Macromolecular crowding meets tissue engineering by self-ssembly: A paradigm shift in regenerative medicine

Abstract

Advancements in molecular and cell biology have led to the development of cell-based therapies to treat injured or degenerated tissues. [1] The rationale of this concept is that functional regeneration can be achieved best by using the innate capacity of cells to create their own tissue-specific extracellular matrix (ECM) avoiding the shortfalls of man-made devices. Although direct cell injections have demonstrated very promising preclinical and clinical outcomes, [2] the mode of administration offers little control over local retention and distribution of the injected cell suspensions[3] leading to scattered therapeutic efficiency. This deficiency has led to the development of living substitutes for skin[4] and blood vessel[5] composed of cells seeded on a collagen scaffold. Notwithstanding the efficacious results in preclinical models and clinical trials, it soon became apparent that the presence of the scaffold hinders tissue remodelling and function. [6] These drawbacks led to the development of the scaffold-free cell-sheet tissue engineering (CSTE)[7] or tissue engineering by self-assembly (TESA), [8] a therapy that offers the fabrication of a contiguous cell sheet that is stabilised by cell-cell contacts and endogenously produced ECM. Despite the documented, in preclinical and clinical setting, positive outcomes for skin, [9] blood vessel, [10, 11] cornea, [12, 13] heart, [14] lung, [15] liver[16] and bone[17] replacement, only Epicel® (Genzyme, USA) for skin and LifeLine™ for blood vessel (Cytograft, USA) have been commercialised so far. This limited technology transfer from bench-top to clinic has been attributed to the substantial long period of time required for ex vivo culture (e.g. 14-35 days for corneal epithelium; [13] 84 days for corneal stromal; [18] 28 days for corneal endothelium;[19] 70 days for lung cell-sheet; [15] and 196 days for blood vessel[11] ) that often leads to loss of native phenotype and cell senescence.[20] Here, we propose a biophysical approach, termed macromolecular crowding (MMC), that increases thermodynamic activities and biological processes by several orders of magnitude,[21] as means to create ECM-rich tissue equivalents. The principle of MMC is derived from the notion that in vivo cells reside in a highly crowded/dense extracellular space and therefore the conversion of the de novo synthesised procollagen to collagen I is rapid. [22] However, in the even substantially more dilute than body fluids (e.g. urine: 36-50g/l; blood: 80g/l) culture conditions (e.g. HAM F10 nutrient medium: 16.55g/l; DMEM/F12 medium: 16.78g/l; DMEM high glucose and L-glutamine medium: 17.22g/l), the rate limiting conversion of procollagen to collagen I is very slow (Figure 1a). We propose that the addition of inert polydispersed macromolecules (presented as spherical objects of variable diameter in Figure 1b) in the culture media will facilitate amplified production of ECM-rich living substitutes.