collagen-coated polylactide microcarriers/chitosan hydrogel composite: injectable scaffold for...

Collagen-coated polylactide microcarriers/chitosan hydrogelcomposite: Injectable scaffold for cartilage regeneration

Yi Hong,1,2 Yihong Gong,1,2 Changyou Gao,1,2 Jiacong Shen1,2

1Key Laboratory of Macromolecule Synthesis and Functionalization, Ministry of Education,Zhejiang University, Hangzhou 310027, China2Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China

Received 2 June 2006; revised 7 May 2007; accepted 14 June 2007Published online 5 September 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.31603

Abstract: A novel structure of injectable scaffold isdesigned and fabricated by combining collagen-coated poly-lactide (PLA) microcarriers and crosslinkable chitosan hydro-gel. The collagen-coated PLA microcarriers were firstlymixed with the hydrogel precursor, a thickening agent ofkonjac glucomannan (KGM), and redox initiators of ammo-nium persulfate and tetramethylethylenediamine (TMEDA).The mixture was then injected into a mold and incubated at378C to obtain the composite scaffold. The hydrogel candeliver the collagen-coated PLA microcarriers to the desiredsite and, after gelation, will prevent them from uncontrolledmovement. On the other hand, the collagen-coated PLAmicrocarriers can substantially enhance the mechanical prop-erties of the composite system. It was found that the micro-carriers suspended stably in 0.6% KGM/1% chitosan deriva-tive (CML) solution at 378C at least for 15 min. The dynamicelastic modulus (G0) of the composite scaffold increased

along with the increase of the microcarrier content. G0 of thecomposite scaffold with 10% microcarriers was measured as0.87–2.15 MPa at a frequency range of 0.1–100 rad/s, whichwas 120–90 times higher than that of its hydrogel systemalone (12.1–24.4 kPa). In vitro culture of chondrocytes/com-posite scaffold showed that the cell metabolic activityincreased rapidly before day 9, then leveled off. Cells in thehydrogel could attach and grow on the surface of the colla-gen-coated PLA microcarriers to form confluent cell layersafter days 9–12. These features make the composite scaffoldto be injectable and applicable in either tissue engineering,or regenerative medicine, and in particular, in orthopaedics.� 2007 Wiley Periodicals, Inc. J Biomed Mater Res 85A: 628–637, 2008

Key words: injectable; collagen; polylactide; microcarriers;chitosan hydrogel; scaffold

INTRODUCTION

Injectable scaffolds are causing much of attentionin tissue engineering and regenerative medicinebecause of the in vivo culture environment for thedelivered cells, minimal invasion, lower cost of sur-gical procedures, and so forth.1,2 According to thephysical formats of the injectable scaffolds, they canbe classified into injectable hydrogels and injectablecell microcarriers.

Injectable hydrogels have been widely used as inject-able scaffolds for their similar structure to the extracellu-lar matrix (ECM) and mild sol-gel transition conditions.The hydrogel precursor is mixed with cells in vitro, andthen the mixture is injected into a demanded site andgelled in situ under physical (such as temperature andUV irradiation) or chemical (such as initiator) inspira-tion to form a three-dimensional scaffold. Natural bio-macromolecules such as collagen,3,4 chitosan,5,6 algi-nate,7–9 hyaluronan,10,11 and so forth, and syntheticmaterials such as poly(ethylene glycol),12,13 poly(propylfumarate),14,15 Pluronic F127,16 and so forth, have beenapplied as the injectable hydrogels. Although biode-gradable and injectable hydrogels possess flowability,shaping ability, and biocompatibility, they generallyhave lower mechanical strength and a higher degrada-tion rate. On the other hand, cell microcarriers withproper shape and size can also be injected into the dam-aged site by delivering within an appropriate liquid me-dium to pile a 3D porous scaffold in vivo. Recently, themicrocarriers made of polylactide (PLA) and poly(lac-tide-co-glycolide) have been used to culture stem cells,smooth muscle cells, and chondrocytes.17–23 In vivo tests

