behavior of articular chondrocytes on ... - World Scientific

Nano LIFE Vol. 3, No. 3 (2013) 1342001 (23 pages) © World Scienti¯c Publishing Company DOI: 10.1142/S1793984413420014

BEHAVIOR OF ARTICULAR CHONDROCYTES ON NANOENGINEERED SURFACES JAMEEL SHAIK Institute for Micromanufacturing Louisiana Tech University, Ruston, LA, USA Biomedical Engineering Program Louisiana Tech University, Ruston, LA, USA Medical Biotechnology Division School of Bio Sciences & Technology VIT University, Vellore, India 632014 [email protected]

JAVEED SHAIKH MOHAMMED Institute for Micromanufacturing Louisiana Tech University, Ruston, LA, USA Biomedical Technology Department King Saud University, Saudi Arabia 14511 [email protected]

MICHAEL J. MCSHANE Institute for Micromanufacturing Louisiana Tech University, Ruston, LA, USA Biomedical Engineering Program Louisiana Tech University, Ruston, LA, USA Department of Biomedical Engineering and Materials Science & Engineering Program Texas A&M University, College Station, TX 77843, USA [email protected]

DAVID K. MILLS* Institute for Micromanufacturing 1 Adams Boulevard, Louisiana Tech University Ruston, LA 71272, USA School of Biological Sciences 1 Adams Boulevard, Louisiana Tech University Ruston, LA 71272, USA [email protected]

Received 1 February 2013 Accepted 10 May 2013 Published 1 July 2013 *Corresponding

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In tissue engineering, surface modi¯cation has becomes one of the leading methods to enhance initial cell attachment and subsequent cellular growth, di®erentiation and tissue formation. This work studied growth and behavior of primary bovine articular chondrocytes on self-assembled multilayer nano¯lms composed of: polyelectrolytes [poly(styrene sulfonate) (PSS), poly-L-lysine (PLL), poly-D-lysine (PDL), chondroitin sulfate (CS), poly(ethyleneimine) (PEI), poly(dimethyldiallylammonium chloride) (PDDA), poly(ethylene glycol) amine (PEG NH2 )] and proteins [bovine serum albumin (BSA), collagen, ¯bronectin, laminin]. These biomaterials were used to build mono-, bi-, and tri-layer nano¯lm architectures. Potential cytotoxic e®ects were assessed using Live/Dead assay and cell proliferation was quanti¯ed using MTT assay. Bright ¯eld and °uorescence microscopy were used to analyze chondrocyte morphology. ImageJ software was used to analyze the number, mean area, circularity and Feret's diameter of viable cells. Cumulative results demonstrated that chondrocyte growth; proliferation and functionality were dependent on initial cell density, nano¯lm thickness and material composition of nano¯lms. Keywords: Layer-by-Layer (LbL) self-assembly; polyelectrolyte multilayer nano¯lms; proteins; nanoscale; surface modi¯cation; nanoengineered surfaces; nano¯lm architectures; cell characterization; chondrocytes.

1. Introduction The study of the growth and behavior of cells on di®erent biomaterials has important implications for tissue engineering. Critical cellular behaviors, such as cell attachment,1,2 proliferation3,4 and di®erentiation,5,6 have been studied and important facts are emerging about surface features and cell adhesion, growth and functionality. It has long been well established that the type of material in°uences initial cell attachment, subsequent cellular growth and preservation of cellular phenotype, thus di®erent materials have di®erent in°uences on cellmatrix interactions. These di®ering cellular behaviors can be taken advantage of in tissue engineering and recent studies have led to the conclusion that the type of material has a major in°uence on the di®erentiation of cells.7,8 The maintenance of cell shape, i.e., the phenotype is very important. Changes in cellular phenotype and function have been associated with cytoskeletal alterations9,10 cell density,11 topography,11 adhesiveness12,13 and diseases, for example, in metastatic cancer.14 The cardiovascular system responds to mechanical forces by changes in cell shape and gene expression, which is dependent on the low molecular weight guanisine triphosphatases of the Rho family.15 Chondrocyte metabolism, in particular, is very sensitive to changes in cell morphology.10,1618 The very labile phenotype of chondrocytes is well demonstrated in the loss of the rounded morphology of chondrocytes with succeeding passages, which

