Acta Biomaterialia xxx (2010) xxxxxx

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Bone nodules on chitosanpolygalacturonic acidhydroxyapatite nanocomposite lms mimic hierarchy of natural bone
Rohit Khanna, Kalpana S. Katti , Dinesh R. Katti
Department of Civil Engineering, North Dakota State University, Fargo, ND 58105, USA

a r t i c l e

i n f o

a b s t r a c t
The ultimate goal of bone tissue engineering is to develop bony tissues on tissue engineered constructs that mimic the native bone. Nanoscale characterization of in vitro generated bony tissues on engineered scaffolds is essential to understanding both the physical and mechanical characteristics of the engineered bone. Bone nodule formation, a typical early indicator of bone formation was observed on chitosan polygalacturonic acidhydroxyapatite (ChiPgAHAP) nanocomposite lms without the use of differentiating media. Thus, the ChiPgAHAP substrates designed are osteoinductive and provide an appropriate microenvironment for cell organization and tissue regeneration. Imaging using atomic force microscopy revealed several levels of hierarchical structures of bone in the bone nodules, consisting of mineralized collagen bers, brils and mineral deposits in extrabrillar spaces. The nanoscale elastic properties of the collagen and mineral crystals were found to be in close agreement with the experimental and simulations results on natural bone reported in the literature. Fourier transform infrared spectroscopy experiments indicate the presence of collagen and biological apatite in bone nodules exhibiting the characteristics of newly precipitated, immature bone. Collectively, our structural, chemical, and mechanical analyses support the conclusion that synthetic bone nodules mimic the hierarchy of natural bone. 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Article history: Received 20 May 2010 Received in revised form 15 October 2010 Accepted 22 October 2010 Available online xxxx Keywords: Bone nodule Hierarchy Collagen Mineral Modulus mapping

1. Introduction In vitro regeneration of biological tissues such as liver, heart, cartilage, skin, and bone is an active area of research in tissue engineering. Tissue engineering offers a promising alternative to traditional transplants by employing synthetic or naturally derived engineered biomaterials to replace damaged tissues [13]. Materials used for hard tissue regeneration such as bone are made mostly from biodegradable and biocompatible natural and synthetic polymers, which are used in combination with hydroxyapatite (HAP) to develop three-dimensional (3D) porous matrices [47]. Porous matrices serve as a template to guide cell organization and growth and allow diffusion of nutrients to transplanted cells, which ultimately leads to generation of complex architectures which mimic native tissues [8]. Appropriate mechanical properties of the scaffold are also required to maintain cellscaffold load transfer mechanisms during tissue regeneration, which is one of the key factors in the selection and design of biomaterials, especially in generating hard tissues such as bone. Various nanoscale reinforcements, including silicates, have also been investigated to enhance the mechanical response of the scaffolds [9,10].

Corresponding author. Tel.: +1 701 231 9504; fax: +1 701 231 6185.
E-mail address: kalpana.katti@ndsu.edu (K.S. Katti).

Biological tissues are complex and exhibit structural hierarchy, and tissue regeneration on synthetic 3D scaffolds requires appropriate design and control of the physical, mechanical, and chemical behavior over the macro-, micro-, and nanoscale of scaffolds, to mimic the 3D hierarchical architecture of tissues [3,11]. For bone mineralization studies, scaffolds are often treated with various growth factors, such as ascorbic acid, dexamethasone, b-glycerophosphate, and bone morphogenetic protein-2 (BMP-2), to stimulate osteogenesis as they would otherwise not form bone [1214]. Thus, the engineered scaffolds are required to possess osteogenic and osteoinductive properties [15]. Extensive studies of the tissue structure, function, and physiology of bone exist in the literature. Human bone exhibits seven orders of hierarchical structure that spans from sub-nanostructures to macrostructures [1619]. At lower levels of hierarchy, bone is a nanocomposite of collagen and mineral crystals of carbonated HAP, also termed biological apatite [20]. The organic matrix of bone consists of mainly type I collagen and non-collagenous proteins. These are the smallest structural building blocks of bone structure, which are repeated several times leading to functional bone. Hierarchical structures serve many biological functions, such as providing mechanical support to cells and promoting and regulating cellular functions such as cell adhesion, proliferation, differentiation, and vascularization in a physiological environment [21]. The unique mechanical properties of bone are derived primarily

1742-7061/$ - see front matter 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2010.10.028

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from its hierarchical structure and organization at all levels of hierarchy [17,22]. The size, shape, orientation, and organization of mineral crystals in collagen brils regulate the mechanical function of bone. Often, small misorientations of the mineral crystals in bone can lead to bone diseases like osteogenesis imperfecta, a brittle bone disease. Hierarchical structures of various calcifying tissues (turkey and chicken bone) have been studied by various researchers using modern experimental techniques such as high voltage electron microscopy (HVEM), computed tomography [19,23,24], and small angle X-ray scattering (SAXS) [2530]. SAXS and HVEM experiments have revealed the size, geometry, orientation, and spatial distribution of mineral crystals both within and on the surface of collagen brils. These studies examined thick sections of calcifying tissues of chicken, turkey, etc., and performed 3D imaging using HVEM or SAXS to evaluate their structure, and also conducted micro-mechanical analyses. In our prior work we designed and developed chitosanpolygalactouronic acidhydroxyapatite (ChiPgAHAP) biopolymer nanocomposites for bone tissue engineering. Biomimetically synthesized ChiPgAHAP nanocomposite samples exhibited improved mechanical strength due to stronger interfacial interactions [31]. These nanocomposites also exhibited swelling characteristics over time when soaked in cell culture medium, as revealed by in situ physico-chemical and mechanical analyses on soaked substrates [32]. Further, 3D porous ChiPgAHAP scaffolds were found to be osteogenic and osteoinductive and exhibited formation of bone nodules in the absence of differentiating medium [33]. The current work reports nanoscale characterization of bone nodules generated by human osteoblasts on ChiPgAHAP nanocomposite lms. The specic objectives of the present work are: (a) nanoscale structural analysis of bone nodules using AFM to characterize the size, morphology, and spatial distribution of phases present on the surface of bone nodules; (b) to determine the nanoscale elastic properties of different constituents of bone nodules by making shallow indentations ($23 nm) using a modulus mapping technique; (c) to characterize the chemical structures of bone nodules using Fourier transform infrared (FTIR) spectroscopy.

