Artificial membrane-binding proteins stimulate oxygenation of stem cells during engineering of large cartilage tissue

Abstract

Restricted oxygen diffusion can result in central cell necrosis in engineered tissue, a problem that is exacerbated when engineering large tissue constructs for clinical application. Here we show that pre-treating human mesenchymal stem cells (hMSCs) with synthetic membrane-active myoglobin-polymer–surfactant complexes can provide a reservoir of oxygen capable of alleviating necrosis at the centre of hyaline cartilage. This is achieved through the development of a new cell functionalization methodology based on polymer–surfactant conjugation, which allows the delivery of functional proteins to the hMSC membrane. This new approach circumvents the need for cell surface engineering using protein chimerization or genetic transfection, and we demonstrate that the surface-modified hMSCs retain their ability to proliferate and to undergo multilineage differentiation. The functionalization technology is facile, versatile and non-disruptive, and in addition to tissue oxygenation, it should have far-reaching application in a host of tissue engineering and cell-based therapies.

Oxygen delivering biomaterials for tissue engineering

“…76 To produce the protein-surfactant complex, they conjugated a cationized myoglobin protein with an anionic surfactant, yielding an amphiphilic molecule capable of anchoring to the cell membrane. They combined the [Mb_C][S] with hMSCs and tested the ability of the hMSCs to form cartilage within non-woven polyglycolic acid (PGA) scaffolds.…”

Vibrational spectroscopy and imaging: applications for tissue engineering

“…Similarly, for nerve regeneration, the sample sections are stained with monoclonal antibodies for Schwann Cells (GFAP, S-100 protein), axonal regrowth (β-III tubulin, neurofilaments and GAP-43, PGP9.5) and Myelin (myelin basic protein) (Carriel et al 2014, 1657–1660). For quantification of the proteins of interest, components are assessed by image analysis, which can involve thresholding and segmentation based on specific staining 28–30 . Scanning and transmission electron microscopy (SEM, TEM) are frequently performed on engineered constructs for evaluation of nanometer and micron level structural features, such as collagen fibril size and progressive tissue formation during cultivation 25, 26, 31 .…”

Strategies for cell membrane functionalization

“…Conformational rearrangement of the amphiphilic polymer surfactant promotes protein stability and aqueous solubility (due to the hydrophilic PEG segment), 97,98 and mediates membrane tethering for around one week in culture (via the hydrophobic tail). 13 Myoglobin conjugates retained their oxygen-binding capacity 99 and were delivered to hMSCs to provide an in situ oxygen reservoir to enhance the production of matrix fibres at the Armstrong and Perriman Strategies for cell membrane functionalization 1101 centre of engineered cartilage constructs. 13 Importantly, both cell priming and protein lipidation necessitate careful modification of the protein surface, as aggressive bioconjugation strategies can lead to denaturation and subsequent loss of biological function.…”

“…13 Myoglobin conjugates retained their oxygen-binding capacity 99 and were delivered to hMSCs to provide an in situ oxygen reservoir to enhance the production of matrix fibres at the Armstrong and Perriman Strategies for cell membrane functionalization 1101 centre of engineered cartilage constructs. 13 Importantly, both cell priming and protein lipidation necessitate careful modification of the protein surface, as aggressive bioconjugation strategies can lead to denaturation and subsequent loss of biological function. 100 With this in mind, orthogonal or site-specific modifications are an attractive option, however, these approaches are not feasible for all proteins.…”

“…This cell functionalization approach has allowed us, for instance, to provide cells with additional binding sites 12 and nutrients, 13 protection in harsh environments, 14 increased adhesion to scaffolds 15 and magnetic contrast. 16 Compared to genetic modification, these strategies are simpler, faster and can be used to deliver a greater variety of materials to a wider range of cells.…”