Controlled shear flow directs osteogenesis on UHMWPE based hybrid

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Controlled shear flow directs osteogenesis on UHMWPE based hybrid nano-biocomposites in custom designed PMMA microfluidic device Sharmistha Naskar, Asish Kumar Panda, Viswanathan Kumaran, Bhupesh Mehta, and Bikramjit Basu ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00147 • Publication Date (Web): 10 Jul 2018 Downloaded from http://pubs.acs.org on July 12, 2018

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ACS Applied Bio Materials

Controlled shear flow directs osteogenesis on UHMWPE based hybrid nano-biocomposites in custom designed PMMA microfluidic device Sharmistha Naskar, †, §, # Asish Kumar Panda, § Viswanathan Kumaran, †, # Bhupesh Mehta, ‡ Bikramjit Basu*, †,§

†Centre for Biosystems Science and Engineering, Indian Institute of Science, Bangalore-560012, India #Microfluidics Laboratory, Department of Chemical Engineering, Indian Institute of Science, Bangalore-560012, India ‡Department of Biophysics, National Institute of Mental Health, Bangalore-560029, India §Laboratory for Biomaterials, Materials Research Centre, Indian Institute of Science, Bangalore-560012, India *Corresponding author: Email- [email protected]

Abstract The combinatorial influence of biophysical cue (substrate stiffness) and biomechanical cue (shear flow) on the osteogenesis-modulation of human mesenchymal stem cells (hMScs) is studied for bone regenerative applications. In this perspective, we report stem cell differentiation on Ultra High Molecular Weight Polyethylene (UHMWPE) based hybrid nano-biocomposite [reinforced with multiwalled carbon-nanotube (MWCNT) and/or nanohydroxyapatite (nHA)] under physiologically relevant shear flow (1 Pa) in a custombuilt microfluidic device. Using genotypic assessment with qRT-PCR and phenotypic assessment through analysis of cytoskeletal remodelling and marker proteins, the role of shear on the progression of osteogenesis modulation has been quantitatively established with statistically significant differences between nHA reinforced and MWCNT reinforced UHMWPE. Early-stage event (alkaline phosphatase activity at day 8), middle-stage event (matrix collagenation at day 14) and late-stage event (matrix calcification at day 20) were

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analysed using mRNA expression changes of limited cell volume after microfluidic culture experiments. The conventional petridish culture (static) exhibited an increased osteogenesis for nanoparticle reinforced UHMWPE, irrespective of the type nanoparticle. The shear mediated culture experiments resulted in noticeable differences in the degree of osteogenesis with MWCNT being more effective than nHA reinforcement. The shear mediated osteogenesis has been attributed to the skewed cellular morphology with higher cell adhesion (vinculin expression) on UHMWPE+nHA than that of UHMWPE+MWCNT. The signatures of the cytoskeletal changes are reflected in terms of left to right chirality as well as alignment and pattern of actin fibres. Moreover, stemness (vimentin expression) was found to be decreased because of differentiation. The electrophysiological analysis using whole clamp experiments also reveal higher inward calcium current and intracellular calcium activity for the cells grown on UHMWPE+nHA nanobiocomposite under shear. Overall, the present study conclusively establishes the synergistic role of substrate stiffness and shear on osteogenesis of hMScs, in vitro. Keywords: Lab-on-a-chip, regenerative medicine, osteogenesis, electrophysiology, L-R chirality, biomaterials


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Table of Contents 1. Introduction ........................................................................................................................ 4 2. Result.................................................................................................................................. 7 2.1. Analysis of microflow properties ................................................................................ 7 2.2. Biomaterial properties ................................................................................................. 8 2.2.1. Surface topography and wettability study............................................................. 8 2.3. In vitro cellular response ............................................................................................. 9 2.3.1. Cytocompatibility analysis.................................................................................... 9 2.3.2. Human mesenchymal stem cell differentiation................................................... 11 2.3.2.1. Flowcytometry analysis to determine G1/G0 arrest ..................................... 11 2.3.2.2. Analysis of focal adhesion ........................................................................... 11 2.3.2.3. Analysis of Actin remodeling ...................................................................... 13 2.3.2.4. Osteogenesis of hMScs marked by ALP activity, collagen deposition, matrix mineralization ............................................................................................................ 15 2.3.2.5. Expression of osteogenic markers proteins: induction and maintenance of the osteogenesis ......................................................................................................... 18 2.3.2.6. Compensation of stemness: evaluation of vimentin expression ................... 20 2.3.2.7. Quantitative mRNA detection for osteogenic gene expression: .................. 21 2.3.3. Analysis of cell functionality .............................................................................. 23 2.3.3.1. Patch clamp study......................................................................................... 23 2.3.3.2. Calcium activity ........................................................................................... 24 3. Discussion ........................................................................................................................ 25 3.1 Surface topography and wettability ............................................................................ 26 3.2. Correlation of the cellular response with biomaterial property ................................. 28 3.3. Left-to-right chirality during cellular orientation under shear ................................... 30 3.4. Shear stress as potent mechano-stimulator of osteogenesis ...................................... 30 3.5. Runx2 is the master gene for osteogenesis ................................................................ 32 3.6. Actin remodeling is critical for osteogenic conversion ............................................. 32 3.7. Membrane ionic currents in hMScs and differentiated cells ..................................... 33 3.8. Compromising the stemness of hMScs at an expense of cellular commitment towards osteogenesis ........................................................................................................ 34 3.9. Correlation of osteogenic marker genes Expression ................................................. 35 4. Conclusions ...................................................................................................................... 36 5. Materials and methods ..................................................................................................... 37



1. Introduction The application of microfluidics as lab-on-a-chip (LOC) is widely accepted as a powerful tool capable of reconstructing cell-level and tissue-level microenvironment and thus, it has drawn much attention to the biomedical community.1 The LOC technology has enabled the researchers to conduct the cell culture experiments within the expanse of only a few square centimeters (or sometimes lesser) with a precise control of culture parameters, like nutrient concentration, spatial allocation, and mechanical stimuli. Between conventional petridish culture and the unconventional microfluidic culture, the physiological cues, in particular mechanical stimuli in form of shear, are completely absent in the former case. The plethora of published studies to understand the role of biophysical cues largely use petridish culture, which do not provide physiologically relevant results. The dynamic culture study with controlled shear flow and using biomaterials as cell growth substrates are rarely reported. This not only demands careful design of microfluidic device but also the clinically relevant biomaterials. The biomaterial chosen is UHMWPE and to realise the role of substrate stiffness, two different types of nano-reinforcements (nHA and MWCNT) are used. Since such materials are used for bone tissue engineering, one has to carefully consider the shear flow in carefully consider the magnitude of shear flow in a microfluidic device. In physiological environment, the cells inside cell inside the body are continuously the cells inside the body are continuously bathed by the interstitial fluid (IF). The hydrostatic and osmotic pressure differences between blood, interstitium, and lymphatics are the driving forces for slow/fast, but constant flow of the IF in various tissues.2 In contrast to soft tissues, the bone tissue experiences substantially higher flow of IF and shear stress as a consequence of muscle contractions, inducing alteration of blood pressure and mechanical loading.3 Due to the small dimensions of the canaliculi (small channel-like structures in bone), the wall shear stress can be as high as 0.8 Pa and 3 Pa,4 which can be comparable to the high vascular-wall shear stress of 2-4 Pa.


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Many researchers reported that subtle shear, exerted by the dynamic fluid environment, played a pivotal role to direct the cells towards proliferation, differentiation, the formation of functional structures, and release chemical factors. For example, Yourek et al. investigated that shear stimulation (0.9 Pa) of hMSc continuously for 3 days in osteogenic media is sufficient to induce osteogenesis.5 Liu et al. reported that intermittent fluid shear stress (IFSS) acts as a potent and physiologically relevant cue to differentiate the hMSc towards osteogenesis, through transient receptor potential melastatin 7 (TRPM7)-Osterix signaling pathway.6 Other than biophysical cues, specific biomaterials support osteogenesis through biophysical cues to the cells. Hydroxyapatite (HA), being a component of the natural bone matrix, is found to be an ideal material for bone regenerative medicine, due to its osteoconductivity and osteoinductivity. Li et al. reported that nano-hydroxyapatite/polyamide composite can exhibit exemplary biomimetic properties in view of osteogenesis.7 Recent advancements in biomaterial science allow carbon nanotube embedded polycarbonate urethane (PCU) to mediate the osteogenic differentiation depending on the degree of integrin activity. 8 Moreover, the nanoscale surface pattern can regulate the integrin arrangement thus influencing cell functionality.9 Oria et al. has described that the cells can sense the density as well as the distribution of the ECM molecule in order to establish the focal adhesion points.10 The same authors also have established hypothetical molecular-clutch model to emphasize the increased cellular adhesion on disordered molecular cues. the spatial arrangements of the adhesive dots result in relax and stretched conformation of the cells by limiting cell attachment and spreading. more the spacing in one direct lesser is the formation of focal adhesion which may be associated to alleviate actin stress fibers formation.11 The present study corroborates with the reports extended by aforementioned authors that presence of nanoparticles in the matrix has been sensed by the cells and have expressed more vinculin as



an obvious outcome. Moreover, the 0.5 wt% MWCNT reinforcement in the polymer matrix has a compelled the cell to manifest stretched feature while increased amount of nHA (6 wt%) has been observed with a relaxed morphology. The wear debris particles generated from an antioxidant impregnated ultrahigh molecular weight polyethylene (UHMWPE) display marked bone remodeling and a higher osteogenic: osteolytic profile compared to conventional UHMWPE particles. As reported in the same study, pure UHMWPE failed to exhibit enough efficacies to fulfill bone regenerative purposes. Hence, it is logical enough to enhance the performance of the polymer as a bone regenerative material by introducing osteoinductive nanoparticles (like MWCNT and nHA). As mentioned earlier, the niche-dependency of the cell fate processes is an unopposed phenomenon, experienced by all the cells in the culture. The effect of the cultureniche can be contributed from the chemokine microenvironment and cell-substratum as well.12 The rigorous interplay between the material-cell interaction and the biophysical cues can drive the conditional cellular decision towards any of the three fates as cell proliferation, cell differentiation or cell-death through apoptosis and/or autophagy. This aspect is investigated in this study. In the above backdrop, human mesenchymal stem cell (hMSc), which is capable of differentiating into different cell types such as osteoblasts, myoblasts, chondrocytes, neurons, and stromal cells, was used in the current study.13 In fact, we have developed a new generation of LOC device incorporating a biocompatible substrate to determine the biomaterial efficacy under the influence of shear stimulus to drive hMScs into osteogenic path(fig. 1a-1b). This would help to reconstruct a physiological mimicry. The biomaterial surface was further patterned into small pits (100 µm diameter) to mimic the physiologically specific space constraint condition (fig. 1c). The shear application onto the cells has been deduced by FE analysis (fig. 1d-1f). A host of biochemical assays and differentiation markers


