research article the potential use of platelet-rich plasma...

Research ArticleThe Potential Use of Platelet-Rich Plasma toReconstruct the Microtia Chondrocyte in HumanAuricular Cartilage Regeneration

Wei-Hong Chen12 Hen-Yu Liu12 Ching-Yu Tsai12 Chia-che Wu3 Hong-Jian Wei12

Alice Liu12 Ming-Tang Lai3 Chiung-Fang Huang4 and Win-Ping Deng12

1Stem Cell Research Center Taipei Medical University Taipei 110 Taiwan2Graduate Institute of Biomedical Materials and Tissue Engineering Taipei Medical University Taipei 110 Taiwan3Department of Otolaryngology Wan-Fang Hospital Taipei Medical University Taipei 116 Taiwan4Department of Dentistry Taipei Medical University Hospital Taipei 110 Taiwan

Correspondence should be addressed to Chiung-Fang Huang d642078yahoocomtwand Win-Ping Deng wpdengms41hinetnet

Received 14 November 2014 Revised 4 March 2015 Accepted 4 March 2015

Academic Editor Ilaria Armentano

Copyright copy 2015 Wei-Hong Chen et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Microtia is characterized as an incomplete auricular development and surgical reconstruction for microtia is still limited even withemerging developments This study aimed to apply bionanomaterials (PRPcollagen scaffold) for human auricular neocartilagereconstruction by using microtia chondrocytes The results showed that PRP (TGF-1205731 750 pgmL and 1 ngmL) increased cellviability of microtia chondrocytes during in vitro 9-day cultures Additionally chondrogenic-specific mRNA of Aggrecan and typeII collagen (Col II) was significantly and continuously expressed with PRP treatment during the 21-day in vitro expansion Tissueengineering of auricular neocartilagewas performed by seedingmicrotia chondrocytes in bionanomaterials (PRPcollagen scaffold)3-dimensional (3D) cultures Immunohistochemistry (IHC) ofCol II showed intensive signals between cells andmatrix after 4-weekculturesConclusion Our results demonstrated that PRP promotes proliferation and redifferentiation of microtia chondrocytes andprovides regenerative potentials in auricular neocartilage reconstruction

1 Introduction

Microtia a malformed appearance of auricle arises fromincomplete skeleton development and results in structuralabnormalities of auricle The prevalence rate of microtiavaries from 083 to 174 per 10000 of birth in different popu-lation [1] and its accurate etiology is still not well understoodIn addition more than 80 of microtia patients also sufferfrom aural atresia [2] Clinical correction ofmicrotia requiresspecialized surgical procedures Autologous cartilage implan-tation has been considered as an ideal approach to avoidimmunological rejection and disease transmission Chon-drocytes from an autologous auricle have been successfullyapplied for auricular cartilage reconstruction in vitro and invivo [3 4] For example auricular reconstruction using costal

cartilage from a rib has been widely applied for microtiarepair [5 6]However complex surgical procedureswill causemultiple risks including initial postoperation pain chest-walldeformity implantation site scarring and gradual resorptionof the implanted costal cartilage Hence modified techniquesare needed for advanced microtia reconstruction

Tissue-engineered cartilage provides extra cartilaginoustissue for cosmetic surgery of the head and neck [7]Among components for tissue engineering specific cell typeplays an important role in cartilage reconstruction In ourprevious studies human mesenchymal stem cells (MSCs)articular chondrocytes and nucleus pulposus were employedin collagen scaffold to regenerate neocartilage [8ndash10] Toaddress auricle repair chondrocytes isolated from normalauricle were generally used [7 11 12] A tissue-engineered

Hindawi Publishing CorporationJournal of NanomaterialsVolume 2015 Article ID 250615 7 pageshttpdxdoiorg1011552015250615

2 Journal of Nanomaterials

auricular cartilage was successfully created in a xenograftnude mouse model [4] However the source of human auric-ular chondrocytes from normal tissue is limited Geneticandmorphologic differences of elastic chondrocytes betweennormal auricle and microtia have not been clarified Tissue-engineered cartilage formation bymicrotia chondrocytes alsoshowed similar elastic features of normal auricular chondro-cytes [13] Thus cells isolated from autologous cartilage aredefinitely ideal for repair that also prevents immune rejectionand transmittable disease To harvest sufficient cell numberswithout losing chondrogenic phenotypes is important forrepair of cartilaginous defects Moreover microtia obtainedfrom patients has limitations such as loss of cell potentialwith high passage number In cell culture the doubling timeand cellular senescence of primary microtia chondrocytescan increase and be insufficient for repeated or researchuse

Hence culture medium supplemented with growth fac-tors or cytokines has been developed Human ear chondro-cytes cultured in fibroblast growth factor 2 (FGF2) prolifer-ated higher than serum containing medium and expresseda more differentiated phenotype [14] The addition of TGF-1205731 insulin-like growth factor (IGF) and FGF-2 was alsoreported to induce chondrogenic redifferentiation in scaffoldreconstruction resembling native auricular cartilage [7 1516] Recently platelet-released growth factors comprised inplatelet-rich plasma (PRP) were demonstrated to encouragewound healing in musculoskeletal injuries [17 18] PRP stim-ulated human nucleus pulposus (NP) regeneration throughTGF-1205731 has been demonstrated in our previous study [10]We have also examined the fact that PRP promotes ex vivoand in vivo NP regeneration [19] In addition the role ofPRP combined embryonic fibroblast in the treatment ofosteoporosis was also confirmed [20] We have evaluated theefficacy of PRP on microtia chondrocytes to regenerate tis-sue-engineered auricular cartilage An advanced cellgrowthfactor-based therapeutic strategy was developed for futurehead and neck surgical procedure