Correspondence to: C. Gao; e-mail: [email protected] grant sponsor: Major State Basic Research Pro-

gram of China; contract grant number: 2005CB623902Contract grant sponsor: National High-Tech Research

and Development Program; contract grant number:2006AA03Z442Contract grant sponsor: Ph.D. Programs Foundation of

Ministry of Education of China; contract grant number:20050335035Contract grant sponsor: National Science Fund for Dis-

tinguished Young Scholars of China; contract grant num-ber: 50425311

' 2007 Wiley Periodicals, Inc.

proved that new tissues can be formed on the injectablecell microcarriers.20,23 Nonetheless, the scaffold piled bymicrocarriers still has limited mechanical strength.Moreover, by this way it is difficult to form a scaffold fit-ting to the shape and size of the damaged tissue. Some-times, the technique also suffers the uncontrolled move-ment of the microcarriers in vivo (observed during theexperiments).

Combination of these two kinds of injectable scaf-folds may solve these problems. The composite scaf-fold may also have synergetic properties withrespect to enough strength, shape persistence ability,good biocompatibility, and a suitable degradationrate. The uncontrolled movement of the microcar-riers may also be avoided, and the problem of deliv-ering the microcarriers with attached viable cells canalso be solved simultaneously. After gelation in situ,the hydrogel/microcarriers forms a three-dimen-sional structure. It can be further predicted that themicrocarriers can still preserve a three-dimensionalporous structure to provide necessary space for cellgrowth and tissue regeneration after the hydrogelhas been completely degraded. Therefore, this con-ception may not only provide a yet largely unex-plored methodology for biomaterial fabrication, butmay also yield scaffolds with a novel structure forapplications in tissue engineering and regenerativemedicine. Recently, gelatin microspheres loadedwith TGF-b1 (MPs) and chondrocytes were mixedwith oligo(poly(ethylene glycol) fumarate), forming aMPs/cell/hydrogel composite under 378C. Chondro-

cytes survived, proliferated, and kept their activityin this composite scaffold.24

To demonstrate the idea, herein collagen-coatedPLA microspheres (the so called microcarriers) aremixed with a water-soluble and crosslinkable chito-san hydrogel to form a composite scaffold in mildconditions. The chitosan derivative is gelled by aredox initiating system composed of ammoniumpersulfate (APS) and tetramethylethylenediamine(TMEDA) at 378C. At the same time, to ensure thesuspension of the collagen-coated PLA microcarriersfor a fluent injection, a thickening agent, konjac glu-comannan (KGM), is incorporated into the compositesystem to increase the viscosity of the solution.Chondrocytes isolated from rabbit ears are furtherseeded into the scaffold, both on the surface of themicrocarriers and in the hydrogel, and culturedin vitro to estimate the efficacy of this strategy.

EXPERIMENTAL

Materials

PLA microspheres coated with a layer of collagen ontheir surfaces (the so-called PLA microcarriers) were fabri-cated by a grafting–coating method described previously.25

The collagen content is *6.4 lg/mg microspheres. Awater-soluble and crosslinkable chitosan derivative [CML,Fig. 1(a)] with grafting ratios of 23% and 52% for metha-crylic acid (MAA) and lactic acid (LA), respectively, was

Figure 1. The molecular structures of (a) water-soluble chitosan (CML) grafted with methylacrylic acid and lactic acidand (b) konjac glucomannan (KGM).

COLLAGEN-COATED POLYLACTIDE MICROCARRIERS/CHITOSAN HYDROGEL COMPOSITE 629

Journal of Biomedical Materials Research Part A

synthesized by grafting MAA and LA under the catalysisof water-soluble carbodiimide according to Ref. 26. KGMpowder (>98%) was gifted by Meiligao Keji Company,Hubei, China. APS (>98%; Yixing Second Chemical Rea-gent Company, China) was purified via recrystallization.N,N,N0,N0-tetramethylethylenediamine (TMEDA, >98%; Qian-jin Chemical Reagent Factory, Shanghai, China) was used asreceived.