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are used to quantitatively assess the extent of osteogenesis with different biophysical cues (fig. 1g). 2. Result 2.1. Analysis of microflow properties FEM solution towards flow pattern: The cell culture medium is considered as an incompressible Newtonian fluid, whose flow inside the channels was simulated using finite element software (FES; COMSOL Multiphysics version 4.3). It is crucial to determine the flow properties of the cell culture medium as the altered behavior of the cell growth was largely claimed to be dependent on the shear stress exerted. The algorithm which was used to develop the solution of the fluid flow is Eulerian, where the mesh is fixed, and the material particles change their position with respect to the fixed mesh. The flow problem inside the microfluidic channel was solved by the continuity equation and Navier-Stokes equations for the steady flow of an incompressible fluid, in which the inertial terms are neglected because the flow is unidirectional and at low Re, ∇ .v = 0

(1)

-∇p + µ ∇2.v = 0

(2)

where, p is the pressure and µ is the viscosity. The shear at the bottom floor was calculated to be ~1 Pa. This is in accordance with the FE solution (fig. 1c-1d). The shear value was deliberately kept as 1 Pa. as hMSc is extremely shear sensitive.5 Liu et al. have mentioned that 1.2 Pa of intermittent shear for a long time drives the cells in osteogenic direction.6 Though, Hong et al. reported the differentiation of hMScs in 3D tissue constructs by perfusion shear flow with shear stresses ranging within 1×10-5 to 1×10-4 Pa.14 In numerous studies, the sensitivity of mammalian cells to shear forces has been investigated.



Turbulent shear stress is generally thought to be more damaging to cells than the laminar shear stress of the same magnitude.15 In this study, microfluidic devices were used to ensure the flow in laminar regime. In terms of the critical level of shear stress that the cells can withstand, variation in opinion renders a diversified conclusion. One notable study reported that cell loss at high shear stress (100 Pa) and low shear stress (1 Pa) is greater than that at intermediate shear stress (10 Pa) for several animal cell lines.16 2.2. Biomaterial properties 2.2.1. Surface topography and wettability study Surface morphology of UHMWPE composite samples was determined using the highresolution atomic force microscopy technique (AFM). The topographic image for surface roughness and morphology analysis was generated by force-induced deflection in the cantilever. Figure 2 illustrates the surface morphology and wettability of UHMWPE along with UHMWPE reinforced with MWCNT and nHA nanofillers. The topographic image of control substrate was shown to have maximum peak height in the order of 0.05nm, which is far less than the agglomerated nanoparticles in the polymer matrix. A uniform distribution of reinforcement is shown in figure 2c, 2d. In figure 2c, the bundle of MWCNT fibrils was clearly observed on the UHMWPE surface with varying diameter of 20 nm to 50 nm and length of ~450 nm. The agglomeration of hydroxyapatite nanoparticle was found with the peak height of topographic image ranging from 20 nm to 35 nm (fig. 2d). The surface roughness was quantitatively analyzed by the root mean squared area roughness (Sq) to investigate the effect of nanofillers in the polymer matrix leading to change in roughness parameter. The roughness of the control sample was found to be 0.2 nm, whereas the value has been increased by ~96% in UHMWPE. The roughness parameter increases nearly by three times with Sq value of 16.04 nm, when MWCNT is incorporated in UHMWPE matrix. UHMWPE+nHA composite was observed to exhibit roughness value of 27.03 nm (fig. 2e).


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The significance of surface roughness properties in cell-material interaction was supported by a quantitative study of surface wettability of polymer composite. Surface wettability property of polymer samples was quantitatively analyzed by measuring water-substrate contact angle using goniometry technique (fig. 2f). The average contact angle of pure UHMWPE was found to be 89.6o which confirms less spreading of the water droplet on the polymer substrate, than control glass coverslip with contact angle value of 70.1o. UHMWPE was observed to have better hydrophilic property (contact angle 80.7o) with hydroxyapatite nanofiller. The effect of hydrophobic nature of UHMWPE with MWCNT nanofiller was assessed as 100.7o contact angle between the composite surface and the water droplet. 2.3. In vitro cellular response The cells require to interact with the implant material in such a way that they retain or gain the potential to simulate the regenerative/reparatory mechanisms, restoring cellular functionality in its original state and function. In the present work, the cellular performance was assessed in two aspects; (i) Fixed cell behavior in view of phenotype and (ii) Cell functionality in view of electrophysiology, while keeping the cells alive and functional. 2.3.1. Cytocompatibility analysis According to Sigot-Luizard et al., cytotoxicity is considered as the detrimental criteria such as cellular alterations, cell death, hampered growth etc. 17 Hence, the minimum criterion to be qualified by any unconventional cell culture approach is to determine the cyto-viability. In this study, we have used a patterned microfluidic device which is displayed in figure 3a-3b. The rationale behind the cytocompatibility study in the present study is to ensure the maintenance of constrained cells in functional condition throughout the culture duration (fig. 3c-3d). WST-1 assay for cell viability: The stem cell differentiation depends on the microenvironment, wherein the cells grow. In order to describe it quantitatively, we have



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used the microfluidic device to grow cell in dynamic shear stress condition after the 3rd day of incubation. Differentiation and proliferation are the two opposing processes, which are inversely related to the aspect of the cell viability values. As the cells switch from proliferation to differentiation, the viability measurement also decreases. Our results have also reflected this biological fact (fig. 3e-3f). As a preliminary assessment of cytocompatibility, all the samples and the device supported cellular growth with a considerable number of viable cells. Maximum cell viability was recorded for the pure UHMWPE on day 3 with the flow. This is perhaps due to the renewal of nutrient media. The percentage of viable cells for UHMWPE was observed to be consistent on day 3 and day 12 in the static culture (fig. 3e). But the application of shear has decreased the viability on day 12 by ~9% than the initial day 3, which continues till day 20 with percentage difference with the initial reading appears to be ~10% (fig. 3f). This denotes the onset of cellular differentiation. The difference in cell viability is ~28% for nHA reinforced UHMWPE with and without shear on day 20. The same sample at the static culture has exhibited a decrease of ~25%when compared to the initial day3.

On day 20, nHA containing polymers with shear achieved the lowest viability. This essentially reflects the cellular decision to drive themselves into the differentiative path. On day 20, UHMWPE+nHA samples were observed with a marked decrease in viability by ~30% than that of the pure polymer. This supports the role of the hydroxyapatite nanoparticles

in

guiding

the

stem

cells

towards

osteogenesis.

In

comparison

UHMWPE+nHA, UHMWPE+MWCNT substrate also exhibited cell viability decrease by ~22% from the starting day of differentiation media. As an inference of the above discussion, it is evident that dynamic flow has supported cell growth, irrespective of sample substrate.


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2.3.2. Human mesenchymal stem cell differentiation 2.3.2.1. Flowcytometry analysis to determine G1/G0 arrest Osteogenic differentiation is associated with cell stagnation at G1 / G0 as the cells are withdrawn from the cycle. We optimized the flow cytometry-based experimentation of the cell cycle status with a low number of cells, which is an obvious challenge for any microfluidic experiments. PI based FACS analysis shows a representative comparison of the cell cycle status of the hMSc. Experiments with flow along with the nanoparticle embedded UHMWPE have synergistically refrained the cells to perform normal cell cycle events (fig. S1a-S1b). It is worthwhile to mention that among all the samples; the static controls had evinced with highest S-phase cells (~63%). As a clear complementary result (fig. S1a-S1b), the cell population of the same showed the smallest fraction of cells in G1/G0 (36%) with only ~1% cells in the G2/M phase. On the contrary, dynamic UHMWPE+nHA samples were the highest among all the samples to have G1/G0 phase cells (~62%). The dynamic culture mode has set a different trend in the cell cycle status of the growing hMScs. All the dynamic groups have a characteristic elevation in the G1 / G0 phase cells than the static group which was atleast lower by c.a. 10% (fig. S1a-S1b) than the former. Comparing within the static group, the nHA based polymer was able to establish the effect of the cell-substratum onto the cells by exhibiting significantly higher G1/G0 phase cells (~50%) than that of the control group (~36%). The cells growing on the UHMWPE+MWCNT were significantly higher in number to be arrested at G1/G0 phase (~43%) than the control. The above discussion indicates that the biomaterial substrate together with the applied shear stress had resulted in cell cycle arrest at G1/G0 phase. 2.3.2.2. Analysis of focal adhesion Considering the unopposed phenomenon regarding cell adhesion that modification of cell functionality is preceded by cell adhesion, we now analyze the formation of the focal



adhesion points. Focal adhesion is a structural union between integrin biomolecule, vinculin and multiple signaling molecules. This also connect the actin cytoskeleton for downstream conduction of the extracellular signal. Cells modify focal adhesion in order to execute cellular events, like differentiation. Vinculin was targeted with anti-vinculin monoclonal antibodies to acquire immunofluorescence images (fig. 4). The attachment of hMSc to nanoparticulated polymeric composites depends on the number of focal adhesions. Cell-to-substrate interaction with a synergistic effect of shear had brought out subsequent alteration in cell behavior under the precise regulation of vinculin expression and in turn focal adhesion formation. Vinculin, being the intermediate between the cytoskeleton and the integrins plays a pivotal role in downstream signal transduction. Such an important member of the focal point was thus decided to be monitored for 4 selected days. The culture duration of 20 days has essentially demarked with four important culture timepoints. Those were day 3- completion cell proliferative phase (day 4 onwards the proliferation media was changed to differentiation media), day 8- an early stage of osteogenesis, and day 14- middle stage of osteogenesis, day 20- terminal stage of osteogenesis. According to figure 4a, the day 3 results exhibit the presence of vinculin in a diffuse manner. The progression of the differentiation process has led to the formation of more and more number of focal adhesions. As an expected outcome, day 20 results have expressed the highest quantity of vinculin molecules. A clear discrepancy between the samples was created due to the substrate modification and application of an external biophysical cue. The cells, which were exposed to nHA containing substrates along with the shear, were able to recruit the highest amount of vinculin. The static culture has expressed lesser vinculin than the corresponding dynamic culture experiments (fig. 4b). The vinculin expression was increased by ~15% to 20% with the application of the shear, irrespective of the substrates. A significant increase has been particularly noticed, while comparing the


