research article a controlled release codelivery...

Research ArticleA Controlled Release Codelivery System of MSCsEncapsulated in DextranGelatin Hydrogel with TGF-1205733-LoadedNanoparticles for Nucleus Pulposus Regeneration

Yibo Gan1 Sukai Li1 Pei Li1 Yuan Xu2 Liyuan Wang1 Chen Zhao1 Bin Ouyang1

Bing Tu1 Chengmin Zhang1 Lei Luo1 Xiangdong Luo3 Xiumei Mo4 and Qiang Zhou1

1National amp Regional United Engineering Laboratory of Tissue Engineering Department of Orthopedics Southwest HospitalThird Military Medical University 30 No Gao Tan Yan Street Shapingba District Chongqing 400038 China2Department of Orthopaedics Xinqiao Hospital Third Military Medical University Chongqing 400038 China3Institution of Burn Research Southwest Hospital Third Military Medical University Chongqing 400038 China4College of Chemistry and Chemical Engineering and Biological Engineering Donghua University Shanghai 201620 China

Correspondence should be addressed to Xiumei Mo xmmdhueducn and Qiang Zhou zq tlh163com

Received 14 June 2016 Accepted 22 August 2016

Academic Editor Kai Tao

Copyright copy 2016 Yibo Gan et alThis is an open access article distributed under theCreative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Mesenchymal stem cell- (MSC-) based therapy is regarded as a potential tissue engineering strategy to achieve nucleus pulposus(NP) regeneration for the treatment of intervertebral disc degeneration (IDD) However it is still a challenge to induce MSCdifferentiation in NP-like cells whenMSCs are implanted into the NPThe purpose of this study was to construct poly(DL-lactide-co-glycolide) (PLGA) nanoparticles as carriers for TGF-1205733 controlled release and establish a codelivery system of a dextrangelatinhydrogel with the nanoparticles for long-term processing of discogenesis differentiation TGF-1205733-loaded PLGA nanoparticles wereprepared by the double-emulsion solvent evaporationmethod and seeded uniformly into the hydrogelMorphological observationsan assessment of the release kinetics of TGF-1205733 a cytotoxic assay a cell proliferation test a biochemical content assay qRT-PCRand immunohistological analyses of the codelivery systemwere conducted in the studyThe results showed that the TGF-1205733-loadednanoparticles could release TGF-1205733 gradually The codelivery system exhibited favorable cytocompatibility and the TGF-1205733 thatwas released could induce MSCs to NP-like cells while promoting ECM-related biosynthesis These results suggest this codeliverysystem may be employed as a promising carrier for discogenesis of MSCs in situ

1 Introduction

Intervertebral disc degeneration (IDD) is a common diseasethat causes lower back pain neck pain and even disabilities[1] IDD affects patientsrsquo quality of life and leads to a largeeconomic burden Currently the common treatments of IDDconsist of bed rest exercise physical therapy and surgery [2]However all of these treatments only relieve the symptomswithout targeting the etiology of IDD Thus there is noapproved therapy for IDD and there is an urgent need fortreatments that target the etiology of IDD to repair theintervertebral disc (IVD)

The IVD is a complex joint that consists of three parts thecentral hydrated nucleus pulposus (NP) the outer lamellar

annulus fibrosus (AF) and the cartilage endplates (CEP)connecting the adjacent vertebras IDD is believed to orig-inate in NP and improper load on IVD leads to NP dehy-dration and subsequently weakens IVDrsquos capacity to absorbcompression [3] Thus rehydration of the degenerative NPmay be the key to treating IDD Previous studies haveindicated that the bioactivity and amounts of NP cells (NPCs)are decreased significantly in IDD resulting in a lowersecretion of extracellular matrix- (ECM-) related proteinsincluding aggrecan and type II collagen [4] Specifically themain function of aggrecan is to absorb water moleculesinto the NP contributing to the compression absorptionfunction of the IVDTherefore increasing NPC quantity andbioactivitymay promote the restoration ofNPhydrationDue

Hindawi Publishing CorporationStem Cells InternationalVolume 2016 Article ID 9042019 14 pageshttpdxdoiorg10115520169042019

2 Stem Cells International

to the poor self-regeneration capability of NPCs cell-basedtherapy could be a promising strategy for the treatment ofIDD [5] Of all of the cell-based approaches implantation ofmesenchymal stem cells (MSCs) encapsulated in hydrogelshas received the greatest attention [6]

An advantage of the MSC-based strategy is that autol-ogous cells are easily harvested and expanded rapidly invitro There is greater potential to regenerate the NP withlarger amounts of implanted cells Another advantage is thatMSCs are able to differentiate to an NP-like phenotype withsome proper biochemical directions such as transforminggrowth factor 120573 (TGF-120573) [7] and differentiation factor 5(GDF5) [8] The TGF-120573 family is one of the exogenousgrowth factors widely used to induce MSCs to discogenesisdifferentiation Generally high-density MSCs in a three-dimensional (3D) carrier were cultured with a serum-freeinduction medium with TGF-120573 isoforms in vitro for chon-drogenic differentiation especially with TGF-1205733 [9] andsubsequently the hybrid was implanted for NP regeneration[10] Hydrogels are considered proper carriers because oftheir similar rheological property to the NP and favorablecytocompatibility [11] However this strategy is difficult toapply for the treatment of early stage IDD Due to thestructural integrity of AF in early stage IDD IVD fenestrationshould be made before implanting a bulk cell-hydrogelhybrid which could possibly accelerate IDD [12] Thereforea novel alternative may involve using an injectable hydrogelas a carrier of MSCs The injectable hydrogels are in theliquid state before implantation and harden after injectionin vivo The process allows minimal invasion through theAF It is beneficial for avoiding extrusion after implantationwhile adapting to the irregular shape of the defect in the NPcavity [13]The precursor solution can also be combined withgrowth factors and cells to reverse disc degeneration [14]However it is still a challenge to maintain long-term MSCdifferentiation in situ [15] The half-life of TGF-1205733 is onlytens of minutes in vivo and it can be easily diluted in bodyfluid diffusion If growth factors were combined directly withthe hydrogel they would soon be degraded or eliminatedTherefore it is difficult to affect TGF-1205733 by physical mixing[15] Thus it is particularly important to construct a suitabledelivery system for the sustained release of growth factors

The rapid development of the FDA-approved poly(DL-lactide-co-glycolide) (PLGA) may be useful for long-termdifferentiation [16] PLGA is a copolymer of PLA and PGAwith favorable biocompatibility favorable biodegradabilityand low immunogenicity Recently PLGA was applied todrug delivery and tissue engineering applications among themost attractive polymeric candidates [17]The preparation ofnanosized particles has increasedwith advances in traditionalmethods because nanoparticles could be delivered to variousbody parts with a wide range of drugs for sustained release[18] PLGA nanoparticles (PLGANPs) can be targeted to aspecific tissue and release the drug locally by endocytosis[19] Such PLGANPs have been widely used to develop anti-cancer drug delivery system such as paclitaxel doxorubicin5-fluorouracil and dexamethasone which were effectivelyformulated via PLGANPs [20] It has also been incorporatedin tissue engineering scaffold for control over spatial and

temporal release of growth factors For instance glial cell-line derived neurotrophic factor (GDNF) was encapsulatedin PLGANPs when implanted into the spinal cord to inducean increase in neuronal survival reported by Wang et al [21]If PLGANPs are used as carriers for a gradual release ofgrowth factors into the IVD local maintenance may lead tosufficient concentrations of TGF-1205733 in the long-term processof NP regenerationThe injectable hydrogel coupled with thesustained release nanoparticles could meet the clinical needsof an ideal MSC-based minimally invasive implantation

In this study TGF-1205733-loaded PLGANPs were preparedand seeded into a dextrangelatin hydrogel to constructa novel codelivery system for inducing long-term MSCdifferentiation in situ The overall objective of this studywas to investigate the potential of the codelivery systemfor NP regeneration To achieve this we first evaluated therelease kinetics of the TGF-1205733 and confirmed that the TGF-1205733-loaded PLGANPs could release enough TGF-1205733 withinan appropriate discogenesis time frame Second we investi-gated the cytocompatibility of the TGF-1205733-loaded PLGANPsFinally we investigated whether the codelivery system couldsupport cell proliferation discogenesis differentiation andfunctional biosynthesis of MSCs

2 Materials and Methods

21 Preparation of the TGF-1205733-PLGANPs The TGF-1205733-loaded PLGANPs were prepared using the double-emulsionsolvent evaporation method (WOW) as previouslydescribed [22] In order to reach the concentration ofTGF-1205733 as previously recommended for the discogenesisdifferentiation of MSCs the target release amount of theTGF-1205733 was 10 ng in 1mL culture medium [23] Briefly 10 120583gof the TGF-1205733 (PeproTech USA) was dissolved in 01mLof distilled water (W

1) Subsequently the PLGA 50 50

(200mg 24ndash38 kD Sigma-Aldrich USA) was dissolvedin 1mL dichloromethane (O Sigma-Aldrich) Later theTGF-1205733 solution was added dropwise to the PLGA solutionThen the solution was treated ultrasonically in an ice bathThis primary emulsion was added dropwise to 6mL of1 polyvinyl alcohol (PVA Sigma-Aldrich USA) aqueoussolution (W

2) and treated ultrasonically again The obtained

W1OW

2emulsion was transferred into 20mL of the 03

PVA aqueous solution and stirred for 4 hours with a propellerstirrer Subsequently the solid particles were collected aftercentrifugation Finally the obtained precipitates were washedfor three times with deionized water The nanoparticles wereharvested by lyophilization and stored at 20∘C in desiccativecondition

22 Morphological Observations The morphology of theTGF-1205733-loaded PLGANPs was examined by the scanningelectron microscopy (SEM S3400N II Hitachi Japan) andthe transmission electron microscope (TEM TECNAI 10Philips USA) Briefly 20120583L of the PLGANPs suspension wasdropped on the mica followed by being air-dried overnightThen morphology of the TGF-1205733-loaded PLGANPs wasexamined by the SEM after spraying For the TEM examina-tion the PLGANPs suspension was dropped on the copper

