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March 2010 , published 22 , doi: 10.1098/rsta.2010.0013 368 2010 Phil. Trans. R. Soc. A Devendra Verma, Kalpana S. Katti and Dinesh R. Katti nanocomposites hydroxyapatite - polyelectrolyte complex Osteoblast adhesion, proliferation and growth on References related-urls http://rsta.royalsocietypublishing.org/content/368/1917/2083.full.html# Article cited in: l.html#ref-list-1 http://rsta.royalsocietypublishing.org/content/368/1917/2083.ful This article cites 32 articles, 1 of which can be accessed free Subject collections (191 articles) materials science (146 articles) biomedical engineering collections Articles on similar topics can be found in the following Email alerting service here in the box at the top right-hand corner of the article or click Receive free email alerts when new articles cite this article - sign up http://rsta.royalsocietypublishing.org/subscriptions go to: Phil. Trans. R. Soc. A To subscribe to on November 5, 2014 rsta.royalsocietypublishing.org Downloaded from on November 5, 2014 rsta.royalsocietypublishing.org Downloaded from

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Page 1: Osteoblast adhesion, proliferation and growth on polyelectrolyte complex-hydroxyapatite nanocomposites

March 2010, published 22, doi: 10.1098/rsta.2010.0013368 2010 Phil. Trans. R. Soc. A

 Devendra Verma, Kalpana S. Katti and Dinesh R. Katti nanocomposites

hydroxyapatite−polyelectrolyte complex Osteoblast adhesion, proliferation and growth on

  

References

related-urlshttp://rsta.royalsocietypublishing.org/content/368/1917/2083.full.html#

Article cited in: l.html#ref-list-1http://rsta.royalsocietypublishing.org/content/368/1917/2083.ful

This article cites 32 articles, 1 of which can be accessed free

Subject collections

(191 articles)materials science   � (146 articles)biomedical engineering   �

 collectionsArticles on similar topics can be found in the following

Email alerting service herein the box at the top right-hand corner of the article or click Receive free email alerts when new articles cite this article - sign up

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Page 2: Osteoblast adhesion, proliferation and growth on polyelectrolyte complex-hydroxyapatite nanocomposites

Phil. Trans. R. Soc. A (2010) 368, 2083–2097doi:10.1098/rsta.2010.0013

Osteoblast adhesion, proliferation and growthon polyelectrolyte complex–hydroxyapatite

nanocompositesBY DEVENDRA VERMA, KALPANA S. KATTI* AND DINESH R. KATTI

Department of Civil Engineering, North Dakota State University,Fargo, ND 58105, USA

In this work, we have investigated osteoblast adhesion, proliferation and differentiationon nanocomposites of chitosan, polygalacturonic acid (PgA) and hydroxyapatite. Thesestudies were done on both two- and three-dimensional (scaffold) samples. Atomicforce microscopy experiments showed nanostructuring of film samples. Scaffolds wereprepared by freeze-drying methods. The mechanical response and porosity of thescaffolds were also determined. The compressive elastic modulus and compressivestrength were determined to be around 0.9 and 0.023 MPa, respectively, and theporosity of these scaffolds was found to be around 97 per cent. Human osteoblastcells were used to study their adhesion, proliferation and differentiation. Optical imageswere collected after different intervals of time of seeding cells. This study indicatedthat chitosan/PgA/hydroxyapatite nanocomposite films and scaffolds promote cellularadhesion, proliferation and differentiation. The formation of bone-like nodules wasobserved after 7 days of seeding cells. The nodule size continues to increase with time, andafter 20 days the size of some nodules was around 735 mm. Scanning electron microscopeimages of nodules showed the presence of extracellular matrix. The alizarin red S stainingtechnique was used to confirm mineralization of these nodules.