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MWCNT and nHA containing samples on the application of flow. The static culture was characterized by a narrow difference for the above-mentioned samples (fig. 4b). There was a marked difference in the pattern of expressed vinculin which can be distinctly observed from. Dynamic flow stress on the cells has caused clumping of the focal proteins (fig 4a). This is because of the adaptation towards the stress originated due to flow. The cells try to bind themselves more strongly onto the substrates and thereby express more focal points. It has been justified with the appearance of ruffle border at the cell edges with shear. Such feature is quite unusual to be found on hMSc / differentiated hMScs, while the cells without shear have formed filopodial extension along with lamellipodium holdfasts (fig. 4c). 2.3.2.3. Analysis of Actin remodeling The cell behavioral parameters are regulated by cytoskeletal arrangement. Not only the cell shape but also the cytoskeletal organization is responsible for the cell fate processes. When the cells were chosen for a definitive fate process like differentiation, it is essential for the cell to undergo multiple deformations without losing their integrity. Cellular aligning and in particular, actin alignment are some of the qualifying processes to lead a cell towards differentiation. The detection of cellular alignment was performed on the basis of microtubule organizing centre (MTOC) locations by α-tubulin staining, whereas actin studies were done with phalloidin based actin staining (fig. 5). The dynamics of the actin of the differentiating cells was precisely studied, selecting threetime-points at an interval of 10 days (fig. 5a-5b). In particular, the culture time point of day 1, day 10 and day 20 were selected as they represent the cellular events designating the attachment of the cell, midway towards differentiation, peak differentiation, respectively. Unlike the vinculin expression, the actin remodeling is a slower process as it requires the spontaneous participation of several proteins. That is the reason that actin study was decided at an interval of 10 days instead, to visible appreciable change. The cell chirality has been observed on the day 4, once the flow



of the media was started (fig. 5c). The application of the biophysical cue in form of shear reveals that chiral morphogenesis is generated because of polarity created for the cells by the directionality of the shear.18 It is a function of intact active actin.19 The polarization of the cells is essentially left to right twisting as also observed by several researchers.20 The radial fibers (RFs) and the transverse fibers (TFs) are clearly observed and these play a pivotal role in cellular L-R (Left-to-Right) asymmetry. While describing the events regarding cell alignment onto the surfaces, it has been noticed that UHMWPE+MWCNT has a tendency to align the cells better than the other substrates. The static culture was able to exhibit a pattern of aligned cells for the MWCNT reinforced samples (fig. 5e). The application of shear has oriented the cells of all samples towards the flow direction (fig. 5f). As an extension of the previous discussion, the behavior of the actin is independent with respect to the cellular arrangement. In case of the MWCNT containing polymers under static culture, the actin cytoskeletons were neither solely aligned nor as random as the other samples, rather exhibit a semi-aligned pattern. When the flow was introduced, all the cells achieved an aligned pattern of the actin cytoskeleton. The actin and cellular alignment has been found as specific combinations of (i) random cell with random actin for all the samples in the static culture other than UHMWPE+MWCNT, (ii) random cell with semi-aligned actin for UHMWPE+MWCNT in static culture, (iii) aligned actin with aligned cells in all samples at the dynamic culture (fig. 5d). The quantification of the actin alignment using ImageJ plugin has revealed that ~ 3700 fibers of the selected ROI (region of interest) have unidirectionality at an angular direction of ~75°. This distribution was quite diffuse for the semi-aligned and highest variability in angular distribution has been observed for the random fibers. The nature of the actin fibershas been observed to alter with the progression of osteogenesis. After attachment, the cells showed a


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pattern of intersecting crossed fibers (fig. 5a-5b). On day 10, hMSc were noticed with thick stress fibers which corroborate with the reports published by Titushkin et. al.21 As the cells started to differentiate along the osteogenic path, the thick fibers were replaced by more thin mesh-likefibers. The actin structure in hMSc is quite different from that in terminally differentiated osteoblasts.21 Indeed, it has been found that during the osteogenic differentiation of hMScs the thick stress fibers were replaced with finer actin mesh, which denoted the successful differentiation process of osteogenesis (fig. 5a-5b). The day 20 observation contains characteristic thinner actins, though in case of dynamic culture, some thick stress fibers still persisted because of the shear stress. 2.3.2.4. Osteogenesis of hMScs marked by ALP activity, collagen deposition, matrix mineralization The three different stages (early, middle, terminal) of osteogenesis can be delineated by the sequential occurrence of specific biological activities favourable for the maintenance of osteogenic microenvironment.22 The ALP activity starts at an early differentiation stage, whereas the collagen deposition designates the middle stage of the differentiation process. The late events, like matrix mineralization, specific marker protein expression establish the terminal osteogenesis. Early osteogenesis: Alkaline phosphatase activity due to osteogenic induction One of the early markers of stem cell differentiation along the osteogenic path is the increased alkaline phosphatase activity of the differentiating cells. The enzyme is crucial for the matrix maturation through the course of osteogenic differentiation from other cell-types.23 The detection of ALP was performed based on the enzyme histochemistry technique using BCIP-NBT. The reaction in BCIP-NBT staining gives rise to blue to brownish product depending upon the medium pH according to manufacturer’s protocol. The procedure occurs in two steps; (i) dephosphorylation of BCIP and (ii) oxidative dimerization of



dephosphorylated BCIP by NBT. Figure S2a shows the alkaline phosphatase (ALP) levels detected by BCIP-NBT staining in hMScs cultured on UHMWPE-nanoparticle (nHA and MWCNT) composites for 8 days. A distinct dark brownish precipitate can be observed in the cells adhered to the samples, because of the enzymatic activity of ALP after culturing the cells in differentiation mode. In accordance with the alizarin red staining based results, the ALP activity happened to be the highest in and UHMWPE+nHA substrates, next usurped by UHMWPE+MWCNT substrates. The cells show a distinct difference in ALP activity between the static and dynamic mode of differentiation. The dynamic mode of cell differentiation supported the cellular decision towards osteogenic path with a significantly higher exhibition of ALP activity, compared to the static mode. It is worthwhile to mention that the trend of expressing the osteogenicspecific genes had been exhibited more on the nanoparticle-UHMWPE, when compared to the control and UHMWPE substrates. Middle stage osteogenesis: Collagen deposition as a marker of osteogenic differentiation Matrix deposition of collagen I by the committed stem cells in osteogenic pathway enables positive feedback signaling, which acts as a vicious process in driving more cells towards the differentiation.24 Figure S2b depicts the level of deposited/secreted collagen by hMScs cultured on UHMWPE nanobiocomposites for 14 days, following the differentiation protocol. Sirius red staining method was followed to determine the degree of collagen deposition. After staining with Sirius red, the bright field optical microscope images suggest that hMScs cultured on UHMWPE-nanoparticle secrete a greater amount of collagen as extracellular matrix (fig. S2). Moreover, the dynamic culture appeared to be more efficient in inducing the cellular collagen secretion than that of the static cultures. Comparing the two nanofillers, nHA vs MWCNT, the former was more competent for osteogenic induction than the later. Nevertheless, both had successfully driven the osteogenic differentiation process in contrast


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tothe control and UHMWPE. This observation in the present study is consistent with the results reported by other groups which involves the synergistic effect of shear on the cells grown on UHMMWPE and HDPE.25 Terminal osteogenesis: matrix mineralization by the differentiating cells The terminal stage of osteogenesis is indicated by mineralization of calcium phosphate on the extracellular matrix (ECM) secreted by the hMScs committed towards osteoblastic lineage. The calcium deposits were visualized by Alizarin Red S (ARS) staining after 20 days of culture on MWCNT and nHA included UHMWPE composites. Figure 6a presents brightfield optical images, which reveal the extent of calcium deposition on the nanoparticle reinforced composites. Clearly, the calcium mineral deposition is significantly higher on nHA composites in comparison to the control and pure UHMWPE. However, MWCNT reinforced UHMWPE also support matrix mineralisation to a comparable extent as of nHA. The biomineralisation started off with almost negligible calcium deposition (day3). As the time proceeded in differentiation-culture, the hMSc oriented themselves in an altered manner with the secretion of calcium salts. Finally, on day 20, the cells with shear had a visible amount of mineral deposition, especially for the nanoparticle-containing materials. The static cultures also exhibited matrix mineralization but, quite lesser than the dynamic one. The nHA reinforced UHMWPE, being the leading substrate with mineralizing effect in the dynamic culture, was still able to maintain similar mineralisation in the static culture too. Nonetheless, the MWCNT filler-UHMWPE also had stimulated the cells towards depositing the mineral in the static culture. This was further confirmed by quantification of the extracellular calcium bound to alizarin red dye, detected by elution of the alzarin red extracts and recording the absorbance at 450 nm, as shown in figure 6b. The calcium-bound dye content was maximum in the differentiated hMSc, cultured in dynamic mode. Highest calcification capability could be



distinctly conferred to the nHA composites, in both dynamic and static culture, next followed by the MWCNT composites. The bilayered sample was also capable to express similar properties as the respective single layered composites. The amount of calcification was ~3 fold in case of UHMWPE+nHA composite compared to its static experiment. From the perspective of extensive quantitative analysis, the cell samples, differentiated on nHA and MWCNT containing UHMWPE, had ensued enhanced dye-binding than the UHMWPE. The other substrates too had higher matrix mineralization, when dynamic versus static results were compared. This is commensurate with the outcome of alzarin red staining of the cellsamples, imaged using an optical microscope. Additionally, the day 3 results revealed a low calcium amount, which seemed to be the basal level of calcium secretion/present to maintain the normal microenvironment of the cells. EDS data also corroborate the similar scenario (fig. 6c). The nHA added UHMWPE samples in dynamic culture has the highest calcium deposition. 2.3.2.5. Expression of osteogenic markers proteins: induction and maintenance of the osteogenesis The process of osteogenesis commences with the transformation of multipotent stem cell to committed osteoprogenitor cells and then differentiation into pre-osteoblasts, which eventually get converted into mature osteoblasts.26 This requires the interplay of multiple factors, which through up/down regulation shape the microenvironment, that recruits the stem cells for osteogenic differentiation. BMP2 expression at the induction of osteogenesis: Osteoblast differentiation is tightly regulated by a range of hormones, cytokines, and multiple transcription factors.27 BMP2 is one of the most important cytokines in this regard and plays several important roles in a variety of cellular functions, ranging from cell growth, and differentiation to bone development and the repair of bone fractures.28 The expression of BMP2 occurs as the late