2 Materials and Methods

21 Chondrocyte Isolation and Culture The auricular carti-lage fragments were harvested from microtia patients Thesevolunteers provided informed consent for the use of their car-tilage as required by the Ethics Committee of Taipei MedicalUniversity Municipal Wan Fang Hospital Microtia cartilagewas minced into 1mm3 pieces and washed three timesin phosphate-buffered saline (PBS) After being digestedwith 03 type I collagenase (sigma) containing penicillin(100 unitsmL GIBCO-BRL) and streptomycin (01mgmLGIBCOBRL) for 15 hours at 37∘C the suspended cells werepelleted and then resuspended in DMEMF12 (Dulbeccorsquosmodified Eaglersquos mediumF12 Ham GIBCO-BRL GrandIsland NY) containing 10 (vv) fetal bovine serum (FBSGIBCO-BRL) in an incubator at 37∘C with 5 CO

2 Culture

medium was changed every 3 days before subsequent exper-iments In addition we counted the cell numbers of primarymicrotia chondrocytes at different passage number (P1 P2

and P3) in cultures with or without PRP treatment to deter-mine the cell proliferation

22 PRP Preparation and TGF-1205731 Concentration Evalua-tion PRP was extracted from total blood and activated aspreviously described [10 20] Subsequently the TGF-1205731 wasused as indicator and quantitatively analyzed using a Quan-tikine enzyme-linked immunosorbent assay (ELISA) kit(DB100 RampD Diagnostics Wiesbaden Germany) in orderto confirm the consistency of PRP for in vitro utilizationand to determine the most appropriate concentration forstudy We prepared a dilution series of TGF-1205731 standards(890207) in 100 120583L volumes in 96-well microtiter platescoated with TGF-1205731-receptor II Conversion of TGF-1205731 toits active form was necessary to estimate total TGF-1205731 sincea large proportion of TGF-1205731 is often present in a latentform The 01mL PRP solution was then mixed with 01mL25N acetic acid10M urea incubated at room temperaturefor 10min and neutralized by an addition of 01mL of 27NNaOH1M HEPES (N-[2-hydroxyethyl] piperazine-N0-[2-ethanesulfonic acid] Sigma) (H-7523)

23 MTT Assay The analytic reagent tetrazolium salt MTT[(3-(45-dimethylthiazol-2-yl)-2 5-diphenyl tetrazoliumbro-mide) Roche Mannheim Germany] assay was used fordetermining cell viability The microtia chondrocytes wereseeded into 96-well plate at a density of 2 times 104 cellsmL andtreated with or without PRP while the experimental controlswere cultured in 1 FBS containing DMEMF-12 The MTTreagent was added into each well on days 1 3 5 7 and 9 Atthe time points OD values (OD 595ndashOD 690) were analyzed4 h after the MTT reaction using Multiskan PC (ThermoLabsystem Franklin MA) and cell survival curves were thenplotted against time

24 Gene Expression Analysis Expressions of type II collagen(Col II) and Aggrecan mRNA of PRP and control groupswere determined by semiquantitative reverse transcriptionpolymerase chain reaction (RT-PCR) of 7- 14- and 21-daymonolayer cultures Total RNA was isolated using TRIzolreagent (Invitrogen Life Technologies Carlsbad CA USA)and used for first stand cDNA synthesis RNA was reverse-transcribed utilizing SuperScript III (Invitrogen Life Tech-nologies) and anOligo d(T)12ndash18 primerThe reactionmixturecontained 10mM dNTP mix 10X RT buffer 25mM MgCl

2

01M DTT RNase inhibitor and RNase H in a total vol-ume of 20120583L PCR was performed using primers for typeII collagen Aggrecan and gyceraldehde-3-phosphate dehy-drogenate (GAPDH) The primer sequences are listed asfollows Col II-forward-51015840-GAA CAT CAC CTA CCA CTGCAAG-31015840 reverse-51015840-GCAGAG TCC TAGAGTGAC TGAG-31015840 Aggrecan-forward 51015840-GCC TTG AGC AGT TCA CCTTC-31015840 reverse-51015840-CTC TTC TAC GGG GAC AGC AG-31015840PCR amplification in a final volume of 50 120583L containing25mMdNTP 25mMMgCl

2 specific primers and TaqDNA

polymerase (Invitrogen Life Technologies) The PCR wasinitiated for 5min at 95∘C and cycled 35 times at 94∘C for1min 72∘C for 1min and 72∘C for 5min

Journal of Nanomaterials 3

25 Reconstruction of Tissue-Engineered Auricular Chondro-cytes in Bionanomaterials and Immunohistochemical AnalysisThe three-dimensional (3D) constructs were formed by bio-nanomaterials (2mgmL purified type I and type II collagenandmixed withwithout PRP) as previously described in thislaboratory [9 10 21] Suspended cells were seeded at adensity of 1 times 106 cellmL onto the collagen matrix and thencultured in an incubator at 37∘C with 5 CO