gives the chondrocytes a ¯broblastic morphology in vitro i.e., they dedi®erentiate and re-express their phenotype when placed in an environment that promotes a rounded cellular morphology.18 Loss of matrix molecules, such as aggrecan and changes in type II collagen ¯ber architecture and associated changes in cell shape, are typical signs of osteoarthritic cartilage and linked to an increase in matrix catabolism.19,20 It has been hypothesized that phenotypic modulation of chondrocytes can be a therapeutic strategy for the treatment of osteoarthritis.21,22 Stabilization of the chondrocytic phenotype, coupled with redifferentiation of the osteoarthritic chondrocytes, is required to help correct matrix anabolism.22 Despite increasing advances in tissue engineering, there is still a lack of biomaterials that mimic the in vivo rounded phenotype of chondrocytes in vitro. Hydrogels such as agarose and alginate have been used to stabilize the phenotype of chondrocytes. Benya and Sha®er demonstrated that chondrocytes regain their rounded morphology when grown on agarose.18 Multilayer nano¯lms are akin to soft hydrogels23 and have emerged as versatile platforms for the growth of cells.1,2426 In a recent demonstration,27 we studied the growth and behavior of primary bovine articular chondrocytes (PBACs) on layer-by-layer (LbL) selfassembled nano¯lms of 11 di®erent biomaterials, including poly(styrene sulfonate) (PSS), ¯bronectin, poly-L-lysine (PLL), poly-D-lysine, laminin, bovine serum albumin (BSA), chondroitin sulfate,

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Behavior of Articular Chondrocytes

poly(ethyleneimine), poly(dimethyldiallylammonium chloride) (CPDDA), collagen and poly(ethylene glycol) amine (PEG-NH2). Mono-, bi-, and trilayer nano¯lm architectures were deposited on polystyrene surfaces. Immunocytochemistry was used to assess chondrocyte functionality. Statistical analyses of chondrocyte viability and metabolic activity results on mono-, bi-, and tri-layer nano¯lm architectures indicated the signi¯cant in°uence of cell seeding density and number of nano¯lm layers on the viability and metabolic activity of chondrocytes.27 In the current study, we extended the above-mentioned work to examine the in°uence of di®erent multilayer nano¯lm surfaces on the phenotype of PBACs. Bright ¯eld microscopy and °uorescence microscopy techniques were used to capture the morphology of chondrocytes on nano¯lm surfaces before and after the cells underwent LiveDead viability assay, respectively. ImageJ software was used to analyze the number, mean area, circularity and Feret's diameter of the live cells on the di®erent nano¯lm surfaces.

2. Materials and Methods 2.1. Substrate preparation Tissue culture 24-well plates (BD Falcon, BD Biosciences, San Jose, CA) were used for absorbance measurements on the assembled multilayer nano¯lms. Tissue culture 96-well black well plates (BD Falcon, BD Biosciences, San Jose, CA) were used in °uorescence measurements involving multilayer nano¯lms. Tissue culture well plate surfaces were pre-treated with a cleaning solution [60% deionized (DI) water, 39% ethyl alcohol (70%) and 1% potassium hydroxide (KOH)] to induce a negative charge on the surface of these plates.

2.2. Biomaterials (polyelectrolyte and protein) preparation All the chemicals were purchased from SigmaAldrich (St. Louis, MO) unless otherwise speci¯ed. PDDA (Mw 150 kDa), PSS (Mw 1 MDa) solutions were prepared at concentrations of 2 mg mL 1 with 0.5 M KCl. PEI (Mw 750 kDa) solution was prepared at concentration of 2 mg mL 1 . PLL (Mw 100 kDa), PDL (Mw 100 kDa), BSA (Mw 66 kDa), PEG-NH2 (Mw 10 kDa), and CS (Mw 500 Da) solutions were prepared at a

concentration of 125 g mL 1 . Type I collagen (Mw 100 KDa, Cohesion, Palo Alto, CA) solution was prepared at a concentration of 1 mg mL 1 . Laminin (Mw 900 kDa) and ¯bronectin (Mw 450 kDa) solutions were prepared at concentrations of 86 g mL 1 and 100 g mL 1 , respectively. All solutions were prepared; using deionized water with a resistivity of 18.2 M cm (Millipore systems), and the pH was adjusted to 7.4 (except a pH of 4 for collagen and a pH of 5 for PEG-NH2 Þ.