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markers of the osteogenesis. The immunofluorescence images illustrate the nature of the BMP2 expression as a marker protein (fig. S3a). As mentioned earlier, BMP2 monoclonal primary antibodies were used to obtain the protein expression. As the intensity of the emitted fluorescence signal was low, hence software-based analysis was required for acquiring values of the pixel intensity (fig. S3b-S3e). Figure S3e shows the quantitative comparison of the BMP2 expression in terms of pixel intensity. There is a distinct elevation of BMP2 protein expression, when the cells were exposed to shear with an osteogenic induction medium. The same substrates, without shear, has lower BMP2 expression. The cell growth on nanoparticle reinforced UHMWPE exhibited upregulation of the osteogenic marker protein. All the samples exhibited a good amount of protein elevation, when compared to the control. The UHMWPE+nHA samples have the maximum expression of BMP2, when the MWCNT-reinforced UHMWPE has subsisted in a similar fashion. The cells, which have grown on nanofiller samples, without shear, have moderately higher BMP2 activity than the control, whereas pure UHMWPE has failed to manifest such characteristics. A basal level of BMP2 expression in osteogenic induction medium could be easily appreciated from figure S3a. In summary, the influence of the biomaterial with a prescribed amount of the nanoparticles has successfully driven the multipotent cells to a committed path of osteogenesis. RUNX2 expression denotes onset of osteogenesis: At the most basic level, osteogenic differentiation requires the expression of the key transcription factor, Runt-related transcription factor 2 (RUNX2).29 Mesenchymal stem cells (MSC) are multipotent cells, functioning as precursors to a variety of cell types including adipocytes, osteoblasts, and chondrocytes. Between osteogenic and adipogenic lineage commitment and differentiation, a theoretical inverse relationship exists, such that differentiation towards an osteoblast phenotype occurs at the expense of an adipocytic phenotype.30 This balance is regulated by



numerous intersecting signaling pathways that converge on the regulation of two main transcription factors: peroxisome proliferator-activated receptor-γ (PPARγ) and Runt-related transcription factor 2 (RUNX2). The expression of Runx2 dictates the osteogenic path, whereas the PPARγ drives the adipogenesis. The selection of the pathway depends on the extracellular cues. The substrate composition and the mechanical stress in form of shear appoint the RUNX2 pathway and as a result, osteogenesis has occurred. While deducing the expression of RUNX2 at the protein level, we have observed that the same transcription factor is present in the cells at a perceptible quantity (fig. 7a). The addition of the nanoparticle fillers into the UHMWPE matrix has increased the expression of RUNX2. As an obvious incidence, highest expression was secured by the UHMWPE+nHA samples cultured under shear (fig. 7b). The static culture also displayed increased expression of RUNX2, which accounts ~32% higher pixel intensity than the control for the same setup. Comparing the dynamic culture results, around ~63% increase in RUNX2 expression was recorded for the same group, than that of the control. The MWCNT containing samples were also efficient enough to bring out a pronounced expression of RUNX2. In this case, the static cultures manifested ~12% increase, whereas the application of shear has elevated the % difference upto ~53%. It clearly postulates that osteogenesis is modulated as a consequence to the synergistic effect of shear and substrates stiffness. 2.3.2.6. Compensation of stemness: evaluation of vimentin expression Vimentin is a type III intermediate filament (IF) protein, which is a marker for epithelial to mesenchymal transition that occurs during embryogenesis and cancer metastasis. A decline in the expression of vimentin, a marker specific for mesenchymal stem cells (MSCs) along with the enhancement in the expression of osteogenic proteins was observed, when hMScs were cultured on nanoparticulate reinforced composites. Figure S4a distinctly denotes a sharp fall


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in protein expression at the day 20. The qRT-PCR analysis of mRNA of vimentin also corresponds well with the protein study (fig. S4b). Importantly, all the samples had failed to maintain the stemness in terms of vimentin gene expression at the end of the culture. 2.3.2.7. Quantitative mRNA detection for osteogenic gene expression: Quantitative RT-PCR analysis is supposed to be the confirmatory test for the above-discussed parameters. The present study also analysed, mRNA expression of the some of the crucial genes which are inevitable for osteogenesis at different points in culture (fig. 8). The initial level of osteoblast differentiation was confirmed by runx2 expression. No significant change in expression of runx2 was recorded for the UHMWPE and UHMWPE+MWCNT samples w.r.t. control in static culture (no shear flow), which shows ~10% increase than nHA reinforced UHMWPE. The expression of the transcription factor exhibits similar behavior in controlled samples both in static and dynamic culture. The value was found to be increased by ~40%, when hMScs were subjected to shear flow than static culture on the UHMWPE. The effect of nanofillers in early osteogenic differentiation was more significant with ~65% and ~125% increase in runx2 expression in dynamic culture for MWCNT and nHA reinforced UHMWPE, respectively. The quantification of Alkaline phosphatase activity of the differentiating cells is one of the key characteristics of stem cell differentiation to follow the osteogenic pathway. In static culture, the expression of ALP is increased consistently by ~10% from control to UHMWPE samples. Unlike runx2, the expression of ALP in control has increased by ~75%, in culture with shear stress. This behavior is found to be similar in case of UHMWPE, but a sharp increase in the alp expression was increased by ~90% was recorded in dynamic than static culture. In case of UHMWPE+nHA, there is ~6% decrease in alp expression from UHMWPE+MWCNT substrate.



Collagen I deposition is generally considered as a checkpoint for the commitment of stem cells towards the osteogenic differentiation. The increased value of colIA expression was observed from static to dynamic culture in all the samples including control sample (fig. 8). In the control sample, colIA expression has increased by ~20% from static to dynamic culture. The percentage of increase in collagen I deposition is more (~27%) in both UHMWPE and UHMWPE+MWCNT substrate. Interestingly, ~55% increase in value for colIA expression was found, when cells are cultured under controlled shear stress vis-à-vis static culture. It can be noted from figure 8, that nHA incorporation in UHMWPE has shown to have the highest colIA expression among all other substrates, when the cell was under shear flow. In the final stage of osteogenesis, osteoblast formation from stem cell was confirmed from quantitative analysis of osteocalcin (ocn) and osteopontin (opn) expression by qRT-PCR. The effect of nanofiller in polymer matrix towards ocn expression is not greatly significant, still, the value has increased by ~6% from virgin UHMWPE to MWCNT and nHA reinforced UHMWPE. The difference in expression of ocn from static to dynamic culture condition decreases from control to polymer substrates with negligible difference in nHA reinforced UHMWPE. Osteopontin expression has shown to have the least effect of shear flow to induce osteogenic differentiation in control samples (fig. 8). The effect of dynamic culture leads to ~20% increase in opn expression value from static culture condition on UHMWPE, whereas the value is significantly increased by two-fold in MWCNT reinforced UHMWPE. The expression of opn is also found to increase by ~83% for the dynamic culture from the static on UHMWPE+nHA composites. The highest value of osteopontin and osteocalcin expression was observed in the case of nHA reinforced UHMWPE. (fig. 8)


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The vinculin gene expression incurs well with the protein level study of the same (fig. 8). The dynamic culture has increased the expression significantly than the static culture for all the samples. While comparing with other genes, vinculin gene seems to be maximally impacted by the shear. As an obvious manifestation, the cells adapt to the shear flow by increasing the number of adhesion points with the substrates. This denotes that unlike the other genes, the regulation of vinculin gene is not solely due to the differentiation, but shear also has played a simultaneous role in the gene expression. 2.3.3. Analysis of cell functionality Importantly, hMScs should be able to express enough calcium activity to get qualified to enter the last stages of osteogenesis towards the formation of osteoblast. It is reported that voltage-directed calcium channels (VDCCs) (including L-type calcium channels) are involved in controlling osteogenic functionalities.31 Therefore, we investigated the presence of VDCCL in the differentiated hMScs as a functional marker of the differentiation process. Apart from this, the intracellular calcium activity was also determined as a consequence of the osteogenesis. 2.3.3.1. Patch clamp study The evolution of the membrane currents in undifferentiated and differentiated cells in dynamic cultures were acquired using whole cell patch-clamp technique (fig. 9). Currentvoltage (I-V) relationships recorded in the potential range from -80 to +80 mV, revealed a family of currents. The undifferentiated cells for both control and UHMWPE showed sufficiently large and cleaner currents with a reversal potential of -60 mV, whereas the differentiated cells show stronger inward currents that were more prominent for nHA reinforced UHMWPE cultures. With the replacement of sodium ion with barium in the bath solution, the undifferentiated cells, both control,and UHMWPE showed very less inward current, whereas the differentiated cells for both UHMWPE+MWCNT and nHA reinforced



UHMWPE showed a stronger shift in the permeability of ions. The MWCNT composites have increased Ba2+currents than that of the Ca2+ currents, with mean peak current amplitudes of −62.3 and −79.8 pA, respectively (fig. 9b). The nHA composites were able to exhibit higher inward current due to calcium and barium conductance. It amounted to −62.8 and −80.3 pA, respectively. As suggested earlier the inward current in osteoblasts is a result of conduction of calcium ions through L-type Ca2+ channels.32 To investigate the presence of L-type calcium channel (CACNA1C) in our cultures, immunocytochemical and mRNA amplification for the α-subunit were analysed for both static and dynamic cultures. Figure 9c shows relatively higher expression of voltagedependent L-type Ca2+ channels (VDCCL) in the UHMWPE nanobiocomposites. The flow has a positive effect on the expression of these channels. The gene expression pattern also concurred the fact of increased mRNA expression for the dynamic culture (fig. 9d). The nHA reinforced UHMWPE has expressed a significantly higher expression, which was not present in the static case. All these parameters suggest that the current is indeed conducted through Ltype Ca2+ channels (VDCCL) and flow has been successfully able to bring a different response among the investigated UHMWPE nanobiocomposites. 2.3.3.2. Calcium activity In order to further confirm the expression of functional voltage-gated calcium channels in dynamic cultures, intracellular calcium activity was analysed using the low-affinity calcium indicator, Fluo4. The voltage-sensitive dihydropyridine (DHP) calcium channels (L-Type) agonist ((±)-Bay K8644 increased the intracellular calcium for both undifferentiated hMScs and for the differentiated osteoblasts (fig. 9b), with the highest rise in fluorescence for the nHA composites (fig. 9d). Moreover, the normalized value of the final slope also experienced an elevation in case of nHA reinforced UHMWPE. Figure 10provides the graphical and pictorial view of the ground state calcium activity of undifferentiated sample cells. Thus, the