2 Medium

withwithout PRP was changed every 3 days before subse-quent experiments After 2 and 4 weeks the 3D constructswere then histologically analyzed They were fixed with 10phosphate buffered formalin for 24 h embedded in paraffinand sectioned for hematoxylin and eosin (HampE) stainingFor immunohistochemical (IHC) analyses tissue sectionswere stained with mouse anti-human type II collagen mon-oclonal antibody (Chemicon International Temecula CAUSA) anti-human SOX9 polyclonal antibody (Abcam Cam-bridge UK) and anti-human Aggrecan monoclonal anti-body (Millipore Darmstadt Germany)

26 Senescence-Associated 120573-Galactosidase (SA-120573-Gal) Stain-ing SA-120573 gal activity is a biomarker for developing stainingin senescent cells in which there is a lysosomal enzymethat catalyzes the hydrolysis of 120573-galactosides into monosac-charides only in senescent cells All cells were fixed in the4 formaldehyde and then the SA-120573-gal assay was used todetermine the cell senescenceThe 120573-galactosidase activity atpH 60 produces blue perinuclear staining in senescent cellsTherefore the blue staining in positive cells was quantitatedby microscopy

27 Statistical Analysis The results of all experiments areshown as the mean plusmn standard deviation (SD) Expressionlevels in the response to PRP were compared to 1 FBSmedium and statistically evaluated using the paired 119905-test ineach group

3 Results

31 Proliferative Response of Microtia Chondrocytes Treatedwith PRP Platelet-rich plasma (PRP) containing variousgrowth factors was extracted from total blood as describedin our previous studies [10 20] TGF-1205731 in PRP was rec-ognized as the core ingredient [22 23] and was used as anindicator for quantifying PRP in the following experimentsThe DMEMF12 with 1 FBS medium was designated as thebasal medium and as a control group while basal mediumcontaining 750 pgmL or 1 ngmL TGF-1205731 in PRP was recog-nized as PRP-conditionalmedium PRP-conditionalmediumwith 750 pgmL or 1 ngmL TGF-1205731 showed the same time-dependent increase on the proliferation of microtia chon-drocytes (Figure 1(a)) Hence PRP-conditional mediumwith750 pgmLTGF-1205731 was chosen for the following experimentsThemorphologies ofmicrotia chondrocytes treatedwith PRPshowed a higher aggregation compared to those in controlgroup on day 9 (Figure 1(b))

Time (days)0 2 4 6 8 10

OD

val

ue

00

02

04

06

08

10

ControlPRP (TGF-120573 750pgmL)PRP (TGF-120573 1ngmL)

lowast

lowast

lowast

lowast

(a)

Control PRP

200120583m 200120583m

(b)

Figure 1 Proliferative response of microtia chondrocytes treatedwith PRP during 9-day cultivation (a) Proliferation of microtiachondrocytes cultured in PRP-conditional mediumwith 750 pgmLTGF-1205731 1000 pgmL TGF-1205731 and 1 FBS basal medium (controlgroup) by MTT assay Data was expressed as mean plusmn SD (119899 = 3)(b) Morphology of microtia chondrocytes treated with and withoutPRP on day 9

32 Aging-Related Changes in Cell Potential of Microtia Chon-drocytes We investigated the passage-dependent senescenceand proliferation over multiple passages (P1 P3 and P5) ofmicrotia chondrocytes with PRP treatment relative to thecontrol which were cultured under normal culture condi-tions Results showed significantly higher cell number in thePRP treatedmicrotia chondrocytes in 5 passages numbers P1P3 and P5 when compared with control group (Figure 2(a))Cellular senescence was measured by staining and quan-tification of senescence-associated beta-galactosidase (SA 120573-gal) in passage 5 cells in PRP treated and control microtiachondrocytes cultures The percentage of SA 120573-gal positivecells was notably decreased in the PRP treated cells relative tothe control (Figure 2(b)) Taken together PRP treatment wasvery effective at increasing microtia chondrocytes in serialpassage as shown in passage numbers P1 P3 and P5 andlowering cell senescence as indicated by SA 120573-gal staining

33 Induced Redifferentiation of Microtia Chondrocytes byPRP To further determine the chondrogenic-specific geneexpression of microtia chondrocyte induced by PRP cells


Cell number

P1 P3 P5

Cel

l num

ber

ControlPRP

0

1e + 5

2e + 5

3e + 5

4e + 5

5e + 5

(a)

Control PRP0

20

40

60

80

100 P5-SA 120573-galactosidase staining

120573-g

al p

ositi

ve ce

lls (

) lowast

(b)

Figure 2 Aging effect on cell potential ofmicrotia chondrocytes (a)Comparative profiles of cell proliferation for microtia chondrocytespassage number in P1 P3 and P5 (b) For cellular senescencesenescence-associated beta-galactosidase activity of microtia chon-drocytes during serial passage (P1 P3 and P5) in cell culturewas examined in the upper panel Quantitative representations ofstaining intensities were observed in the lower panel Representativeresults of 3 experiments were demonstrated lowast119875 lt 005

were cultured in PRP-conditional medium (containing750 pgmL TGF-1205731) or basal medium Expressions of chon-drogenic genes such as Aggrecan and type II collagen (ColII) were diminished during 21-day in vitro expansion inbasal medium (without PRP) However specific mRNA inboth Aggrecan and Col II was significantly and continuouslyexpressed on day 21 in PRP treatment group (Figure 3(a))Quantitatively an approximately 2-fold increase in Aggrecanand 3-fold increase in Col II were observed in PRP treat-ment group when compared to control group (Figure 3(b))These results indicated that chondrogenic redifferentiation ofmicrotia chondrocytes can be induced by PRP