Keywords: biomaterials; chitosan; hydroxyapatite; scaffold; polygalacturonic acid

1. Introduction

The replacement of biological human tissues with living and non-living constructsfalls into the realm of a large portion of the biomaterials industry. Tissue engi-neering remains the most favourable technique, with the potential for replacementof biocompatible tissues and organs (Vacanti 2006), although materials that areinvestigated for hard-tissue replacements (Katti 2004) are made using advancedtechniques in materials science, engineering and nanotechnology. Optimizedgrowth, proliferation and the osteogenic nature of the cells as well as appropriatescaffold degradation remain the challenging features of successful tissue-engineered bone. Cell morphology, cytoskeletal structure, adhesion and differen-tiation can strongly depend on substrate stiffness under conditions where chemicalsignals are constant. It has been shown that, for proper functioning, the matrix*Author for correspondence ([email protected]).

One contribution of 14 to a Theme Issue ‘Advanced processing of biomaterials’.

This journal is © 2010 The Royal Society2083

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should provide a similar mechanical response to the host environment for cellsin the body. Osteoblast cells reside in the rigid matrix provided by collagenfibres and hydroxyapatite crystals. Hence, for growth of functional bone tissue, itis of the utmost necessity that scaffolds provide a mechanical response similarto that of bone matrix. Bone serves as the primary mechanical support forthe body. Thus, scaffolds used for bone tissue engineering should also provideadequate mechanical support to prevent the collapse of neonatal tissue. Materialrequirements for scaffold design are stringent and, in the quest to develop newmaterials, researchers often have to deal with multi-objective problems. Thescaffolds should be biocompatible, biodegradable and show adequate mechanicalresponse to support cellular adhesion, proliferation and differentiation (Sun et al.2004; Ma 2008; Stevens 2008). The mechanical response of the scaffold is animportant factor especially for bone tissue engineering (Hollister 2006). If thescaffold cannot provide a mechanical modulus in the range of that of hard tissue(10–1500 MPa), then any nascent tissue formation will probably also fail owing toexcessive deformation (Goulet et al. 1994). The mechanical response of scaffoldsis inversely proportional to the porosity. A porosity of scaffold around 90 per centhas been considered optimal for tissue engineering (Karageorgiou & Kaplan2005; Mohamad Yunos et al. 2008). At such high porosity, mechanical strengthand elastic modulus decrease significantly. Various methods such as porogenleaching, freeze-drying, gas foaming and rapid prototyping have been used for thefabrication of scaffolds (Randolph et al. 2003; Salgado et al. 2004; Harris et al.2008; Peltola et al. 2008; Stevens et al. 2008). Although scaffolds made of syntheticpolymers such as polycaprolactone and polylactic acid show better mechanicalresponse than scaffolds made from natural polymers, they are hydrophobic innature and also not biofunctional (Ma & Choi 2001; Sherwood et al. 2002;Bhowmik et al. 2007; Verma et al. 2008a,b). Also, the recent literature indicatesthat the size and scale of the scaffold structures also affect the morphologyand functionality of cells. Further, it is also known that cells often adhere andproliferate well in nanostructured materials (Laurencin et al. 1999; Teixeira et al.2003; Jayaraman et al. 2004).

Although biopolymers such as chitosan, chitin, collagen, etc. are biofunctional,their inadequate mechanical strength and deteriorating structural integrity underwet conditions are major drawbacks. In our previous work, for enhancementof the mechanical properties of biopolymer-based composites, we developedcomposites of hydroxyapatite (HAP) with chitosan (Chi) and polygalacturonicacid (PgA) (Verma et al. 2009) and also of hydroxyapatite with clays and chitosan(Katti et al. 2008). The two biopolymers, chitosan and PgA, are biocompatible,biodegradable and also electrostatically complementary to each other. Thisstrategy led to significant improvement in the mechanical properties of the newcomposites. We observed increases of more than 100 per cent in the values of theelastic modulus and ultimate compressive strength (Verma et al. 2009). Analysisof the nanostructure using atomic force microscopy (AFM) revealed a multi-level organization in these composites. The enhancement in mechanical responsewas attributed to stronger interfaces due to strong electrostatic interactionbetween the oppositely charged chitosan and PgA. In the present work, wesynthesized scaffolds made from two electrostatically complementary biopolymersand hydroxyapatite. The biopolymers used for scaffold fabrication are chitosanand PgA. PgA is a de-esterified form of pectin. The suitability of these scaffolds