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rise in intracellular calcium upon ((±)-Bay K8644 application further confirms the presence of L-type voltage-gated calcium channel in the differentiated cells. This corroborates with other reports, where inclusion of L-type Ca2+ channels (VDCCL) was designated as a milestone for osteogenic differentiation.33 3. Discussion The virtue of microfluidic culture is that it can recreate the artificial microenvironment by integrating micropatterned surface for entrapping a low number of cells, which gives unbiased output. For example, Halldorsson et al. reported the advantages of microfluidic cell culture to closely mimic the natural microenvironment of a cell by continuous perfusion along with the benefit of maintaining a small population.34 Justifying the relevance of introducing biomaterial together with the microfluidic device, it can be said that this novel endeavor impelled an unbiased analytical output towards biocompatibility based cell study. Our biocompatibility testing in microfluidic model ensures a controlled flow velocity of ~15.6 ml/h to provide continuous supply of fresh medium and removal of waste similar to the tissues bathed with IF.35 The cells were allowed to grow in 100 µm-wide and 50 µm-deep pits situated in 1 mm wide and 200 µm-deep channels. The base of the channels was constituted with the experimental biomaterials to enable the cells to grow on a substratum. This can be correlated with a simulative approach of the biomaterials being implanted inside the body. hMSc were found to be invigorated to have minimal amount of osteogenesis, when they were subjected to material interaction. However, the intrigued synergistic effect of shear on top of the biomaterial has escorted the stem cells into various degrees of osteogenesis. nHA has been proven to have maximal potency in performing cellular differentiation. This had been justified with the expression of early, middle, and late stage differentiation characteristics like alkaline phosphatase activity, collagen synthesis, and matrix mineralization, respectively. The osteoinductive gene expression has been evaluated with



qRT-PCR to support the marker protein expression in view of osteogenic differentiation. As revealed by several researchers, differentiated hMSc towards osteogenesis expresses L-type of calcium channels with an increased inward calcium current.31 Further, we have performed the functionality tests of the cells by patch clamping in whole cell mode to confirm the presence of calcium and barium current. The intracellular calcium activity was afterward deduced using calcium channel agonist. The static mode of determination of material impact on the cells revealed with the similar outcome for both nHA and MWCNT composites. Here lies the motivation of applying shear while determining the cell fate. The static culture represents the conventional biocompatibility experiments, whereas the dynamic one can simulate the in vivo scenario. The application of the mechanical stimulation has successfully brought a discernible influence of biophysical cue vis-à-vis biomechanical cue. An embodiment of such device along with biopolymers enhances the cellular activity by getting them exposed to mechanical stimulation from the shear flow and physical cue from the material interaction, simultaneously. The dynamic nature of the cellular decisions originates as the cells sense and monitors the microenvironmental chemical and physical factors through sensory structures capable of detecting and transducing extracellular stimuli downstream to the interior of the cell. 3.1 Surface topography and wettability AFM scanning probe technique detects the force profiles of individual atoms of cantilever needle to the surface of the material under study. Apart from chemical properties of materials, surface topography also plays a vital role in the understanding the mechanism of cell-material interaction.36 Surface topographic 3D images of polymer nanocomposite were found to have agglomerated nanoparticles in the polymer matrix. Nanoparticles have high surface energy due to the high surface area to volume ratio. They always tend to diminish their higher surface energy by clumping together. The maximum height of the MWCNT aggregate was


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found to be ~46.4 nm with the length of ~450 nm, giving the value 10 nm for height to length aspect ratio (fig. 2c). This value of aspect ratio is characteristic of MWCNT, indicating the agglomeration is MWCNT bundle. The agglomerate found in case of nHA reinforced sample has a length of less than 200nm without any long chain-like morphology rather appears to be particle type structure with a maximum height of ~35nm (fig. 2d). The topographic image of the control sample was shown to have peaks in picometer level, which does not contribute to the significant value of surface roughness. The surface roughness values increase with the addition of nanofillers in UHMWPE matrix. The reason for the surface property is the agglomerate structures present on the polymer surface. The roughness value increases from MWCNT to nHA by ~68%, because of the presence of higher amount of nHA than MWCNT in the polymer matrix. The surface wettability dependency on the roughness parameters was analyzed by the measurement of water-polymer contact angle. Surface wettability plays a major role in the adsorption of protein and subsequent cellular adhesion. The controlled sample was shown to have hydrophilic behavior with 70.1o contact angle (fig. 2f). UHMWPE depicts very little hydrophilic nature, having the contact angle value of 89.4o. Because of the absence of functional group in polyethylene chain(-CH2-CH2-)n, the water molecule does not have any stronger interaction with the surface of UHMWPE. It shows little hydrophilic behavior only due to the Vander Waal interaction water with the polymer chain. The behavior of the water droplet depicts hydrophobic nature of UHMWPE+MWCNT composite with a contact angle of 100.03o. Weak Vander Waal interaction of MWCNT with water molecule may be the cause for higher contact angle in MWCNT reinforced UHMWPE than virgin UHMWPE. Also, nHA reinforced UHMWPE exhibits hydrophilic behavior because of the presence of polar groups like PO43- and OH-. The contact angle of 81.10o is due to the formation of H-



bond between the electronegative atoms of these polar groups of hydroxyapatite with the water molecules. 3.2. Correlation of the cellular response with biomaterial property This work demonstrates that small changes in matrix hydrophobicity through the addition of nanoparticles can dramatically alter cell-matrix interactions and in turn have a profound impact on various cellular behaviour, such as adhesion, cytoskeletal organization, and differentiation. The difference in wettability between nHA and MWCNT containing UHMWPE allows deconvolution of more cell adhesion on the hydrophilic substrates than the hydrophobic one

37

In addition to modulating cell adhesion, hydrophobicity also influences

the cytoskeletal organization of hMScs in a cell-density dependent manner. Previous studies have shown that the cytoskeletal changes that stem cells allow them to respond to their extrinsic/intrinsic cues.36 By harnessing the changes in the cytoskeletal organization of hMScs on nanoparticulated nHA composite, we were able to direct the highest amount of differentiation into osteogenic lineages. Moreover, AFM study reveals higher roughness for the nHA-composites which has helped the cells to differentiate more on those materials.38 The nanotopography appears to influence the process of actin remodeling. At an early stage of cell growth, the cells exhibit random arrangement of actins with thick fibers which govern cellular attachment (fig. 5a). The cells at that initial phase seek for survival by spreading onto the substrates. More the compatibility of the substrate, faster is the alignment and growth of the cells. Bao et al. reported that the alignment of the cell is dependent on the substrate rigidity. As far as the tensile properties are concerned, the yield strength (the transition of linear to non-linear deformation) of UHMWPE+0.5 wt% MWCNT is 22 MPa, whereas that of UHMWPE+6 wt% nHA is 18MPa. When compared to baseline value of 14 MPa for unreinforced UHMWPE, the above values do establish positive role of nanoscale reinforcement on strength enhancement. Concerning the elastic stiffness property, which is


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reported to be a significant biomaterial physical property to influence cell functionality, the addition of 6 wt% nHA or 0.5 wt% MWCNT enhances elastic modulus from 625 MPa (unreinforced UHMWPE) to 783 MPa or 748 MPa, respectively. The low volume fraction of reinforcement is This could be a possible able to modulate elastic modulus by 20-25%. The differenced in elastic stiffness properties can explain why the differentiating cells were more aligned onto the MWCNT containing UHMWPE substrates. Then HA is more biocompatible than the MWCNT because hydroxyapatite is a component of the bone matrix. Regarding the biocompatibility of UHMWPE, Macuvele et al. have designated UHMWPE as the gold standard, w.r.t. an articulating counterface during arthroplasties.39 As a reason, the authors have highlighted the material’s superior wear resistance along with high fracture toughness and biocompatibility compared to other polymers. Wang et al. performed in vivo experiments, where it was observed that the pure MWCNT has very little osteoinduction, whereas a composite of hydroxyapatite has increased the efficacy of bone formation. It was also mentioned by Wang et al. in their review article about the surface charges of a material can promote protein adsorption and cell attachment.40 Li et al. also justified their observations by reporting hydroxyapatite coated scaffold as a better candidate for osteointegration and osteogenesis.41 Moreover, Müller et al. suggested that calcium phosphate surfaces are ideal for osteogenic differentiation by escalating the MAPK-ERK1/2-RUNX2 pathway.42 Hydroxyapatite, being a biomimetic calcium phosphate ceramic,

it

promotes

osteogenesis

more

than

MWCNT.

Therefore,

the

UHMWPE+0.5wt%MWCNT, being a stiffer material than UHMWPE+6wt%nHA, has helped the cells to align more, but the nHA containing samples have overcome the stiffness difference in terms of osteogenesis. Several studies have inferred that osteogenesis depends on the 43, but according to our study narrow difference of the stiffness has been neglected by the cells and the biological mimicry of the nHA has dictated a better degree of osteogenesis.



