Evaluation of Nanostructured Composite Collagen Chitosan Matrices for Tissue Engineering

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1 TISSUE ENGINEERING Volume 7, Number 2, 2001 Mary Ann Liebert, Inc. Evaluation of Nanostructured Composite Collagen Chitosan Matrices for Tissue Engineering WEI TAN, B.S., 1 RAJ KRISHNARAJ, Ph.D., 2 and TEJAL A. DESAI, Ph.D. 1 ABSTRACT The development of suitable three-dimensional matrices for the maintenance of cellular viability and differentiation is critical for applications in tissue engineering and cell biology. The structure and composition of the extracellular matrix (ECM) has been shown to modulate cell behavior with respect to shape, movement, proliferation, and differentiation. Although collagen and chitosan have separately been proposed as in vitro ECM materials, the influence of chitosan collagen composite matrices on cell morphology, differentiation, and function is not well studied. To this end, gel matrices of different proportions of collagen and chitosan were examined ultrastructurally and characterized for their ability to regulate cellular activity. A three-chamber system with circulating hydraulic fluids was used to evaluate the gel stability under fluid force. Results indicated that overall matrix integrity increased with the proportion of chitosan. Scanning electron microscopy indicated that the addition of chitosan greatly influences ultrastructure and changes collagen fiber cross-linking, reinforcing the structure and increasing pore size. K562 cells cultured in three-dimensional gels were examined for cell proliferation and differentiation. Although cell proliferation was inhibited with an increasing proportion of chitosan, cell function based on cytokine-release was greatly augmented. Results suggest that a hybrid chitosan collagen matrix may have potential biological and mechanical benefits for use as a cellular scaffold. T HE INTRODUCTION DEVELOPMENT of suitable synthetic extracellular matrices (ECM) that can recreate three-dimensional cell cell interactions and control tissue formation in vitro and in vivo is important for tissue engineering and cell culture applications. Natural ECM can be used as a model for the development of synthetic matrices for tissue engineering. The two main classes of extracellular macromolecules that form the natural ECM are proteoglycans and fibrous proteins. Proteoglycans, containing long unbranched polysaccharide side chains covalently tethered to a fibrous backbone, form three-dimensional networks of hydrated gels in which cells can be embedded. Structural fibrous proteins such as collagen provide the tensile strength of the ECM, whereas proteoglycans provide compressive strength. The ECM plays a critical role in tissue development, support, and cellular functions such as growth, differentiation, and motility. Currently, collagen is one of the most common ECM materials used for culturing cells in vitro. Collagen molecules assemble to form microfibrils, which, in turn, form fibrils to create the collagen fiber. Col- 1 Department of Bioengineering and 2 Department of Medicine, University of Illinois at Chicago, Chicago, Illinois. 203