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for bone tissue engineering has been evaluated by studying the cellular responseof human osteoblast cells. Hydroxyapatite-containing chitosan/PgA scaffoldsmaintained their structural integrity under wet conditions. These scaffolds showedan extremely porous (approx. 97%) and interconnected architecture. Thesescaffolds also promoted cell adhesion, proliferation and differentiation.

2. Materials and methods

(a) Materials

Na2HPO4, ultrapure bioreagent grade, was obtained from J. T. Baker. GR gradeCaCl2 was obtained from EM Science. Chitosan and PgA were obtained fromSigma-Aldrich Chemicals. All these materials are used as supplied.

(b) Synthesis of films and scaffolds

The chitosan solution was prepared by dissolving 1 g chitosan in 100 mlof deionized water, and the PgA solution was prepared by dissolving 1 gPgA in 100 ml of deionized water. These two solutions were mixed togetherby adding chitosan solution drop-wise to PgA solution. Further, the mixedsolution was sonicated and centrifuged to remove extra water, followed byfreeze-drying. The freezing of the solution was done by immersing a beakercontaining the solution into liquid nitrogen. Composite scaffolds of ChiPgAand HAP were also made. ChiPgAHAP composite scaffolds were made byadding HAP solution to ChiPgA solution prior to freeze-drying. After theaddition of HAP, the resulting solution was further sonicated for proper mixing.Scaffolds with two different concentrations of HAP were made: 10 per cent(ChiPgAHAP10) and 20 per cent (ChiPgAHAP20). In order to synthesize filmsof ChiPgA, ChiPgAHAP10 and ChiPgAHAP20, these solutions were diluted in1 : 10 ratio and 3 ml of the resultant solution of each composition was air-driedon tissue culture polystyrene petri dishes. Films of each composition were madein triplicate. Films of ChiPgAHAP10 and ChiPgAHAP20 were also preparedon a 24-well plate for an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) cell growth study.

(c) Porosity determination

The porosity of the porous scaffolds was determined by measuring thedimensions and mass of the scaffolds and comparing the calculated density withthe density of the solid nanocomposites. The apparent density r (Hoemann et al.2005) of the scaffolds was calculated as r = mV −1, where m is the mass and Vis the volume of the porous scaffolds. The porosity of the porous scaffolds wascalculated from

porosity =(

1 − r

rs

),

where rs is the density of the solid nanocomposite, determined to be 1.5 g cm−3.

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(a) (b)

Figure 1. AFM phase images acquired at tapping mode of (a) ChiPgA and (b) ChiPgAHAP20.Scale bars, 500 nm.

(d) Cell culture

Human foetal osteoblast hFOB 1.19 (hFOB) cells were propagated on ChiPgA,ChiPgAHAP10 and ChiPgAHAP20 films. Cells were also seeded in porousscaffolds of ChiPgAHAP20. The cell culture medium used for these studies wasa 1 : 1 mixture of Ham’s F12 Medium and Dulbecco’s Modified Eagle’s Mediumwith 2.5 mM L-glutamine (without phenol red). To make complete growth media,0.3 mg ml−1 G418 antibiotic and 10 per cent foetal bovine serum was added to the1 : 1 mixture of Ham’s F12 Medium and Dulbecco’s Modified Eagle’s Medium.