3.3. Left-to-right chirality during cellular orientation under shear One of the most important cellular responses is mediated through the cytoskeletal remodelling which provides the cell with the provision of changing cell shape and adapting them in the altered situation. Helical cytoskeletal polymers, such as actin filaments and microtubules, are the subcellular candidates which mediate the reorientation of the cells in the direction of the mechanical force (flow of fluid).44 A scientific attribute towards categorizing several classes of actin filaments, can be based on thickness and protein composition. Weakly motile cells exhibited a left-right asymmetry in their actin arrangement under mechanical force along shifting of the actin morphology of the actins between thick-bundled and thindiffuse fibers. In the present study, the application of the flow condition has portrayed similar left-right cellular chirality, which acts as the evidence of the cells, responding to the mechanical stimulus (fig. 5c). According to McConnell et al., the Class I myosins are the molecular motors and those are responsible to link the cellular membranes to the actins.45 These proteins play active roles in generating membrane tension and mechano-signal transduction. Liu et al. described that L-R chirality is controlled by unidirectional twisting of α-actinin rich radial actin fibers (RF).46 The torque generated by the radial actins manifests tangential shift in the retrograde movement of transverse actin fibers (TFs). In the current study, figure 5c clearly depicts the RF twisting in order to align the cell along the applied shear. 3.4. Shear stress as potent mechano-stimulator of osteogenesis Coming to the role of shear, it acts as a biophysical cue onto the cells for which the cells experience in vivo like physiological condition and response accordingly. Yourek et al. observed the osteogenic effect of the shear on the hMScs.5 The shear stress in the current microfluidic system could have stimulated nuclear localization of TAZ (transcriptional coactivator with PDZ-binding motif), a transcriptional modulator of MSCs. This was also


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reported by Kim et al. that activated TAZ expresses osteogenic genes such as CTGF and Cyr61, and induced osteogenic differentiation.47 Transient receptor potential melastatin 7 (TRPM7), which is known to be a mechanosensitive protein, is upregulated due to shear. Expressed TRPM7 then helps in localizing increased amount of vinculin to the membranebound integrins.6 This could be a possible explanation of getting increased vinculin expression, apart from the differentiation triggered elevation. This plays a two-way role by serving an adaptor to form the focal adhesion sites to remain attached to the substrate amidst of shear. On the other hand, the vinculin mediates MAPK-ERK1/2 signaling pathways involving Focal adhesion kinase (FAK).48 This, in turn, expresses the RUNX2 which is the designated as a “master transcription factor of osteoblast differentiation”. Extending the role of shear further, it also targets expression of BMP2 which has also osteogenic effect.

49

The

BMP-2-induced signaling pathway, for example, upregulates the expression of the three central osteogenic transcription factors Osterix, RUNX2, and DLX5.50 BMPs are also activated through the upstream via binding to homomeric type II receptors on the membrane, which transphosphorylases homomeric type I receptor to start the downstream Smaddependent

and

non-Smad-dependent

signaling.

Both

the

pathways

converge

to

phosphorylating the RUNX2.26 The expression of runx2 genes was maintained consistently till the formation of preosteoblast from the hMSc.51 Also, the experimental outcome in the current study corroborates with the above explanation that the nanoparticle-containing samples having higher BMP2, RUNX2 expression, elicit more osteogenic characteristics. We got an increased expression of BMP2 and RUNX2 for dynamic culture than the static culture, which concurs with the justification that shear stress stimulates osteogenic differentiation process. The nHA samples with shear exhibit highest degree of osteogenesis due to the synergistic effect of shear and substrate stiffness.



3.5. Runx2 is the master gene for osteogenesis The expression of runx2 gene is regulated at multiple levels. There are several signaling pathways and those modulate the function of this transcription factor. RUNX2 are subjected to humoral regulation as well as growth factors and cytokines, including TGF-β, BMP, FGF, sonic hedgehog, vitamin D3, and estrogen.52 Runx2 controls the expression of major genes related to bone matrix protein through a direct binding site, called osteoblast-specific cisacting element (OSE2), which is present in the promoter of several osteoblast-specific genes such as ocn, opn,53 and collagen-type I.54 Also, Jang et al. reported the indirect regulation of RUNX2 of ocngene through ATF6 DNA binding factor.55 In our study, the above explanation synchronizes the elevated level of RUNX2 both as protein and mRNA. The higher the osteogenesis, more is the runx2 expression. Our result is in agreement with other outcomes, where along with runx2 expression, other bone-specific genes were also expressed. Also, the expression of runx2 and all the associated genes were mostly unregulated in case of nHA composites under the shear. This holds a strong reliance towards the postulate of nHA being an invigorating factor for osteogenesis. 3.6. Actin remodeling is critical for osteogenic conversion The remodeling of actin cytoskeleton takes place as the hMScs differentiate into bone-like cells. Robust wire-like actin fibers slowly get replaced by thin threads of actin mesh. (fig. 5) There was a difference in actin arrangement for dynamic and static samples. The cell samples, after differentiation in dynamic culture, contain a trace amount of thick actins as stress fibers. The thinning of the actin fibres can be due to nuclear relocalization. Ono et al. mentioned the intranuclear actin filaments in cultured muscle cells, which remain colocalized with ADF/cofilin.56 Furthermore, Gao et al. reported that cofilin-mediated nucleus-cytosol shuttle occurs in osteoblast-like cells in response to cyclic stretch.57 This is in agreement with the experiments performed by Sen et al., that nuclear-relocalized actins by the cofilin can


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efficiently drive osteogenesis.58 On the other hand, OPN has been found to increase the turnover number of actin depolymerization and repolymerization.59 The possible molecular mechanism of the osteogenic actin remodeling can also be due to OPN mediated depolymerization of F-actin. The concurring outcome of an increase in OCN may be justified by this explanation. The depolymerisation of robust actins threads causes probably remodeling and thinning. The released G-actins, which are then transported to the nucleus, further enhance the master gene (Runx2) expression. As a justification of the shear inducing osteogenesis, which was clearly evident from our study, it can be also concluded that shear increases the depolymerization of the actin through Ca2+ and protein kinase C.60 This increases the availability of the G-actins in the cells to get translocated inside the nucleus. 3.7. Membrane ionic currents in hMScs and differentiated cells The cells were differentiated to bone-like cells by incorporating a number of changes, which prepare the cells to respond to the calcium regulatory hormones and growth factor. It has been suggested that intracellular Ca2+, in addition to cAMP not only acts as an effector to mediate the action of parathyroid hormone (PTH), but plays an important role in physiological calcium regulation.61 The PTH acts on the cells through the free Ca2+ available inside the cell. Thus, free availability of the Ca2+ is crucial for bone cell performance. The cells try to incorporate more number of calcium channels as they differentiate from stem cells.62 The current study has a homology with the results published by other researchers, that osteogenic differentiation changes the electrophysiological response of the cells.31 The hMSc exhibited low inward calcium current which was increased after the induction of the osteogenesis. The existence of the L-type calcium channels was proven by the patch clamp technique in whole cell mode, which was further validated by qRT-PCR and immunofluorescence staining of CACNA1C. Several researchers have mentioned the



importance of L-type calcium channel expression in osteogenic cells. Wen et al. stated that Ltype calcium channels are critical for the osteogenic process. It not only marks the transformed cells with bone cell-like characteristics, but also dictates the differentiation process, efficiently. Wen et al. demonstrated that the VDCC L is central for Ca2+-signalling in the cells, which is crucial for mechano-induced bone formation in rats.33 This reinforces the hypothesis of self-preparation of osteoblast-like cells to respond to calcium regulatory hormones. As a correlation to the increased calcium channels, the calcium activity has also been found to be increased in the cases, where the maximum differentiation has been commenced. We found the agonist (±)-Bay K8644 has increased the entry of calcium more in the dynamic cultures than the static as the former contains increased number of calcium channels. This is in corroboration with the other osteogenic characteristics being expressed more on nanoparticulated samples under dynamic mode. 3.8. Compromising the stemness of hMScs at an expense of cellular commitment towards osteogenesis The assessment of stem cell marker expression reveals that the stemness of the hMSc compensated in lieu of osteogenicity. The immunofluorescence study of vimentin strengthened with the mRNA expression has concomitantly exhibited a decline in the vimentin expression with progression of the differentiation processes. Vimentin has been reported to inhibit the activation of transcription factor 4 (ATF-4). Lin et al. reported the significant role of ATF4 as a transcriptional factor in osteogenesis63, which seem to have a detrimental effect on the osteogenic process if blocked.64 Furthermore, vimentin is known to down-regulate the expression of OCN and delay matrix mineralization in immature osteoblasts.65 Hence, we have evaluated the expression of vimentin at mRNA and protein level, after 14 days of hMSc culture in osteoinductive media, both in static and dynamic mode. Considering the denouements in tandem, there is a dynamic interplay between


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vimentin as an intermediate filament and the osteogenesis. Supporting the fact of diminished stemness, FACS results have also demonstrated to have G1/G0 arrest more for the nanoparticle-containing samples under shear. This substantiates decreased proliferation of the stem cells. The repudiated nature of the hMScs towards proliferation and stemness proves them to be committed in a particular lineage. 3.9. Correlation of osteogenic marker genes Expression The runx2 gene is considered as the master gene to establish the osteogenic behavior. Other than the runx2 gene, transcription of other osteo-lineagespecific genes was increased when the cells were cultured on nanoparticle containing UHMWPE. In addition, the shear stress acts as a mechanical stimulation to drive the cell into osteogenesis. Morinobu et al. reported the same incidence of the shear stress which plays a role as a biophysical cue to increase the expression of the accessory genes.66 Ocn is one of the bone-specific gene which codes transcript for osteocalcin, which is a secretory protein.67 It is a well-known fact about the bone playing the role as an endocrine organ and it secretes osteocalcin which is considered as the only hormone secreted.67 As a representative of the bone, osteoblasts exhibit endocrine activity which stimulates insulin secretion and osteocalcin are molecularly responsible to carry out this function.68 As a preparatory measure to qualify as an endocrine cell, the osteogenic differentiation leads to upregulation of ocn gene in the committed cells, which satisfy the properties of an osteoblast. The upregulation of the ocn gene has been found to have direct correlation with the degree of osteogenesis. UHMWPE nanocomposites under shear flow mediated culture exhibited the highest transcription of the gene. D’Alonzo et al. have demonstrated osteopontin as a transcriptional regulator for ocn gene.69 Again, it has been previously discussed that osteopontin actively takes part in regulating the



actin network. As a consequence, it is well defined to have an increased opn mRNA in the nanoparticle-embedded polymer having the shear stimulation. Unraveling the importance of alkaline phosphatase other than acting as a phosphoryl transferase, it is deduced by Golub et al. that ALP participates in matrix mineralization. 70 As an osteogenic marker, hence alp expression in the current study is unconfronted, exhibiting increased expression when an incidence of higher mineralization has occurred (in case of UHMWPE nanocomposites with shear). The summary of the results of this work is encrypted through schematics in figure 11. The sequential events were displayed in view of changing the cellular microenvironment (fig. 11a). The biomaterials exhibited variation in influencing the cells under shear (fig. 11b). Analysing the stages of the osteogenic process, each step requires the intervention of a new set of proteins, which are the definite outcomes of shear and material stiffness. 4. Conclusions As an innovative modification of the conventional system of biocompatibility testing, we implemented a lab-on-a-chip (LOC) device for a better understanding of the stem cell-based differentiation under the influence of the polymer composites in a dynamic physiological environment. This chip design allows us to study differentiation by mimicking the physiological in vivo scenario and to deduce the biomaterial efficacy in supporting osteogenesis, in vitro. The conventional petridish culture experiments revealed better osteogenesis on UHMWPE nanobiocomposite than that on unreinforced UHMWPE. With application of physiologically relevant shear of 1 Pa, the cells were influenced to exhibit higher extent of osteogenesis on nHA reinforced UHMWPE than the MWCNT reinforced polymer. The epigenetic regulation of the gene by the combined effect of shear and biomaterial is validated with higher expression of osteogenic factors. The stemness marker (vimentin)