2.3. Multilayer nano¯lm deposition Unmodi¯ed tissue-culture polystyrene (TCPS, MidScienti¯c, St. Louis, MO) was used as a standard control. PEI served as the precursor layer in all cases of multilayer nano¯lms. With the pre-treatment step, a negative charge is formed on the surface of the tissue culture well plates. Therefore, an alternately charged material (PEI) was chosen as the precursor layer that provides the structural basis for further ¯lm deposition. Monolayers, bilayers and trilayers of the 11 biomaterials were deposited onto substrates with nano¯lm architectures shown in Table 1. The nano¯lm deposition was performed manually using pipettes. For each layer deposition, 100 L and 200 L biomaterial solution was used in the case of 96- and 24-well plates, respectively. The incubation times for the di®erent biomaterial solutions were set as follows for optimum nano¯lm deposition: PSS, PDDA, PEI-10 min; (PEG-NH2 Þ, BSA, PDL, CS, collagen, PLL-30 min; laminin, ¯bronectin-overnight. After each deposition step, DI water rinsing was performed to remove the loosely bound polymers. Multiple layers of alternately charged polyelectrolytes/proteins were deposited to achieve the desired multilayer nano¯lm architectures. After the completion of multilayer deposition process, the plates were kept under the UV light overnight in a laminar °ow-hood. Next, these plates were stored at 4 C until further use. Each of the multilayer nano¯lm architectures shown in Table 1 was created in triplicate to allow statistical analysis of nano¯lm characteristics and cell behavior on the nano¯lm surfaces.

2.4. Cell culture of PBACs PBACs were obtained from bovine knee joints. The joints were obtained from the Meats Plant at Louisiana Tech University. All necessary aseptic

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J. Shaik et al. Table 1. Multilayer architectures of mono-, bi- and tri-layer nano¯lms of 11 di®erent biomaterials used in this study. Multilayer nano¯lm architectures Biomaterial

Monolayer

Bilayers

Trilayers

PDDA BSA PDL CS PEI Collagen PLL Fibronectin PSS Laminin PEG-NH2

PSS/PDDA BSA PSS/PDL CS PSS/PEI PSS/Collagen PSS/PLL Fibronectin PSS Laminin PSS/PEG-NH2

(PSS/PDDA)2 (BSA/PEI)/BSA (PSS/PDL)2 (CS/PEI)/CS (PSS/PEI)2 (PSS/Collagen)2 (PSS/PLL)2 (PSS/PEI)/Fibronectin (PSS/PEI)/PSS (PSS/PEI)/Laminin (PSS/PEI)/PSS/PEG-NH2

(PSS/PDDA)3 (BSA/PEI)2 /BSA (PSS/PDL)3 (CS/PEI)2 /CS (PSS/PEI)3 (PSS/Collagen)3 (PSS/PLL)3 (PSS/PEI)2 /Fibronectin (PSS/PEI)2 /PSS (PSS/PEI)2 /Laminin (PSS/PEI)2 /PSS/PEG-NH2

precautions were taken during the isolation of cells. Articular cartilage was removed via dissection, placed in a calcium and magnesium-free HBSS and mined into approximately 10 mm pieces. Cartilage tissues were then transferred to a Nalgener 25 mL tissue digestion °ask provided with a stir bar. The tissues were rinsed several times again with HBSS/ PenStrep. The HBSS/PenStrep solution was removed and approximately 15 mL of sterile, ¯ltered 0.1% pronase (Sigma Aldrich, St. Louis, MO) was added to each °ask. Flasks were placed into a 5% CO2/95% air humidi¯ed incubator on a stir plate incubated for 90 min at 37 C. At the end of 90 min, °asks were removed from the incubator and 15 mL of sterile, ¯ltered 0.4% Type II collagenase was added. Flasks were returned to the incubator and incubated for 180 min at 37 C. At the end of the incubation, the contents of the °asks were centrifuged at 1000 g for 5 min and the cells were collected by centrifugation. The supernatant was plated in 100 20 mm tissue culture dishes with Dulbecco's Modi¯ed Eagle's Medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, penicillin 100 units mL 1 and streptomycin (complete DMEM, Sigma Aldrich, St. Louis, MO). The PBACs were passaged three times on tissue culture plastic (TCP, Midsci, St. Louis, MO). Before seeding cells, tissue culture plates with multilayer nano¯lms were treated with 70% ethanol for 30 min followed by a 5 min wash with HBSS without calcium and magnesium (Fisher Scienti¯c, Pittsburgh, PA). Cells from passage 3 were used in the experiments. Ham's F-12 medium (GibcoBRL, Grand