2 lagen can be reconstituted to form hydrated gels that are similar to loose connective tissue in vivo. Cell behavior in three-dimensional hydrated collagen gels is more typical of in vivo behavior than those grown on two-dimensional plastic culture surfaces. Several ECM-like materials that combine synthetic polymers with three-dimensional collagen gels have been investigated. For example, polyethylene glycol (PEG)/collagen composites have been used as in vivo scaffolds for connective tissue reconstruction, 1 whereas fibronectin collagen and laminin collagen composites have been used to grow cells in vitro. Such composite materials incorporating collagen have been explored to obtain matrices with improved biomimetic and biomechanical properties for cell growth and immobilization. Chitosan is a (1-4)2-amino-2-deoxy-B-D-glucan, a unique polysaccharide derived from chitin that has structural characteristics similar to glycosaminoglycans. 2 In the natural ECM, proteoglycans and glycosaminoglycans have important roles in intertwining with the fibrous structure of collagen to obtain mechanical stability and compressive strength. Likewise, proteoglycans have been shown to influence cell behavior. Therefore, it may be important to develop collagen composites with chitosan or other glycosaminoglycanlike components to create more suitable biomimetic microenvironments for cells. To this end, collagen chitosan matrices of varying proportions and concentrations were prepared and seeded with K562 cells to examine the effect of chitosan on cell behavior in collagen gels. K562, a human hemopoietic cell line, was used to test cell viability and proliferation in these three-dimensional matrices. These cells exhibit migratory function, express integrin alpha4beta1 and alpha5beta1 and are shown to adhere to fibronectin and purified extracellular matrix components. 3 Moreover, these cells are able to attach firmly to collagen preparations. 4 Although collagen and chitosan have separately been proposed as in vitro ECM materials, the influence of composite chitosan collagen matrices on cell viability and differentiation has not been thoroughly examined. We have studied the ultrastructure and stability of composite chitosan/collagen gel matrices via scanning electron microscopy (SEM) and their effects on cell trapping, cell viability, and cell proliferation. Collagen chitosan gel matrix preparation MATERIALS AND METHODS The composite gel matrices were formed from collagen and varying amounts of chitosan. Briefly, Type I collagen (Sigma) was solublized in 1 M of acetic acid, with a final concentration of 2 mg/ml. Chitosan (Sigma) was made soluble in 1 M of acetic acid by stirring for 1 day. The insoluble portion was filtered with a 0.2-mm filter, thus forming a viscous and clear solution. The final concentration was 8 mg/ml. To prevent contamination of these stock solutions, 1% of streptomycin and penicillin was added. When reconstituting the gel matrix, the ph and osmolarity of the solution were raised to physiological levels (ph 7.4; osmolarity 300 mosm) by gently and thoroughly mixing the following solutions in a microcentrifuge tube on ice: (1) 100 ml of 10 3 Hanks balanced salt solution (HBSS); (2) a predetermined volume of collagen solution to obtain the desired final concentration in a final volume of 1 ml; (3) a predetermined volume of chitosan solution to obtain the desired proportion with collagen; (4) 7 ml 5% wt/vol NaHCO 3 ; (5) a predetermined amount of 1 M of NaOH so that the final solution ph is 7.4; and (6) pure water or complete media to make the final solution volume equal to 1 ml. The solution was mixed well, distributed into 24-well plates, and put into a 37 C incubator for min. Because chitosan tends to form small capsules very easily when the ph is raised to near-neutral levels, it was found to be better to mix all the components before adding NaOH to mix chitosan and collagen thoroughly. Chitosan collagen gel matrices were then seeded with 100 ml of cell suspension for biological testing. Cell proliferation and viability TAN ET AL. K562 cells were incubated at 37 C with 5.0% CO 2. K562 cells were fed with RPMI media with 10% fetal bovine serum (FBS) and 1% glutamine every other day. Chitosan collagen gel matrices of varying pro- 204

3 COLLAGEN CHITOSAN MATRICES portions were seeded with 100 ml of cell suspension and were examined for 8 days. Cell numbers were quantified with hemocytometry. In addition, AlamarBlue was used to track the cell proliferation of the cells cultured in three-dimensional matrices. The method is similar to that described by Zhi-Jun et al. 5 Briefly, 10% vol/vol AlamarBlue was added into culture media (typically 25 ml added to 250 ml of culture media). AlamarBlue reduction was then measured spectrophotometrically at 570 nm and 600 nm or fluorometrically. Cell viability is measured using trypan blue exclusion. The blue-dyed cells are subtracted from the living cell number. Cell viability studies were carried out for 8 days. The stability of reconstituted matrices The stability of the matrices under fluid flow was monitored using a three-chamber system. Collagen chitosan solutions prepared as described above were injected into the middle chamber. The sample was left undisturbed for 30 min to allow gel formation. After 30 min, the sample was subjected to hydraulic flow by pumping media through the chamber at 40 ml/min. After 4 h, the gel thickness was measured and the gel was fixed for SEM examination as described below. SEM of reconstituted gel matrices Collagen chitosan extracellular matrices were prepared by physical mixing before gelation; the mixtures were incubated at 37 C to form gels. The concentration of collagen was kept at 8 mg/ml and influence of chitosan concentration (8 mg/ml, 16 mg/ml, 24 mg/ml, and 32 mg/ml) on collagen structure was evaluated. Collagen gels and collagen chitosan gels with different proportions were fixed, dehydrated, and dried using the following methods. Gels were fixed with 2.5% glutaraldehyde in cacodylate buffer (ph 4) for 1 h at room temperature. The specimens were washed three times with cacodylate buffer three times prior to dehydration through a series of graded alcohol solutions. Instead of critical point drying, small pieces of matrix were put into hexamethyldisilazane (HMDS; Electron Microscopy Sciences) in small wells of a glass dish for 15 min. Then, pieces were transferred to a dry well and air-dried for about 15 min. Matrix pieces were mounted and silver paint was added to the edges of each specimen. The resulting samples were sputter coated and examined using a scanning electron microscope. RESULTS Ultrastructural examination of the matrix using SEM Using SEM to examine the ultrastructure of collagen chitosan gels at a magnification of 3500 (Fig. 1), the matrix macrostructure and pore distribution could be seen. With magnification set at 43,000 (Fig. 2), the detailed microstructure of the matrices could be more clearly examined. The figures show that when chitosan was introduced into collagen solution, the formation of collagen fibers was not perturbed by the tiny chitosan clumps, because the diameter of fibers in the pure collagen and those in gels with chitosan are very similar. However, fibrous organization was significantly changed by chitosan, and pore sizes of gel matrices were greatly affected by different chitosan concentrations. SEM micrographs of 1:3 collagen:chitosan matrices without cells were very unclear because chitosan carries a positive charge and deviates the electron route. However, cells carry a negative charge and can neutralize the positive charge produced by chitosan; thus, the micrographs of the 1:3 collagen:chitosan matrix with cells produced much clearer images than the unseeded matrix. With increasing amounts of chitosan added to the collagen gel, more collagen fibers were sequestered together and greater numbers of clumps and knobs were formed, as shown in Fig. 2D. The collagen fibers were more dense and the distribution of all fibers was more heterogeneous with the addition of chitosan. Moreover, the cell distribution became increasingly uneven in these matrices. The stability of the reconstituted matrices under fluid force was examined using the three-chambered system described earlier. These tests revealed that the higher the chitosan concentration in collagen chitosan gels, the thicker the gel remained after circulating media for 4 h (Table 1). The testing was performed under the media flow rate of 14 ml/min, and the circulation lasted for 4 h. 205