Fabrication of KGM/CML hydrogel

CML was dissolved in water to obtain 1% (w/v) CMLsolution, into which varying amount of KGM powder wasadded to obtain a KGM/CML solution. APS and TMEDAwere added sequentially with a final concentration of5 mM. Finally this mixture was injected into a mold by asyringe at 378C to obtain the KGM/CML hydrogel afterincubation for varying time periods. Gelation time of theKGM/CML hydrogels was recorded.

Suspension ability of collagen-coated PLAmicrocarriers in KGM/CML solutions

The collagen-coated PLA microcarriers were mixed with1% (w/v) CML solution containing different amounts ofKGM. The mixtures were placed in a water bath at 378C.After 15 min, the macroscopic suspension property of thecollagen-coated PLA microcarriers in the KGM/CML solu-tions was recorded by a digital camera.

Fabrication of collagen-coated PLA microcarriers/chitosan hydrogel composite scaffold

CML was dissolved in water to obtain 1% (w/v) CMLsolution, into which KGM powder was added to obtain0.6% (w/v) KGM/1% (w/v) CML solution. APS andTMEDA were added sequentially with a final concentra-tion of 5 mM. The collagen-coated PLA microcarriers werethen mixed with this solution. The mixture was injectedinto a mold by a syringe and incubated at 378C for 8 minto obtain the collagen-coated PLA microcarriers/chitosanhydrogel composite scaffolds. After lyophilization, themicrocarriers/chitosan hydrogel composite scaffolds werecoated with a thin gold layer and observed by scanningelectron microscopy (SEM) (SIRION, FEI).

Dynamic elastic modulus measurement

One percent (w/v) CML solution, KGM/CML solutions,and PLA microspheres/KGM/CML mixtures with 5 mMinitiators were placed in a cylinder mold at 378C to obtainhydrogels with a height of 1.8 mm and a diameter of25 mm, respectively. Dynamic elastic modulus was meas-ured on an Advanced Rheometric Expansion System(ARES) using parallel board mode at 378C. The sampleswere firstly scanned at a strain of 0.1–100% and a fre-quency of 1 rad/s to ensure the linear region, and then

scanned at a frequency of 0.1–100 rad/s and at a definitestrain. It is worth mentioning that here water is usedinstead of PBS, although ions from PBS may also haveinfluence on the hydrogel’s mechanical properties. Yetthis influence should be very minimal since the presentgelation mechanism is covalent bonding between chitosanmolecules, and the ionic concentration of PBS is not veryhigh.

Chondrocyte culture

Chondrocytes were isolated from cartilage tissue of rab-bit ears (Japanese big ear white). Briefly, cartilage tissuewas cut into small pieces. Chondrocytes were isolated byincubating the cartilage pieces in Dulbecco’s minimumessential medium (DMEM) containing 0.2% collagenasetype II (Sigma) at 378C for 6 h under agitation. The iso-lated chondrocytes were centrifuged, resuspended inDMEM supplemented with 10% fetal bovine serum (FBS),100 U/mL penicillin, and 100 lg/mL streptomycin. Thecell suspension was then seeded in an 11-cm plastic tissueculture dish (Falcon) and incubated in a humidified atmos-phere of 95% air and 5% CO2 at 378C. After a confluentcell layer was formed, the cells were detached using 0.25%trypsin in PBS and were resuspended in PBS, and used forthe experiments.

Chondrocytes seeding in the collagen-coatedPLA microcarriers/chitosan hydrogel scaffold

Chondrocytes were seeded on the collagen-coated PLAmicrocarriers as described previously25 and culturedin vitro for 1 week, and then used for the following experi-ments.

