHTs

1

(minus20

∘ C)

aP-P

RPP

CHTs

2

(minus20

∘ C)

aP-P

RPP

CHTs

3

(minus20

∘ C)

aP-P

RPP

CHTs

3

(minus80

∘ C)

aP-P

RPP

CHTs

3

(minus196∘ C)







Acknowledgments


References













4































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















25000

20000

15000

10000

5000

0

Cum

ulat

ive T

GF-120573

(pg

mL

PRP)

Time (hours)0 10 20 30 40 50 60 70 80

(a)

Time (hours)0 10 20 30 40 50 60 70 80

12000

10000

8000

6000

4000

2000

0

Cum

ulat

ive P

DG

F-A

B (p

gm

L PR

P)

(b)

25000

20000

15000

10000

5000

0

Cum

ulat

ive T

GF-120573

1 (p

gm

L PR

P)

Time (hours)0 10 20 30 40 50 60 70 80

(c)

Time (hours)0 10 20 30 40 50 60 70 80

15000

12000

9000

6000

3000

0

Cum

ulat

ive P

DG

F-A

B (p

gm

L PR

P)

(d)











Cel

l num

ber

wel

l

5

6

4

3

2

1

0

times105

Time (days)2 3 4 5 6 7 8 9 10 11

(a)

Cel

l num

ber

wel

l

times105

Time (days)

25

20

15

10

50

002 4 6 8 10

(b)






4 Conclusions



ALP

activ

ity in

405

nm (A

U)

16

14

12

10

08

06

04

02

00

Blan

k

aP-P

RPP

CHTs

1

(minus20

∘ C)

aP-P

RPP

CHTs

2

(minus20

∘ C)

aP-P

RPP

CHTs

3

(minus20

∘ C)

aP-P

RPP

CHTs

3

(minus80

∘ C)

aP-P

RPP

CHTs

3

(minus196∘ C)







Acknowledgments


References













4































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Cel

l num

ber

wel

l

5

6

4

3

2

1

0

times105

Time (days)2 3 4 5 6 7 8 9 10 11

(a)

Cel

l num

ber

wel

l

times105

Time (days)

25

20

15

10

50

002 4 6 8 10

(b)






4 Conclusions



ALP

activ

ity in

405

nm (A

U)

16

14

12

10

08

06

04

02

00

Blan

k

aP-P

RPP

CHTs

1

(minus20

∘ C)

aP-P

RPP

CHTs

2

(minus20

∘ C)

aP-P

RPP

CHTs

3

(minus20

∘ C)

aP-P

RPP

CHTs

3

(minus80

∘ C)

aP-P

RPP

CHTs

3

(minus196∘ C)







Acknowledgments


References













4































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Acknowledgments


References













4































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Performance of PRP Associated with Porous Chitosan as a