TAN ET AL. A B C FIG. 1. SEMs of collagen with chitosan of different proportions where collagen concentration is always 8.

collagen chitosan matrices with 1:3 proportion and with K562 cells in a three-dimensional matrix. (Original magnification: 33,500.

Figure 3 shows cell proliferation on pure collagen gels and collagen chitosan gels of varying proportions.

As the chitosan proportion increased, the inhibitory effect increased.

When the proportions of chitosan were above that level, the further increase in chitosan had no detectable influence on cell growth.

When the proportion was kept constant, but the concentrations of both components were adjusted by either increasing or decreasing them, cell proliferation was

4 TAN ET AL. A B C FIG. 1. SEMs of collagen with chitosan of different proportions where collagen concentration is always 8.0 mg/ml: (A) pure collagen matrices; (B) collagen chitosan composite matrices with 1:1 proportion; (C) collagen chitosan matrices with 1:3 proportion; and (D) collagen chitosan matrices with 1:3 proportion and with K562 cells in a three-dimensional matrix. (Original magnification: 33,500.) Cell proliferation and viability The influence of the matrix on cell behavior was evaluated by cell proliferation and viability assays. Figure 3 shows cell proliferation on pure collagen gels and collagen chitosan gels of varying proportions. As indicated by the results, cell growth was greatly inhibited on collagen chitosan gels compared to pure collagen gels. As the chitosan proportion increased, the inhibitory effect increased. However, this only applied to situations where the proportion of chitosan to collagen was greater than 3 (i.e., 1:3 collagen chitosan matrices). When the proportions of chitosan were above that level, the further increase in chitosan had no detectable influence on cell growth. It was the proportion between collagen and chitosan that influenced cell growth rather than the concentration of both collagen and chitosan. When the proportion was kept constant, but the concentrations of both components were adjusted by either increasing or decreasing them, cell proliferation was not affected. Cell growth rates on the collagen/chitosan composite matrices with 0.8 mg/ml and 1.6 mg/ml chitosan concentrations and matrices with 0.4 mg/ml and 0.8 mg/ml chitosan concentrations were similar, respectively. Figure 4 shows cell viability in the various matrices. Cell viability was found to always be above 95%, and there was little difference among all samples. This seems to indicate that chitosan does not significantly affect cell viability in matrices, but does affect cell proliferation. D 206

COLLAGEN CHITOSAN MATRICES A B C FIG. 2. SEMs of collagen with chitosan of different proportions where collagen concentration is always 8.

chitosan matrices with 1:3 proportion and with K562 cells in three-dimensional gel. (Original magnification: 343,000.