1:10 with DI water and 3 ml of the resulting solution was air dried on TCPS petri dishes. Films made of both compositions were found to be biocompatible. ChiPgA20%HAP was selected for biocompatibility studies in the present work. 3D porous scaffolds were prepared using a freeze drying technique described elsewhere [35]. 2.2. Cell culture and Alizarin Red S staining Human osteoblasts (CRL 11,732, ATCC) were cultured in Dulbeccos modied Eagles medium (DMEM/F12, Hyclone), supplemented with 2.5 mM l-glutamine (without phenol red), 10% fetal bovine serum (FBS, ATCC) and 1% antibiotic (G418, JR Scientic Inc.), referred as complete medium. Film samples were UV sterilized for 1 h. Subsequently, 1 105 cells were seeded onto lm samples and placed under standard cell culture conditions (37 C, 5% CO2) in a humidied atmosphere. The complete medium was replaced every 3 days. Cell cultures were monitored for 2 and 12 h and 10 and 22 days. At the end of predetermined time, phase contrast/uorescence micrographs were recorded using an inverted microscope (Axiovert, Zeiss, Germany). For mineralization studies, cell seeded samples were removed from the incubator after 22 days of culture, washed three times with phosphate-buffered saline (PBS), and xed with 2.5% glutraldehyde for 6 h. Fixed cultures were washed twice with PBS and then stained with Alizarin Red S dye (ARS) (5 mg ml1) for 5 min. Cultures were rinsed thoroughly with water to reduce non-specic ARS staining. The presence of red color is an indicator of calcium deposits in the culture. For nanoscale characterization of bone nodules four nodule samples were carefully obtained from the same culture before xing the samples for ARS staining. Unstained and unxed bone nodule samples were immediately taken for structural, mechanical and chemical analyses. 2.3. Cell proliferation determined by MTT assay Cell proliferation studies were carried out using MTT reagent (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-terazolium bromide) obtained from EMD Biosciences. The MTT assay reduces the yellow tetrazolium salt to purple formazan crystals by mitochondrial dehydrogenase enzymes of metabolically active cells. ChiPgA HAP nanocomposite lms were prepared directly on 24-well plates by solvent evaporation. Completely dried samples were then subjected to UV sterilization for 1 h. Subsequently, 1.5 ml of cell culture medium containing 2 104 cells were seeded into each of the 24 wells. Cell proliferation experiments on ChiPgAHAP lms were carried out for 1, 4, 8, and 22 days. For cell proliferation studies on ChiPgAHAP scaffolds, 1.5 ml of cell culture medium containing 1 105 cells were seeded onto scaffolds and incubated for 2, 4, 8, 12, and 16 days. At predetermined times 150 ll of MTT solution (5 mg ml1 in PBS) was added to each of the wells and incubated for 6 h. The solution was aspirated and 500 ll of dimethylsulfoxide (DMSO) (Mediatech Inc., USA) was added to dissolve formazan crystals. Mean absorbance values were read at 570 nm using a microplate reader (Bio-Rad Laboratories, USA). The resulting absorbance values give a quantitative measure of viable cells in terms of the cell population. All the experiments were run in triplicate. 2.4. Nanostructural analysis The size, morphology, and spatial distribution of the constituents of the bone nodule were characterized by multimode AFM (Veeco Metrology Group, Santa Barbara, CA), equipped with a Nanoscope IIIa controller and J-type piezo scanner. The equipment had a z-axis resolution of around 0.5 . Silicon nitride cantilever probes (model NP-S20) with pyramidal tips of $20 nm radius of

2. Materials and methods 2.1. Synthesis of nanocomposite lms Disodium hydrogen phosphate (J.T. Baker) and calcium chloride (EM Sciences) were used to prepare nanohydroxyapatite particles by the wet chemistry route. The detailed processing route is discussed elsewhere [34]. Chitosan (molecular weight 190,000, >85% deacetylation) and polygalacturonic acid (molecular weight 25,000) were obtained from SigmaAldrich chemicals. For lm preparation, chitosan and PgA powders were dissolved separately in 100 ml of deionized (DI) water. The solution pH values were adjusted to 4.5. The pH of the chitosan and PgA solutions were adjusted by adding acetic acid and NaOH, respectively. Chitosan PgA solution was prepared by drop-wise addition of chitosan solution to PgA solution followed by sonication for 3 min. To synthesise the ChiPgAHAP nanocomposite, HAP nanoparticles (1 g per 100 ml of DI water) were sonicated for 45 min and then mixed with sonicated ChiPgA solution. The resulting solution was sonicated again for 90 s to obtain a uniform dispersion of HAP nanoparticles in the nanocomposite. Further, lms of two compositions were made (ChiPgA10% HAP and ChiPgA20%HAP) on tissue culture polystyrene (TCPS) petri dishes by the solvent evaporation route in a clear air laboratory environment at room temperature. For lm preparation ChiPgAHAP solution was diluted in the ratio