34 Three-Dimensional Culture of Auricular Neocartilagefor Microtia Chondrocytes with Bionanomaterials To recon-struct tissue-engineered auricular cartilage three-dimen-sional (3D) cultures were performed by seeding microtiachondrocytes in bionanomaterials (collagen scaffold mixedwith or without PRP) and then chondrogenic-specific matri-ces were histologically determined (Figure 4) From grossmorphology there were no significant differences betweentissue-engineeredmicrotia cartilages cultured with and with-out PRP on days 14 and 28 (Figures 4(a)ndash4(d)) Hematoxylinand eosin (HampE) staining showed that chondrocyte-likerounded cells of auricle in 3D culture and a higher cell densitywere observed in microtia chondrocytes cultured with PRP(Figures 4(f) and 4(h)) compared to control group onday 28 (Figure 4(g)) Immunohistochemical (IHC) stainingdemonstrated that staining signal for type II collagen SOX9and Aggrecan synthesis was more intensive and extensive inmicrotia chondrocytes cultured with PRP (Figures 4(j) 4(l)4(n) 4(p) 4(r) and 4(t)) compared to their control groups(Figures 4(i) 4(k) 4(m) 4(o) 4(q) and 4(s)) The resultsindicate that PRP promotes chondrogenic differentiation ofmicrotia in 3D cultures supported by theirmRNAexpressionof monolayer cultures

4 Discussion

We have previously demonstrated that platelet-rich plasma(PRP) promoted chondrogenic redifferentiation in interver-tebral disc (IVD) and bone regeneration [10 19 20] In thisstudy we showed the role of PRP in auricle regeneration byusing microtia chondrocytes For tissue reconstruction cellsnumbers need to bemultiplied to obtain the required amountHowever it is challenging to prevent dedifferentiation duringin vitro cell expansion We and others have previouslydemonstrated that PRP could be recognized as a mitogenfor human nucleus pulposus stromal stem cells osteoblastic-like cells and fibroblast proliferation [10 24ndash26] We firstdemonstrated the important role of transforming growthfactor-1205731 (TGF-1205731) in PRP and assigned TGF-1205731 as a quantityindicator for PRP concentration [10] This is essential andaccurate to determine the dosage of PRP for controlling bothproliferation and redifferentiation in cartilage regenerationTGF-1205731-induced specific pSMAD23 pathway was activatedby PRP [10] Similar to our previous study [10 19 20] PRPincreased cell viability of microtia chondrocyte by 10-foldat TGF-1205731 750 pgmL and 1 ngmL compared with 1 FBScontrol (Figure 1) Microtia chondrocytes also showed anaggregated morphology in the presence of PRP indicatingthe upregulated chondrogenic redifferentiation phenotypesfollowed by abundant cell-to-cell contact [27]Therefore ourresult also showed that PRP could recover proliferation andreplicative potential during cellular senescence (Figure 2)

Chondrogenic-specific genes were subsequently exam-ined for chondrogenic redifferentiation potentials of microtiachondrocytes in the presence of PRP From RT-PCR resultsspecific marker gene expression such as Aggrecan and typeII collagen (Col II) was diminished during in vitro cultureIt has been reported as a particular feature referring to


PRPDay 7 Day 14 Day 21

GAPDH

Collagen type II

Aggrecan

minus + minus + minus +

(a)

Aggrecan

Days6 8 10 12 14 16 18 20 22

Expr

essio

n ra

tio

04

06

08

10

12

14

16

18

20

ControlPRP

ControlPRP

Col II

Days6 8 10 12 14 16 18 20 22

Expr

essio

n ra

tio

02

04

06

08

10

12

14

16

18

20

(b)

Figure 3 Chondrogenic-specific mRNA of microtia chondrocytes by semiquantitative RT-PCR (a) Microtia chondrocytes were culturedwith (+) and without (minus) PRP during 21 days Chondrogenic-specific mRNA including Aggrecan and type II collagen was examined and(b) their expression ratio was normalized with respective glyceraldehydes phosphate dehydrogenase (GAPDH) lowast119875 lt 005 as PRP groupcompared to 1 FBS control group at the same time point using paired 119905-test

dedifferentiation of primary chondrocytes during expansion[28] In Figure 3 specific mRNA of Aggrecan and Col IIwas significantly and continuously expressed up to 21 dayscompared with the normal control (119875 lt 005) van Oschet al reported that type II collagen in human and rabbitauricular chondrocytes was increased byTGF-1205731 and insulin-like growth factor (IGF-I) [7] Basic fibroblast growth factor(bFGF) was also demonstrated to promote in vitro and in vivogrowth of human pediatric auricular cartilage [3] Moreoverhuman ear chondrocytes expanded in medium containingTGF-1205731 fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor bb (PDGF-bb) displayed a superiorpostexpansion chondrogenic potential [29] Interestingly allthese growth factors were the core constituents of PRP Takentogether PRP stimulates microtia chondrocyte proliferationwithout loss of chondrogenic-specificmarkers in themicrotiachondrocyte expansion cultures