CML and KGM powder were sterilized under UV irra-diation for 3 h. CML (50 mg) was dissolved in PBS toobtain 5 mL (1%, w/v) CML/PBS solution, into which30 mg KGM powder was added to obtain 5 mL (0.6%, w/v)KGM/(1%, w/v) CML/PBS solution. One molar solutionsof APS and TMEDA in PBS were prepared and sterilizedby filtering with 0.22-lm membranes, respectively. Fiftymicroliters of APS and 50 lL TMEDA were added into theKGM/CML/PBS solution sequentially. The final initiatorconcentration was 5 mM. Two-hundred microliters ofcells/PBS suspension was mixed with the KGM/CML/PBS solution under gentle agitation. The final cell densityin the mixture was 8 3 106/mL. Finally, 250 mg collagen-coated PLA microcarriers with attached chondrocytes wereadded into the cells/KGM/CML solution. Collagen-coatedPLA microcarriers/hydrogel precursor mixture (250 ll)was injected into a mold by a 1-mL syringe after the mix-ture was uniformly stirred using a magnetic stirrer. Afterplaced into an incubator at 378C for *8 min, a compositescaffold containing chondrocytes was formed. Then 1 mLDMEM supplemented with 10% FBS was added. After30 min, the composite scaffold was transferred to a well ofa 24-well culture plate and then 2 mL DMEM supple-mented with 10% FBS was added. Over the following 3 h,the culture medium was changed three times to removethe unreacted and soluble hydrogel components. During

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the next 12-day culture period, the medium was changedevery 3 days.

Chondrocytes encapsulated in the composite scaffoldswere stained by fluorescein diacetate (FDA, 20 lg/mL)/ethidium bromide (EB, 40 lg/mL) and observed by confo-cal laser scanning microscopy (CLSM, Bio-Rad Radiance2100). FDA and EB were used to stain the viable and deadcells, respectively. Chondrocyte morphology was alsoobserved by SEM (Stereoscan 260, Cambridge) after rou-tine fixation, sequential dehydration, and critical pointdrying.

MTT assay

Three composite scaffolds with cells were taken out andplaced in a 12-well culture plate at desired time intervals.After addition of 100 lL MTT (3-(4,5-dimethyl)thiazol-2-yl-2,5-dimethyl tetrazolium bromide, 5 mg/mL) into thehydrogels, the cells were continually cultured for another4 h. During this period, viable cells could reduce the MTTto formazan pigment, which was dissolved by 1 mL di-methyl sulphoxide (DMSO) after removal of the culturemedium. The hydrogel was smashed and centrifuged at11,000 rpm to ensure the complete dissolution of formazanpigment by DMSO and to remove the suspension productsin the solution. Then the absorbance at 570 nm (150 lL so-lution) was recorded under a microplate reader (Bio-Rad550).

Statistical analysis

Data from all studies were analyzed using one-wayANOVA. Results are reported as mean 6 standard devia-tion. The significant level was set as p < 0.05.

RESULTS AND DISCUSSION

KGM/CML hydrogels and microcarrier suspension

To ensure that the collagen-coated PLA microcar-riers and hydrogel precursor can be successfullyinjected into a damaged site, the microcarriers mustbe suspended in the hydrogel precursor for a suffi-cient long time. The density of the collagen-coatedPLA microcarriers (*1.26 g/cm3) is quite high,while the viscosity of the 1% (w/v) CML solution(*40 cp) is quite low, making that the PLA spherescould not be steadily suspended for enough longtime. As shown in Figure 2 (first set), most of thecollagen-coated PLA microcarriers had sedimentatedon the vessel bottom after mixing with the CML so-lution for 15 min at 378C. Therefore, measures mustbe taken to improve the suspension ability of the col-lagen-coated PLA microcarriers.