Island, NY) containing 10% FBS (Biosource, Camarillo, CA) supplemented with 1% PenicillinStreptomycin (Pen-Strep) (Biosource, Camarillo, CA) was used to culture the cells. PBACs were seeded at di®erent seeding densities onto the multilayer nano¯lm surfaces.

2.5. Cell morphological observations Bright ¯eld microscopy and °uorescence microscopy techniques were used to capture the morphology of chondrocytes on nano¯lm surfaces before and after the cells underwent LiveDead viability assay, respectively. ImageJ software was used as an adjunct to the LiveDead viability analysis for counting cells from the LiveDead Images. Each image was divided into three randomly selected regions, analyzed and the mean of the resultant cell counts were obtained. Also, ImageJ software was used to calculate the mean area, circularity and Feret's diameter of the live cells from the LiveDead Images.

3. Results and Discussion 3.1. Morphological observations of PBACs on nano¯lm surfaces using bright ¯eld microscopy Morphological observations demonstrate that chondrocytes can be successfully grown on the nano¯lms. Chondrocytes tolerated nearly all the materials favorably. A representative sample of cells on the di®erent biomaterials is provided in Figs. 112. All the images displayed below unless

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(a) Fig. 1.

(b)

PBACs on TCPS: (a) after 36 h in culture and (b) after 72 h in culture.

(a)

(b)

(c)

(d)

Fig. 2. PBACs on PEG-NH2 after 36 h: (a) on monolayer, (b) on bilayers, (c) on trilayers; after 72 h (d) on monolayer, (e) on bilayers and (f ) on trilayers.

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(e)

(f ) Fig. 2.

(Continued )

otherwise speci¯ed are of chondrocytes seeded at a cell density of 5000 cells mL 1 . The cells on TCPS (see Fig. 1) have a typical ¯broblastic phenotype. On PEG-NH2 (see Fig. 2), it can be observed that the cells tend to acquire a far less ¯broblast

phenotype and tend towards a slightly rounded to orthogonal phenotype (SROP). PDDA (see Fig. 3), a polyelectrolyte, tends to stabilize the phenotype of the chondrocytes to a SROP. Unlike PEG-NH2, the SROP is more pronounced on both bilayers and

(a)

(b)

(c) Fig. 3.

PBACs on PDDA after 36 h: (a) on bilayers; after 72 h (b) on bilayers and (c) on trilayers. 1342001-6


(a)

(b)

(c) Fig. 4.

PBACs on BSA after 72 h: (a) on monolayer, (b) on bilayers and (c) on trilayers.

(a) Fig. 5.

(b)

PBACs on PDL after 36 h: (a) on bilayers, (b) on trilayers, after 72 h (c) on monolayer, (d) on bilayers and (e) on trilayers.

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(c)

(d)

(e) Fig. 5.

trilayers after 72 h. BSA (see Fig. 4) appears to stabilize the phenotype of the chondrocytes toward a more rounded phenotype. The roundedness is more pronounced on monolayer and bilayers after 72 h whereas one can observe SROP on trilayers. The chondrocytes on PDL (see Fig. 5) exhibit SROP on bilayers while it is slightly less pronounced on the trilayers. On CS (see Fig. 6), as in the case of other proteins used, chondrocytes display the similar trend of increased SROP and decreased ¯broblast phenotype compared with the other materials. Figure 7 displays the SROP of chondrocytes on PEI. Figure 8 exhibits the SROP of chondrocytes on the di®erent layers. Figure 9 displays the SROP of chondrocytes on PLL. Very few cells are present on trilayers with marked cell death. The chondrocytes on ¯bronectin (see Fig. 10) exhibit SROP. The chondrocytes on PSS (see Fig. 11) show a SROP on all the layers. Cells on laminin (see Fig. 12) exhibit SROP along with their typical characteristics.