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curvature and a nominal stiffness of 0.06 N m1, were used for imaging under contact mode in a clear air laboratory environment (room temperature, 4050% relative humidity). Height and deection images were obtained from several regions of bone nodule samples to evaluate the topography and sub-surface structures. Sectional analysis was used to measure the feature sizes of constituents on nodule surfaces using nanoscope software (AFM Technical Manual, Veeco Metrology, Santa Barbara, CA). 2.5. Nanoscale modulus mapping A Hysitron triboscope nanomechanical instrument (Minneapolis, MN) equipped with a Nanoscope IIIa controller (Veeco Metrology, Santa Barbara, CA) was used to obtain modulus maps of the surface of bone nodule samples with a Berkovich (threesided pyramid, 100200 nm tip radius) diamond indenter tip. Modulus mapping is a surface nanomechanical characterization technique that enables measurement of the purely elastic properties of a material surface by applying extremely shallow displacements ($23 nm). Detailed descriptions of the principle of modulus mapping, its mechanistic formulation and application can be found in the literature [36,37]. In a typical experiment at regions of sample surface were identied by imaging in contact mode and height images were obtained. Further, modulus maps were acquired from the same area by applying a quasi-static force of 3 lN with a superimposed 1 lN sinusoidal force at a frequency of 200 Hz. High resolution modulus maps were obtained on scan areas of 5 5 lm and 2.5 2.5 lm. Tip calibration was performed using a standard quartz sample of known elastic modulus. 2.6. Chemical analysis using micro-FTIR Micro-FTIR was used to evaluate the chemical structure on the surface of bone nodule samples. In many mineralization studies, bone specimens are xed to prevent bacterial growth and degradation of the sample before the data is acquired. However, this leads to concerns that bone sample treatment with a xative solution can potentially alter the chemistry of the mineral phase [38]. In our study, bone nodules were not treated with xative solutions. Spectra were acquired on various regions of the bone nodule samples using a Nicolet micro-FTIR spectrometer equipped with a Continulm spectroscopy accessory with an internal liquid nitrogen cooled MCT/B detector. Spectra were collected at a frequency resolution of 4 cm1 using Omnic software and processed using Grams/AI7 software from Galactic Industries. 3. Results 3.1. ChiPgAHAP microstructure and cellular response to the Chi PgAHAP nanocomposite Fig. 1 shows AFM height image (100 100 lm) of a ChiPgA HAP nanocomposite lm. Bundles of ChiPgA bers were observed to be uniformly distributed in the ChiPgAHAP microstructure. The ChiPgA bers were found to be several microns long and 1 2 lm in diameter. Nano-HAP particles with a mean particle size of 50 nm were observed as before [39]. A phase contrast image of osteoblasts seeded on ChiPgAHAP nanocomposite lms after 2 h culture is shown in Fig. 2a. As can be seen, the majority of cells are observed to have a rounded morphology, indicating poor cell attachment to the substrate, although most of the seeded cells remained attached. Fluorescence images were obtained to ascertain cell density and cell viability after 12 h culture. Fig. 2b shows the uorescence image of a cell seeded ChiPgAHAP nanocomposite after 12 h culture obtained after live/dead cell assay. As can be

Fig. 1. AFM height image indicating the topography of micro-sized bundles of Chi PgA bers randomly distributed on the surface of the ChiPgA20HAP nanocomposite.

seen, the cells appear to be viable on the synthetic ChiPgAHAP substrate. A rounded cell mass, as observed in Fig. 2b, is indicative of cell assemblage and organization. The same cell density was obtained after 2 h and 12 h culture. This observation suggests that cells migrate, assemble and organize themselves during 12 h culture to form rounded cell masses (Fig. 2b). With an increase in cell culture time to 810 days the cells appear to-collect themselves towards a rounded cell mass from all directions as revealed in the phase contrast image taken on the cell seeded ChiPgAHAP nanocomposite after 10 days of culture (Fig. 2c). Cellular growth on the ChiPgAHAP nanocomposite was continuously monitored throughout the cell culture duration. Osteoblasts generated dense structures (described as bone nodules) on the ChiPgAHAP nanocomposites without the use of a differentiating medium, which were observed and analyzed for mineralization after 22 days of culture. Fig. 2d shows a phase contrast image of an unstained bone nodule on the ChiPgAHAP substrate after 22 days of culture. A large number of cells with rounded morphology appear to surround bone nodule. A phase contrast image of a stained bone nodule is shown in Fig. 2e. Here the appearance of red color on the nodule indicates the presence of calcium deposits. Bone nodules appear to grow in size from less than 200 lm to a maximum size in the range 500800 lm. Cell proliferation studies were performed for cell culture durations of 1, 4, 8, and 22 days, using the MTT assay (Fig. 2f). Mean absorbance values increase gradually from day 1 to day 8, followed by higher absorbance values observed after 22 days of culture. 3.2. Hierarchical micro/nanostructures of bone nodule AFM analyses were performed on unstained bone nodule samples. Fig. 3a shows the 3D AFM height image of a 10 10 lm scan area on the surface of a bone nodule sample. The observed height image reveals a topography of hierarchical structures in the extracellular matrix of a bone nodule synthesized by osteoblasts on the ChiPgAHAP nanocomposites after 22 days of culture. As seen in Fig. 3a, lower levels of hierarchical bone structure are observed, consisting of an array of repeating collagen bers ($1.7 lm width) and collagen brils ($300 nm width) aligned along the long axes of the collagen bers. Mineral deposits are observed to lie on the surfaces of collagen bers and brils along the long axes of the collagen bers. Collagen brils and the spatial distribution of mineral

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Fig. 2. Phase contrast image of osteoblasts after 2 h incubation. (a) A uorescence image indicates that cells are viable and assemble into a cell mass after 12 h cell culture. (b) A phase contrast image indicating cell assemblage and alignment of cells as a rounded cell mass, observed after 10 days culture. (c) A phase contrast image of an unstained bone nodule after 22 days cell culture. The periphery of the bone nodule appears to be surrounded by a large cell mass with a rounded morphology. (d). A phase contrast image of a stained bone nodule on the substrate after 22 days culture. (e) Red color on the nodule indicates the presence of calcium deposits. (f) MTT assay results indicating cell proliferation after 1, 4, 8, and 22 days cell culture.

deposits in these regions were closely examined by taking high resolution height and deection images (5 5 lm) of the regions indicated by a box in Fig. 3a. The height image indicated a topography of mineralized collagen brils with a banded appearance, aligned parallel to each other and to the long axes of the collagen bers, as revealed in Fig. 3b. Irregularly shaped micro/nanosized mineral patches ($501200 nm) were observed on the surface of collagen brils. A schematic representing regions (1)(4) is shown

in Fig. 3b. The illustration demonstrates the hierarchical organization of mineralized collagen brils (300 nm diameter) in a single collagen ber (1.7 lm diameter), aligned parallel to each other and to the long axes of the collagen brils. The illustration demonstrates the spatial arrangement of mineral crystals/patches, their shape, size, location, and orientation on the collagen brils. The schematic representation is scaled relative to the AFM images obtained on the regions indicated in Fig. 3a. An AFM deection image

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Fig. 3. (a) 3D AFM height image of the surface of a bone nodule sample indicating the topography of hierarchical structures in the extracellular matrix of the bone nodule. (b) Hierarchical structures of a bone nodule consisting of an array of repeating collagen bers ($1.7 lm width) and collagen brils ($300 nm width) aligned along the long axes of collagen bers. Schematic diagram of the hierarchical organization of collagen brils in a single collagen ber. The illustration demonstrates the spatial arrangement of mineral patches, their shape, size, location and orientation on the collagen brils. (c) Mineral patches were observed to lie on the surfaces of collagen brils parallel and perpendicular to the long axes of collagen brils.