Here, KGM was added into the CML solution topronouncedly improve its viscosity, so that sedimen-

tation of the collagen-coated PLA microcarrierscould be slowed down. KGM is a natural polysac-charide obtained from the Amorphophallus konjacplant, having water-soluble and nonionic characteris-tics.27 Its main chain consists of b-1,4 linked man-nose and glucose units [Fig. 1(b)]. Its solution pos-sesses very high viscosity. For example, the viscosityof 0.6% (w/v) KGM solution is as high as *2500 cp(50 rpm, 258C). Because of its biocompatibility andbiodegradability, KGM and its derivatives have beenapplied in the fields of food engineering as foodadditive and thickening agent, in pharmaceuticalsfor drug delivery and cell therapy, in biotechnology,in fine chemistry, and so forth.28 As shown in Figure2 (except for the first set), addition of KGM couldeffectively improve the suspension property of thecollagen-coated PLA microcarriers, in particularwhen its content was larger than 0.6% (w/v), abovewhich no sedimentation of the collagen-coated PLAmicrocarriers was observed during a 15-min storageat 378C.

The gelation time is an important issue of thehydrogel system, which may influence the shapingproperty and cytoviability. Therefore, the gelationtime as a function of KGM concentration was stud-ied. As shown in Figure 3, the gelation time waskept almost constant at 5.5 min when the KGM con-tent was lower than 0.6% (w/v). Above this value,for example at 0.8% (w/v), the gelation time wassignificantly prolonged (p < 0.05). At a higher con-tent of KGM, diffusion of the small free radicals issurely slowed down because of the high viscosity ofthe system, leading to a slower reaction rate of theC¼¼C double bonds, which are responsible for thegelation.

Incorporation of the KGM may also influenceother properties like mechanical strength. Figure 4shows the dynamic elastic modulus (G0) of theKGM/CML hydrogels measured by ARES. In Figure4(a), when the strain was lower than *1%, G0 keptalmost unchanged along with the strain for eachhydrogel system. When the strain reached a criticalpoint (gc), the linear viscoelastic region (LVR) disap-peared and G0 decreased rapidly. This phenomenonis understood as the Payne effect, indicating that thenetwork structure of the KGM/CML hydrogel isdamaged at higher strain.29 Along with the increaseof KGM content, gc shifted to a lower value. Accord-ing to the result of LVR, the G0 values for all thesamples were set at 1% strain. Thus one can observethat the G0 increased from *550 Pa to *6.6 kPawhen the KGM content was changed from 0 to 0.8%(w/v). Figure 4(b) shows that the G0 of the KGM/CML hydrogels increased also along with anincrease of the KGM content. For example, at aKGM content of 0.8% (w/v), the G0 was improvedfrom 1.5 to 93 kPa, when the frequency was changed



from 10�1 to 102 rad/s. These results reveal thataddition of KGM can enhance the mechanicalstrength of the hydrogel system, but can result inunstable hydrogel structure which is more sensitiveto larger strain or higher frequency.

Taking into consideration of the suspension prop-erty of the collagen-coated PLA microcarriers, gela-tion time, and mechanical strength of the hydrogel,the KGM content was set as 0.6% (w/v) for the nextexperiments. At this content, the hydrogel systemhas a fast enough gelation time and adequate me-chanical strength, while the sedimentation of the col-lagen-coated PLA microcarriers can be effectivelyavoided.

Collagen-coated PLA microcarriers/chitosanhydrogel composite scaffold

By fixing the composition of the hydrogel systemat 0.6% (w/v) KGM/1% (w/v) CML supplementedwith 5 mM APS/TMEDA, influence of the collagen-coated PLA microcarriers’ content on the propertiesof the resulting composite scaffold was studied. Themixtures containing 2.5–10% (w/v) collagen-coatedPLA microcarriers could be easily injected into amold with a syringe. After gelation at 378C, compos-ite scaffolds with a predefined shape were easilyformed. As shown in Figure 5(a–c), both the micro-carriers and the hydrogel were assembled into onescaffold with a fixed 3D shape. Collagen-coated PLAmicrocarriers were encapsulated and dispersedevenly through the hydrogel. From the enlargedimage shown in Figure 5(d), one can find that thecollagen-coated PLA microcarriers were tightly

Figure 3. The gelation time of KGM/1% CML hydrogelas a function of KGM concentration initiated by 5 mMAPS/TMEDA at 378C (n ¼ 3).