(Continued )

Lamellopodia and ¯lopodia of the chondrocytes are more pronounced on this material than any other. However, these are more pronounced on the bi- and trilayers as compared to monolayers. Table 2 displays the phenotypes observed on monolayer, bilayers and trilayers of the di®erent biomaterials used in this study. These observations are from a mixture of the images acquired after 36 h and 72 h. It can be observed from the table that PBACs exhibit SROP on most of the biomaterials. This raises the exciting possibility of the utilization of LbL-assembled multilayer nano¯lms for the stabilization of the phenotype of chondrocytes. Despite our results, there is a necessity of testing the growth of chondrocytes on multilayer nano¯lms having more number of bilayers than the ones used in this study to check if increased number of bilayers will help in stabilization of the phenotype of chondrocytes. Morphological observations are summarized in Table 2.

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(a)

(a)

(b)

(b)

(c)

(c)

Fig. 6. PBACs on CS after 36 h: (a) on bilayers, (b) on trilayers; after 72 h and (c) on trilayers.

Fig. 7. PBACs on PEI after 36 h: (a) on monolayer, (b) on bilayers; after 72 h and (c) on trilayers.

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Fig. 8.

(a)

(b)

(c)

(d)

PBACs on collagen after 36 h on: (a) trilayers; after 72 h on (b) monolayer, (c) bilayers and (d) trilayers.

(a) Fig. 9.

(b)

PBACs on PLL: after 36 h on: (a) monolayer, (b) bilayers, (c) trilayers; after 72 h and (d) on trilayers.

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(c)

(d) Fig. 9.

3.2. Morphological observations of PBACs on nano¯lm surfaces using °uorescence microscopy Representative samples of the growth of bovine articular chondrocytes on di®erent materials assessed using LiveDead viability assay are displayed in Figs. 13 and 14. Only images of live cells are shown below to highlight the di®erences in morphology of the cells on di®erent layered nano¯lms. All the images displayed below are from the cells seeded at cell seeding density of 5000 cells mL 1 . Figure 13 depicts the PBACs on TCPS. The cells have a ¯broblast phenotype. Cells on PEG-amine (see Fig. 14) have SROP and exhibit spread-out morphology. It can be deduced that PEG-amine is favorable for the growth and viability of chondrocytes. Figure 14 also

(Continued )

displays the cells on monolayer, bilayers and trilayers of PDDA. Cells on monolayer and trilayers are round and °oating, but there are many live cells displaying SROP on bilayers. It can be observed that there is a di®erence in the size of the chondrocytes in between the di®erent layers; cells on bilayers of PDDA are larger in size as compared to the cells on monolayer and trilayers of PDDA. Cells on BSA are round and °oating in a few wells. Cells on bilayers are better attached than cells on other layers. Cells on PDL have more SROP than PEG-amine, are well attached and display spread-out morphology. It can be observed that the highest number of cells is on trilayers, which display a ¯broblast phenotype while there are very few cells on bilayers. Cells on CS are well attached and widespread. There are a few rounded cells, and some of the cells display SROP. The cellular extensions

(a) Fig. 10.

(b)

PBACs on ¯bronectin after 36 h on: (a) monolayer, (b) bilayers, (c) trilayers; after 72 h on (d) monolayer and (e) bilayers. 1342001-11

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(c)

(d)

(e) Fig. 10.

are longer and in some ways resemble cells on laminin. Cell viability is greater indicating that CS is more favorable for chondrocyte growth and viability. Cells on PEI are well attached; display SROP and well spread in most cases indicating that PEI is favorable for chondrocyte growth and viability.

(Continued )

Cells on monolayer and bilayers of collagen are few in number while there are a large number of cells present on the trilayers. It can be observed that cells on the trilayers are well attached and show SROP. Cells on PLL display SROP, are well attached and wide spread. Cells display slightly longer extensions

(a) Fig. 11.

(b)

PBACs on PSS after 36 h on: (a) monolayers, (b) bilayers, (c) trilayers; after 72 h on (d) bilayers and (e) trilayers. 1342001-12


(c)

(d)

(e) Fig. 11.

on this material as well. Cell viability is more indicating that PLL is favorable for chondrocyte growth and viability. Cells on bilayers appear to be bigger in size compared to the cells on other layers. Cells on

(Continued )

¯bronectin display SROP, are well attached and widespread. The viability of cells is the best on ¯bronectin among all the biomaterials used in this study indicating that ¯bronectin is one of the most

(a) Fig. 12.