Stem Cells International 3

mesh with the film Then the excess liquid on copper meshedge was absorbed with filter paper Next drop phospho-tungstic acid negative stain solution for 2 minutes followedby gently washing for three times The nanoparticles wereexamined by the TEM after air drying The particle size wasdetermined by laser particle size analyzer (Zetasizer V702Malvern UK) (119899 = 3)

23 Encapsulation Efficiency (EE) The EE in the PLGANPswas estimated by a ldquotwo-steprdquo extractionmethod [24] Brieflyabout 5mg of the lyophilized TGF-1205733-PLGA nanoparticleswas added to 1mL of acetonitrile The supernatant wasdiscarded and the precipitation was pelleted down aftercentrifugation The pellet was dissolved in the PBS and thesamples (in duplicate) were centrifuged again The super-natant was preserved after centrifugation and its residuewas dissolved in NaOH The protein amount in aqueoussolution and NaOH (the actual theoretical loading amountof TGF-1205733 encapsulated in the 5mg of PLGANPs 119872actual)was determined by the ELISA kit (Thermo Fisher ScientificUSA) (119899 = 3) The EE of the TGF-1205733 in the PLGANPs wascalculated as follows

EE =119872actual119872theoretical

times 100 (1)

where 119872theoretical is the theoretical loading amount of theTGF-1205733 encapsulated in 5mg PLGANPs

24 Release Kinetics of the TGF-1205733 The release kinetics ofthe TGF-1205733 were determined in vitro as previously described[25] Briefly the TGF-1205733-loaded nanoparticles (50mg) weresuspended with 3mL of the PBS shaking at 100 rpm at 37∘CThe supernatant was collected and stored at minus20∘C on thepredetermined time points (days 1 3 7 10 14 21 and 28) aftercentrifugation Then an equal amount of the fresh PBS wasadded and incubated as previously described The releasedquantity of the TGF-1205733 was determined by the ELISA kitThe nanoparticlesrsquo release kinetic curve was obtained usingthe accumulated release percentage (119899 = 3)

25 Isolation and Culture of Mouse MSCs The usage ofthe animal followed the guidelines of Local Animal EthicsCommittee (SYXK (YU) 2012-0012) Bone marrow-derivedMSCs were collected from 6-week-old Balbc mouse as in aprevious study [26] Briefly the mouse was killed by cervicaldislocation Bone marrow was harvested by flushing thefemurs and tibiae with the complete culture medium [Dul-beccorsquos modified Eaglersquos medium (DMEM HyClone USA)supplemented with 10 fetal bovine serum (FBS GibcoUSA) and 1 penicillinstreptomycin (Gibco USA)] MSCswere isolated frommarrow cells followed by density-gradientcentrifugation (1077 gcm3) Remove the nonadherent cellsafter 3 days and collect the adherent cell by trypsinization(005 trypsin-EDTA Gibco USA) when reaching 90confluence The medium was changed every 2 days

26 LiveDead Assay Four groups were chosen for thecytotoxicity assay (1) TGF-1205733-PLGANPs supplemented

with the TGF-1205733-loaded PLGANPs at the concentration of1000 120583gmL in the culture medium (2) TGF-1205733 group sup-plemented with the TGF-1205733 at the concentration of 10 ngmLin the culture medium (3) PLGANPs group supplementedwith the blank PLGANPs at the concentration of 1000120583gmLin the culture medium (4) control group no supplement inthe culture medium Except for the TGF-1205733 group the MSCsor MSCs-seeded hybrids were cultured with the completeculture medium Cells cultured with 01 Triton (BeyotimeBiotechnology China) were chosen as negative control Inorder to evaluate the cytotoxicity of the PLGANPs MSCswere seeded on the 24-well plates at the density of 1 times104well After 1 day the culture medium was removedand 1mL culture medium containing the PLGANPs at theconcentration of 1000 120583gmL was added to the wells after thecell attachment The cytotoxicity was evaluated after 7 daysrsquococulture in vitro by a LIVEDEAD ViabilityCytotoxicityAssay Kit (Invitrogen USA) according to the manufac-turerrsquos instructions The stained cells were observed with afluorescent microscope (LSM 510 Zeiss Germany) Livingcells percentage was calculated by Image J software (WayneRasband National Institute of Health USA) Three imagesfrom three samples were evaluated for each group

27 Establishment of the Codelivery System The dextran andgelatin were obtained from Sigma-Aldrich The oxidativedextran (Oxi-Dex) and the amino gelatin (amino-Gel) wereprepared as our previous study [27] The resulting solutionwas dialyzed (MWCO 7000 JinKeHongDa China) againstdistilledwater for 3 daysThen it was lyophilized to obtain theproducts Then the dextran and the gelatin were dissolved inthe PBS to reach the concentration of 20The encapsulationand the sustained release process were exhibited in Figure 1Briefly the TGF-1205733-loaded PLGANPs or the blank PLGANPswere suspended by the Oxi-Dex solution Next the amino-Gel solution was added to the mixture at a mass ratioof dextran versus gelatin to 3 5 at the concentration of20 The final solution was transferred to a mold rapidlyfor gelation for 5min The density of PLGANPs in thehydrogel was 1000120583gmL which was set to reach the TGF-1205733 concentration of 10 ngmL in the system according tothe release kinetics 1mL of culture medium was addedand changed every day for the following tests For the cellsencapsulation the MSCs (P3ndashP5) were premixed with theprepolymer solutions at a density of 5 times 106 cellssdotmLminus1 Andthe hybrid was shaped to a cylinder (Φ = 10mm height= 6mm) and cultured in our bioreactor with a circulatingsystem The hybrids were divided into four groups (1) TGF-1205733-PLGANPs the MSCs encapsulated in the hydrogel withthe TGF-1205733-loaded PLGANPs at the density of 1000120583gmLcultured with the complete medium (2) TGF-1205733 groupthe MSCs encapsulated in the hydrogel with no supple-ment cultured with the medium containing TGF-1205733 at theconcentration of 10 ngmL (3) PLGANPs group the MSCsencapsulated in the hydrogel with the blank PLGANPs at thedensity of 1000120583gmL cultured with the complete medium(4) control group the MSCs encapsulated in the hydrogelwith no supplement cultured with the complete medium


WOW

Oxi-Dex Amino-Gel

MSCs

PLGA nanoparticles

Nanoparticlesencapsulation

Schiff-basedcrosslink

CH

NOO

OO

yx

HHO

Discogenesis

Cell surface receptor

TGF-1205733

PLGA 50 50

Figure 1 Schematic diagram of establishing the codelivery system of MSCs encapsulated in the dextrangelatin hydrogel with the TGF-1205733-loaded nanoparticles After the TGF-1205733-loaded PLGANPs were prepared using the WOW method the MSCs and the TGF-1205733-loadedPLGANPs were incubated with the Oxi-Dex and amino-Gel solution The codelivery system was formed by the Schiff-based reactionbetween the Oxi-Dex and amino-GelThe TGF-1205733 could be released gradually with degradation of the PLGANPs for long-term discogenesisdifferentiation

28 Cell Proliferation in 3D To detect the cell proliferationof MSCs in the codelivery system the hydrogels were trans-ferred to a 24-well plate at the time point of days 7 14 and28 Then fresh medium containing 10 CCK-8 was addedfor 3 h incubationThe absorbance of incubation solutionwasmeasured at 450 nm using a microplate reader (Model 550Bio-Rad USA) (119899 = 4)

29 Biochemistry Assays For testing the cell viability andECM deposits the MSCs-seeded hybrids were lyophilized atdays 7 14 and 28 DNA and glycosaminoglycan (GAG) con-tent were analyzed as described before [28]The hydroxypro-line (HYP) content was quantified according to a previousmethod [29] (119899 = 3)

210 Real-Time PCRAssay After 28 days of postseeding cell-seeded hybrids were treated with TRIzol (Geno TechnologyInc USA) RNA was extracted according to the manufac-turerrsquos instruction cDNA was generated by applying cDNAreverse transcription kit (Life TechnologiesUSA) anddilutedto 5 ng120583L Gene expression was analyzed by quantitativereal-time PCR (Applied Biosystems 7500 Thermo FisherScientific USA) Data were calculated by the 2minusΔΔCt methodThe primers used in this study are shown in Table 1

211 Immunohistochemical Analysis For histological analysisof cell morphology and ECM secretion hybrids from each

time point (7 14 and 28 days) were moved into paraffinThen in order to visualize the ECM deposition of thehybrids the sections were immunostained by collagen IIand aggrecan as in reported method [30] The aggrecan(1 400 sc-16492 Santa Cruz Biotechnology USA) typeII collagen (1 400 ab34712 Abcam UK) and goat anti-mouse horseradish peroxidase-conjugated secondary anti-body (1 1000 CW0102 Cwbiotech USA) were applied in theanalysis

212 Statistical Analysis Themeanplusmn standard deviation (SD)was applied to present the quantitative data in this study Thedifferences between the groups were analyzed by ANOVAwith SPSS (version 150 IBM USA) Student-Newman-Keulstest was adopted to compare pairwise between the groupsobtained Differences were accepted as significant when two-sided probabilities were less than 005

3 Results

31 Morphology and Release Kinetics of TGF-1205733-LoadedPLGANPs In Vitro The SEM of the TGF-1205733-loadedPLGANPs is shown in Figure 2(a) The particles exhibitedthe smooth surfaces of the nanosize sphere As shown inFigure 2(b) the TEM image showed that nanoparticles weredispersed well and that their size was uniform The particlesize of the tested sample presented a normal distribution(Figure 2(c)) and the diameter of the nanoparticles was


(a) (b)

01 100 1000

Inte

nsity

()

25

20

15

10

5

0

Size (d nm)

(c) (d)

(e)

Figure 2 (a) Scanning electron microscopy micrographs of the TGF-1205733-loaded PLGANPs (magnification 50000x scale = 2 120583m) (b)transmission electronmicroscopy of the TGF-1205733-loaded PLGANPs (magnification 500000x scale = 02 120583m) showing good dispersibility ofthe PLGANPs (c) size distributions of the TGF-1205733-loaded PLGANPs (d) general views of the codelivery system of the MSCs encapsulateddextrangelatin hydrogel with the TGF-1205733-loaded PLGANPs after 28 daysrsquo in vitro culture (e) a custom-made bioreactor system includingintegrated servomotor and circulating system for promoting TE-NP tissue formation