For further clinical applications of auricle surgical recon-struction PRP could be viewed as a good natural source ofgrowth factor cocktails In addition the cell adhesion proteinincluding fibrin fibronectin and vitronectin comprised inPRP also provides 3-dimensional (3D) architecture for fixingseeded chondrocytes [30] Previously we have seeded chon-drocytes into collagen scaffold to reconstitute human engi-neered nucleus pulposus tissue and human articular carti-lage [8ndash10] PRP was subsequently employed in the auric-ular reconstruction system containing microtia chondro-cytecollagen scaffold In Figure 4 higher cell density wasonly shown in 4-week PRP treated groupsNumerous lacunaesurrounded cells were also observed by HampE staining Inten-sive signals of type II collagen SOX9 and Aggrecan stainingby immunohistochemistry also indicated that PRP stimulatesthe major chondrogenic matrix synthesized by microtiachondrocytes in 3D environment (Figure 4(l)) Both PRP


PRPControl PRPControl

Day 14 Day 28

HampE

(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

(m) (n) (o) (p)

(q) (r) (s) (t)

Gross morphology

IHC of Col II

SOX9

Aggrecan

200120583m200120583m200120583m200120583m

200120583m200120583m200120583m200120583m

Figure 4 Engineered auricle tissue by microtia chondrocytes reconstructed in 3D collagen scaffold with and without PRP Neocartilage wasanalyzed by their gross morphology ((a)ndash(d)) HampE stain ((e)ndash(h)) (200x) and IHC of Col II ((i)ndash(l)) SOX9 ((m)ndash(p)) and Aggrecan stain((q)ndash(t)) (200x rarr 400x) on days 14 and 28

and collagen framework rendered essential compositions forauricular neocartilage formation

5 Conclusion

In this study we demonstrated that PRP increased the poten-tial of microtia chondrocytes to effectively regenerate auricletissue Our findings not only provide a more advanced appli-cation but also contribute to a new cellgrowth factor-basedtherapeutic strategy for clinical microtia reconstruction

Conflict of Interests

Wei-Hong Chen Hen-Yu Liu Ching-Yu Tsai Chia-che WuHong-Jian Wei Alice Liu Ming-Tang Lai Chiung-Fang

Huang andWin-PingDeng declare that they have no conflictof interests with the mentioned trademarks or companies

Authorsrsquo Contribution

Wei-Hong Chen and Hen-Yu Liu contributed equally to thiswork Chiung-Fang Huang and Win-Ping Deng contributedequally to this work

References

[1] S Suutarla J Rautio A Ritvanen S Ala-Mello J Jero and TKlockars ldquoMicrotia in Finland comparison of characteristicsin different populationsrdquo International Journal of PediatricOtorhinolaryngology vol 71 no 8 pp 1211ndash1217 2007


[2] F Alasti and G van Camp ldquoGenetics of microtia and associatedsyndromesrdquo Journal of Medical Genetics vol 46 no 6 pp 361ndash369 2009

[3] C A Arevalo-Silva Y Cao M Vacanti Y Weng C A Vacantiand R D Eavey ldquoInfluence of growth factors on tissue-engi-neered pediatric elastic cartilagerdquo Archives of OtolaryngologymdashHead and Neck Surgery vol 126 no 10 pp 1234ndash1238 2000

[4] Y Cao J P Vacanti K T Paige J Upton and C A VacantildquoTransplantation of chondrocytes utilizing a polymer-cell con-struct to produce tissue-engineered cartilage in the shape of ahuman earrdquo Plastic and Reconstructive Surgery vol 100 no 2pp 297ndash304 1997

[5] D E Johns and K A Athanasiou ldquoGrowth factor effects oncostal chondrocytes for tissue engineering fibrocartilagerdquo Celland Tissue Research vol 333 no 3 pp 439ndash447 2008

[6] V Kizkner and A Barak ldquoFramework changes using costal car-tilage formicrotia reconstructionrdquoArchives of OtolaryngologymdashHead and Neck Surgery vol 134 no 7 pp 768ndash770 2008

[7] G J V M van Osch S W van der Veen and H L Verwoerd-Verhoef ldquoIn vitro redifferentiation of culture-expanded rabbitand human auricular chondrocytes for cartilage reconstruc-tionrdquo Plastic and Reconstructive Surgery vol 107 no 2 pp 433ndash440 2001

[8] W-H Chen M-T Lai A T H Wu et al ldquoIn vitro stage-specific chondrogenesis of mesenchymal stem cells committedto chondrocytesrdquo Arthritis and Rheumatism vol 60 no 2 pp450ndash459 2009

[9] W-H Chen W-F Lai W-P Deng et al ldquoTissue engineeredcartilage using human articular chondrocytes immortalizedby HPV-16 E6 and E7 genesrdquo Journal of Biomedical MaterialsResearch Part A vol 76 no 3 pp 512ndash520 2006

[10] W-H Chen W-C Lo J-J Lee et al ldquoTissue-engineeredintervertebral disc and chondrogenesis using human nucleuspulposus regulated through TGF-1205731 in platelet-rich plasmardquoJournal of Cellular Physiology vol 209 no 3 pp 744ndash754 2006