(b)

PBACs on laminin after 36 h on: (a) monolayer, (b) bilayers, (c) trilayers; after 72 h on and (d) bilayers. 1342001-13

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(c)

(d) Fig. 12.

(Continued )

favorable among the di®erent biomaterials used in this study for chondrocyte growth and viability. Cells on PSS display SROP, are attached and well spread in most cases indicating that PSS is favorable for chondrocyte growth and viability. The cells on the bilayers are bigger in size compared to the cells on monolayer and trilayers. Cells on laminin are attached in some cases and display SROP on monolayer and bilayers. There were a large number of dead cells on trilayers hence they have been displayed here. PBACs grew on all the LbL-modi¯ed biomaterials favorably. Morphological observations indicated that all the biomaterials tolerated the chondrocytes fairly well. On most of the biomaterials tested, chondrocytes exhibited SROP. On some of the

materials, the chondrocytes showed a rounded morphology for a few days after seeding. The most important observations were that cells on bilayers of PDDA, PLL and PSS were bigger in

Table 2. Summary of morphological observations of phenotypes exhibited by PBACs on mono-, bi- and trilayer nano¯lms (Cell seeding density 5000 cells mL 1 Þ.

(a)

Multilayer nano¯lm architectures Biomaterial

Monolayer

Bilayers

Trilayers


SROP Rounded SROP SROP SROP SROP SROP SROP SROP SROP SROP

SROP Rounded SROP SROP SROP SROP SROP SROP SROP SROP SROP

SROP SROP Fibroblast SROP SROP SROP Cell death SROP SROP SROP SROP

(b) Fig. 13. 1342001-14

(a), (b) Live PBACs on TCPS.


Fig. 14.

Live PBACs on mono-, bi- and tri-layer nano¯lm surfaces of 11 di®erent biomaterials (Cell seeding density 5000 cells mL 1 Þ.

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Fig. 14.

size compared to cells on monolayers and trilayers of PDDA, PLLA and PSS. Also, cells on bilayers of BSA showed better cell attachment when compared to cells on other layers of BSA. From the Live/Dead morphological observations, it is suggested that PEG-amine, PDL, CS, PEI, PLL, ¯bronectin and PSS were more favorable for the growth and

(Continued )

viability of chondrocytes than other materials used and were even an improvement over the control (TCPS). Table 3 summarizes the morphological observations of LiveDead Images. On comparing Tables 2 and 3, it can be seen that after LiveDead analysis PBACs on monolayer and trilayers of PDDA, bilayers and trilayers of BSA, and

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Cell Seeding Density 5000 cells mL 1 : PDL exhibits increasing number of live cells with increasing layers. PEI, collagen, ¯bronectin and PSS too exhibit the same behavior: increasing number of live cells with increasing layers except for a slight drop in case of bilayers. Laminin and PDDA exhibit the opposite behavior — decreasing number of cells with increasing layers. In case of PEG-NH2, BSA and CS, the bilayers exhibit the largest number of live cells. Cell Seeding Density 15 000 cells mL 1 : PDDA, BSA, PDL, PEI, PLL and PSS exhibit an increase in the number of live cells with density but exhibit large drop in numbers for the bilayers, especially PDDA, BSA and PEI. Fibronectin exhibits a steady decline in the number of cells with increasing layers while PEG-NH2, CS, collagen and laminin exhibit the same behavior except for a drop in the number of cells for bilayers. Cell Seeding Density 25 000 cells mL 1 : PDDA, collagen and PSS exhibit increasing number of live cells with increasing layers while BSA, CS and laminin exhibit the same behavior except for a drop in the number of live cells for bilayers. In the case of PDL, PEI, PLL and ¯bronectin, bilayers show the largest number of live cells.

Table 3. Summary of morphological observations of phenotypes exhibited by PBACs after LiveDead analysis on mono-, bi- and tri-layer nano¯lms (Cell seeding density 5000 cells ml 1 Þ. Multilayer nano¯lm architectures Biomaterial

Monolayer

Bilayers

Trilayers


Rounded Rounded SROP SROP SROP Rounded SROP SROP SROP SROP SROP

SROP SROP SROP SROP SROP Rounded SROP SROP SROP SROP SROP

Rounded Rounded Fibroblast SROP SROP SROP SROP SROP SROP Cell death SROP

monolayers and trilayers of collagen show quite di®erent cellular morphologies. The exact reason behind these di®erences is not completely understood, and need further investigation.