21836 plusmn 276 nm The gross morphology of the dextrangelatin hydrogel with TGF-1205733-loaded PLGANPs is shown inFigure 2(d)

The release kinetics of TGF-1205733 are shown in Figure 3On day 1 the TGF-1205733 was released in a burst with a releasepercentage of 63 plusmn 09 Subsequently the sustained releasepercentage of the drug decreased and stabilized graduallyduring the middle stage of release from day 2 to day 14 and212plusmn08of theTGF-1205733was released from the nanoparticlesat this stage After 28 days the cumulative release percentage

reached 564 plusmn 24The EE of the TGF-1205733 in the PLGANPswas 641 plusmn 257 It indicated that the TGF-1205733 loaded in thePLGANPs could be released stably for the long term

32 Cytotoxicity of PLGANPs and Cell Proliferation of MSCsin the Codelivery System in Long-Term Culture A fluorescentlivedead stain was used to assess the effects of PLGANPinternalization Living cells are stained by calcium AMwhich yields a green fluorescence Membranes of dead cellscomprise EthD-1 yielding a red fluorescence Fluorescent


Table 1 Real-time polymerase chain reaction primers

Gene Sequence Size

Aggrecan Forward 51015840 ATTTCCACACGCTACACCCTG 31015840 164 bpReverse 51015840 TGGATGGGGTATCTGACTGTC 31015840

Type II collagen 1205721 Forward 51015840 CAGGATGCCCGAAAATTAGGG 31015840 132 bpReverse 51015840 ACCACGATCACCTCTGGGT 31015840

KRT18 Forward 51015840 TCCATCAGGGTGACTCAGAAA 31015840 250 bpReverse 51015840 CCAGCTTCAAGGGGCTCAA 31015840

Shh Forward 51015840 AAAGCTGACCCCTTTAGCCTA 31015840 123 bpReverse 51015840 TTCGGAGTTTCTTGTGATCTTCC 31015840

GAPDH Forward 51015840 GGAGTTGCTGTTGAAGTCGCA 31015840 532 bpReverse 51015840 GGAGTTGCTGTTGAAGTCGCA 31015840

Primers for aggrecan type II collagen 1205721 Shh and GAPDHwere designed frommus musculus gene sequences obtained from the NCBI GenBank and RefSeqdatabases using primer 50 software

0 5 10 15 20 25 30

Accu

mul

atio

n pe

rcen

tage

Release time (day)

0

10

20

30

40

50

60

70

of T

GF-1205733

()

Figure 3 Release kinetics of the TGF-b3 from the PLGANPs Datawere expressed as means plusmn SD (119899 = 3)

images of MSCs obtained 7 days after PLGANP exposureare shown in Figure 4 There is no significant difference inMSC viability among all of the groups on day 7 (119901 gt 005)although almost all the dead cells were found in the groupincubated with PLGANPs The results demonstrated thatPLGANPshad little cytotoxicity in the exposed concentrationand good cytocompatibility when PLGANPs were loadedwith the TGF-1205733

The CCK-8 assay was conducted to quantify the prolif-eration activity of MSCs within the codelivery system Thehigher optical density (OD) indicated greater cell numbersAs shown in Figure 5 the cell proliferation activity of MSCsof the TGF-1205733 group was statistically higher than all of theother groups after 7 days of postseeding (119901 gt 005) On day14 theMSCs supplied with TGF-1205733 in medium or with TGF-1205733-loaded PLGANPs exhibited a higher OD value than thatof the other groups (119901 lt 005)The cell metabolic activity washigher in the TGF-1205733 group than in the TGF-1205733-PLGANPsgroup but the difference was not significant (11-fold 119901 gt005) After 28 days of postseeding the medium containingTGF-1205733 and the TGF-1205733-loaded PLGANPs could promotesignificantly greater cell proliferation than the other groups

(119901 lt 005) but there was no obvious difference that could befound between the two groups (119901 gt 005)The results indicatethat TGF-1205733-loaded PLGANPs could release active TGF-1205733in the codelivery system and TGF-1205733 could promote long-term cell metabolism

33 Effect of the Codelivery System on MSC Phenotype Thediscogenesis phenotype of seeded MSCs on day 28 wasinvestigated by qRT-PCR as shown in Figure 6 In generalthe TGF-1205733 could induce MSCs to differentiate into NP-likecells After 28 days of postseeding the expression of KRT18was upregulated significantly more in the TGF-1205733 and TGF-1205733-PLGANP groups than in the other groups (119901 lt 005) Inthe TGF-1205733 group the expression was significantly elevatedrelative to the TGF-1205733-PLGANP group (184-fold 119901 lt 005)For constructs cultured in TGF-1205733-loaded PLGANPs theexpression of Shh was 37- 28- and 19-fold greater thanthe control PLGANP and TGF-1205733 groups respectively (119901 lt005) Aggrecan expression in the TGF-1205733-PLGANPs groupwas 247- 133- and 14-fold greater after 28 days of culturethan the control PLGANP and TGF-1205733 groups respectively(119901 lt 005) The expression of type II collagen 1205721 in theTGF-1205733-PLGANPgroupwas 21- and 22-fold higher than thecontrol and PLGANP groups respectively (119901 lt 005) after 28days of culture however there was no significant differencebetween the TGF-1205733-PLGANP and TGF-1205733 groups (119901 gt005) The phenomenon demonstrated that TGF-1205733-loadedPLGANPs could induce differentiation toward NP-like cellseffectively and promote the expression of NP-selective ECM-related genes

34 Biochemical Content of the Codelivery System As shownin Figure 7(a) the results of the DNA content assay agreedwith the CCK-8 assayThe enhanced cell proliferation activitywas also evidenced by greater elevated DNA content at day28 than at days 7 and 14 in the hybrid with TGF-1205733-loadedPLGANPs (19- and 12-fold resp 119901 lt 005)

As shown in Figure 7(b) the GAG content was elevatedconstantly during the culture period in the hybrid with theTGF-1205733-PLGANPs (19-fold to 24-fold higher than it on day7 119901 lt 005) The GAG content was significantly greater in


(a) (b)

(c) (d)

200120583m

(e)

Perc

enta

ge o

f liv

ing

cells

()

012

9493

9596979899

100C

ontro

l

cont

rol

Neg

ativ

e

PLG

AN

Ps

TGF-1205733

TGF-1205733-

PLG

AN

Ps

(f)

Figure 4 Cytotoxicity assay of the MSCs cocultured with (a) 01 Triton-X (negative control) complete culture medium containing (b) nosupplement (control) (c) 1000120583gmL blank PLGANPs (PLGANPs) (d) 1000120583gmL TGF-1205733-loaded PLGANPs (TGF-1205733-PLGANPs) and(e) 10 ngmL TGF-1205733 (TGF-1205733) in the medium (magnification 100x scale = 200 120583m) Data were expressed as means plusmn SD (119899 = 3)

the TGF-1205733-PLGANPs group compared with the control andthe PLGANPs group after 14 and 28 days (119901 lt 005) butnot 7 days of postseeding (119901 gt 005) The GAG content wassignificantly higher in the TGF-1205733-PLGANPs group than theTGF-1205733 group on day 28 (12-fold higher 119901 lt 005)

As shown in Figure 7(c) the HYP content was 10- and11-fold greater after 14 and 28 days of culture in the controlgroup respectively compared with day 7 (119901 lt 005) For theTGF-1205733-PLGANPs group the HYP content was 15- and 23-fold greater respectively compared with day 7 (119901 lt 005)after 14 and 28 days of culture And there is significantlyhigher HYP content in the TGF-1205733-PLGANPs groups than

the PLGANPs group during the whole period (119901 lt 005)After 28 days of postseeding the HYP content was highersignificantly in the TGF-1205733-PLGANPs group than in theTGF-1205733 groups (11-fold 119901 lt 005)

As shown in Figure 8 the histological staining showedMSCs in all the groups exhibiting the rounded morphologyas the native NPCs There are more cell clusters in the hybridwith TGF-1205733-loaded PLGANPs and medium containingTGF-1205733 Immunohistochemical analysis revealed the ECMdistributed in the cytoplasm and the pericellular regionHigher deposition of aggrecan and type II collagen could befound in the TGF-1205733-PLGANPs and TGF-1205733 groups The


Day 7 Day 14 Day 28

Negative controlControlPLGANPs

0

1

2

3

4

TGF-1205733TGF-1205733-PLGANPs

AB

AB

ABAB

A

lowast

lowast

lowast

lowast

lowast

OD

(450

nm)

Figure 5 CCK-8 result of the MSCs cultured in the dextrangelatin hydrogel encapsulated with no supplement (control) 1000120583gmL blankPLGANPs (PLGANPs) 1000120583gmL TGF-1205733-loaded PLGANPs (TGF-1205733-PLGANPs) and the hydrogels with no supplements cultured inthe medium containing 10 ngmL TGF-1205733 (TGF-1205733) for 7 14 and 28 days The hydrogels with no seeded cell were chosen as the negativecontrol The data were expressed as the mean plusmn SD (119899 = 4) The samples indicated with (lowast) had a significant difference between the twogroups (119901 lt 005) The samples indicated with (A) had a significant difference compared to the same group on day 7 (119901 lt 005) and (B) hada significant difference compared to the same group on day 14 (119901 lt 005)

lowest deposition of ECM could be found in the PLGANPsand the control group These results indicated the cell pro-liferation and ECM-related biosynthesis of MSCs could bepromoted by the TGF-1205733-PLGANPs

4 Discussion

Due to the accelerated aging population therapy for IDD hasbeen widely considered in recent years Biological therapy forIDDmay be better suited to alleviate pain repair degenerativeNP and restore the biomechanical function of the IVD thantraditional treatments [31] However appropriate implan-tation and delivery strategies are still being explored Theadvantage of the codelivery system established in the presentstudy is that it could be applied as a minimally invasiveinjection and may promote long-term differentiation intoNP-like cells in situThe purpose of this study was to evaluatethe potential of a hydrogel with TGF-1205733-loaded PLGANPs toinduce long-term differentiation of MSCs and promote NPregeneration