Figure 4. Plots of dynamic storage modulus G0 versus (a) strain g and (b) frequency at 378C for KGM/CML hydrogels with dif-ferent KGM content marked in the insets. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 2. Photo images to show the suspension of colla-gen-coated PLA microcarriers in KGM/CML solution withdifferent KGM content as marked on the bottles after mix-ing for 15 min at 378C. [Color figure can be viewed in theonline issue, which is available at www.interscience.wiley.com.]

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bound within the hydrogel, demonstrating the gela-tion would be helpful to keep the microcarriers inplace. The microcarriers can in turn help shaping thehydrogel.

Similarly to the CML/KGM hydrogel system, thePLA microspheres/hydrogel system presents no sig-nificant difference of G0 for each sample before thecritical strain point (gc). When the strain reached thegc, LVR disappeared and G0 decreased rapidly asshown in Figure 6(a). Again, at this point the struc-

ture of the continuous phase of KGM/CML hydro-gel was damaged.29 The gc shifted to lower strain athigher content of PLA microspheres, which meansthat incorporation of the microcarriers may weakenthe stability of the hydrogel, revealing a largeramount of defects in the composite. Figure 6(a)shows also that the G0 increased tremendously withthe increase of the content of PLA microspheres atlower strain (�1%). For example, when the microcar-riers’ content increased from 0 to 10% (w/v), the G0

Figure 5. SEM images to show the microstructures of collagen-coated PLA microcarriers and 0.6% KGM/1% CML hydro-gel composite with different content of the microcarriers: (a) 2.5%, (b) 5%, and (c) 10%; (d) is higher magnification of (c).

Figure 6. Plots of dynamic storage modulus G0 versus (a) strain g and (b) frequency at 378C for PLA microspheres and0.6% KGM/1% CML hydrogel composites with different content of microspheres marked in the insets. [Color figure canbe viewed in the online issue, which is available at www.interscience.wiley.com.]



of the microcarriers/hydrogel composite scaffoldsincreased from 1.3 to 11.0 kPa. More relevantimprovement of the G0 was found at a higher con-tent of PLA microspheres under a dynamic forceinteraction at a fixed train of 1%, especially in theregion at lower frequency (about 0.1 rad/s) [Fig.6(b)]. For example, when the microcarriers’ contentwas 10% (w/v), the G0 of the composite scaffold was0.87–2.15 MPa at a frequency ranging from 0.1 to100 rad/s. This value was a hundred times higherthan that of the 0.6% KGM/1% CML hydrogel (12.1–24.4 kPa). This enhancement of the mechanicalstrength is of course attributed to the PLA micro-spheres, which can bear very high load than thehydrogel.

In summary, combination of the microcarrierswith the hydrogel has a synergetic effect on the fol-lowing aspects: (1) the hydrogel endows the mixturewith good injectability; (2) after gelation, the hydro-gel can encapsulate and localize the microcarriers toinhibit uncontrolled movement of the later; and (3)the microcarriers significantly enhance the mechani-cal strength of the composite system.