[11] T de Chalain J H Phillips and A Hinek ldquoBioengineering ofelastic cartilage with aggregated porcine and human auricularchondrocytes and hydrogels containing alginate collagen and120581-elastinrdquo Journal of Biomedical Materials Research vol 44 no3 pp 280ndash288 1999

[12] H Yamaoka H Asato T Ogasawara et al ldquoCartilage tissueengineering using human auricular chondrocytes embedded indifferent hydrogel materialsrdquo Journal of Biomedical MaterialsResearch Part A vol 78 no 1 pp 1ndash11 2006

[13] S H Kamil M P Vacanti C A Vacanti and R D EaveyldquoMicrotia chondrocytes as a donor source for tissue-engineeredcartilagerdquo Laryngoscope vol 114 no 12 pp 2187ndash2190 2004

[14] E W Mandl H Jahr J L M Koevoet et al ldquoFibroblast growthfactor-2 in serum-free medium is a potent mitogen and reducesdedifferentiation of human ear chondrocytes in monolayerculturerdquoMatrix Biology vol 23 no 4 pp 231ndash241 2004

[15] P B Saadeh B Brent B J Mehrara et al ldquoHuman cartilageengineering chondrocyte extraction proliferation and charac-terization for construct developmentrdquoAnnals of Plastic Surgeryvol 42 no 5 pp 509ndash513 1999

[16] C A Arevalo-Silva Y Cao Y Weng et al ldquoThe effect offibroblast growth factor and transforming growth factor-betaon porcine chondrocytes and tissue-engineered autologouselastic cartilagerdquo Tissue Engineering vol 7 no 1 pp 81ndash88 2001

[17] S Mehta and J T Watson ldquoPlatelet rich concentrate basic sci-ence and current clinical applicationsrdquo Journal of OrthopaedicTrauma vol 22 no 6 pp 433ndash438 2008

[18] S Sampson M Gerhardt and B Mandelbaum ldquoPlatelet richplasma injection grafts for musculoskeletal injuries a reviewrdquoCurrent Reviews in Musculoskeletal Medicine vol 1 no 3-4 pp165ndash174 2008

[19] W H Chen H Y Liu W C Lo et al ldquoIntervertebral discregeneration in an ex vivo culture system using mesenchymalstem cells and platelet-rich plasmardquo Biomaterials vol 30 no29 pp 5523ndash5533 2009

[20] W-C Lo J-F Chiou J G Gelovani et al ldquoTransplantation ofembryonic fibroblasts treated with platelet-rich plasma inducesosteogenesis in SAMP8micemonitored bymolecular imagingrdquoJournal of Nuclear Medicine vol 50 no 5 pp 765ndash773 2009

[21] W-F Lai J-R Tang and C-T Chen ldquoFibrication of a cartilageimplantrdquo US Patent 20030152556 A1 2003

[22] R Landesberg M Roy and R S Glickman ldquoQuantificationof growth factor levels using a simplified method of platelet-rich plasma gel preparationrdquo Journal of Oral and MaxillofacialSurgery vol 58 no 3 pp 297ndash300 2000

[23] G Weibrich W K G Kleis G Hafner and W E HitzlerldquoGrowth factor levels in platelet-rich plasma and correlationswith donor age sex and platelet countrdquo Journal of Cranio-Max-illofacial Surgery vol 30 no 2 pp 97ndash102 2002

[24] P Arpornmaeklong M Kochel R Depprich N R Kublerand K K Wurzler ldquoInfluence of platelet-rich plasma (PRP) onosteogenic differentiation of rat bone marrow stromal cells Anin vitro studyrdquo International Journal of Oral and MaxillofacialSurgery vol 33 no 1 pp 60ndash70 2004

[25] T Kawase K Okuda L F Wolff and H Yoshie ldquoPlatelet-rich plasma-derived fibrin clot formation stimulates collagensynthesis in periodontal ligament and osteoblastic cells in vitrordquoJournal of Periodontology vol 74 no 6 pp 858ndash864 2003

[26] Y Liu A Kalen O Risto and O Wahlstrom ldquoFibroblastproliferation due to exposure to a platelet concentrate in vitrois pH dependentrdquo Wound Repair and Regeneration vol 10 no5 pp 336ndash340 2002

[27] S T Yoon K S Kim J Li et al ldquoThe effect of bone morpho-genetic protein-2 on rat intervertebral disc cells in vitrordquo Spinevol 28 no 16 pp 1773ndash1780 2003

[28] K von der Mark V Gauss H von der Mark and P MuellerldquoRelationship between cell shape and type of collagen synthe-sised as chondrocytes lose their cartilage phenotype in culturerdquoNature vol 267 no 5611 pp 531ndash532 1977

[29] A G Tay J Farhadi R Suetterlin G Pierer M Heberer and IMartin ldquoCell yield proliferation and postexpansion differenti-ation capacity of human ear nasal and rib chondrocytesrdquoTissueEngineering vol 10 no 5-6 pp 762ndash770 2004

[30] R E Marx ldquoPlatelet-rich plasma evidence to support its userdquoJournal of Oral andMaxillofacial Surgery vol 62 no 4 pp 489ndash496 2004