In this codelivery system the hydrogel described iscrosslinked by a Schiff-based reaction The compositions ofdextran and gelatin include natural materials providing anadhesive site for encapsulated cells [32] Their biodegrad-ability also provides space for the cell proliferation andECM secretion Meanwhile the Oxi-Dex and amino-Gelprepolymers are in a liquid state while the gelation timewas approximately 58 seconds after being mixed which is

feasible for a clinical injectable application Although thedextrangelatin hydrogel is mechanically weak as an implantfor NP an implanted scaffold would not carry toomuch com-pression in the early stage of IDD because the mechanicalstructure is not damaged Additionally the aldehyde groupsof the material could react with the surface of the tissueresulting in the hydrogel fixing to the tissue surface through acovalent bond [33] The tissue integration would prevent theleakage of encapsulated MSCs and PLGANPs and promotethe long-term beneficial effect of the codelivery systemThusthe dextrangelatin hydrogel is a suitable carrier for MSCs

The release percentage and time frame of the drug dependon the carriersrsquo degradation rate PLGA is a well-known car-rier for drug delivery systems In general the lowermolecularweight of PLGA with a higher ratio of PGA is degradedrapidly because of its hydrophilic and amorphous state causedby a higher glycolic acid content On the contrary the highermolecular weight of PLGA with a higher ratio of PLA makesnanoparticles more hydrophobic which slows degradation[34] Thus the release time of the drug could be easilycontrolled from weeks to months by adjusting the PLAPGAratio According to the chondrogenesis process TGF-1205733 isrequired for sustained release for more than one month [35]Meanwhile TGF-1205733 is a hydrophilic protein similar to PGATherefore PLGA with a relatively low molecular weight anda higher PGA content (PLA PGA = 50 50) was chosen toreach relatively rapid degradation as the carrier material usedin this study


0

1

2

3

4

5Re

lativ

e gen

e exp

ress

ion

Control

A

AB

ABC

PLGANPs TGF-1205733TGF-1205733-PLGANPs

(a)

0

1

2

3

4

5

Relat

ive g

ene e

xpre

ssio

n

AB

ABC

ControlPLGANPs TGF-1205733

TGF-1205733-PLGANPs

(b)

0

10

20

30

40

Relat

ive g

ene e

xpre

ssio

n

AB

ABC


TGF-1205733-PLGANPs

(c)

0

1

2

3

4

Relat

ive g

ene e

xpre

ssio

n

ABAB


TGF-1205733-PLGANPs

(d)

Figure 6 The expressions of (a) KRT18 (b) Shh (c) aggrecan and (d) type II collagen 1205721 of MSCs in the dextrangelatin hydrogel withno supplement (control) 1000 120583gmL blank PLGANPs (PLGANPs) 1000 120583gmL TGF-1205733-loaded PLGANPs (TGF-1205733-PLGANPs) and thehydrogels with no supplements cultured in the medium containing 10 ngmL TGF-1205733 (TGF-1205733) for 28 days The expression levels werequantified using real-time PCR and normalized to the housekeeping gene GAPDHThe data were expressed as the mean plusmn SD (119899 = 3) Thesamples indicated with (A) had a significant difference compared to the control group (119901 lt 005) (B) had a significant difference comparedto the TGF-1205733-PLGANPs group (119901 lt 005) and (C) had a significant difference compared to TGF-1205733 group (119901 lt 005)

SEM scanning showed that the TGF-1205733-loaded nanopar-ticles were spherical with a smooth appearance As theTEM image showed there is no obvious adhesion betweenparticles indicating proper dispersion It would be beneficialif nanoparticles evenly and uniformly distributed the TGF-1205733 concentration in the hydrogel In previous studies ahydrogel with TGF-120573-loaded microspheres was constructedand exhibited favorable chondrogenesis activity of MSCs[36 37] In contrast with these micron-sized microspheresthe injectability could be significantly improved by using ananosized PLGA in the codelivery system Needle puncturecan aggravate IDD and is even used to establish an animalmodel of IDD [38] Rabbit IVDs subjected to annular needle

punctures using 16 to 21 G needles led to degeneration after8 weeks [38 39] Owing to the intact structure of the IVDin the early stage of IDD the annulus structure should beprotected effectively However injection could not be avoidedwhen cell-based therapy was applied to early stage IDD Itis encouraging that PLGANPs could be injected with muchfiner needles than micron-sized microspheres alleviatingannular injury Therefore PLGANPs are particularly suitableas drug carriers for the treatment of early stage IDD

A desired drug delivery rate is one essential criterionfor a successful drug delivery system Generally the drugrelease could be roughly divided into the following threestages the burst release period the stable period and the


Day 7 Day 14 Day 280

05

1

15

2

25


TGF-1205733-PLGANPs

A A A A

ABAB

lowast

lowast

lowast

lowast

lowastD

NA

cont

ent (120583

gco

nstr

uct)

(a)


50

100

150

200

250

300


TGF-1205733-PLGANPs

A

A A

ABAB

lowast

lowast

lowast

lowast

lowast

GAG

cont

ent (120583

gco

nstr

uct)

(b)


2

4

6

8

10

12

14

16


TGF-1205733-PLGANPs

A A

ABAB

B

lowast

lowast

lowastlowast

lowastlowast

Hyd

roxy

prol

ine c

onte

nt (120583

gco

nstr

uct)

(c)

Figure 7 Measurement of the biochemical content of the hybrids including (a) DNA (b) GAG and (c) HYP of the MSCs-seededdextrangelatin hydrogels encapsulatedwith no supplement (control) 1000 120583gmL blank PLGANPs (PLGANPs) 1000 120583gmLTGF-1205733-loadedPLGANPs (TGF-1205733-PLGANPs) and the hydrogels with no supplements cultured in the medium containing 10 ngmL TGF-1205733 (TGF-1205733) for7 14 and 28 days Data were expressed as means plusmn SD (119899 = 3) The samples indicated with (lowast) had a significant difference between the twogroups (119901 lt 005) The samples indicated with (A) had a significant difference compared to the same group on day 7 (119901 lt 005) and (B) hada significant difference compared to the same group on day 14 (119901 lt 005)

accelerated period The nanoparticles exhibited a relativelylower burst release on the first day Then TGF-1205733 wasstably released from the nanoparticles from days 2 to 14Cumulative release was approximately 564 in the first28 days In general the TGF-1205733-loaded PLGANPs in thisstudy established a depot with a low burst release which isideal for long-term continuous drug release and would meetthe requirement of discogenesis differentiation in situ In aprevious study TGF-1205733 could also be immobilized on thescaffold for controlled release [40] The drug-immobilizedscaffold could release TGF-1205733 in a long-term culture and

induce chondrogenic differentiation However the TGF-1205733crosslinked on the scaffold usually exhibited a higher burstrelease High concentration of TGF-1205733 above 10 ngmL maybe detrimental to chondrogenesis of MSCs as reported byClarke et al [23] Therefore the stable release system ofPLGANPs in this study is probably a suitable alternative forlong-term chondrogenesis

The cell viability in all of the groups was greater than94 indicating that the TGF-1205733-loaded nanoparticles andblank nanoparticles both exhibited favorable cytocompatibil-ity To investigate the cell proliferation of the hydrogel with


(a1) (d1)(c1)(b1)

Agg

reca

nTy

pe II

colla

gen

Control PLGANPsNegative control

(e1)

(a2) (d2)(c2)(b2) (e2)

50120583m


Figure 8 Photomicrographs showed the immunohistological staining for aggrecan (a1ndashe1) and type II collagen (a2ndashe2) of neo-NP tissueformation in the codelivery system of the MSCs-seeded dextrangelatin hydrogel encapsulated with no supplement (control (b1-2))1000 120583gmL blank PLGANPs (PLGANPs (c1-2)) 1000120583gmL TGF-1205733-loaded PLGANPs (TGF-1205733-PLGANPs (d1-2)) and the hydrogelswith no supplements cultured in the medium containing 10 ngmL TGF-1205733 (TGF-1205733 (e1-2)) for 28 days (a1-2) were negative controls forimmunostaining (magnification 400x scale = 50 120583m)

TGF-1205733-PLGANPs during the period of MSC discogenesisthe proliferation of encapsulated MSCs was assessed for28 days As shown in Figure 4 the PLGANPs alone hadno obvious impact on cell proliferation during the testingperiod In the TGF-1205733-PLGANPs and TGF-1205733 groups thecell proliferation of MSCs was enhanced significantly whichhas been described by Shi and Massague [41] The phe-nomenon could be attributed to the fact that TGF-1205733 couldactivate the Smad complex and subsequently activate thep15Ink4b promoter [42] This could also be evidenced by thegradually increasing DNA content in the codelivery systemThe results demonstrated that the high metabolite activity ofMSCs was maintained in the hydrogel with TGF-1205733-loadednanoparticles laying a foundation for the differentiation

Due to the low cell density present in the NP transplant-ing a large number of autologous cells such asMSCs with anappropriate carriermay be an effective strategy for promotingnew tissue formation [43] For this strategy a properlydifferentiated phenotype is the basis of the regeneration ofthe NP MSCs could differentiate toward a chondrogeniclineage with the assistance of TGF-1205733 which is proven bythe upregulation of aggrecan (ACAN) and type II collagen(Col2) 1205721 However due to a lack of NPC phenotypesnative NPCs are always treated as chondrocyte-like cells andidentified by the expression of chondrogenesis markers suchas SOX-9 Col2 and ACANThus it could not be determinedwhether MSCs actually differentiated into NPCs in manystudies However the composition and biological functionof chondrocytes and NPCs are obviously different [44] TheECM secreted by chondrocytes is much stiffer than thatreleased by NPCs and is not suitable for fluid pressurizationin the NP Thus improper differentiation may result in afailure to restore IVD function If MSCs are chosen as the cellsource to promote NP regeneration it is necessary to identify

the cell phenotype of differentiated cells to ensure properECM deposits [45] Some markers identified in rat arraysshowed relatively significant differential expression in humanNPCs versus articular cartilage cells especially KRT18 [46]These results were also confirmed at the protein level withimmunohistochemistry [47] Additionally sonic hedgehog(Shh) is a ligand of the hedgehog family and it was selectivelyexpressed in young NP as Dahia et al reported [48] Shhis required for the proper functioning of NPCs and is apotential vital phenotype of NPCs [49] Encapsulating MSCsin a hydrogel with TGF-1205733-PLGANPs led to a significantincrease in the NP phenotypic marker genes of KRT18 andShh relative to the control and PLGANPgroups In particularthere was a much higher expression of Shh in the TGF-1205733-PLGANP group than in the TGF-1205733 group These positiveresults indicated that the codelivery system could efficientlyinduce the discogenesis differentiation of MSCs triggeredby TGF-1205733 in situ establishing a foundation for enhancedbiosynthesis of implanted cells