(Fig. 3c) resolved the sharp surface features of the irregular mineral patches on the surface of the collagen brils. Some micro-sized mineral patches were observed to extend over adjoining group of brils, as indicated by an arrow in the deection image of Fig. 3c. Mineral patches on the surface of collagen brils were observed to be very thin (1325 nm) and overlay the collagen brils, as revealed by the banded signatures of collagen brils underneath the mineral patch, as shown in Fig. 3c. A hierarchical organization of collagen brils and mineral crystals oriented along the collagen brils was also observed on the bone nodule surface, as shown in the deection image in Fig. 4. Individual collagen brils were oriented parallel to each other and to the long axes of the collagen brils. The mineral crystals appear to be nearly at (length 167 19 nm, width 129 6.0 nm) and were oriented parallel to each other in each bril and aligned along the long axes of the collagen brils. A schematic representation illustrates the spatial arrangement, shape, size, location, and orientation of mineral crystals along the long axes of the collagen brils, as observed in Fig. 4. Mineral crystals are demonstrated to be oriented parallel to each other in each bril and aligned along the collagen bril. In the schematic, mineral crystals are shown to be parallel to spatially spaced brils with an interbrillar distance of $130 nm. The illustration has been drawn for the regions shown by a dotted box in the deection image.

Hierarchical bone nodule structures were observed on the Chi PgAHAP brous scaffold after 16 days of culture, as shown in Fig. 5a. Oriented collagen bers are observed in the height image. Nanostructured mineral crystals appear to lie along the long axes of the collagen brils. Fig. 5b shows the cell proliferation results for the ChiPgAHAP brous scaffold for cell culture durations of 2, 4, 8, 12, and 16 days, respectively. The MTT assay results indicate that the cell population was almost constant until day 8 of cell culture. Rapid cell proliferation was observed after 12 and 16 days cell culture. 3.3. Nanoscale elastic properties of bone nodule Fig. 6 shows the height image and corresponding modulus map (5 5 lm) of the surface of a bone nodule sample. The diagonal arrows in both images indicate the direction of the long axes of the collagen brils. The height image (Fig. 6a) indicates an apparent tapered topography of an array of cylindrical collagen bers aligned parallel to the long axes of the collagen bers. The modulus map image (Fig. 6b) represents the elastic responses of the bone nodule constituents, spatially distributed over an area of 5 5 lm. Modulus values were in the range 460 GPa. In Fig. 6 a lighter color is related to the elastic responses of the soft collagenous phase with a lower modulus while a darker color corresponds to stiffer

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Fig. 4. (a) AFM deection image indicating the individual collagen brils oriented parallel to each other. Flat mineral crystals are parallel to each other in each bril and aligned along the long axis of the collagen brils. Mean repeat distance between the mineral crystals oriented along the individual brils is observed to be 74 nm. (b) Schematic representation illustrating the spatial arrangement, shape, size, location and orientation of mineral crystals along the sides of collagen brils enclosed in a single collagen ber.

Fig. 5. (a) AFM height and corresponding deection image of the surface of a bone nodule on a ChiPgAHAP brous scaffold after 16 days cell culture. Oriented collagen bers are revealed in the height image. (b) Nanostructured mineral crystals appear to lie along the long axes of collagen brils. MTT assays indicate cell proliferation after 2, 4, 8, and 16 days cell culture.

mineral components with a higher elastic modulus. In between grayscale shades on the modulus map are indicative of collective elastic responses of the constituents of the bone nodule, mainly collagen and mineral structures. Further, selected modulus vs. position plots were used to separate the individual responses of the

constituents distributed spatially on the surface of a bone nodule. Plot 1 (40005000 nm regions, dotted arrows on modulus map) and plot 4 (34005000 nm regions, dotted arrows on modulus map) indicate moduli in the ranges 6.412.7 and 5.310.4 GPa, respectively as also indicated by arrows in plots 1 and 4. These

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Fig. 6. Height image (a), and corresponding modulus map image (b) of a 5 5 lm scan area on the surface of a bone nodule sample. Arrows shown in both the images indicate the direction of the long axes of the collagen brils. (c) Modulus map image showing the elastic responses of the bone nodule constituents, spatially distributed over an area of 5 5 lm. The lighter color is related to the elastic response of a soft collagenous phase with a lower modulus while the darker color corresponds to a stiffer mineral component with a higher modulus. Mixed colors on the modulus map are related to the collective elastic responses of the constituents of the bone nodule, mainly collagen and mineral structures. Selected modulus vs. position plots (14) with dotted and solid arrows indicate the elastic responses of the major components of the bone nodule, i.e. collagen brils, collagenmineral complexes and mineral lying parallel (Mk) and perpendicular (M\) to the long axes of the collagen brils.

regions are represented by lighter colors on the modulus map and indicate the nanoscale elastic responses of collagen with a minimal inuence of mineral. On these plots the lowest values of $4 GPa indicate the elastic property of pure collagen brils/molecules. Close assessments of both the height and modulus map images revealed the signatures of parallel arrays of collagen bers and brils, oriented parallel to the long axes of the collagen bers. Although, mineral deposits were not resolved in the height image, their elastic responses parallel and perpendicular to the long axes of the collagen bers were distinctly marked by darker colored regions (higher modulus) along the diagonal bands in the modulus map

image. Plot 2 indicates the elastic properties of constituents spatially distributed along the sectional prole 2. Sectional prole 2 in the modulus map represents the elastic responses of mineral crystals in the parallel and perpendicular directions, as indicated by the dotted arrows. Along the collagen bril axes mineral deposits (Mk) exhibited elastic moduli of 3060 GPa, with most of the high modulus values centered around 3040 GPa (plot 2). In addition, evaluation of the elastic properties of mineral deposits on the surface of the collagen brils oriented in the perpendicular direction was also done. The surfaces of mineralized collagen brils were resolved in the regions enclosed by stars in the modulus