These matrices should ideally create biomimetic microenvironments for cell growth, immobilization, and differentiation, as well as provide appropriate physical support.

The composite ECM described in this paper is reconstituted from networks of collagen fibers and chitosan.

Chitosan collagen matrices were prepared in our lab to be used as a culture substrate, as well as a reconstituted three-dimensional matrix.

5 COLLAGEN CHITOSAN MATRICES A B C FIG. 2. SEMs of collagen with chitosan of different proportions where collagen concentration is always 8.0 mg/ml: (A) pure collagen matrices; (B) collagen chitosan composite matrices with 1:1 proportion; (C) collagen chitosan matrices with 1:3 proportion; and (D) collagen chitosan matrices with 1:3 proportion and with K562 cells in three-dimensional gel. (Original magnification: 343,000.) DISCUSSION The design of appropriate cell matrices is essential for creating long-term constructs for applications in tissue engineering and cellular delivery. These matrices should ideally create biomimetic microenvironments for cell growth, immobilization, and differentiation, as well as provide appropriate physical support. Furthermore, the matrix should not elicit short- or long-term immunological or deleterious responses when placed in vivo. The composite ECM described in this paper is reconstituted from networks of collagen fibers and chitosan. The influence of chitosan on the collagen gel matrices was examined in terms of structural and biological properties. Chitosan collagen matrices were prepared in our lab to be used as a culture substrate, as well as a reconstituted three-dimensional matrix. The main components of this matrix, consisting of collagen I fibers and a largely amorphous interfibrillar matrix (chitosan polysaccharides), are similar to that of the natural ECM. Structural modifications in collagen chitosan composites Collagen molecules assemble to form microfibrils, which in turn form fibrils to give rise to the collagen fiber. In their natural state, the collagen trihelical configuration is held in place by direct chemical bonds, D 207

6 TAN ET AL. TABLE 1. VOLUME OF THE MATRIX AS A FUNCTION OF WEIGHT % COLLAGEN:CHITOSAN AFTER 4 H OF FLUID FLOW Collagen; chitosan (weight %) No chitosan 1; 1 1; 2 1; 3 Volume after 4 hr/ 0 1/10 1/3 1/4 1/3 original volume hydrogen bonds, and water-bridged cross-links. Associated with collagen in native tissue are elastin, proteoglycans, and mucopolysaccharides attached covalently to protein cores. These components are thought to modulate collagen fibrillogenesis, fill space, as well as bind and organize water. Our experiments revealed that with increasing amounts of chitosan added to the collagen gel, more collagen fibers were sequestered together, the collagen fibers were more dense, and the distribution of all fibers was more heterogeneous. It has been shown that chitosan interacts with collagen in the mixture solution, forming ionic bonds when mixing. 6 Our study suggest these collagen chitosan interactions also exist in gel conditions and influence collagen fiber configurations. Ultrastructural examination of the matrices show small chitosan beads tightly tethered to a collagen fiber backbone. Several collagen fibers or different portions of one fiber seem to be woven and linked together through chitosan. The microstructure created by the addition of chitosan may prevent collagen fibers from shifting, induce formation of interchain crosslinks, and reinforce the fibers by anchoring them in place. The collagen chitosan interaction also influences the pore density and pore size of the matrix. A greater concentration of chitosan in the matrix results in a lower pore density and a smaller pore size of the matrix. FIG. 3. K562 cell proliferation on collagen chitosan matrices of different proportions. The mean values are used in the plot. Error bars represent the standard deviation (n 5 3). 208