3.3. Viability analysis of PBACs on nano¯lm surfaces ImageJ software was used as an adjunct to the LiveDead viability analysis for counting cells from the LiveDead Images. The results for live cells for the di®erent seeding densities and layers are shown in Figs. 15(a)15(c).

3.4. Area of PBACs on nano¯lm surfaces ImageJ software was used to calculate the area of cells from the LiveDead Images. The results for

(a) Fig. 15. LiveDead Image analysis results of PBACs on di®erent biomaterials: Cell seeding density of (a) 5000 cells mL 1 , (b) 15 000 cells mL 1 and (c) 25 000 cells mL 1 . 1342001-17

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(b)

(c) Fig. 15.

live cells for the di®erent seeding densities and layers are shown in Figs. 16(a)16(c). Cell Seeding Density 5000 cells mL 1 : On visual inspection, one can clearly observe that chondrocytes on bilayers of ¯bronectin have the highest area followed by chondrocytes on bilayers of laminin and PDDA. But at the same time, the standard deviations are too high for both of these bilayers so the results have to be interpreted with caution. In some cases, for example, chondrocytes on trilayers of laminin and chondrocytes on bilayers of CS have very small mean areas when compared to the areas of chondrocytes on other bilayers but these results are pretty much consistent with the low number of chondrocytes on these bilayers. These layers have more dead cells than live cells. Chondrocytes on

(Continued )

PSS, collagen and BSA (to an extent) exhibit increasing area with increasing number of layers while chondrocytes on TCPS, PEG-NH2 and PSS (to an extent) exhibit decreasing area with increasing number of layers. Chondrocytes on bilayers of PDL, CS has very small mean areas. Cell Seeding Density 15 000 cells mL 1 : Chondrocytes on PEG-NH2, PDDA, collagen, PSS and ¯bronectin, PEI (to an extent) exhibit decreasing mean area with increasing number of layers. Chondrocytes on bilayers of laminin, CS, PLL and PDL possess the maximal mean area compared to the mean area of chondrocytes on monolayer and trilayers of these materials. Chondrocytes on bilayers of TCPS exhibit the least mean area when compared to the mean area of chondrocytes on all

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(a)

(b)

(c) Fig. 16. Areas of PBACs on di®erent biomaterials measured using ImageJ: Cell seeding density of (a) 5000 cells mL 1 , (b) 15 000 cells mL 1 and (c) 25 000 cells mL 1 .

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the layers of the materials for this cell seeding density. Cell Seeding Density 25 000 cells mL 1 : Chondrocytes on bilayers of PDDA and trilayers of PEI exhibit the maximum area when compared to the mean area of chondrocytes on all other layers of all the materials for this cell seeding density. Chondrocytes on CS, PDL, ¯bronectin, PSS and PEGNH2 (to an extent) exhibit decreasing mean area with increasing number of layers. Chondrocytes on bilayers of PEI and laminin exhibit the least mean area compared to the mean area of chondrocytes on other layers of these materials.

3.5. Circularity of PBACs on nano¯lm surfaces ImageJ software was used to calculate the circularity of cells from the LiveDead Images. The results for live cells for the di®erent seeding densities and layers are shown in Figs. 17(a)17(c). Cell Seeding Density 5000 cells mL 1 : Chondrocytes on TCPS, PDL and collagen exhibit decreasing circularity with increasing number of layers. Chondrocytes on bilayers of PLL, laminin, PEG-NH2 and ¯bronectin exhibit greater circularity compared to the circularity of chondrocytes on other layers of these materials. At the same time, chondrocytes on bilayers of PDDA, BSA, CS, PEI and PSS exhibit lesser circularity compared to the circularity of chondrocytes on other layers of these materials. Cell Seeding Density 15 000 cells mL 1 : Chondrocytes on PLL, ¯bronectin and PSS (to an extent) exhibit decreasing circularity with increasing number of layers. Chondrocytes on bilayers of TCPS, BSA, CS, PEI and collagen exhibit greater circularity compared to the circularity of chondrocytes on other layers of these materials. On the other hand, chondrocytes on bilayers of PEG-NH2, PDDA, PDL and laminin exhibit lesser circularity compared to the circularity of chondrocytes on other layers of these materials. Cell Seeding Density 25 000 cells mL 1 : Of all the materials and cell seeding densities used, PEG-NH2 is the sole material, which exhibits increasing circularity of chondrocytes with increasing number of layers. For this density, several materials exhibit decreasing circularity with increasing number of