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expression has been lower, which evokes the onset of osteogenic differentiation of the hMSCs. The osteogenesis is further supported by early processes, like increased alkaline phosphatase activity, followed by matrix mineralisation and collagination deposition. These markers for the osteogenesis were elevated due to nanoparticle addition to the UHMWPE matrix when the cells were grown without shear. In the presence of shear, nHA reinforced UHMWPE has exhibited higher ALP activity, calcium deposition and collagen formation by the differentiating cells. The shear has a definitive effect on cell orientation by modulating the actin arrangement, characterised by L-R chirality as an initiation of actin remodeling process. Importantly, this study revealed the alteration of electrophysiological property of preosteoblast-differentiated-hMScs. The whole cell clamping experiments clearly reveal higher expression of L-type calcium channels, with larger inward calcium current. The presence of the particular class of calcium channel was also confirmed by immunofluorescence and qRT-PCR. Overall, the protein level expression of the osteogenic markers like RUNX2, BMP2 and calcium channel has been significantly increased for the shear exposed cells, especially for the cells grown on UHMWPE+nHA polymers. Summarising, the combinatorial effect of the mechanical and the physical stimuli has enabled the hMScs to differentiate profoundly to generated pre-osteoblast like cells, more on the nHA reinforced than the MWCNT reinforced composites. Therefore, it can be inferred that in physiologically simulated condition, biomechanical and biophysical cues act synergistically and thereby, synchronize the stem cell behaviour and functionality towards osteogenesis. 5. Materials and methods Experimental protocols followed to obtain the results towards determining osteogenesis have been described in details in the supporting information.



Supporting Information Materials and Methods: Biomicrofluidic-Biomaterial setup, Fluid flow parameters, Material property characterization, Cell Culture, Cytocompatibility assessment, Experimental Procedures towards determining osteogenesis, Fluorophore tagged antibody-based staining for actin and microtubules, Determination of osteogenic markers, Cell physiological analysis by functional experiments, Statistical analysis; List of figures: FACS analysis, Alkaline phosphatase activity, Collagen-I deposition, Immunofluorescence labeling of BMP2, Vimentin expression.

Acknowledgments The authors acknowledge the Department of Science and Technology (DST) and major funding support from Department of Biotechnology (DBT), Government of India via the ‘Translational Centre on Biomaterials for Orthopaedic and Dental Applications’. Further, we thank Biosystems Science and Engineering (BSSE), IISc for the immense financial support. A special note of thanks is due to Dr.Kanagaraj from the Department of Material Science and Engineering, IIT Guwahati, for kindly providing us the samples. The authors also thank, Prof. B. Padmanabhan, Head of the Department of Biophysics, NIMHANS, for permitting us to conduct the electrophysiology-based studies. The authors gratefully acknowledge the support of Dr. Uttara Chakraborty for flowcytometry, Monisha Mohandas for AFM images, Srimanta Barui for SOLIDWORKS, Usama Abbasi for FEM simulation and Nidhesh for the calcium study.


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List of Table Table 1: Details of the Primers Used for the Gene Expression Study



Table 2. Summary of the events leading to osteogenesis as an effect of biomaterials substrate and shear


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List of figures:

Figure 1. LOC devices enable the cell culture to grow under dynamic conditions. Schematic illustration of device fabrication and in silico shear determination: (a) Three sheets of PMMA and biomaterial substrate with superimposable patterns of PSA sheets alternatively fixed together to form the microfluidic device; (b) Cell culture experimental setup using a syringe pump to plunge cell media; (c)-(f) Stepwise FES simulation towards achieving the solution of the flow problem of the patterned channelled microfluidic device; (g) The experimental protocol with samples analyzed for the different osteogenic markers.


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Figure 2. The topography of a surface is known to substantially affect the biocompatibility properties. Substrate parameters, like wettability, roughness play vital role in cell-material interaction, and thereby exert influences on the cellular functionality. Surface topography study depicting the formation of nanofillers agglomerate; (a) Morphology of glass slide used as a control in cell culture experiments; (b)UHMWPE with uniform surface morphology; (c) clumped MWCNT bundle formation (d) agglomerated nHA showing particle aggregation; (e) Surface area roughness parameter (Sq) depicting the



roughness increase with the amount of nanofiller in polymer matrix; (f) Surface wettability study showing the influence of nanoparticle present in the UHMWPE with polar group in the nHA phase leading to more hydrophilic behavior of polymer matrix than MWCNT phase


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Figure 3. Cytocompatibility is perhaps the preliminary study which reveals the cellular health and progression rate. Cytocompatibility of the device (a),(b) Patterned channels used for differentiation of hMScs; (c) Proliferation of stem cell on micropatterned channel for day3, the time period given for cellular adaptation to microfluidic environment (d) Cell growth inside the device with static and dynamic culture at various timepoints; (e),(f) The



plots of WST-1 assay exhibit measured percentages of cell proliferation for day3, day12 and day20 occurred in the differentiation culture conditions.

Figure 4. Cell attachment to biomaterial substrate is mediated by focal adhesion complexes. (a) Focal adhesion complexes on the UHMWPE-composites were visualized by immunostaining for vinculin (focal adhesion marker protein) at four-time points of culture. Vinculin was labeled with Alexa Fluor 488 (green), nuclei with DAPI (blue) and actin


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cytoskeleton by rhodamin-phalloidin (red); (b) Number of focal adhesions per cell was quantified from a minimum of 25 cells per sample. Three replicates were used per experiment and data showed corresponds to the mean ± SD of two independent experiments. * indicates statistically significant difference (p < 0.05) of dynamic w.r.t. static. # indicates statistically significant difference (p < 0.05) of the composites w.r.t. control; (c) Formation of ruffle borders on the dynamic culture has occurred as a consequence of continuous shear. The cells in static only exhibit lamellipodium and filopodium.



Figure 5. Differentiation process is an affair of actin reorganization along with other marker expression. Actin remodeling showing differentiation of the cells. (a),(b) The static and dynamic culture has different actin arrangement. Also, differentiation of the cells causes changes in actin characteristics; (c) L-R asymmetry during cytoskeletal organization under the influence of shear. The TF and RF torsion are responsible for the cellular orientation; (d)


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Representative images of actin orientation. Quantitative estimation manifests three arrangements of actins: random cell with random actin for all the samples in the static culture other

than

UHMWPE+MWCNT,

random

cell

with

semi-aligned

actin

for

UHMWPE+MWCNT in static culture, aligned actin with aligned cells in all samples of the dynamic culture. (e),(f) Cellular orientation was determined using the microtubule organization. Dynamic culture showing more oriented cells than the static culture. α-Tubulin was labeled with Alexa Fluor 488 (green), nuclei with DAPI (blue) and actin cytoskeleton by rhodamin-phalloidin.

Figure 6. Biomineralization, an indicator of terminal stage osteogenesis of differentiating hMScs. (a) Bright-field optical micrographs showing alizarin red stained calcium deposits formed on UHMWPE composites after 20 days of hMSC culture in



osteoinductive media; (b) Quantification of extracellular calcium deposits by elution of calcium-ARS complex extracts and recording the absorbance at 450 nm. * indicates statistically significant difference (p < 0.05) of dynamic w.r.t. static. # indicates statistically significant difference (p < 0.05) of the composites w.r.t. control. (c) The EDAX spectra recorded from the matrix of the cells on the material surfaces.


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Figure 7. The master factor of osteogenesis, RUNX2 is essentially expressed prior to the osteogenic differentiation which commits the cells towards bone formation. This was estimated both qualitatively and quantitatively; (a) Immunofluorescence labeling of RUNX2, a mesenchymal stem cell marker with Alexa Fluor 488 (green) and inset image of nuclei with DAPI (blue) indicates an upregulation of RUNX2 expression in hMSCs cultured on UHMWPE-composites after 14 days of culture; (b) The quantitative intensity showing higher expression of the protein for nanoparticulated UHMWPE. * indicates statistically significant difference (p < 0.05) of dynamic w.r.t. static. # indicates statistically significant difference (p < 0.05) of the composites w.r.t. control.



Figure 8. Quantitative RT-PCR analysis of gene expression could be considered as the confirmatory test for the immunofluorescence methods.The mRNA specific for the osteoblast differentiation was quantified for both the static and dynamic culture. The mRNA expression levels of Runx2, ALP, ColIA, OCN, OPN, and vinculin were normalized to the expression level of GAPDH. Data shown here are the 2-ΔΔCt values of the targeted genes. Three samples were used for each experiment and mean ± SD wasplotted. All the osteogenic genes have increased expression due to differentiation. The nanoparticulated UHMWPE reflects the consistent higher expression of the osteogenic genes. If compared among the static and dynamic, the dynamic shows significantly higher expression. *indicates statistically significant difference (p < 0.05) of dynamic w.r.t. static. # indicates statistically significant difference (p < 0.05) of the composites w.r.t. control.


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Figure 9. The electrophysiology defines the functional state of the cells. The higher calcium currents for differentiated hMSc confirms osteoblastic characteristics. Whole cell clamping for detection of inward calcium current for the dynamic culture; (a) Representative multi-voltage traces of voltage response of the cells showing the presence of



inward current; (b) Inward calcium current was detected for nanoparticle reinforced composites. The I-V plot showing non-linear relationship due to the modulation of the current across the membrane by the present calcium channels; (c) Presence of L-type calcium channel was confirmed by immunodetection. The anti-human L-type α1C subunit (CaV1.2) (green) was used to localize the channel proteins and nuclear chromatin was stained with DAPI. (d) qRT-PCR reveals increased expression of CACNA1C gene which signifies upregulated L-type calcium channels.