7 COLLAGEN CHITOSAN MATRICES FIG. 4. K562 cell viability on collagen-chitosan matrices of different proportions. The mean values are used in the plot. Error bars represent the standard deviation (n 5 3). The collagen chitosan gels are biological composites that have the crystalline chitosan component as the main support material in an amorphous interfibrillary collagen matrix. This structure allows the collagen chitosan gel to maintain structural integrity as compared to collagen alone under fluid flow, as shown by our tests. This structural integrity may be advantageous in applications requiring long-term maintenance of three-dimensional cell-seeded matrices in vivo or in vitro. The ultrastructure of collagen chitosan composites greatly influences the microenvironment for cell growth. When the collagen-to-chitosan ratio is more than 1:2, the greater amount of chitosan beads causes collagen fibers to clump together and fill in pores within the fiber structure. This high-density microstructure stabilizes the porous collagen structure and helps to maintain matrix microarchitecture. Microenvironments for cell growth in collagen chitosan gel matrices Cell proliferation was inhibited in collagen chitosan gel matrices compared to pure collagen gels. The reason may be due to the fact that the addition of chitosan increases matrix density and cell matrix contact, therefore limiting the space for cell proliferation. Cells become trapped more deeply into hydrated chitosan gels rather than just attached to collagen fibers. The negativity and adhesivity of chitosan may result in greater interaction between cells and the matrix. However, data on cell proliferation also suggests that this interaction saturates when the chitosan proportion goes beyond three times collagen in weight. The natural ECM is composed of a complex network of macromolecules that hold cells together in an organized fashion to form complex tissues. These organized networks allow cells within to migrate and interact with the matrix and one another. Nutrients, metabolites, and hormones from neighboring blood vessels diffuse into the ECM to provide nourishment and mediate communication between the cells. The in- 209

8 creased pore size in chitosan collagen matrices with lower proportions of chitosan may be beneficial for the diffusion of nutrients and metabolites in three-dimensional matrices. Biocompatibility TAN ET AL. Lastly, the addition of chitosan does not take away from the beneficial properties of using collagen as a substrate for cell culture or tissue engineering ECM. Chitosan is biologically sourced, biodegradable in vivo, and biocompatible as well, so that the addition of this component will not affect the overall biocompatibility of the composite matrices. CONCLUSION In this study, gel matrices of different proportions of collagen and chitosan were examined ultrastructurally and characterized for their ability to regulate cellular activity. The influence of chitosan collagen composite matrices on cell viability and proliferation was examined. Addition of chitosan to traditional collagen-based matrices alters the cross-linking pattern of the collagen fibers, increases the integrity of the matrix by gluing the fibers together, and alters cell proliferation. Although cells maintain viability in all collagen chitosan composite matrices, those matrices with a 1:1 collagen:chitosan proportion seem most suitable as synthetic matrices, because at higher chitosan levels, the porosity of the matrix is low and cell proliferative capacity is minimal. The addition of chitosan to the matrix is beneficial in that it promotes greater cell matrix interaction and leads to cell trapping and immobilization of cells. The addition of chitosan also stabilizes the fibrous structure and prevents deformation of these matrices under fluid flow. REFERENCES 1. Tan, J., and Saltzman, M. Influence of synthetic polymers on neutrophil migration in three-dimensional collagen gels. J. Biomed Mater Res. 46, 465, Chandy, T., and Sharma, C. Chitosan as a biomaterial. Biomater. Art. Cells Art. Org. 18, 1, Turner, M.L., Masek, L.C., Hardy, C.L., Parker, A.C., and Sweetenham, J.W. Comparative adhesion of human haemopoietic cell lines to extracellular matrix components, bone marrow stromal and endothelial cultures. Br. J. Haematol. 100, 112, Klein, G., Kibler, C., Schermutzki, F., Brown, J., Muller, C.A., and Timpl, R. Cell binding properties of collagen type XIV for human hematopoietic cells. Matrix Biol. 16, 307, Zhi-Jun, Y., Sriranganathan, N., Vaught, T., Arastu, S.K., and Ahmed, S.A. A dye-based lymphocyte proliferation assay that permits multiple immunological analysis: mrna, cytogenetic, apoptosis, and immunophenotyping studies. J. Immun. Meth. 210, 25, Taravel, M.N., and Domard, A. Relation between physicochemical characteristics of collagen and its interactions with chitosan: I. Biomaterials 14, 930, Address reprint requests to: Tejal A. Desai, Ph.D. Department of Bioengineering (MC 063) University of Illinois at Chicago 851 South Morgan Street Chicago, IL Tdesai@uic.edu 210

Lab Module 7: Cell Adhesion

Lab Module 7: Cell Adhesion Tissues are made of cells and materials secreted by cells that occupy the spaces between the individual cells. This material outside of cells is called the Extracellular Matrix