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Journal ofNanomaterials


auricular cartilage was successfully created in a xenograftnude mouse model [4] However the source of human auric-ular chondrocytes from normal tissue is limited Geneticandmorphologic differences of elastic chondrocytes betweennormal auricle and microtia have not been clarified Tissue-engineered cartilage formation bymicrotia chondrocytes alsoshowed similar elastic features of normal auricular chondro-cytes [13] Thus cells isolated from autologous cartilage aredefinitely ideal for repair that also prevents immune rejectionand transmittable disease To harvest sufficient cell numberswithout losing chondrogenic phenotypes is important forrepair of cartilaginous defects Moreover microtia obtainedfrom patients has limitations such as loss of cell potentialwith high passage number In cell culture the doubling timeand cellular senescence of primary microtia chondrocytescan increase and be insufficient for repeated or researchuse

Hence culture medium supplemented with growth fac-tors or cytokines has been developed Human ear chondro-cytes cultured in fibroblast growth factor 2 (FGF2) prolifer-ated higher than serum containing medium and expresseda more differentiated phenotype [14] The addition of TGF-1205731 insulin-like growth factor (IGF) and FGF-2 was alsoreported to induce chondrogenic redifferentiation in scaffoldreconstruction resembling native auricular cartilage [7 1516] Recently platelet-released growth factors comprised inplatelet-rich plasma (PRP) were demonstrated to encouragewound healing in musculoskeletal injuries [17 18] PRP stim-ulated human nucleus pulposus (NP) regeneration throughTGF-1205731 has been demonstrated in our previous study [10]We have also examined the fact that PRP promotes ex vivoand in vivo NP regeneration [19] In addition the role ofPRP combined embryonic fibroblast in the treatment ofosteoporosis was also confirmed [20] We have evaluated theefficacy of PRP on microtia chondrocytes to regenerate tis-sue-engineered auricular cartilage An advanced cellgrowthfactor-based therapeutic strategy was developed for futurehead and neck surgical procedure

2 Materials and Methods

21 Chondrocyte Isolation and Culture The auricular carti-lage fragments were harvested from microtia patients Thesevolunteers provided informed consent for the use of their car-tilage as required by the Ethics Committee of Taipei MedicalUniversity Municipal Wan Fang Hospital Microtia cartilagewas minced into 1mm3 pieces and washed three timesin phosphate-buffered saline (PBS) After being digestedwith 03 type I collagenase (sigma) containing penicillin(100 unitsmL GIBCO-BRL) and streptomycin (01mgmLGIBCOBRL) for 15 hours at 37∘C the suspended cells werepelleted and then resuspended in DMEMF12 (Dulbeccorsquosmodified Eaglersquos mediumF12 Ham GIBCO-BRL GrandIsland NY) containing 10 (vv) fetal bovine serum (FBSGIBCO-BRL) in an incubator at 37∘C with 5 CO

2 Culture

medium was changed every 3 days before subsequent exper-iments In addition we counted the cell numbers of primarymicrotia chondrocytes at different passage number (P1 P2

and P3) in cultures with or without PRP treatment to deter-mine the cell proliferation

22 PRP Preparation and TGF-1205731 Concentration Evalua-tion PRP was extracted from total blood and activated aspreviously described [10 20] Subsequently the TGF-1205731 wasused as indicator and quantitatively analyzed using a Quan-tikine enzyme-linked immunosorbent assay (ELISA) kit(DB100 RampD Diagnostics Wiesbaden Germany) in orderto confirm the consistency of PRP for in vitro utilizationand to determine the most appropriate concentration forstudy We prepared a dilution series of TGF-1205731 standards(890207) in 100 120583L volumes in 96-well microtiter platescoated with TGF-1205731-receptor II Conversion of TGF-1205731 toits active form was necessary to estimate total TGF-1205731 sincea large proportion of TGF-1205731 is often present in a latentform The 01mL PRP solution was then mixed with 01mL25N acetic acid10M urea incubated at room temperaturefor 10min and neutralized by an addition of 01mL of 27NNaOH1M HEPES (N-[2-hydroxyethyl] piperazine-N0-[2-ethanesulfonic acid] Sigma) (H-7523)

23 MTT Assay The analytic reagent tetrazolium salt MTT[(3-(45-dimethylthiazol-2-yl)-2 5-diphenyl tetrazoliumbro-mide) Roche Mannheim Germany] assay was used fordetermining cell viability The microtia chondrocytes wereseeded into 96-well plate at a density of 2 times 104 cellsmL andtreated with or without PRP while the experimental controlswere cultured in 1 FBS containing DMEMF-12 The MTTreagent was added into each well on days 1 3 5 7 and 9 Atthe time points OD values (OD 595ndashOD 690) were analyzed4 h after the MTT reaction using Multiskan PC (ThermoLabsystem Franklin MA) and cell survival curves were thenplotted against time

24 Gene Expression Analysis Expressions of type II collagen(Col II) and Aggrecan mRNA of PRP and control groupswere determined by semiquantitative reverse transcriptionpolymerase chain reaction (RT-PCR) of 7- 14- and 21-daymonolayer cultures Total RNA was isolated using TRIzolreagent (Invitrogen Life Technologies Carlsbad CA USA)and used for first stand cDNA synthesis RNA was reverse-transcribed utilizing SuperScript III (Invitrogen Life Tech-nologies) and anOligo d(T)12ndash18 primerThe reactionmixturecontained 10mM dNTP mix 10X RT buffer 25mM MgCl