The biosynthetic activity of MSCs in the presence ofTGF-1205733-loaded nanoparticles was evidenced by increasingGAG and HYP with an extended culture time Interestinglyproteoglycan synthesis was slightly higher in the hydro-gel with TGF-1205733-loaded PLGANPs than in the TGF-1205733-supplemented culture medium on day 28 There are twopotential explanations for this phenomenon On the onehand due to the bulk size of the hydrogel the TGF-1205733 in themedium may be unable to penetrate into the central regionof the hydrogel The concentration gradient of TGF-1205733 coulddecline from the outer to the inner area On the contrary thenanoparticles could distribute uniformly in the hydrogel asmentioned aboveThus TGF-1205733 could reach the inner regionof the hydrogel and activate more MSCs in the hydrogel Onthe other hand the half-life of TGF-1205733 in the medium was


relatively short With the extension of the culture time thebiological effects observed with the shorter duration of TGF-1205733 were gradually increased

Clusters of MSCs in a chondrocyte-like rounded shapein the codelivery system indicated cells were in a state ofproliferation in the area of the degraded scaffold and weremoving toward discogenesis differentiationThe findings alsoindicated that aggrecan and type II collagen expression wereupregulated in the TGF-1205733-PLGANP and TGF-1205733 groupsThe ECM-related proteins started to distribute in the pericel-lular region indicating new NP tissue formation Due to therelatively short culture period the scaffold was not degradedenough to provide more space for tissue formation Furtherexperiments would need to be conducted to determine theeffect of long-term culture

In the present study a limitation was that the hybridwas cultivated in vitro where the medium condition issuitable for MSC proliferation This may have influencedMSC viability and affected the long-term regeneration inthe harsh microenvironment of the NP in vivo which is ofhigh acidity and low nutrient supply [50 51] In addition wefound that the structure of the hydrogel at the concentrationof 10 was easily damaged following long-term culture invitro in our previous study Thus we applied the hydrogelwith a higher concentration of 20 and found that thestructure was stable enough but this may compromise itsinjectable property In the future an in vivo study should beconducted to explore the viability and biosynthetic situationof MSCs and test the feasibility of the codelivery system forNP injection

5 Conclusion

In summary in this series of in vitro studies we successfullyestablished a codelivery system of a dextrangelatin hydrogelwith TGF-1205733-loaded PLGANPs The nanoparticles couldstably release active TGF-1205733 for long termThedifferentiatingpotential of this novel hybridwas validated by inducingMSCsinto discogenesis cells in situ during the period of 28 daysIt is possible that the codelivery system may be applied forthe treatment of early stage IDD using autologous MSCs Infuture studies we will investigate the long-term regenerativeeffect of MSCs using the delivery hybrid in a large-animalmodel of IDD

Competing Interests

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

Authorsrsquo Contributions

Yibo Gan and Sukai Li contributed equally to this study

Acknowledgments

The authors thank Deying Chen for excellent technicalsupport This study was funded by the NSFC of China

(Grant no 81272029) Key Project of Military Logistics(Grant no BWS13C014-1) and Preresearch Fund of MilitaryMedicine of Third Military Medical University (Grant noSWH2013JS04)

References

[1] M A Adams and P J Roughley ldquoWhat is intervertebral discdegeneration andwhat causes itrdquo Spine vol 31 no 18 pp 2151ndash2161 2006

[2] B I Martin R A Deyo S K Mirza et al ldquoExpenditures andhealth status among adults with back and neck problemsrdquo TheJournal of the American Medical Association vol 299 no 6 pp656ndash664 2008

[3] B Peng J Hao S Hou et al ldquoPossible pathogenesis of painfulintervertebral disc degenerationrdquo Spine vol 31 no 5 pp 560ndash566 2006

[4] A H Hsieh and J D Twomey ldquoCellular mechanobiology of theintervertebral disc new directions and approachesrdquo Journal ofBiomechanics vol 43 no 1 pp 137ndash145 2010

[5] D Oehme T Goldschlager P Ghosh J V Rosenfeld and GJenkin ldquoCell-based therapies used to treat lumbar degenerativedisc disease a systematic review of animal studies and humanclinical trialsrdquo Stem Cells International vol 2015 Article ID946031 16 pages 2015

[6] A Hiyama J Mochida T Iwashina et al ldquoTransplantation ofmesenchymal stem cells in a canine disc degeneration modelrdquoJournal of Orthopaedic Research vol 26 no 5 pp 589ndash6002008

[7] M V Risbud T J Albert A Guttapalli et al ldquoDifferentiationof mesenchymal stem cells towards a nucleus pulposus-likephenotype in vitro implications for cell-based transplantationtherapyrdquo Spine vol 29 no 23 pp 2627ndash2632 2004

[8] C Bucher A Gazdhar L M Benneker T Geiser and BGantenbein-Ritter ldquoNonviral gene delivery of growth and dif-ferentiation factor 5 to humanmesenchymal stem cells injectedinto a 3D bovine intervertebral disc organ culture systemrdquo StemCells International vol 2013 Article ID 326828 9 pages 2013

[9] E Steck H Bertram R Abel B Chen A Winter and WRichter ldquoInduction of intervertebral disc-like cells from adultmesenchymal stem cellsrdquo STEM CELLS vol 23 no 3 pp 403ndash411 2005

[10] S M Richardson N Hughes J A Hunt A J Freemont and JA Hoyland ldquoHuman mesenchymal stem cell differentiation toNP-like cells in chitosan-glycerophosphate hydrogelsrdquo Bioma-terials vol 29 no 1 pp 85ndash93 2008

[11] P Roughley C Hoemann E DesRosiers FMwale J Antoniouand M Alini ldquoThe potential of chitosan-based gels containingintervertebral disc cells for nucleus pulposus supplementationrdquoBiomaterials vol 27 no 3 pp 388ndash396 2006

[12] C-Q Zhao L-M Wang L-S Jiang and L-Y Dai ldquoThe cellbiology of intervertebral disc aging and degenerationrdquo AgeingResearch Reviews vol 6 no 3 pp 247ndash261 2007

[13] L M Boyd and A J Carter ldquoInjectable biomaterials andvertebral endplate treatment for repair and regeneration of theintervertebral discrdquo European Spine Journal vol 15 no 3 ppS414ndashS421 2006

[14] W-Y Su Y-C Chen and F-H Lin ldquoInjectable oxidizedhyaluronic acidadipic acid dihydrazide hydrogel for nucleuspulposus regenerationrdquo Acta Biomaterialia vol 6 no 8 pp3044ndash3055 2010


[15] S M Richardson J A Hoyland R Mobasheri C CsakiM Shakibaei and A Mobasheri ldquoMesenchymal stem cellsin regenerative medicine opportunities and challenges forarticular cartilage and intervertebral disc tissue engineeringrdquoJournal of Cellular Physiology vol 222 no 1 pp 23ndash32 2010

[16] D-H Kim and D C Martin ldquoSustained release of dexametha-sone from hydrophilic matrices using PLGA nanoparticles forneural drug deliveryrdquoBiomaterials vol 27 no 15 pp 3031ndash30372006

[17] H K Makadia and S J Siegel ldquoPoly Lactic-co-Glycolic Acid(PLGA) as biodegradable controlled drug delivery carrierrdquoPolymers vol 3 no 3 pp 1377ndash1397 2011

[18] M L Hans and A M Lowman ldquoBiodegradable nanoparticlesfor drug delivery and targetingrdquo Current Opinion in Solid Stateand Materials Science vol 6 no 4 pp 319ndash327 2002

[19] M S Cartiera K M Johnson V Rajendran M J Caplan andW M Saltzman ldquoThe uptake and intracellular fate of PLGAnanoparticles in epithelial cellsrdquo Biomaterials vol 30 no 14 pp2790ndash2798 2009

[20] S Acharya and S K Sahoo ldquoPLGA nanoparticles containingvarious anticancer agents and tumour delivery by EPR effectrdquoAdvancedDrugDelivery Reviews vol 63 no 3 pp 170ndash183 2011

[21] Y-CWang Y-T Wu H-Y Huang et al ldquoSustained intraspinaldelivery of neurotrophic factor encapsulated in biodegradablenanoparticles following contusive spinal cord injuryrdquo Biomate-rials vol 29 no 34 pp 4546ndash4553 2008

[22] G-K Zou Y-L Song W Zhou et al ldquoEffects of local deliveryof bFGF from PLGA microspheres on osseointegration aroundimplants in diabetic ratsrdquo Oral Surgery Oral Medicine OralPathology and Oral Radiology vol 114 no 3 pp 284ndash289 2012

[23] L E Clarke J C McConnell M J Sherratt B Derby S MRichardson and J A Hoyland ldquoGrowth differentiation factor6 and transforming growth factor-beta differentially medi-ate mesenchymal stem cell differentiation composition andmicromechanical properties of nucleus pulposus constructsrdquoArthritis Research andTherapy vol 16 no 2 article R67 2014

[24] M Diwan and T G Park ldquoPegylation enhances protein stabilityduring encapsulation in PLGA microspheresrdquo Journal of Con-trolled Release vol 73 no 2-3 pp 233ndash244 2001

[25] F B Basmanav G T Kose and V Hasirci ldquoSequential growthfactor delivery from complexed microspheres for bone tissueengineeringrdquo Biomaterials vol 29 no 31 pp 4195ndash4204 2008

[26] G Yoshimatsu N Sakata H Tsuchiya et al ldquoDevelopmentof polyvinyl alcohol bioartificial pancreas with rat islets andmesenchymal stem cellsrdquo Transplantation Proceedings vol 45no 5 pp 1875ndash1880 2013