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Fig. 7. Micro-FTIR spectrum of a bone nodule sample in the region 2350800 cm1. The major bands for collagen are amide I, amide II and amide II and those of biological apatite m1, m3 PO43, m3 HPO43, and m2 CO32, respectively.

map image, which represents the elastic responses of constituents on the surface of the collagen brils (1060 GPa). Although individual collagen brils were not resolved in the height image, the widths of these regions were observed to be same as that of a single collagen ber (Fig. 3b). Also, more than 50% of the area enclosed by stars in the modulus map are represented by darker colors in the modulus map, indicating the presence of mineral crystals in several regions on the surfaces of the collagen brils. Elastic moduli on the surface of the collagen brils (28004000 nm regions on sectional prole 2) varied in the range 12.536 GPa, which is indicative of responses from primarily stiff mineral deposits (M\ = 2040 GPa) and a composite response of collagen brilmineral with a mean modulus of 15 GPa. Combined elastic responses ($15 GPa) from collagen and mineral constituents were observed in several regions of the modulus map, as represented by mixed colors, mostly lying in the regions between the collagen bers. These regions are selectively shown in sectional proles 1, 3, and 4 (solid arrows) in the modulus map image and have also been marked on plots 1, 3, and 4, respectively.

Table 1 FTIR band assignment for a nodule sample. Band position (cm1) 2315 2027 1805 1678 1560 1470 1415 1273 1188 1127 1079 976 873
a

Band assignment NH vibrations of aminea NH3+ symmetric stretchinga Amides containing CONHCOa Amide I Amide II Type A carbonate m COO and m3 CO32, type AB carbonate Amide III m3 HPO43 m3 PO43, non-stoichiometric HAP, newly precipitated apatites Weak m3 PO43 m1 PO43 m2 CO32

Bands from cell culture medium.

3.4. Chemical analysis of bone nodule A FTIR spectrum of a bone nodule sample in the region 800 2350 cm1 is shown in Fig. 7. Band assignments are shown in Table 1. The bands at 2315, 2027, and 1805 cm1 are atrributed to NH vibrations, NH3+ symmetric stretching, and amides containing CONHCO, respectively. All the three bands originate from the cell culture medium. To conrm this fact, separate experiments were carried out by soaking a clean gold substrate in cell culture medium. All the bands in the bone nodule were compared with micro-FTIR spectroscopy results reported in the literature for pediatric human bone [40]. The bands at 1678, 1560, and 1273 cm1 were assigned to amide I, amide II and amide III, respectively. In biological apatite carbonate groups substitute for PO43 (type A substitution) or OH (type B substitution) groups in HAP (Ca10(PO4)6(OH)2). The band at 1470 cm1 can be assigned to type A carbonate and the band at 1415 cm1 can be assigned to molecular vibrations of m COO and m3 CO32 type AB carbonate species. Phosphate bands are observed in the region 9001200 cm1. Both inorganic orthophosphate ions, i.e. PO43 and HPO42 ions, have been reported to

occur in biological apatites of mineralized tissue [40]. The presence of a band at 1188 cm1 indicates the existence of HPO42 ions in apatite. The band at 1127 cm1 has been previously observed in the spectra of newly precipitated apatites, and is attributed to m3 PO43, which is a non-stoichiometric HAP. A weak band at 1079 cm1 can be attributed to molecular vibrations of m3 PO43 ions. The band at 976 cm1 can be assigned to the m1 vibration mode of PO43 ions. The bands from 850 to 900 cm1 are assigned to m2 CO32; in the spectrum shown this band was observed at 873 cm1. Our microspectroscopic analysis indicates that all the major bands of the bone nodule correspond to bands observed in human bone [40]. This is suggestive of the fact that bone nodules generated on ChiPgAHAP nanocomposite lms are chemically similar to human bone. 4. Discussion Previously we synthesized ChiPgAHAP nanocomposite lms and scaffolds at solution pH values of 7.0 and 8.5 that resulted in a nano-colloidal morphology of the ChiPgA material system [41]. The ChiPgAHAP nanocomposite lms and scaffolds

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R. Khanna et al. / Acta Biomaterialia xxx (2010) xxxxxx Table 2 Elastic properties of collagen. Tissue type/experimental conditions Bone nodule Technique Modulus mapping Elastic modulus (GPa) $48 (Collagen) $15 (Collagen-mineral) 2060 (Mineral) 4.56.2 3.7511.5 2.9 0.1 4.8 1.0 57 Reference Present work Present work Present work [43] [40] [42] [44] [45]

Collagen Collagen of rat tail tendon (varying dehydration states) Collagen of bovine Achilles tendon (0.15 M NaCl solution) Collagen-like peptide Hydrated cross-link-free collagen bril

Steered molecular dynamics AFM indentation X-ray diffraction Simulation Simulation: atomistic and steered molecular dynamics

synthesized at a solution pH of 4.5 resulted in a brous morphology of the ChiPgA material system (Fig. 1). Bone nodules were formed on both kinds of nanocomposites, i.e. on nano-colloidal [33] as well as brous ChiPgAHAP nanocomposites, in the absence of any osteogenic supplements. AFM imaging of bone nodules formed on the brous ChiPgAHAP nanocomposite revealed a hierarchical structural organization of mineralized collagen bers and brils (Figs. 3 and 4). The structural hierarchy, molecular structures. and nanomechanical properties of the bone nodules can be further discussed in the light of the existing literature.