(e) MTT detection of viable cells

The MTT solution was used to analyse the growth rate of osteoblastcells on ChiPgAHAP10 and ChiPgAHAP20 films. MTT is a dye that iscaptured by the viable cells and reduced by a mitochondrial reaction toformazan. This product accumulates inside the cells and, since it is unableto pass through the cell membrane, the cytoplasm acquires a violet colour.The amount of formazan formed is directly proportional to the number oflive cells. For this study, osteoblast cells were seeded on ChiPgAHAP10 andChiPgAHAP20 films grown in 24-well plates. To each well, 0.5 ml of cell-containing medium was added and, after specific intervals of time (1, 3,7 and 10 days), the MTT solution (100 ml) was added to four wells andincubated for 3 h followed by the addition of 0.5 ml of dimethyl sulphoxideto each well containing MTT solution and incubated at room temperaturefor 1 h. After 1 h, absorbance values were recorded at l = 550 nm using amicroplate reader.

3. Experimental

Compression tests of composites were done at a loading rate of 2 N min−1 using anMTS materials testing servo-mechanical test frame (Materials Testing Solutions,Eden Prairie, MN, USA). AFM phase imaging was performed using a multi-mode

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15 kV

Figure 2. SEM image of ChiPgAHAP20 scaffold. Scale bar, 100 mm.

compression regime

deformationregime

elastic regime

strain, e

stre

ss, s

Figure 3. A representative plot depicting the stress–strain behaviour of the ChiPgAHAP scaffoldunder load.

AFM having a Nanoscope-IIIa controller equipped with a J-type piezo scanner(Veeco Metrology Group, Santa Barbara, CA, USA). The morphology of theporous composites was analysed using secondary electron imaging on a JEOL6000, JSM 6300 V scanning electron microscope (SEM).

4. Results and discussion

(a) AFM images of thin films

Figure 1 shows AFM images of ChiPgA and ChiPgAHAP film samples. Bothsamples exhibit nanostructured surfaces. This nanostructuring of surfaces occursdue to the fact that ChiPgA forms nanoparticles upon mixing of chitosan andPgA in solution. Additionally, HAP particles are also nanosized. Coalescing ofoppositely charged polymers into nanoparticles in solution is characteristic of

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(a)

(b)

Figure 4. Optical images obtained from inverted microscope after 1 day of seeding cells on(a) ChiPgAHAP10 and (b) ChiPgAHAP20. Scale bars, 100 mm.

polyelectrolyte complexes. The nanoparticles have core–shell structures, with thecore containing both polymers, and the shell made up of the polymer in excess.The size and surface characteristics of these nanocolloids are dependent on therelative molecular weight of the polymers, order of mixing, etc. AFM images showthat ChiPgAHAP samples have larger particle sizes; however, a closer look atthese particles shows that they are made of smaller particles and have a similarsize to that observed in the case of ChiPgA samples. Hydroxyapatite particleswere also observed in ChiPgA samples.

(b) Structure and mechanical properties of scaffolds

Figure 2 shows an SEM image of the ChiPgAHAP20 scaffold. The pore sizesin this scaffold were found to be in the range of 100 mm. The porosity of thesescaffolds is around 97 per cent. Previous studies on chitosan/calcium phosphate(30% calcium phosphate) scaffolds showed compressive elastic modulus andstrength around 1.7 and 0.17 MPa, with porosity around 87.5 per cent (Zhang &Zhang 2001). In another study, compressive elastic modulus and mechanicalstrength were reported to be around 0.46 and 8.16 MPa for chitosan–alginatescaffold, with 91.9 per cent porosity (Li et al. 2005). The ChiPgAHAP20 scaffold

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(a)

(b)

Figure 5. Optical images obtained from inverted microscope after 4 days of seeding cells on(a) ChiPgAHAP10 and (b) ChiPgAHAP20. Scale bars, 100 mm.

showed three distinct regions in compressive stress–strain plots (figure 3). In thefirst region, the response was linear and the elastic modulus was calculated fromthe slope of this region. In the second region, the strain–stress response resultedfrom the collapsed scaffold and, in this region, the slope of the curve decreasedsignificantly. Compressive mechanical strength was determined from the onsetof the second region. Further compression caused densification of the scaffold,and this region showed an increase in the slope of the stress–strain curve. Thecompressive elastic modulus and mechanical strength of these scaffolds are foundto be around 0.9 and 0.023 MPa, respectively. Porosity has a major influenceon the mechanical response of scaffolds. The low mechanical strength of theChiPgAHAP20 scaffold could be attributed to its highly porous structure.