In vitro chondrocyte growth behavior within thecomposite scaffold

As the composite scaffold has been successfullyconstructed, its biofunctionality was further eval-uated by in vitro culture of chondrocytes. In thisstudy, the composite scaffold contained 5% collagen-coated PLA microcarriers loaded with chondrocytes,and a hydrogel of 0.6% KGM/1% CML and 5 mMAPS/TMEDA, and suspended chondrocytes. As

shown in Figure 7, cell viability measured by theMTT assay initially increased with prolongation ofthe culture time (p < 0.05), and then leveled off after9 days (p > 0.05). This is substantially different withthe pure hydrogel system,30 in which the cytoviabil-ity increased initially and then decreased again atlonger culture time (12 days). In that case, progres-sive cell death rather than cell proliferation as meas-ured by DNA assay was concluded, implying thatthe hydrogel does not have enough cytocompatibil-ity to induce cell growth. In contrast, the hugeincrease of the cytoviability in the composite scaffoldindicates that the chondrocytes can retain their meta-bolic activity, which is further confirmed by viablecell staining (Fig. 8) and SEM observations (Fig. 9).

Figure 8 shows that at day 1 [Fig. 8(a,d)], roundviable chondrocytes (green regions), with a size ofabout 10 lm, attached on the collagen-coated PLAmicrocarriers (black round regions) and dispersedrandomly in the hydrogel. Meanwhile, a number ofdead cells (red dots) existed in the hydrogel and onthe microcarriers’ surfaces. Generally, cell death islikely to occur during the procedures of cell seeding,encapsulation, and gelation. After culture for 6 days[Fig. 8(b,e)], cellular clusters emerged and sus-pended in the hydrogel, accompanying with the dis-appearance of single cells. It is interesting to noticethat lots of cells are observed on the surfaces of thecollagen-coated PLA microcarriers [Fig. 8(b)] whereonly a few viable cells or no cells existed initially[Fig. 8(a)]. At day 12 [Fig. 8(c,f)], most of the chon-drocytes are observed either in cell clusters or on thesurfaces of the collagen-coated PLA microcarriers.Single cells can hardly be found.

The cell/scaffold constructs were further subjectedfor SEM investigation. At day 1 [Fig. 9(a,d)], the col-lagen-coated PLA microcarriers were encapsulatedin the hydrogel to form a 3D structure. Round cells[Fig. 9(d), light grey] were encapsulated in thehydrogel and few cells existed on the surfaces of themicrocarriers. At day 6 [Fig. 9(b,e)], the dried com-posite scaffold showed a 3D porous structure piledby the microcarriers with low amount of hydrogel.In Figure 9(e), a great number of cells are observedon the surfaces of the microcarriers. Similar resultscan be observed under SEM after the construct wascultured for 12 days [Fig. 9(c,f)]. Figure 10(a) furtherpresents the morphology of a single round chondro-cyte existing within the hydrogel. Some fine villuson the cell surface can be clearly seen. A cell cluster[Fig. 10(b)] was also detected as shown by theCLSM. Again, on the surfaces of the collagen-coatedPLA microcarriers, a chondrocyte monolayer [Fig.10(c)] was unambiguously formed.

These results provide further evidence for the pro-gressive growth of the chondrocytes, in particular onthe collagen-coated PLA microcarriers. It is very

Figure 7. Cytoviability of a composite system containingcollagen-coated PLA microcarriers with attached chondro-cytes and in 0.6% KGM/CML hydrogel loaded with chon-drocytes as a function of culture time (n ¼ 3). The contentof the microcarriers was 5% and the cell seeding densitywas 8 3 106/mL.

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interesting that some microcarriers with no or few ofcells on their surfaces were abundant with chondro-cytes later on, indicating that cell growth ofremained or returned chondrocytes occurred on themicrocarriers’ surface. Since collagen is an important

component of the ECM and contains lots of informa-tional peptide sequences like RGD, it is understand-able that the collagen-enriched surfaces of the micro-carriers are more cytoinductive than that of the chi-tosan hydrogel, although the latter is often regarded

Figure 8. CLSM images to show live/dead chondrocytes encapsulated in composite of collagen-coated PLA microcarriersand 0.6% KGM/1% CML hydrogel for (a) 1 day, (b) 6 days, (c) 12 days. (d), (e), and (f) are higher magnifications of (a),(b), and (c), respectively. Cells were stained by FDA/EB. The content of collagen-coated PLA microcarriers was 5% andthe cell seeding density was 8 3 106/mL. [Color figure can be viewed in the online issue, which is available at www.inter-science.wiley.com.]