served [48]. The major conclusion from the AFM structural analysis is that synthetic bone nodules mimic the hierarchy of natural bone. 4.2. Nanoscale elastic modulus of collagen and mineral of bone nodules In human bone, the basic building blocks are designed at the nanoscale, with hard nanosized mineral crystals embedded in a soft collagenous matrix. Evaluation of the nanoscale mechanical properties of these structural elements generated by human osteoblasts on synthetic biomaterials can serve as guidelines for scaffold design and development. The elastic moduli of collagen and mineral of the bone nodules obtained in the present work are in close agreement with experimental and simulations results reported in the literature for various collagenous and calcifying tissues of different animal species and human bone [4951] (Table 2). In our results, the maximum modulus value of $60 GPa, with a mean value of 35 GPa, was measured, which appears to be a nanoscale mechanical property of mineral crystals at extremely shallow penetration depths of 23 nm. The minimum elastic modulus of 48 GPa for collagen corresponds well with that reported in the case of rat tail tendon (E = 3.7511.5 GPa, for a penetration depth of 25 nm) [50] and human dentin containing interbrilar mineral (E = 2GPa) [52] using AFM and nanoindentation. Our results are also in close agreement with X-ray diffraction results for bovine Achilles tendon (E = 2.9 0.1 GPa) [53]. Steered molecular dynamics (SMD) simulations have been conducted on collagen by our group [5456]. The simulations results also indicate that the elastic modulus of collagen is in the range 4.56.2 GPa. The elastic modulus of collagen is dependent on the rate of pulling, stiffness of the spring used in pulling and the length of the molecule. The elastic moduli of collagen (57 GPa) reported in the literature using SMD simulations [57,58] are in close agreement with the elastic moduli obtained from our experimental and SMD simulation results. 4.3. FTIR spectra of bone nodules Tissue engineered bone should be chemically similar to human bone to regulate bone physiology and mechanical functions during the healing or repair of tissue. For instance, it has been reported that in carbonated or biological apatites carbonate ion substitution for phosphate ions alter the crystal shape and arrangement in the maturing bone, leading to altered mechanical properties [59]. Our FTIR analysis indicates that the developing bone nodule is chemically similar to human bone [40], as revealed by the presence of collagen (amide I, amide II and amide III bands) and carbonated HAP (Fig. 7). Evidence of three major bands i.e. m3 HPO43 (1188 cm1), m3 PO43 (1127 cm1) and m2 CO32 (873 cm1), conrmed the presence of immature biological apatite in bone nodules. Biological apatites are both chemically and structurally different from the synthetic nanohydroxyapatite used in the preparation of the Chi PgAHAP nanocomposites [39,60], as revealed by the distinctive sizes, shapes, and chemistries of the as-synthesized HAP and

4.1. Synthetic bone nodule mimics natural bones hierarchy Nature has designed the complex and mechanically robust hierarchical structures of bone by repeating the smallest structural building blocks (i.e. collagen molecules, brils, and mineral crystals) several times to generate functional bone. In this work, we observed lower levels of hierarchical structures (collagen bers, brils, and mineral crystals) of bone nodules formed on both two-dimensional lm substrates and 3D scaffolds. Mineral deposits on the surface of collagen brils were rst reported by Landis and co-workers in the case of calcifying leg tendons of turkey and chicken bone using 3D electron microscopic tomography [24,42]. For the rst time, we observed mineral crystals on the surface of collagen brils of in vitro generated bone nodules on an articial substrate, as revealed by high resolution AFM images (Figs. 3 and 4). Crystals are deposited in a highly organized manner in the hole and overlap zones of collagen brils with characteristic repeat distances of 67 nm [18,43]. As can be seen in Fig. 4, the mineral crystals occur at almost equi-spaced distances along individual collagen brils with a repeat distance of 74 7.1 nm, which very closely matches the repeat distance of $67 nm between mineral crystals, reported in the case of trabecular bone [44]. This repeat distance between the mineral crystals corresponds to the distance by which adjacent collagen molecules are staggered. In human dentin, the collagen brils are randomly oriented and the diameters of hydrated and dehydrated collagen brils have been found to be in the range 75105 nm [45]. In contrast, collagen brils in bone nodules possess a high degree of structural organization and are oriented perfectly parallel to the long axes of collagen bers with a diameter of $ 300 nm. Mineral crystals in the bone nodule are observed to be larger in size, which suggests that mineral crystals are not only conned to 40 nm hole zones. These mineral crystals are believed to nucleate initially in gap zones and grow in length along their crystallographic c-axis parallel to the collagen bril axes and extend to the surface of the collagen brils and extrabrillar spaces by fusion of smaller crystals into larger ones, as suggested by Landis et al. [23,46]. This suggests that the formation of mineral crystals is site dependent and occurs as a result of independent nucleation events in local zones of collagen, as also observed by Landis et al. [23,42,47]. Recently, mineralized bone nodules generated by osteoblasts were characterized by TEM, but no collagenmineral hierarchical organization was ob-

Please cite this article in press as: Khanna R et al. Bone nodules on chitosanpolygalacturonic acidhydroxyapatite nanocomposite lms mimic hierarchy of natural bone. Acta Biomater (2010), doi:10.1016/j.actbio.2010.10.028