(c) Biocompatibility studies on films

Human osteoblast cells were seeded on films of ChiPgA and hydroxyapatite-containing ChiPgA. The cells do not adhere to ChiPgA films and tend to form aspherical morphology over the substrate surface. Further studies on osteoblastadhesion, proliferation and differentiation were conducted on ChiPgAHAP10and ChiPgAHAP20 films. These films were directly formed on polystyrene

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Figure 6. Optical image obtained from inverted microscope after 1 day of seeding cells on ChiPgAwith 60% chitosan. Scale bar, 200 mm.

(a)

(b)

Figure 7. Optical images obtained from inverted microscope after 7 days of seeding cellson (a) ChiPgAHAP10 and (b) ChiPgAHAP20. Bone nodules were observed after 7 days ofseeding cells. These samples were stained with live/dead assay. Grey colour indicates live cells.Scale bars, 100 mm.

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735 µm

Figure 8. Optical image showing osteoblast bone nodule. Scale bar, 200 mm.

Figure 9. Osteoblast nodule stained with alizarin red S. Red colour of nodule shows mineralization.Substrate also shows red colour because it also contains hydroxyapatite. Scale bar, 200 mm.

petri dishes. Optical images after 1 day of seeding cells indicate that, in bothChiPgAHAP10 and ChiPgAHAP20 samples, cells start adhering to the substrate.The morphology of the cells was observed to be intermediate between flatand spherical shape (figure 4). Figure 5 shows optical images after 4 days ofseeding cells. These images suggest that the osteoblast cells have attained a flatmorphology. This flat morphology of cells is an indicator of good adhesion of cellsonto the substrate.

Adhesion of osteoblast cells onto the substrate is a necessary step beforecells proliferate and differentiate further. Surface charge is a critical factor foradhesion of cells to substrate. Previous studies on osteoblast cells have shownthat the positively charged surface promotes cellular adhesion (Amaral et al.2007). ChiPgA may have a negatively charged surface, which hindered adhesionof cells. The surface charge of ChiPgA was varied by mixing chitosan and PgAin different ratios. Osteoblast cells did not adhere to ChiPgA with chitosanconcentration below 60 per cent. However, ChiPgA containing 60 per centchitosan favoured adhesion of osteoblast cells (figure 6). As discussed earlier,

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Figure 10. Optical image showing that some of the nodules detach from the substrate and start tofloat on the surface. As shown, these nodules coalesce together. Scale bar, 200 mm.

180

160

140

120

100

num

ber

of c

ells

1000

)

80

60

40

20

01 3 7

number of days10

Figure 11. Osteoblast cellular growth evaluated by MTT assay. Osteoblasts showed higher growthrate on ChiPgAHAP20 (dark grey) than on ChiPgAHAP10 (light grey) samples.

ChiPgA samples containing HAP also favoured adhesion of osteoblast cells.The addition of hydroxyapatite to ChiPgA allowed adhesion of osteoblast cells.Hydroxyapatite can chelate with negatively charged polymers. We believe that,in ChiPgAHAP samples, hydroxyapatite neutralized the negative surface chargeand thus favoured osteoblast adhesion.

After 7 days of seeding, it was observed that there is an increase in numberof cells, which shows that these samples promote cellular proliferation. Sampleswere stained with live/dead assay and all cells were found alive. Nodule formationby osteoblast cells was also observed in these samples. This is shown in figure 7.The size of these nodules was found to be below 100 mm. The size and numberof nodules increased with seeding time. After 10 days of seeding cells, most of

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(a)

25 kV ×110 17/AUG/07

15 kV ×9000 17/AUG/07

15 kV ×50 000 17/AUG/07

(b)

(c)

Figure 12. SEM images of (a) osteoblast nodule, (b) osteoblast nodule showing ECM produced byosteoblast cells, and (c) osteoblast nodule showing that ECM produced by osteoblast cells exhibitprimarily fibrous morphology. Scale bars, (a) 100 mm, (b) 2 mm, (c), 0.5 mm.

the nodules have sizes in the range of 250–500 mm, while some of these noduleswere observed to have grown to more than 500 mm and sometimes attainedsizes around 700–900 mm (figure 8). The increases in size and in the number ofnodules were observed in both the ChiPgAHAP10 and ChiPgAHAP20 samples.Mineralization of these nodules was studied by staining them with alizarin red S.