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Figure 10. Calcium signaling is one of the central processes of differentiation which occurssustainedly during osteogenesis. Calcium activity was determined with Fluo4-AM dye. The presence of the L-type calcium channels was assured by applying (±)-Bay K8644. This acts as an agonist of L-type calcium channel. The cells cultured under dynamic setup at day 20 was chosen for determining the intracellular calcium activity. (a),(b) Control and UHMWPE+nHA samples were compared where the nHA particulated samples manifest higher response than the control on applying the ((±)-Bay K8644; (c) The real-time plot of the change in pixel intensity reveals that when the agonist was added (red arrow), the nanoparticulated substrates were able to reflect an elevated response than the control. The recording was done for 180s with capturing the events once at every 3s. At 9th second the agonist was added; (d) The bar plot showing the difference between the initial and the final



pixel intensity for each sample. * indicates statistically significant difference (p < 0.05) w.r.t control. (e) Change in the slope before adding and after adding ((±)-Bay K8644 was plotted takingnormalized value against the initial slope. Inset showing the absolute slope before and after adding ((±)-Bay K8644; (f) The control sample at day 3 was recorded without applying the agonist to determine the ground level of calcium activity in the undifferentiated cells.


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Figure 11. Osteogenic differentiation is a consequence of competitive expression of bone markers by repressing the other differentiation processes like adipogenesis. Schematic representation of the shear triggered osteogenic differentiation of human mesenchymal stem cells on UHMWPE composites; (a) The day wise tentative events were collectively exhibiting the progress of differentiation process under shear; (b) Stemness v/s osteogenesis profile onto various substrates; (c) Onset of osteogenesis progresses with formation of the



committed osteoblast progenitor cells. The markers start to appear at various time points in a cascade manner to drive the complex process of differentiation.


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Graphical abstract



List of figures:

Figure 1. LOC devices enable the cell culture to grow under dynamic conditions. Schematic illustration of device fabrication and in silico shear determination: (a) Three sheets of PMMA and biomaterial substrate with superimposable patterns of PSA sheets alternatively fixed together to form the microfluidic device; (b) Cell culture experimental setup using a syringe pump to plunge cell media; (c)-(f) Stepwise FES simulation towards achieving the solution of the flow problem of the patterned channelled microfluidic device; (g) The experimental protocol with samples analyzed for the different osteogenic markers.


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Figure 2. The topography of a surface is known to substantially affect the biocompatibility properties. Substrate parameters, like wettability, roughness play vital role in cell-material interaction, and thereby exert influences on the cellular functionality. Surface topography study depicting the formation of nanofillers agglomerate; (a) Morphology of glass slide used as a control in cell culture experiments; (b)UHMWPE with uniform surface morphology; (c) clumped MWCNT bundle formation (d) agglomerated nHA showing particle aggregation; (e) Surface area roughness parameter (Sq) depicting the



roughness increase with the amount of nanofiller in polymer matrix; (f) Surface wettability study showing the influence of nanoparticle present in the UHMWPE with polar group in the nHA phase leading to more hydrophilic behavior of polymer matrix than MWCNT phase


Page 66 of 79

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Figure 3. Cytocompatibility is perhaps the preliminary study which reveals the cellular health and progression rate. Cytocompatibility of the device (a),(b) Patterned channels used for differentiation of hMScs; (c) Proliferation of stem cell on micropatterned channel for day3, the time period given for cellular adaptation to microfluidic environment (d) Cell growth inside the device with static and dynamic culture at various timepoints; (e),(f) The



plots of WST-1 assay exhibit measured percentages of cell proliferation for day3, day12 and day20 occurred in the differentiation culture conditions.

Figure 4. Cell attachment to biomaterial substrate is mediated by focal adhesion complexes. (a) Focal adhesion complexes on the UHMWPE-composites were visualized by immunostaining for vinculin (focal adhesion marker protein) at four-time points of culture. Vinculin was labeled with Alexa Fluor 488 (green), nuclei with DAPI (blue) and actin


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cytoskeleton by rhodamin-phalloidin (red); (b) Number of focal adhesions per cell was quantified from a minimum of 25 cells per sample. Three replicates were used per experiment and data showed corresponds to the mean ± SD of two independent experiments. * indicates statistically significant difference (p < 0.05) of dynamic w.r.t. static. # indicates statistically significant difference (p < 0.05) of the composites w.r.t. control; (c) Formation of ruffle borders on the dynamic culture has occurred as a consequence of continuous shear. The cells in static only exhibit lamellipodium and filopodium.



Figure 5. Differentiation process is an affair of actin reorganization along with other marker expression. Actin remodeling showing differentiation of the cells. (a),(b) The static and dynamic culture has different actin arrangement. Also, differentiation of the cells causes changes in actin characteristics; (c) L-R asymmetry during cytoskeletal organization under the influence of shear. The TF and RF torsion are responsible for the cellular orientation; (d)


Page 70 of 79

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Representative images of actin orientation. Quantitative estimation manifests three arrangements of actins: random cell with random actin for all the samples in the static culture other

than

UHMWPE+MWCNT,

random

cell

with

semi-aligned

actin

for

UHMWPE+MWCNT in static culture, aligned actin with aligned cells in all samples of the dynamic culture. (e),(f) Cellular orientation was determined using the microtubule organization. Dynamic culture showing more oriented cells than the static culture. α-Tubulin was labeled with Alexa Fluor 488 (green), nuclei with DAPI (blue) and actin cytoskeleton by rhodamin-phalloidin.

Figure 6. Biomineralization, an indicator of terminal stage osteogenesis of differentiating hMScs. (a) Bright-field optical micrographs showing alizarin red stained calcium deposits formed on UHMWPE composites after 20 days of hMSC culture in



osteoinductive media; (b) Quantification of extracellular calcium deposits by elution of calcium-ARS complex extracts and recording the absorbance at 450 nm. * indicates statistically significant difference (p < 0.05) of dynamic w.r.t. static. # indicates statistically significant difference (p < 0.05) of the composites w.r.t. control. (c) The EDAX spectra recorded from the matrix of the cells on the material surfaces.


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Figure 7. The master factor of osteogenesis, RUNX2 is essentially expressed prior to the osteogenic differentiation which commits the cells towards bone formation. This was estimated both qualitatively and quantitatively; (a) Immunofluorescence labeling of RUNX2, a mesenchymal stem cell marker with Alexa Fluor 488 (green) and inset image of nuclei with DAPI (blue) indicates an upregulation of RUNX2 expression in hMSCs cultured on UHMWPE-composites after 14 days of culture; (b) The quantitative intensity showing higher expression of the protein for nanoparticulated UHMWPE. * indicates statistically significant difference (p < 0.05) of dynamic w.r.t. static. # indicates statistically significant difference (p < 0.05) of the composites w.r.t. control.



Figure 8. Quantitative RT-PCR analysis of gene expression could be considered as the confirmatory test for the immunofluorescence methods.The mRNA specific for the osteoblast differentiation was quantified for both the static and dynamic culture. The mRNA expression levels of Runx2, ALP, ColIA, OCN, OPN, and vinculin were normalized to the expression level of GAPDH. Data shown here are the 2-ΔΔCt values of the targeted genes. Three samples were used for each experiment and mean ± SD wasplotted. All the osteogenic genes have increased expression due to differentiation. The nanoparticulated UHMWPE reflects the consistent higher expression of the osteogenic genes. If compared among the static and dynamic, the dynamic shows significantly higher expression. *indicates statistically significant difference (p < 0.05) of dynamic w.r.t. static. # indicates statistically significant difference (p < 0.05) of the composites w.r.t. control.


Page 74 of 79

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Figure 9. The electrophysiology defines the functional state of the cells. The higher calcium currents for differentiated hMSc confirms osteoblastic characteristics. Whole cell clamping for detection of inward calcium current for the dynamic culture; (a)



Representative multi-voltage traces of voltage response of the cells showing the presence of inward current; (b) Inward calcium current was detected for nanoparticle reinforced composites. The I-V plot showing non-linear relationship due to the modulation of the current across the membrane by the present calcium channels; (c) Presence of L-type calcium channel was confirmed by immunodetection. The anti-human L-type α1C subunit (CaV1.2) (green) was used to localize the channel proteins and nuclear chromatin was stained with DAPI. (d) qRT-PCR reveals increased expression of CACNA1C gene which signifies upregulated L-type calcium channels.


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Figure 10. Calcium signaling is one of the central processes of differentiation which occurssustainedly during osteogenesis. Calcium activity was determined with Fluo4-AM dye. The presence of the L-type calcium channels was assured by applying (±)-Bay K8644. This acts as an agonist of L-type calcium channel. The cells cultured under dynamic setup at day 20 was chosen for determining the intracellular calcium activity. (a),(b) Control and UHMWPE+nHA samples were compared where the nHA particulated samples manifest higher response than the control on applying the ((±)-Bay K8644; (c) The real-time plot of the change in pixel intensity reveals that when the agonist was added (red arrow), the nanoparticulated substrates were able to reflect an elevated response than the control. The recording was done for 180s with capturing the events once at every 3s. At 9 th second the agonist was added; (d) The bar plot showing the difference between the initial and the final



pixel intensity for each sample. * indicates statistically significant difference (p < 0.05) w.r.t control. (e) Change in the slope before adding and after adding ((±)-Bay K8644 was plotted takingnormalized value against the initial slope. Inset showing the absolute slope before and after adding ((±)-Bay K8644; (f) The control sample at day 3 was recorded without applying the agonist to determine the ground level of calcium activity in the undifferentiated cells.

Figure 11. Osteogenic differentiation is a consequence of competitive expression of bone markers by repressing the other differentiation processes like adipogenesis. Schematic representation of the shear triggered osteogenic differentiation of human mesenchymal stem


Page 78 of 79

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cells on UHMWPE composites; (a) The day wise tentative events were collectively exhibiting the progress of differentiation process under shear; (b) Stemness v/s osteogenesis profile onto various substrates; (c) Onset of osteogenesis progresses with formation of the committed osteoblast progenitor cells. The markers start to appear at various time points in a cascade manner to drive the complex process of differentiation.


Controlled shear flow directs osteogenesis on UHMWPE based hybrid

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