layers — PDL, PEI, collagen, PSS and laminin. Chondrocytes on bilayers of TCPS, PDDA, BSA and PLL exhibit greater circularity compared to circularity of chondrocytes on other layers of these materials. On the other hand, chondrocytes on bilayers of ¯bronectin show lesser circularity compared to the circularity of chondrocytes on other layers of ¯bronectin.

3.6. Feret's diameter of PBACs on nano¯lm surfaces ImageJ software was used to calculate the Feret's diameter of cells from the LiveDead Images. The results for live cells for the di®erent seeding densities and layers are shown in Figs. 18(a)18(c). Cell Seeding Density 5000 cells mL 1 : Chondrocytes on CS and PEI exhibit increasing Feret's diameter with increasing number of layers. Chondrocytes on bilayers of PEG-NH2, PDDA, BSA, collagen and PSS exhibit larger Feret's diameter compared to the Feret's diameter of chondrocytes on other layers of these materials. On the other hand, chondrocytes on bilayers of TCPS, PDL and ¯bronectin exhibit smaller Feret's diameter compared to the Feret's diameter of chondrocytes on other layers of these materials. Cell Seeding Density 15 000 cells mL 1 : Chondrocytes on BSA exhibit increasing Feret's diameter with increasing number of layers while chondrocytes on PEI and PSS exhibit decreasing Feret's diameter with increasing number of layers. Chondrocytes on bilayers of PDL, CS, collagen, ¯bronectin and laminin exhibit greater Feret's diameter compared to the Feret's diameter of chondrocytes on other layers of these materials. Cell Seeding Density 25 000 cells mL 1 : Chondrocytes on CS exhibit increasing Feret's diameter with increasing number of layers while chondrocytes on PEG-NH2 and BSA exhibit decreasing Feret's diameter with increasing number of layers. Chondrocytes on bilayers of PDDA, PDL, collagen, PLL and PSS exhibit larger Feret's diameter compared to Feret's diameter of chondrocytes on other layers of these materials. On the other hand, chondrocytes on bilayers of TCPS, PEI, ¯bronectin and laminin exhibit smaller Feret's diameter compared to Feret's diameter of chondrocytes on other layers of these materials.

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(b)

(c) Fig. 17. Circularities of PBACs on di®erent biomaterials measured using ImageJ: Cell seeding density of (a) 5000 cells mL 1 , (b) 15 000 cells mL 1 and (c) 25 000 cells mL 1 . 1342001-21

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(c) Fig. 18. Feret's diameters of PBACs on di®erent biomaterials measured using ImageJ: Cell seeding density of (a) 5000 cells mL 1 , (b) 15 000 cells mL 1 and (c) 25 000 cells mL 1 . 1342001-22


4. Conclusions The growth and behavior of PBACs was examined on monolayers, bilayers and trilayers of 11 di®erent biomaterials. Cells on majority of the materials exhibited SROP with stray instances of rounded and ¯broblast phenotype. Cells exhibited di®erences in number, area, circularity and Feret's diameter — these di®erences varied both with the type of biomaterial and also the number of layers of materials. These di®erences in cellular characteristics can be put to use in tissue engineering applications, in vitro and drug-toxicity assays, etc. While phenotype is one of the major characteristic of interest, the concomitant changes associated with phenotype di®erences of PBACs on di®erent biomaterials need to be analyzed thoroughly in future studies. Future studies should examine the growth of PBACs on multilayer nano¯lms containing more number of layers. Further, the range of biomaterials used too can be expanded to choose the biomaterial exhibiting the best cellular characteristics. Modulation of phenotype to achieve the in vivo phenotype was one of the major aims of this study. Since the interactions observed in this study were only short term, there is a requirement for the analysis of long term interactions of cells on biomaterials.

Acknowledgments This work was supported by a DARPA and NAVY SPAWAR SC N66001-05-1-8903 grant and a National Science Foundation (Grant No. 0092001) awarded to Dr. David K. Mills.

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