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R. Khanna et al. / Acta Biomaterialia xxx (2010) xxxxxx [13] Kawasaki K, Aihara M, Honmo J, Sakurai S, Fujimaki Y, Sakamoto K, et al. Effects of recombinant human bone morphogenetic protein-2 on differentiation of cells isolated from human bone, muscle, and skin. Bone 1998;23:223. [14] Chen D, Harris MA, Rossini G, Dunstan CR, Dallas SL, Feng JQ, et al. Bone morphogenetic protein 2 (BMP-2) enhances BMP-3, BMP-4, and bone cell differentiation marker gene expression during the induction of mineralized bone matrix formation in cultures of fetal rat calvarial osteoblasts. Calcif Tissue Int 1997;60:283. [15] Petite H, Viateau V, Bensaid W, Meunier A, de Pollak C, Bourguignon M, et al. Tissue-engineered bone regeneration. Nat Biotechnol 2000;18:959. [16] Currey JD. Mechanical-properties of mother of pearl in tension. Proc R Soc Lond B Biol Sci 1977;196:443. [17] Rho JY, Kuhn-Spearing L, Zioupos P. Mechanical properties and the hierarchical structure of bone. Med Eng Phys 1998;20:92. [18] Weiner S, Wagner HD. The material bone: Structure mechanical function relations. Annu Rev Mater Sci 1998;28:271. [19] Landis WJ. The strength of a calcied tissuedepends in part on the molecular structure and organization of its constituent mineral crystals in their organic matrix. Bone 1995;16:533. [20] Fratzl P, Weinkamer R. Natures hierarchical materials. Prog Mater Sci 2007;52:1263. [21] Stoddart A, Cleave V, Langer R. The evolution of biomaterials. Nat Mater 2009;8:444. [22] Ji BH, Gao HJ. Mechanical properties of nanostructure of biological materials. J Mech Phys Solids 2004;52:1963. [23] Landis WJ, Song MJ, Leith A, McEwen L, McEwen BF. Mineral and organic matrix interaction in normally calcifying tendon visualized in 3 dimensions by high-voltage electron-microscopic tomography and graphic imagereconstruction. J Struct Biol 1993;110:39. [24] Landis WJ, Hodgens KJ, Song MJ, Arena J, Kiyonaga S, Marko M, et al. Mineralization of collagen may occur on bril surfaces: Evidence from conventional and high-voltage electron microscopy and three-dimensional imaging. J Struct Biol 1996;117:24. [25] Fratzl P, Fratzlzelman N, Klaushofer K, Vogl G, Koller K. Nucleation and growth of mineral crystals in bone studied by small-angle X-ray scattering. Calcif Tissue Int 1991;48:407. [26] Fratzl P, Schreiber S, Klaushofer K. Bone Mineralization as Studied by Smallangle X-ray Scattering. Connect Tissue Res 1996;35:9. [27] Fratzl P, Paris O, Klaushofer K, Landis WJ. Bone mineralization in an osteogenesis imperfecta mouse model studied by small-angle X-ray scattering. J Clin Invest 1996;97:396. [28] Rinnerthaler S, Roschger P, Jakob HF, Nader A, Klaushofer K, Fratzl P. Scanning small angle X-ray scattering analysis of human bone sections. Calcif Tissue Int 1999;64:422. [29] Paris O, Zizak I, Lichtenegger H, Roschger P, Klaushofer K, Fratzl P. Analysis of the hierarchical structure of biological tissues by scanning X-ray scattering using a micro-beam. Cell Mol Biol 2000;46:993. [30] Tesch W, Eidelman N, Roschger P, Goldenberg F, Klaushofer K, Fratzl P. Graded microstructure and mechanical properties of human crown dentin. Calcif Tissue Int 2001;69:147. [31] Verma D, Katti KS, Katti DR, Mohanty B. Mechanical response and multilevel structure of biomimetic hydroxyapatite/polygalacturonic/chitosan nanocomposites. Mater Sci Eng C 2008;28:399. [32] Khanna R, Katti KS, Katti DR. In situ swelling behavior of chitosan polygalacturonic acid/hydroxyapatite nanocomposites in cell culture media. Int J Polym Sci 2010;2010:12. [33] Verma D, Katti KS, Katti DR. Osteoblast adhesion, proliferation and growth on polyelectrolyte complexhydroxyapatite nanocomposites. Philos Trans R Soc London A 2010;368:2083. [34] Katti KS, Turlapati P, Verma D, Bhowmik R, Gujjula PK, Katti DR. Static and dynamic mechanical behavior of hydroxyapatitepolyacrylic acid composites under simulated body uid. Am J Biochem Biotechnol 2006;2(2):73. [35] Verma D, Katti KS, Katti DR. Polyelectrolyte-complex nanostructured brous scaffolds for tissue engineering. Mat Sci Eng C Mat Biological Appl 2009;29:2079. [36] Asif SAS, Wahl KJ, Colton RJ, Warren OL. Quantitative imaging of nanoscale mechanical properties using hybrid nanoindentation and force modulation. J Appl Phys 2001;90:1192. [37] Balooch G, Marshall GW, Marshall SJ, Warren OL, Asif SAS, Balooch M. Evaluation of a new modulus mapping technique to investigate microstructural features of human teeth. J Biomech 2004;37:1223. [38] Pleshko NL, Boskey AL, Mendelsohn R. An ft-ir microscopic investigation of the effects of tissue preservation on bone. Calcif Tissue Int 1992;51:72. [39] Khanna R, Katti KS, Katti DR. Nanomechanics of surface modied nanohydroxyapatite particulates used in biomaterials. J Eng Mech ASCE 2009;135:468. [40] Petra M, Anastassopoulou J, Theologis T, Theophanides T. Synchrotron microFT-IR spectroscopic evaluation of normal paediatric human bone. J Mol Struct 2005;733:101. [41] Verma D. Design of polymerbiopolymerhydroxyapatite biomaterials for bone tissue engineering: Through molecular control of interfaces. Ph.D. dissertation, North Dakota State University, Fargo; 2008. p. 191. [42] Landis WJ, Hodgens KJ, Arena J, Song MJ, McEwen BF. Structural relations between collagen and mineral in bone as determined by high voltage electron microscopic tomography. Microsc Res Tech 1996;33:192.

mineral deposits of bone nodules. The differences in physicochemical characteristics can arise due to the different physiological environments in which they are generated. It suggests that the environment essentially mediates the structural and biological functions of functional tissues. 5. Conclusions 1. The prepared ChiPgAHAP brous nanocomposites are osteoconductive and osteoinductive and thereby provide an appropriate microenvironment for cell organization and bone nodule formation. 2. The bone nodules formed on the biomaterial systems described in this work mimic natural bones hierarchy in the light of following results. a) High resolution AFM imaging revealed hierarchical structures of a bone nodule, consisting of an array of repeating collagen bers ($1.7 lm width) and collagen brils ($300 nm width) aligned along the long axes of collagen bers. Mineral deposits are observed to lie on the surfaces of collagen bers and brils along the long axes of the collagen bers, and are reported for the rst time in an in vitro generated bone nodule. Structural organization of the bone nodule constituents was found to be similar to that of calcifying tissues, which supports the conclusion that synthetic bone nodules mimic the natural bone hierarchy. b) The elastic properties of mineral deposits parallel and perpendicular to the long axes of collagen brils is reported for the rst time using a modulus mapping technique. The nanoscale elastic properties of collagen brils and mineral crystals closely match the experimental and simulation results for bone reported in the literature, which supports the conclusion that bone nodules mimic the nanoscale mechanical properties of natural bone. c) FTIR analysis indicates the presence of collagen and biological apatite in the bone nodules, which supports the conclusion that bone nodules are chemically similar to human bone and possess the characteristics of newly precipitated or immature bone.