[27] X Mo H Iwata S Matsuda and Y Ikada ldquoSoft tissue adhesivecomposed of modified gelatin and polysaccharidesrdquo Journal ofBiomaterials Science Polymer Edition vol 11 no 4 pp 341ndash3512000

[28] B J Dekosky N H Dormer G C Ingavle et al ldquoHierarchicallydesigned agarose and poly(ethylene glycol) interpenetratingnetwork hydrogels for cartilage tissue engineeringrdquo TissueEngineeringmdashPart CMethods vol 16 no 6 pp 1533ndash1542 2010

[29] N Blumenkrantz and G Asboe-Hansen ldquoAn assay for hydrox-yproline and proline on one sample and a simplified methodfor hydroxyprolinerdquo Analytical Biochemistry vol 63 no 2 pp331ndash340 1975

[30] L J Smith D J Gorth B L Showalter et al ldquoIn vitro char-acterization of a stem-cell-seeded triple-interpenetrating-network hydrogel for functional regeneration of the nucleus

pulposusrdquo Tissue Engineering Part A vol 20 no 13-14 pp1841ndash1849 2014

[31] D Sakai ldquoFuture perspectives of cell-based therapy for inter-vertebral disc diseaserdquo European Spine Journal vol 17 no 4 ppS452ndashS458 2008

[32] Y Liu and M B Chan-Park ldquoHydrogel based on interpenetrat-ing polymer networks of dextran and gelatin for vascular tissueengineeringrdquo Biomaterials vol 30 no 2 pp 196ndash207 2009

[33] S Ramaswamy D-A Wang K W Fishbein J H Elisseeffand R G Spencer ldquoAn analysis of the integration betweenarticular cartilage and nondegradable hydrogel using magneticresonance imagingrdquo Journal of Biomedical Materials ResearchPart B Applied Biomaterials vol 77 no 1 pp 144ndash148 2006

[34] J M Anderson and M S Shive ldquoBiodegradation and biocom-patibility of PLA and PLGA microspheresrdquo Advanced DrugDelivery Reviews vol 64 pp 72ndash82 2012

[35] H Koga T Muneta T Nagase et al ldquoComparison of mes-enchymal tissues-derived stem cells for in vivo chondrogenesissuitable conditions for cell therapy of cartilage defects in rabbitrdquoCell and Tissue Research vol 333 no 2 pp 207ndash215 2008

[36] L Bian D Y Zhai E Tous R Rai R L Mauck and J ABurdick ldquoEnhanced MSC chondrogenesis following deliveryof TGF-1205733 from alginate microspheres within hyaluronic acidhydrogels in vitro and in vivordquo Biomaterials vol 32 no 27 pp6425ndash6434 2011

[37] T Ogawa T Akazawa and Y Tabata ldquoIn vitro proliferationand chondrogenic differentiation of rat bone marrow stemcells cultured with gelatin hydrogel microspheres for TGF-1205731releaserdquo Journal of Biomaterials Science Polymer Edition vol 21no 5 pp 609ndash621 2010

[38] K Masuda Y Aota C Muehleman et al ldquoA novel rabbit modelof mild reproducible disc degeneration by an anulus needlepuncture correlation between the degree of disc injury andradiological and histological appearances of disc degenerationrdquoSpine vol 30 no 1 pp 5ndash14 2005

[39] S Sobajima J F Kompel J S Kim et al ldquoA slowly progressiveand reproducible animal model of intervertebral disc degenera-tion characterized by MRI X-ray and histologyrdquo Spine vol 30no 1 pp 15ndash24 2005

[40] H Fan H Tao Y Wu Y Hu Y Yan and Z Luo ldquoTGF-1205733immobilized PLGA-gelatinchondroitin sulfatehyaluronic acidhybrid scaffold for cartilage regenerationrdquo Journal of BiomedicalMaterials Research Part A vol 95 no 4 pp 982ndash992 2010

[41] Y Shi and J Massague ldquoMechanisms of TGF-120573 signaling fromcell membrane to the nucleusrdquo Cell vol 113 no 6 pp 685ndash7002003

[42] X H Feng X Lin and R Derynck ldquoSmad2 Smad3 and Smad4cooperate with Sp1 to induce p151198681198991198964119861 transcription in responseto TGF-120573rdquo The EMBO Journal vol 19 no 19 pp 5178ndash51932000

[43] J Zeckser M Wolff J Tucker and J Goodwin ldquoMultipotentmesenchymal stem cell treatment for discogenic low back painand disc degenerationrdquo Stem Cells International vol 2016Article ID 3908389 13 pages 2016

[44] S Zeiter M van der Werf and K Ito ldquoThe fate of bovine bonemarrow stromal cells in hydrogels a comparison to nucleuspulposus cells and articular chondrocytesrdquo Journal of TissueEngineering amp Regenerative Medicine vol 3 no 4 pp 310ndash3202009

[45] B M Minogue S M Richardson L A H Zeef A J Freemontand J A Hoyland ldquoCharacterization of the human nucleus


pulposus cell phenotype and evaluation of novel marker geneexpression to define adult stem cell differentiationrdquo Arthritisand Rheumatism vol 62 no 12 pp 3695ndash3705 2010

[46] F Lv V Y L Leung S Huang Y Huang Y Sun and K MC Cheung ldquoIn search of nucleus pulposus-specific molecularmarkersrdquo Rheumatology vol 53 no 4 pp 600ndash610 2014

[47] D Sakai T Nakai J Mochida M Alini and S Grad ldquoDif-ferential phenotype of intervertebral disc cells microarray andimmunohistochemical analysis of canine nucleus pulposus andanulus fibrosusrdquo Spine vol 34 no 14 pp 1448ndash1456 2009

[48] C L Dahia E J Mahoney A A Durrani and C WylieldquoIntercellular signaling pathways active during intervertebraldisc growth differentiation and agingrdquo Spine vol 34 no 5 pp456ndash462 2009

[49] C L Dahia E Mahoney and C Wylie ldquoShh signaling fromthe nucleus pulposus is required for the postnatal growth anddifferentiation of themouse intervertebral discrdquo PLoSONE vol7 no 4 Article ID e35944 2012

[50] KWuertz K Godburn and J C Iatridis ldquoMSC response to pHlevels found in degenerating intervertebral discsrdquo Biochemicaland Biophysical Research Communications vol 379 no 4 pp824ndash829 2009

[51] M J Farrell E S Comeau and R L Mauck ldquoMesenchymalstem cells produce functional cartilage matrix in three- dimen-sional culture in regions of optimal nutrient supplyrdquo EuropeanCells and Materials vol 23 pp 425ndash440 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


to the poor self-regeneration capability of NPCs cell-basedtherapy could be a promising strategy for the treatment ofIDD [5] Of all of the cell-based approaches implantation ofmesenchymal stem cells (MSCs) encapsulated in hydrogelshas received the greatest attention [6]

An advantage of the MSC-based strategy is that autol-ogous cells are easily harvested and expanded rapidly invitro There is greater potential to regenerate the NP withlarger amounts of implanted cells Another advantage is thatMSCs are able to differentiate to an NP-like phenotype withsome proper biochemical directions such as transforminggrowth factor 120573 (TGF-120573) [7] and differentiation factor 5(GDF5) [8] The TGF-120573 family is one of the exogenousgrowth factors widely used to induce MSCs to discogenesisdifferentiation Generally high-density MSCs in a three-dimensional (3D) carrier were cultured with a serum-freeinduction medium with TGF-120573 isoforms in vitro for chon-drogenic differentiation especially with TGF-1205733 [9] andsubsequently the hybrid was implanted for NP regeneration[10] Hydrogels are considered proper carriers because oftheir similar rheological property to the NP and favorablecytocompatibility [11] However this strategy is difficult toapply for the treatment of early stage IDD Due to thestructural integrity of AF in early stage IDD IVD fenestrationshould be made before implanting a bulk cell-hydrogelhybrid which could possibly accelerate IDD [12] Thereforea novel alternative may involve using an injectable hydrogelas a carrier of MSCs The injectable hydrogels are in theliquid state before implantation and harden after injectionin vivo The process allows minimal invasion through theAF It is beneficial for avoiding extrusion after implantationwhile adapting to the irregular shape of the defect in the NPcavity [13]The precursor solution can also be combined withgrowth factors and cells to reverse disc degeneration [14]However it is still a challenge to maintain long-term MSCdifferentiation in situ [15] The half-life of TGF-1205733 is onlytens of minutes in vivo and it can be easily diluted in bodyfluid diffusion If growth factors were combined directly withthe hydrogel they would soon be degraded or eliminatedTherefore it is difficult to affect TGF-1205733 by physical mixing[15] Thus it is particularly important to construct a suitabledelivery system for the sustained release of growth factors

The rapid development of the FDA-approved poly(DL-lactide-co-glycolide) (PLGA) may be useful for long-termdifferentiation [16] PLGA is a copolymer of PLA and PGAwith favorable biocompatibility favorable biodegradabilityand low immunogenicity Recently PLGA was applied todrug delivery and tissue engineering applications among themost attractive polymeric candidates [17]The preparation ofnanosized particles has increasedwith advances in traditionalmethods because nanoparticles could be delivered to variousbody parts with a wide range of drugs for sustained release[18] PLGA nanoparticles (PLGANPs) can be targeted to aspecific tissue and release the drug locally by endocytosis[19] Such PLGANPs have been widely used to develop anti-cancer drug delivery system such as paclitaxel doxorubicin5-fluorouracil and dexamethasone which were effectivelyformulated via PLGANPs [20] It has also been incorporatedin tissue engineering scaffold for control over spatial and

temporal release of growth factors For instance glial cell-line derived neurotrophic factor (GDNF) was encapsulatedin PLGANPs when implanted into the spinal cord to inducean increase in neuronal survival reported by Wang et al [21]If PLGANPs are used as carriers for a gradual release ofgrowth factors into the IVD local maintenance may lead tosufficient concentrations of TGF-1205733 in the long-term processof NP regenerationThe injectable hydrogel coupled with thesustained release nanoparticles could meet the clinical needsof an ideal MSC-based minimally invasive implantation

