Controlled pattern of cell growth in modulated protein nanocomplexes Regulating cells spreading in three dimensions

The efficiency of polymeric biomaterials greatly depends on their ability to interact with living cells. Indeed, cell processes, such as proliferation, two-dimensional (2D) adhesion, and three-dimensional (3D) spreading are directly affected by cell–matrix interactions [1][2][3]. Although, knowledge of cell interactions in 2D with different polymers ranging from synthetic biodegradable polymers to natural biopolymers is increasing; well-controlled 3D cell growthfor these types of biomaterials, especially in the presence of stimulus, is seldom a concern. It is also of great importance to know how the 3D pattern of cell growth can be affected by micro- and nanoscale features of polymer-based biomaterials. On the other hand, since tissue engineering therapies usually use 3D biodegradable scaffolds as a short-term substitute for the extracellular matrix (ECM, a complex mixture of proteins and polysaccharides beyond the cells) [3][4], such information would also generate fundamental insights into the materials’ ability to support tissue regeneration while inducing a minimal inflammatory response. Moreover, the ability to control the migration and spreading of cells in 3D model microenvironments potentially provides an opportunity to revolutionize our understanding of cellular behavior in the physiological conditions of the human body.

Having recognized the differences between cells grown on a flat surface versus 3D formats, researchers are now exploring hydrophilic polymer systems with multiple functional domains to create new microenvironments that recapitulate critical biochemical cues of the native ECM [3][4][5][6]. Structurally, this microenvironment should not only be a carrier for the cells to be encapsulated, but should also offer a communication system to underpin cellular events. While the complexity of naturally occurring ECM is very difficult to reproduce, some hydrogels, mainly based on collagen, represent a promising option to mimic the main elements of native ECM. Collagen molecules, as the most abundant protein of ECM, are actually a large family of several protein isoforms [3][6][7]. Highly purified collagen is commercially available, but less expensive gelatin which is a denatured collagen is often used for the 3D cell culture. Some studies also demonstrated that gelatin hydrogels are appealing candidates for the development of versatile tissue engineering scaffolds [3][5]. The current gelatin constructs, however, are still far from those that are expected to be capable of controlling cell growth in 3D while maximizing the likelihood of long-term cell survival. Although, protein hydrogels treated either physically or chemically have recently been shown to be promising candidates for controlling the cell functions in 3D [8][9][10][11][12], but little is known about the gelatin networks that are modified simultaneously by organic chemicals and bioactive nanostructures. The extent of modifications in such a modulated complex is hypothesized to control the architectural features and hence plays a key role to afford a range of cellular functions, including cell binding and spreading. In addition, depending on the type and degree of modification, these engineered ECM-like complexes would be potentially attractive for a variety of tissue engineering applications.

The cover image of this issue is what results when a crosslinked collagen hydrogel in combination with rod-like hydroxyapatite nanoparticles is used to control the spreading of mammalian cells in 3D. The first step is to synthesize rod-like nanoparticles using the chemical precipitation followed by hydrothermal treatment [13][14]. The nanoparticles mimicking bone minerals were then incorporated into a light crosslinkable gelatin-based hydrogel that was chemically modified with methacrylic anhydride in a controlled fashion. The complex network was subsequently formed by the UV-light in the presence of photoinitiator molecules, while, at the same time, cells were thoroughly encapsulated in the resulting complex according to a previously described procedure [10]. The overview of the procedures can be briefly presented by the following schematic reactions:

Controlled pattern of cell growth in modulated protein nanocomplexes Regulating cells spreading in three dimensions

The two-step process exploited in the current study allows for the synergistic combination of high mechanical properties and great potential for cell–matrix interaction in a single construct. Other advantages of the fabricated system include high bioactivity, structural similarity to the ECM, and the potential for providing a flexible reservoir for high density of cells. Moreover, the reaction chemistry is water compatible, allowing incorporation of growth factors and biological agents that promote cellular proliferation and differentiation.