References
[1] Langer R, Vacanti JP. Tissue engineering. Science 1993;260:920. [2] Langer R, Tirrell DA. Designing materials for biology and medicine. Nature 2004;428:487. [3] Khademhosseini A, Vacanti JP, Langer R. Progress in tissue engineering. Sci Am 2009;300:64. [4] Marijnissen W, van Osch G, Aigner J, van der Veen SW, Hollander AP, Verwoerd-Verhoef HL, et al. Biomaterials 2002;23:1511. [5] Mauck RL, Yuan X, Tuan RS. Chondrogenic differentiation and functional maturation of bovine mesenchymal stem cells in long-term agarose culture. Osteoarthritis Cartilage 2006;14:179. [6] Freyman TM, Yannas IV, Yokoo R, Gibson LJ. Fibroblast contraction of a collagenGAG matrix. Biomaterials 2001;22:2883. [7] Hsu SH, Chang SH, Yen HJ, Whu SW, Tsai CL, Chen DC. Evaluation of biodegradable polyesters modied by type II collagen and ArgGlyAsp as tissue engineering scaffolding materials for cartilage regeneration. Artif Organs 2006;30:42. [8] Forte G, Carotenuro F, Pagliari F, Pagliari S, Cossa P, Fiaccavento R, et al. Stem Cells 2008;26:2093. [9] Katti KS, Katti DR, Dash R. Synthesis and characterization of a novel chitosan/ montmorillonite/hydroxyapatite nanocomposite for bone tissue engineering. Biomedical Materials 2008;3. [10] Katti KS, Ambre AH, Peterka N, Katti DR. Use of unnatural amino acids for design of novel organomodied clays as components of nanocomposite biomaterials. Philos Trans R Soc London A 2010;368:1963. [11] Traversa E, Mecheri B, Mandoli C, Soliman S, Rinaldi A, Licoccia S, et al. Tuning Hierarchical Architecture of 3D Polymeric Scaffolds for Cardiac Tissue Engineering, Vol. 3. New York: Taylor & Francis; 2008. p. 97. [12] Franceschi RT, Iyer BS, Cui YQ. Effects of ascorbic-acid on collagen matrix formation and osteoblast differentiation in murine MC3T3E1 cells. J Bone Miner Res 1994;9:843.

Please cite this article in press as: Khanna R et al. Bone nodules on chitosanpolygalacturonic acidhydroxyapatite nanocomposite lms mimic hierarchy of natural bone. Acta Biomater (2010), doi:10.1016/j.actbio.2010.10.028

R. Khanna et al. / Acta Biomaterialia xxx (2010) xxxxxx [43] Landis WJ, Silver FH. The structure and function of normally mineralizing avian tendons. Comp Biochem Physiol A Comp Physiol 2002;133:1135. [44] Hassenkam T, Fantner GE, Cutroni JA, Weaver JC, Morse DE, Hansma PK. Highresolution AFM imaging of intact and fractured trabecular bone. Bone 2004;35:4. [45] Habelitz S, Balooch M, Marshall SJ, Balooch G, Marshall GW. In situ atomic force microscopy of partially demineralized human dentin collagen brils. J Struct Biol 2002;138:227. [46] Landis WJ, Librizzi JJ, Dunn MG, Silver FH. A study of the relationship between mineral content and mechanical properties of turkey gastrocnemius tendon. J Bone Miner Res 1995;10:859. [47] Landis WJ. Mineral Characterization in Calcifying Tissues: Atomic, Molecular and Macromolecular Perspectives, Vol. 35. Gordon & Breach, 1996. p. 1. [48] Gentleman E, Swain RJ, Evans ND, Boonrungsiman S, Jell G, Ball MD, et al. Nat Mater 2009;8:763. [49] Kinney JH, Marshall SJ, Marshall GW. The mechanical properties of human dentin: A critical review and re-evaluation of the dental literature. Crit Rev Oral Biol Med 2003;14:13. [50] Wenger MPE, Bozec L, Horton MA, Mesquida P. Mechanical properties of collagen brils. Biophys J 2007;93:1255. [51] Rho JY, Roy ME, Tsui TY, Pharr GM. Elastic properties of microstructural components of human bone tissue as measured by nanoindentation. J Biomed Mater Res 1999;45:48.

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[52] Balooch M, Habelitz S, Kinney JH, Marshall SJ, Marshall GW. Mechanical properties of mineralized collagen brils as inuenced by demineralization. J Struct Biol 2008;162:404. [53] Sasaki N, Odajima S. Stressstrain curve and Youngs modulus of a collagen molecule as determined by the X-ray diffraction technique. J Biomech 1996;29:655. [54] Bhowmik R, Katti KS, Katti DR. Mechanics of molecular collagen is inuenced by hydroxyapatite in natural bone. J Mater Sci 2007;42:8795. [55] Katti DR, Pradhan SM, Katti KS. Directional dependence of hydroxyapatite collagen interactions on mechanics of collagen. J Biomech 2010;43:1723. [56] Pradhan SM, Katti DR, Katti KS. Steered molecular dynamics study of mechanical response of full length and short collagen molecules. ASCE J Nanomech, Submitted for publication. [57] Lorenzo AC, Caffarena ER. Elastic properties, Youngs modulus determination and structural stability of the tropocollagen molecule: A computational study by steered molecular dynamics. J Biomech 2005;38:1527. [58] Buehler MJ. Nature designs tough collagen: explaining the nanostructure of collagen brils. Proc Natl Acad Sci USA 2006;103:12285. [59] Akkus O, Adar F, Schafer MB. Age-related changes in physicochemical properties of mineral crystals are related to impaired mechanical function of cortical bone. Bone 2004;34:443. [60] Verma D, Katti K, Katti D. Experimental investigation of interfaces in hydroxyapatite/polyacrylic acid/polycaprolactone composites using photoacoustic FTIR spectroscopy. J Biomed Mater Res Part A 2006;77A:59.

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