In this study TGF-1205733-loaded PLGANPs were preparedand seeded into a dextrangelatin hydrogel to constructa novel codelivery system for inducing long-term MSCdifferentiation in situ The overall objective of this studywas to investigate the potential of the codelivery systemfor NP regeneration To achieve this we first evaluated therelease kinetics of the TGF-1205733 and confirmed that the TGF-1205733-loaded PLGANPs could release enough TGF-1205733 withinan appropriate discogenesis time frame Second we investi-gated the cytocompatibility of the TGF-1205733-loaded PLGANPsFinally we investigated whether the codelivery system couldsupport cell proliferation discogenesis differentiation andfunctional biosynthesis of MSCs

2 Materials and Methods

21 Preparation of the TGF-1205733-PLGANPs The TGF-1205733-loaded PLGANPs were prepared using the double-emulsionsolvent evaporation method (WOW) as previouslydescribed [22] In order to reach the concentration ofTGF-1205733 as previously recommended for the discogenesisdifferentiation of MSCs the target release amount of theTGF-1205733 was 10 ng in 1mL culture medium [23] Briefly 10 120583gof the TGF-1205733 (PeproTech USA) was dissolved in 01mLof distilled water (W

1) Subsequently the PLGA 50 50

(200mg 24ndash38 kD Sigma-Aldrich USA) was dissolvedin 1mL dichloromethane (O Sigma-Aldrich) Later theTGF-1205733 solution was added dropwise to the PLGA solutionThen the solution was treated ultrasonically in an ice bathThis primary emulsion was added dropwise to 6mL of1 polyvinyl alcohol (PVA Sigma-Aldrich USA) aqueoussolution (W

2) and treated ultrasonically again The obtained

W1OW

2emulsion was transferred into 20mL of the 03

PVA aqueous solution and stirred for 4 hours with a propellerstirrer Subsequently the solid particles were collected aftercentrifugation Finally the obtained precipitates were washedfor three times with deionized water The nanoparticles wereharvested by lyophilization and stored at 20∘C in desiccativecondition

22 Morphological Observations The morphology of theTGF-1205733-loaded PLGANPs was examined by the scanningelectron microscopy (SEM S3400N II Hitachi Japan) andthe transmission electron microscope (TEM TECNAI 10Philips USA) Briefly 20120583L of the PLGANPs suspension wasdropped on the mica followed by being air-dried overnightThen morphology of the TGF-1205733-loaded PLGANPs wasexamined by the SEM after spraying For the TEM examina-tion the PLGANPs suspension was dropped on the copper





times 100 (1)








WOW

Oxi-Dex Amino-Gel

MSCs

PLGA nanoparticles



CH

NOO

OO

yx

HHO

Discogenesis


TGF-1205733

PLGA 50 50








3 Results



(a) (b)

01 100 1000

Inte

nsity

()

25

20

15

10

5

0

Size (d nm)

(c) (d)

(e)








Gene Sequence Size







0 5 10 15 20 25 30

Accu

mul

atio

n pe

rcen

tage

Release time (day)

0

10

20

30

40

50

60

70

of T

GF-1205733

()









(a) (b)

(c) (d)

200120583m

(e)

Perc

enta

ge o

f liv

ing

cells

()

012

9493

9596979899

100C

ontro

l

cont

rol

Neg

ativ

e

PLG

AN

Ps

TGF-1205733

TGF-1205733-

PLG

AN

Ps

(f)







Day 7 Day 14 Day 28


0

1

2

3

4


AB

AB

ABAB

A

lowast

lowast

lowast

lowast

lowast

OD

(450

nm)



4 Discussion






0

1

2

3

4

5Re

lativ

e gen

e exp

ress

ion

Control

A

AB

ABC


(a)

0

1

2

3

4

5

Relat

ive g

ene e

xpre

ssio

n

AB

ABC


TGF-1205733-PLGANPs

(b)

0

10

20

30

40

Relat

ive g

ene e

xpre

ssio

n

AB

ABC


TGF-1205733-PLGANPs

(c)

0

1

2

3

4

Relat

ive g

ene e

xpre

ssio

n

ABAB


TGF-1205733-PLGANPs

(d)







05

1

15

2

25


TGF-1205733-PLGANPs

A A A A

ABAB

lowast

lowast

lowast

lowast

lowastD

NA

cont

ent (120583

gco

nstr

uct)

(a)


50

100

150

200

250

300


TGF-1205733-PLGANPs

A

A A

ABAB

lowast

lowast

lowast

lowast

lowast

GAG

cont

ent (120583

gco

nstr

uct)

(b)


2

4

6

8

10

12

14

16


TGF-1205733-PLGANPs

A A

ABAB

B

lowast

lowast

lowastlowast

lowastlowast

Hyd

roxy

prol

ine c

onte

nt (120583

gco

nstr

uct)

(c)






(a1) (d1)(c1)(b1)

Agg

reca

nTy

pe II

colla

gen


(e1)

(a2) (d2)(c2)(b2) (e2)

50120583m











5 Conclusion


Competing Interests




Acknowledgments



References































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





times 100 (1)








WOW

Oxi-Dex Amino-Gel

MSCs

PLGA nanoparticles



CH

NOO

OO

yx

HHO

Discogenesis


TGF-1205733

PLGA 50 50








3 Results



(a) (b)

01 100 1000

Inte

nsity

()

25

20

15

10

5

0

Size (d nm)

(c) (d)

(e)








Gene Sequence Size







0 5 10 15 20 25 30

Accu

mul

atio

n pe

rcen

tage

Release time (day)

0

10

20

30

40

50

60

70

of T

GF-1205733

()









(a) (b)

(c) (d)

200120583m

(e)

Perc

enta

ge o

f liv

ing

cells

()

012

9493

9596979899

100C

ontro

l

cont

rol

Neg

ativ

e

PLG

AN

Ps

TGF-1205733

TGF-1205733-

PLG

AN

Ps

(f)







Day 7 Day 14 Day 28


0

1

2

3

4


AB

AB

ABAB

A

lowast

lowast

lowast

lowast

lowast

OD

(450

nm)



4 Discussion






0

1

2

3

4

5Re

lativ

e gen

e exp

ress

ion

Control

A

AB

ABC


(a)

0

1

2

3

4

5

Relat

ive g

ene e

xpre

ssio

n

AB

ABC


TGF-1205733-PLGANPs

(b)

0

10

20

30

40

Relat

ive g

ene e

xpre

ssio

n

AB

ABC


TGF-1205733-PLGANPs

(c)

0

1

2

3

4

Relat

ive g

ene e

xpre

ssio

n

ABAB


TGF-1205733-PLGANPs

(d)







05

1

15

2

25


TGF-1205733-PLGANPs

A A A A

ABAB

lowast

lowast

lowast

lowast

lowastD

NA

cont

ent (120583

gco

nstr

uct)

(a)


50

100

150

200

250

300


TGF-1205733-PLGANPs

A

A A

ABAB

lowast

lowast

lowast

lowast

lowast

GAG

cont

ent (120583

gco

nstr

uct)

(b)


2

4

6

8

10

12

14

16


TGF-1205733-PLGANPs

A A

ABAB

B

lowast

lowast

lowastlowast

lowastlowast

Hyd

roxy

prol

ine c

onte

nt (120583

gco

nstr

uct)

(c)






(a1) (d1)(c1)(b1)

Agg

reca

nTy

pe II

colla

gen


(e1)

(a2) (d2)(c2)(b2) (e2)

50120583m











5 Conclusion


Competing Interests




Acknowledgments



References































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


WOW

Oxi-Dex Amino-Gel

MSCs

PLGA nanoparticles



CH

NOO

OO

yx

HHO

Discogenesis


TGF-1205733

PLGA 50 50








3 Results



(a) (b)

01 100 1000

Inte

nsity

()

25

20

15

10

5

0

Size (d nm)

(c) (d)

(e)








Gene Sequence Size







0 5 10 15 20 25 30

Accu

mul

atio

n pe

rcen

tage

Release time (day)

0

10

20

30

40

50

60

70

of T

GF-1205733

()









(a) (b)

(c) (d)

200120583m

(e)

Perc

enta

ge o

f liv

ing

cells

()

012

9493

9596979899

100C

ontro

l

cont

rol

Neg

ativ

e

PLG

AN

Ps

TGF-1205733

TGF-1205733-

PLG

AN

Ps

(f)







Day 7 Day 14 Day 28


0

1

2

3

4


AB

AB

ABAB

A

lowast

lowast

lowast

lowast

lowast

OD

(450

nm)



4 Discussion






0

1

2

3

4

5Re

lativ

e gen

e exp

ress

ion

Control

A

AB

ABC


(a)

0

1

2

3

4

5

Relat

ive g

ene e

xpre

ssio

n

AB

ABC


TGF-1205733-PLGANPs

(b)

0

10

20

30

40

Relat

ive g

ene e

xpre

ssio

n

AB

ABC


TGF-1205733-PLGANPs

(c)

0

1

2

3

4

Relat

ive g

ene e

xpre

ssio

n

ABAB


TGF-1205733-PLGANPs

(d)







05

1

15

2

25


TGF-1205733-PLGANPs

A A A A

ABAB

lowast

lowast

lowast

lowast

lowastD

NA

cont

ent (120583

gco

nstr

uct)

(a)


50

100

150

200

250

300


TGF-1205733-PLGANPs

A

A A

ABAB

lowast

lowast

lowast

lowast

lowast

GAG

cont

ent (120583

gco

nstr

uct)

(b)


2

4

6

8

10

12

14

16


TGF-1205733-PLGANPs

A A

ABAB

B

lowast

lowast

lowastlowast

lowastlowast

Hyd

roxy

prol

ine c

onte

nt (120583

gco

nstr

uct)

(c)






(a1) (d1)(c1)(b1)

Agg

reca

nTy

pe II

colla

gen


(e1)

(a2) (d2)(c2)(b2) (e2)

50120583m











5 Conclusion


Competing Interests




Acknowledgments



References































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)

01 100 1000

Inte

nsity

()

25

20

15

10

5

0

Size (d nm)

(c) (d)

(e)








Gene Sequence Size







0 5 10 15 20 25 30

Accu

mul

atio

n pe

rcen

tage

Release time (day)

0

10

20

30

40

50

60

70

of T

GF-1205733

()









(a) (b)

(c) (d)

200120583m

(e)

Perc

enta

ge o

f liv

ing

cells

()