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(a)

(b)

15 kV ×1000 17/AUG/07

15 kV ×3300 17/AUG/07

Figure 13. SEM images showing (a) osteoblast cells growing in ChiPgAHAP20 scaffold and (b) thesame cells and scaffold at higher magnification. Scale bars, (a) 10 mm and (b) 5 mm.

The substrate also showed red staining owing to the presence of hydroxyapatitein the films (figure 9). These nodules appear to detach from the substrate, floaton the surface and coalesce together (figure 10). Osteogenic supplements suchas ascorbic acid, dexamethasone and b-glycerophosphate are extensively used toinduce in vitro mineralization (Franceschi et al. 1994; Canalis & Delany 2002). Inthe ChiPgAHAP10 and ChiPgAHAP20 samples, mineralization occurred withoutthe addition of any mineralizing supplement. Mineralization by osteoblast cellsin the absence of any such supplements has also been reported for bioactiveglasses (Jones et al. 2007). In bioactive glasses, silicon is believed to causethe necessary signals for mineralization (Xynos et al. 2001). In ChiPgAHAPnanocomposites, there is no silicon present and no supplement is added to causemineralization. The mineralization mechanism in these nanocomposites is yet tobe understood.

The growth of osteoblast cells over the period of 10 days was studied by MTTassay. This study was done on films of ChiPgAHAP10 and ChiPgAHAP20. Theinitial cell density was 1 × 105 cells per well. Figure 11 shows the average numberof cells determined from MTT tests after 1, 3, 7 and 10 days. The MTT resultsindicate that both ChiPgAHAP10 and ChiPgAHAP20 favour cellular growth, anda higher concentration of hydroxyapatite causes a higher cellular growth rate.

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Figure 12a–c shows SEM images of a nodule. These images show thatcells are bridged by extracellular matrix (ECM). The ECM produced byosteoblasts exhibit a fibrous morphology and so this ECM may primarily becomposed of collagen fibres. Previous study shows that a higher concentration ofhydroxyapatite in ChiPgAHAP nanocomposite favours a higher cellular growthrate. Hence, osteoblast cellular behaviour was only investigated in scaffolds madeof ChiPgAHAP20. In these samples, cells have spherical morphology, in contrastto the flat morphology, observed in film samples (figure 13). Collagen fibres werealso observed within the scaffolds. In addition, in the scaffolds, cells produceECM-containing fibres. However, the fibres observed within the scaffolds arethicker compared with those observed within the nodules on film samples. Thethickness of fibres produced by osteoblasts in film samples was determined to bearound 50 nm, whereas the thickness of fibres on scaffolds was determined to bearound 250 nm.

5. Conclusions

Novel nanocomposite scaffolds were synthesized for bone tissue engineering. Theporosity of the scaffold was determined to be around 97 per cent. However, thisscaffold maintained its structural integrity under wet conditions and providedthe necessary mechanical support for cellular growth. The potential for theirapplication in bone tissue engineering was evaluated by investigating cellularresponses using human osteoblast cells. This study showed that osteoblast cellsadhere, proliferate and produce bone-like nodules on film and scaffold samples.These results indicate that chitosan/PgA/hydroxyapatite-based nanocompositeshave significant potential for bone tissue engineering.

This work was supported in part by a grant from the National Science Foundation (CAREER0132768). D.V. would like to acknowledge support from a NDSU Graduate School DoctoralDissertation Award.

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