2

01M DTT RNase inhibitor and RNase H in a total vol-ume of 20120583L PCR was performed using primers for typeII collagen Aggrecan and gyceraldehde-3-phosphate dehy-drogenate (GAPDH) The primer sequences are listed asfollows Col II-forward-51015840-GAA CAT CAC CTA CCA CTGCAAG-31015840 reverse-51015840-GCAGAG TCC TAGAGTGAC TGAG-31015840 Aggrecan-forward 51015840-GCC TTG AGC AGT TCA CCTTC-31015840 reverse-51015840-CTC TTC TAC GGG GAC AGC AG-31015840PCR amplification in a final volume of 50 120583L containing25mMdNTP 25mMMgCl

2 specific primers and TaqDNA

polymerase (Invitrogen Life Technologies) The PCR wasinitiated for 5min at 95∘C and cycled 35 times at 94∘C for1min 72∘C for 1min and 72∘C for 5min



2 Medium




3 Results


Time (days)0 2 4 6 8 10

OD

val

ue

00

02

04

06

08

10


lowast

lowast

lowast

lowast

(a)

Control PRP

200120583m 200120583m

(b)





Cell number

P1 P3 P5

Cel

l num

ber

ControlPRP

0

1e + 5

2e + 5

3e + 5

4e + 5

5e + 5

(a)

Control PRP0

20

40

60

80


120573-g

al p

ositi

ve ce

lls (

) lowast

(b)




4 Discussion





GAPDH

Collagen type II

Aggrecan


(a)

Aggrecan

Days6 8 10 12 14 16 18 20 22

Expr

essio

n ra

tio

04

06

08

10

12

14

16

18

20

ControlPRP

ControlPRP

Col II

Days6 8 10 12 14 16 18 20 22

Expr

essio

n ra

tio

02

04

06

08

10

12

14

16

18

20

(b)






Day 14 Day 28

HampE

(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

(m) (n) (o) (p)

(q) (r) (s) (t)

Gross morphology

IHC of Col II

SOX9

Aggrecan

200120583m200120583m200120583m200120583m

200120583m200120583m200120583m200120583m



5 Conclusion







References







































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





2 Medium




3 Results


Time (days)0 2 4 6 8 10

OD

val

ue

00

02

04

06

08

10


lowast

lowast

lowast

lowast

(a)

Control PRP

200120583m 200120583m

(b)





Cell number

P1 P3 P5

Cel

l num

ber

ControlPRP

0

1e + 5

2e + 5

3e + 5

4e + 5

5e + 5

(a)

Control PRP0

20

40

60

80


120573-g

al p

ositi

ve ce

lls (

) lowast

(b)




4 Discussion





GAPDH

Collagen type II

Aggrecan


(a)

Aggrecan

Days6 8 10 12 14 16 18 20 22

Expr

essio

n ra

tio

04

06

08

10

12

14

16

18

20

ControlPRP

ControlPRP

Col II

Days6 8 10 12 14 16 18 20 22

Expr

essio

n ra

tio

02

04

06

08

10

12

14

16

18

20

(b)






Day 14 Day 28

HampE

(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

(m) (n) (o) (p)

(q) (r) (s) (t)

Gross morphology

IHC of Col II

SOX9

Aggrecan

200120583m200120583m200120583m200120583m

200120583m200120583m200120583m200120583m



5 Conclusion







References







































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




Cell number

P1 P3 P5

Cel

l num

ber

ControlPRP

0

1e + 5

2e + 5

3e + 5

4e + 5

5e + 5

(a)

Control PRP0

20

40

60

80


120573-g

al p

ositi

ve ce

lls (

) lowast

(b)




4 Discussion





GAPDH

Collagen type II

Aggrecan


(a)

Aggrecan

Days6 8 10 12 14 16 18 20 22

Expr

essio

n ra

tio

04

06

08

10

12

14

16

18

20

ControlPRP

ControlPRP

Col II

Days6 8 10 12 14 16 18 20 22

Expr

essio

n ra

tio

02

04

06

08

10

12

14

16

18

20

(b)






Day 14 Day 28

HampE

(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

(m) (n) (o) (p)

(q) (r) (s) (t)

Gross morphology

IHC of Col II

SOX9

Aggrecan

200120583m200120583m200120583m200120583m

200120583m200120583m200120583m200120583m



5 Conclusion







References







































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





GAPDH

Collagen type II

Aggrecan


(a)

Aggrecan

Days6 8 10 12 14 16 18 20 22

Expr

essio

n ra

tio

04

06

08

10

12

14

16

18

20

ControlPRP

ControlPRP

Col II

Days6 8 10 12 14 16 18 20 22

Expr

essio

n ra

tio

02

04

06

08

10

12

14

16

18

20

(b)






Day 14 Day 28

HampE

(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

(m) (n) (o) (p)

(q) (r) (s) (t)

Gross morphology

IHC of Col II

SOX9

Aggrecan

200120583m200120583m200120583m200120583m

200120583m200120583m200120583m200120583m



5 Conclusion







References







































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





Day 14 Day 28

HampE

(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

(m) (n) (o) (p)

(q) (r) (s) (t)

Gross morphology

IHC of Col II

SOX9

Aggrecan

200120583m200120583m200120583m200120583m

200120583m200120583m200120583m200120583m



5 Conclusion







References







































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials








































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



research article the potential use of platelet-rich plasma...

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