012

9493

9596979899

100C

ontro

l

cont

rol

Neg

ativ

e

PLG

AN

Ps

TGF-1205733

TGF-1205733-

PLG

AN

Ps

(f)







Day 7 Day 14 Day 28


0

1

2

3

4


AB

AB

ABAB

A

lowast

lowast

lowast

lowast

lowast

OD

(450

nm)



4 Discussion






0

1

2

3

4

5Re

lativ

e gen

e exp

ress

ion

Control

A

AB

ABC


(a)

0

1

2

3

4

5

Relat

ive g

ene e

xpre

ssio

n

AB

ABC


TGF-1205733-PLGANPs

(b)

0

10

20

30

40

Relat

ive g

ene e

xpre

ssio

n

AB

ABC


TGF-1205733-PLGANPs

(c)

0

1

2

3

4

Relat

ive g

ene e

xpre

ssio

n

ABAB


TGF-1205733-PLGANPs

(d)







05

1

15

2

25


TGF-1205733-PLGANPs

A A A A

ABAB

lowast

lowast

lowast

lowast

lowastD

NA

cont

ent (120583

gco

nstr

uct)

(a)


50

100

150

200

250

300


TGF-1205733-PLGANPs

A

A A

ABAB

lowast

lowast

lowast

lowast

lowast

GAG

cont

ent (120583

gco

nstr

uct)

(b)


2

4

6

8

10

12

14

16


TGF-1205733-PLGANPs

A A

ABAB

B

lowast

lowast

lowastlowast

lowastlowast

Hyd

roxy

prol

ine c

onte

nt (120583

gco

nstr

uct)

(c)






(a1) (d1)(c1)(b1)

Agg

reca

nTy

pe II

colla

gen


(e1)

(a2) (d2)(c2)(b2) (e2)

50120583m











5 Conclusion


Competing Interests




Acknowledgments



References































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Gene Sequence Size







0 5 10 15 20 25 30

Accu

mul

atio

n pe

rcen

tage

Release time (day)

0

10

20

30

40

50

60

70

of T

GF-1205733

()









(a) (b)

(c) (d)

200120583m

(e)

Perc

enta

ge o

f liv

ing

cells

()

012

9493

9596979899

100C

ontro

l

cont

rol

Neg

ativ

e

PLG

AN

Ps

TGF-1205733

TGF-1205733-

PLG

AN

Ps

(f)







Day 7 Day 14 Day 28


0

1

2

3

4


AB

AB

ABAB

A

lowast

lowast

lowast

lowast

lowast

OD

(450

nm)



4 Discussion






0

1

2

3

4

5Re

lativ

e gen

e exp

ress

ion

Control

A

AB

ABC


(a)

0

1

2

3

4

5

Relat

ive g

ene e

xpre

ssio

n

AB

ABC


TGF-1205733-PLGANPs

(b)

0

10

20

30

40

Relat

ive g

ene e

xpre

ssio

n

AB

ABC


TGF-1205733-PLGANPs

(c)

0

1

2

3

4

Relat

ive g

ene e

xpre

ssio

n

ABAB


TGF-1205733-PLGANPs

(d)







05

1

15

2

25


TGF-1205733-PLGANPs

A A A A

ABAB

lowast

lowast

lowast

lowast

lowastD

NA

cont

ent (120583

gco

nstr

uct)

(a)


50

100

150

200

250

300


TGF-1205733-PLGANPs

A

A A

ABAB

lowast

lowast

lowast

lowast

lowast

GAG

cont

ent (120583

gco

nstr

uct)

(b)


2

4

6

8

10

12

14

16


TGF-1205733-PLGANPs

A A

ABAB

B

lowast

lowast

lowastlowast

lowastlowast

Hyd

roxy

prol

ine c

onte

nt (120583

gco

nstr

uct)

(c)






(a1) (d1)(c1)(b1)

Agg

reca

nTy

pe II

colla

gen


(e1)

(a2) (d2)(c2)(b2) (e2)

50120583m











5 Conclusion


Competing Interests




Acknowledgments



References































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)

(c) (d)

200120583m

(e)

Perc

enta

ge o

f liv

ing

cells

()

012

9493

9596979899

100C

ontro

l

cont

rol

Neg

ativ

e

PLG

AN

Ps

TGF-1205733

TGF-1205733-

PLG

AN

Ps

(f)







Day 7 Day 14 Day 28


0

1

2

3

4


AB

AB

ABAB

A

lowast

lowast

lowast

lowast

lowast

OD

(450

nm)



4 Discussion






0

1

2

3

4

5Re

lativ

e gen

e exp

ress

ion

Control

A

AB

ABC


(a)

0

1

2

3

4

5

Relat

ive g

ene e

xpre

ssio

n

AB

ABC


TGF-1205733-PLGANPs

(b)

0

10

20

30

40

Relat

ive g

ene e

xpre

ssio

n

AB

ABC


TGF-1205733-PLGANPs

(c)

0

1

2

3

4

Relat

ive g

ene e

xpre

ssio

n

ABAB


TGF-1205733-PLGANPs

(d)







05

1

15

2

25


TGF-1205733-PLGANPs

A A A A

ABAB

lowast

lowast

lowast

lowast

lowastD

NA

cont

ent (120583

gco

nstr

uct)

(a)


50

100

150

200

250

300


TGF-1205733-PLGANPs

A

A A

ABAB

lowast

lowast

lowast

lowast

lowast

GAG

cont

ent (120583

gco

nstr

uct)

(b)


2

4

6

8

10

12

14

16


TGF-1205733-PLGANPs

A A

ABAB

B

lowast

lowast

lowastlowast

lowastlowast

Hyd

roxy

prol

ine c

onte

nt (120583

gco

nstr

uct)

(c)






(a1) (d1)(c1)(b1)

Agg

reca

nTy

pe II

colla

gen


(e1)

(a2) (d2)(c2)(b2) (e2)

50120583m











5 Conclusion


Competing Interests




Acknowledgments



References































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Day 7 Day 14 Day 28


0

1

2

3

4


AB

AB

ABAB

A

lowast

lowast

lowast

lowast

lowast

OD

(450

nm)



4 Discussion






0

1

2

3

4

5Re

lativ

e gen

e exp

ress

ion

Control

A

AB

ABC


(a)

0

1

2

3

4

5

Relat

ive g

ene e

xpre

ssio

n

AB

ABC


TGF-1205733-PLGANPs

(b)

0

10

20

30

40

Relat

ive g

ene e

xpre

ssio

n

AB

ABC


TGF-1205733-PLGANPs

(c)

0

1

2

3

4

Relat

ive g

ene e

xpre

ssio

n

ABAB


TGF-1205733-PLGANPs

(d)







05

1

15

2

25


TGF-1205733-PLGANPs

A A A A

ABAB

lowast

lowast

lowast

lowast

lowastD

NA

cont

ent (120583

gco

nstr

uct)

(a)


50

100

150

200

250

300


TGF-1205733-PLGANPs

A

A A

ABAB

lowast

lowast

lowast

lowast

lowast

GAG

cont

ent (120583

gco

nstr

uct)

(b)


2

4

6

8

10

12

14

16


TGF-1205733-PLGANPs

A A

ABAB

B

lowast

lowast

lowastlowast

lowastlowast

Hyd

roxy

prol

ine c

onte

nt (120583

gco

nstr

uct)

(c)






(a1) (d1)(c1)(b1)

Agg

reca

nTy

pe II

colla

gen


(e1)

(a2) (d2)(c2)(b2) (e2)

50120583m











5 Conclusion


Competing Interests




Acknowledgments



References































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

1

2

3

4

5Re

lativ

e gen

e exp

ress

ion

Control

A

AB

ABC


(a)

0

1

2

3

4

5

Relat

ive g

ene e

xpre

ssio

n

AB

ABC


TGF-1205733-PLGANPs

(b)

0

10

20

30

40

Relat

ive g

ene e

xpre

ssio

n

AB

ABC


TGF-1205733-PLGANPs

(c)

0

1

2

3

4

Relat

ive g

ene e

xpre

ssio

n

ABAB


TGF-1205733-PLGANPs

(d)







05

1

15

2

25


TGF-1205733-PLGANPs

A A A A

ABAB

lowast

lowast

lowast

lowast

lowastD

NA

cont

ent (120583

gco

nstr

uct)

(a)


50

100

150

200

250

300


TGF-1205733-PLGANPs

A

A A

ABAB

lowast

lowast

lowast

lowast

lowast

GAG

cont

ent (120583

gco

nstr

uct)

(b)


2

4

6

8

10

12

14

16


TGF-1205733-PLGANPs

A A

ABAB

B

lowast

lowast

lowastlowast

lowastlowast

Hyd

roxy

prol

ine c

onte

nt (120583

gco

nstr

uct)

(c)






(a1) (d1)(c1)(b1)

Agg

reca

nTy

pe II

colla

gen


(e1)

(a2) (d2)(c2)(b2) (e2)

50120583m











5 Conclusion


Competing Interests




Acknowledgments



References































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



05

1

15

2

25


TGF-1205733-PLGANPs

A A A A

ABAB

lowast

lowast

lowast

lowast

lowastD

NA

cont

ent (120583

gco

nstr

uct)

(a)


50

100

150

200

250

300


TGF-1205733-PLGANPs

A

A A

ABAB

lowast

lowast

lowast

lowast

lowast

GAG

cont

ent (120583

gco

nstr

uct)

(b)


2

4

6

8

10

12

14

16


TGF-1205733-PLGANPs

A A

ABAB

B

lowast

lowast

lowastlowast

lowastlowast

Hyd

roxy

prol

ine c

onte

nt (120583

gco

nstr

uct)

(c)






(a1) (d1)(c1)(b1)

Agg

reca

nTy

pe II

colla

gen


(e1)

(a2) (d2)(c2)(b2) (e2)

50120583m











5 Conclusion


Competing Interests




Acknowledgments



References































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a1) (d1)(c1)(b1)

Agg

reca

nTy

pe II

colla

gen


(e1)

(a2) (d2)(c2)(b2) (e2)

50120583m











5 Conclusion


Competing Interests




Acknowledgments



References































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





5 Conclusion


Competing Interests




Acknowledgments



References































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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