In order to monitor the impact of modifications on the cellular response, fluorescence images were obtained after nucleus staining with 4′,6-diamidino-2-phenylindole and fluorescent labeling of F-actin microfilament cytoskeleton with Alexa Fluor 488 phalloidin at the appropriate time points. The strange 3D spreading pattern of fibroblasts in the cover image is a result of the high concentration of bioactive nanoparticles which hinders cell infiltration in some areas at the initial stages of encapsulation, but then induces the cell growththroughout the hydrogel. Indeed, increased particle concentration slows the cell spreading at the initial stages but subsequently promotes the formation of relatively straight actin microfilaments. At lower concentrations of particles, cells can migrate and proliferate more readily leading to a well-interconnected cellular network, although the resulting construct presents some limitations, such as low mechanical strength and suboptimal durability. Interestingly, while fibroblasts exhibited a less homogeneous spreading in the nanocomplexcompared to bare gelatin, preosteoblastic cells in contrast exhibited an even spreading and an increased amount of F-actin fibers, in comparison to unmodified hydrogel. On the other hand, in vitro mineralization studies using simulated body fluid under physiological conditions showed the accumulation of a substantial amount of new minerals in the nanocomplexes, indicating a promising bioactivity. Preliminary examinations revealed that cell responses to this protein construct should be partly affected by the nanoscale features of the rod-like particles and by the matrix stiffness of the crosslinked hydrogel. These findings suggest that modulated protein nanocomplexes could be useful both for creating new 3D cell culture platforms and for the development of tailored tissue-engineering scaffolds with a high cell population in a confined volume. The principles derived from such studies may also help us to improve our understanding on cell–biomaterial interactions in the future.


This work was partially supported by the Iranian Nanotechnology Initiative Council, Iran and the Ministry of Science, Research, and Technology, Iran. Technical assistance from Harvard University, United States, Iran Polymer and Petrochemical Institute, Iran and Sharif University of Technology, Iran is also gratefully acknowledged.

Further reading

[1] L. Cai, A.S. Guinn, S. Wang
Acta Biomater., 7 (2011), pp. 2185-2199

[2] M. Sadat-Shojai, M.T. Khorasani, A. Jamshidi
Nanotech Insights, 4 (2013), pp. 2-5

[3] B.A. Justice, N.A. Badr, R.A. Felder
Drug Discov. Today, 14 (2009), pp. 102-107

[4] M. Sadat-Shojai
J. Mater. Sci. Technol., 32 (2016), pp. 1013-1020

[5] N.E. Fedorovich, et al.
Tissue Eng., 13 (2007), pp. 1905-1925

[6] W.T. Brinkman, et al.
Biomacromolecules, 4 (2003), pp. 890-895

[7] J.H. Miner
Microsc. Res. Techniq., 71 (2008), pp. 349-356

[8] M. Sadat-Shojai
Iran. J. Biotechnol. (2017), pp. 274-275
(special issue)

[9] M. Sadat-Shojai
Mol. Biol. Res. Commun., 3 (Suppl. 1) (2014), p. 67

[10] M. Sadat-Shojai, M.T. Khorasani, A. Jamshidi
Mat. Sci. Eng. C, 49 (2015), pp. 835-843

[11] K. Ulubayram, et al.
J. Biomater. Sci. Polym. Ed., 13 (2002), pp. 1203-1219

[12] Y.C. Chen, et al.
Adv. Funct. Mater., 22 (2012), pp. 2027-2039

[13] M. Sadat-Shojai, M. Atai, A. Nodehi, Method for production of biocompatible nanoparticles containing dental adhesive, US Patent: US 8,357,732 B2, 2013.

[14] M. Sadat-Shojai
Hydroxyapatite: Inorganic Nanoparticles of Bone (Properties, Applications, and Preparation Methodologies)
(first ed.), Iranian Students Book Agency (ISBA), Tehran (2010)

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DOI: 10.1016/j.mattod.2018.06.003