Figure 9. SEM images of a composite system containing collagen-coated PLA microcarriers with attached chondrocytesand in 0.6% KGM/CML hydrogel loaded with chondrocytes after in vitro culture for (a) 1 day, (b) 6 days, (c) 12 days. (d),(e), and (f) are higher magnifications of (a), (b), and (c), respectively. The content of collagen-coated PLA microcarrierswas 5% and the cell seeding density was 8 3 106/mL.



as a biocompatible material too. As previously dem-onstrated,25 upon attachment on the collagen-coatedmicrocarriers, the chondrocytes can then maintaintheir normal phenotype and grow until confluentcell layers are formed on the surfaces. The other rea-son may be due to hydrogel degradation during theculture, which resulted in cellular attachment on themicrocarrier surfaces because the chitosan hydrogelencapsulating the cells is degraded faster.30 At thesame time, from Figure 9 one can conclude that thehydrogel content decreased significantly from 3d[Fig. 9(a)] to 6d [Fig. 9(b)] and 12d [Fig. 9(c)], whichcan possibly be attributed to the degradation of thehydrogel. In a word, by incorporation of the micro-carriers having collagen layers on their surfaces, thecomposite system becomes more cytocompatible, asshown by the steady increase of the cytoviability(Fig. 7) and the large cell population on the surfacesof the collagen-coated PLA microcarriers (Figs. 8 and9). These results indicate that the composite scaffoldcan be potentially applied as an injectable scaffold intissue engineering and regenerative medicine.

CONCLUSIONS

An injectable collagen-coated PLA microcarriers/chitosan hydrogel composite scaffold has been suc-cessfully fabricated. Addition of the thickening agentKGM can effectively increase the viscosity of the

water-soluble and crosslinkable chitosan derivative(CML) solution, thus endowing the collagen-coatedPLA microcarriers with sufficient suspension time inthe hydrogel precursor for delivery and injection.The dynamic elastic modulus of the KGM/CMLhydrogel increases with the increase of the KGMcontent. The gelation time of the CML is kept con-stant when the KGM content is �0.6%. At a fixedhydrogel composition of 0.6% KGM/1% CML and5 mM APS/TMEDA, the dynamic elastic modulus ofthe composite scaffold shows a positive correlationwith the microcarrier content. When the microcarriercontent is 10%, the dynamic elastic modulus of thecomposite scaffold is 0.87–2.15 MPa at a frequencyfrom 0.1 to 100 rad/s, which is 120–90 times higherthan that of the hydrogel (12.1–24.4 kPa). In vitro cul-ture of the construct of chondrocytes/compositescaffold shows that the cytoviability increases rap-idly before 9 days, and then levels off. Cells in thehydrogel can freely move to form cell clusters, andattach and grow on the surfaces of the collagen-coated PLA microcarriers to form confluent celllayers after 9–12 days.

In conclusion, combination of the microcarriersand the hydrogel has a synergetic effect on the fol-lowing aspects: (1) the hydrogel endows the mixturewith good injectability; (2) after gelation, the hydro-gel can encapsulate and localize the microcarriers toinhibit uncontrolled movement of the later; (3) themicrocarriers significantly enhance the mechanicalstrength of the composite system; and (4) cells are

Figure 10. SEM images to show the chondrocyte morphology in the hydrogel (a, b) and on the surface of collagen-coatedPLA microcarriers (c) in the composite scaffold.

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more active than in pure hydrogel. All these featuresensure the potential application of the compositescaffold in areas of either noninvasive tissue engi-neering or regenerative medicine, and in particular,in orthopaedics.

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