Abstract
Biomaterials combining biochemical and biophysical cues to establish close-to-extracellular matrix (ECM) models have been explored for cell expansion and differentiation purposes. Multivariate arrays are used as material-saving and rapid-to-analyze platforms, which enable selecting hit-spotted formulations targeting specific cellular responses. However, these systems often lack the ability to emulate dynamic mechanical aspects that occur in specific biological milieus and affect physiological phenomena including stem cells differentiation, tumor progression, or matrix modulation. We report a tailor-made strategy to address the combined effect of flow and biochemical composition of three-dimensional (3D) biomaterials on cellular response. We suggest a simple-to-implement device comprising (i) a perforated platform accommodating miniaturized 3D biomaterials and (ii) a bioreactor that enables the incorporation of the biomaterial-containing array into a disposable perfusion chamber. The system was upscaled to parallelizable setups, increasing the number of analyzed platforms per independent experiment. As a proof-of-concept, porous chitosan scaffolds with 1 mm diameter were functionalized with combinations of 5 ECM and cell-cell contact-mediating proteins, relevant for bone and dental regeneration, corresponding to 32 protein combinatorial formulations. Mesenchymal stem cells adhesion and production of an early osteogenic marker were assessed on-chip on static and under-flow dynamic perfusion conditions. Different hit-spotted biomaterial formulations were detected for the different flow regimes using direct image analysis. Cell-binding proteins still poorly explored as biomaterials components – amelogenin and E-cadherin – were here shown as relevant cell response modulators. Their combination with ECM cell-binding proteins – fibronectin, vitronectin, and type 1 collagen – rendered specific biomaterial combinations with high cell adhesion and ALP production under flow. The developed versatile system may be targeted at widespread tissue regeneration applications, and as a disease model/drug screening platform. Statement of SignificanceA perfusion system that enables cell culture in arrays of three-dimensional biomaterials under dynamic flow is reported. The effect of 31 cell-binding protein combinations in the adhesion and alkaline phosphatase (ALP) production of mesenchymal stem cells was assessed using a single bioreactor chamber. Flow perfusion was not only assessed as a classical enhancer/accelerator of cell growth and early osteogenic differentiation. We hypothesized that flow may affect cell-protein interactions, and that key components driving cell response may differ under static or dynamic regimes. Indeed, hit-spotted formulations that elicited highest cell attachment and ALP production on static cell culture differed from the ones detected for dynamic flow assays. The impacting role of poorly studied proteins as E-cadherin and amelogenin as biomaterial components was highlighted.
Highlights
The poor isolation of the role of individual factors and their interplay on human tissues healing ability hinders the development of simplified, yet relevant and effective, implantable tissue engineering systems and in vitro testing devices [1]
Human proteins including fibronectin, vitronectin and type-I collagen are presented to cells as adhesive proteins present in native human tissues’ extracellular matrix (ECM), capable of mediating cell-matrix interactions through different membrane integrins [48]
The interactions between each individual biomaterial combination and human Bone marrow-derived mesenchymal stem cells (BMSCs) were tested on static cell culture conditions, as well as under a dynamic perfusion flow
Summary
The poor isolation of the role of individual factors and their interplay on human tissues healing ability hinders the development of simplified, yet relevant and effective, implantable tissue engineering systems and in vitro testing devices [1]. The extracellular matrix (ECM) is accepted as a cornerstone aspect for the maintenance of tissues function and regeneration capability Since it is the three-dimensional (3D) matter that embeds cells, it provides mechanical cues and physical/structural support to tissues, is composed of molecules secreted by the tissues’ own cells, and regulates several signaling pathways [4]. Such phenomena involve cell-cell and cell-matrix adhesion mediation proteins, which have significant roles in the recruitment and binding of growth factors that trigger signaling cascades commonly involved in cell differentiation signaling [5]. Besides the challenging task of understanding the biochemical diversity of the ECM, the use of 3D systems capable of resembling the native ECM architecture and micromechanical environment is of utmost importance to achieve in vitro/in vivo correlating results [7,8,9] and to identify critical aspects to increase the yield of in vitro approaches [10,11,12,13]
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