Biophysics Rules the Cell Culture but Has Yet to Reach the Clinic: Why Is That?

Musculoskeletal injuries are the leading cause of physical disability worldwide, with associated annual direct and indirect healthcare expenditure in excess of $874 billion in the United States alone.1 Current treatments are predominantly based on tissue grafts (autografts are preferred)2,3 and biomaterials.4,5 Given that the former are associated with scarce availability, insufficient remodeling, and adverse immune reactions,6-8 and the latter with substandard stability, poor biologic response, and foreign body response,9-11 their clinical suitability has been questioned and gave rise to the field of cell-based therapies.12 Cell-based therapies advocate that optimal repair and regeneration can be achieved through the utilization of the intrinsic capacity of cells to build native supramolecular assemblies; cells are the natural born extracellular matrix (ECM) builders, after all. Unfortunately, cell-based therapies require in vitro cell expansion in artificial tissue culture media and plastics. Removed from their optimal tissue niche, cells lose their phenotype, function, and therapeutic potency.13,14 Thus, contemporary tissue engineering incorporates high levels of biomimicry in the design of functional and physiologically relevant in vitro microenvironments to recapitulate ex vivo, insofar as possible, the complexity of the in vivo tissue context of the cells. Here, we briefly discuss recent advancements in biophysical aspects of cell culture systems and whether these developments have influenced clinical translation and commercialization of cell-based therapies in the musculoskeletal space. Biophysics and dynamics (in the form of architectural, geometrical, dimensional, and topographical features; biomechanical properties, such as elastic modulus and shear forces and cyclic strains; and localized density) are ubiquitous in nature and determine cell and tissue specificity and function.15,16 For example, tendons are composed of highly ordered, bidirectionally aligned collagen fibrils (up to 100 nm to 1,000 nm in diameter), which, bundled together, form collagen fibers (1 μm to 20 μm in diameter) and collagen fiber bundles (20 μm to 500 μm in diameter).17 Bone exhibits a radial gradient porous structure from the outside: the cortical bone has outer porosity of approximately 5%, while the inner part can reach porosity up to approximately 10%; porosity of the cancellous bone starts at approximately 50% in the outer layer and can reach approximately 90% in the inner layer.18 Articular cartilage has a zonal architecture, and the organization and alignment of the collagen fibrils/fibers is different in every zone (eg, parallel, perpendicular, diagonal, radial).19 Advancements in engineering have made available numerous nano- and microfabrication technologies (eg, electrospinning, imprinting) that have enabled control of permanently differentiated cells and stem cells.20,21 For example, electrospun and/or imprinted substrates have been shown not only to maintain tenocyte,22,23 chondrocyte,24 and osteoblast25 phenotype, but also to direct stem cells toward tenogenic,26 chondrogenic,27 and osteogenic28 lineages. The term durotaxis is used to describe the ability of cells to migrate directionally toward areas of high ECM rigidity. ECM elasticity/mechanical compliance governs numerous in vivo biologic processes, including cellular spreading, migration, and differentiation; morphogenesis; wound healing; and disease progression.29,30 In the last decade, numerous in vitro studies have demonstrated the positive influence of substrate rigidity in tendon-,31 cartilage-,32 and bone-derived33 cell phenotype maintenance and in stem cell differentiation toward tenogenic,34 chondrogenic,35 and osteogenic36 lineages. Static or dynamic uniaxial or multiaxial tensile, compressive, or shear mechanical loads are also crucial for the development, function, and healing of musculoekeltal tissues.37,38 It is not a coincidence, after all, that exercise is an integral element of any orthopaedic rehabilitation regime.39,40 Several bioreactor systems of variable complexity have been used as means to control tenocyte,41 chondrocyte,42 and osteoblast43 phenotype in vitro and to direct stem cells toward tenogenic,44 chondrogenic,45 and osteogenic46,47 lineages. Musculoskeletal tissues, like any other tissue, are highly dense ECM assemblies. Yet again, traditional cultures are conducted in dilute culture media that barely imitate the density of body fluids, let alone compact tissues. To emulate this dense ECM microenvironment in vitro, macromolecular crowding, also known as localized density or excluding volume effect, has been proposed and has been shown to substantially modulate nuclear processes, such as gene transcription, RNA splicing and DNA replication, and protein properties, such as diffusion coefficients, folding kinetics, and thermodynamic activities, both intracellularly and extracellularly.48,49 In vitro data have shown macromolecular crowding to maintain tenocyte and osteoblast phenotype50 and to enhance chondrogenesis in stem cell culture.51 Despite the significant volume of work in the in vitro setting, only a handful of studies have assessed in preclinical models the influence of mechanical preconditioning in tissue regeneration. However, in all cases, the cells were seeded into/onto a scaffold, the cell/scaffold system was subjected to mechanical loading in vitro for a period of time, and then the cell/scaffold system was implanted.52 To date, no study has assessed in preclinical models or in a clinical setting the influence of surface topography, substrate rigidity, mechanical stimulation, or macromolecular crowding preconditioning in permanently differentiated or stem cell–only implantation. What has hampered preclinical/clinical translation and commercialization of these game-changing technologies? Financial issues may be the first reason. There are only a few companies that manufacture bioreactor systems with the capacity to apply loads, and the systems available are not only far too expensive, but they also have limited capacity for cell expansion. Reproducibility issues may be the second reason. Although electrospinning is widely available in the laboratory setting, only a handful of companies have industrialized the process, and it is still challenging to control precisely the dimensionality of electrospun mats. Scalability issues may be the third reason. Although imprinting has solved the problem of reproducible scaffold dimensionality, we are still far away from producing economically the likely trillions of imprinted cell culture substrates required per year to expand cells for education, research, development, and clinical purposes. Lack of sufficient evidence may be the fourth reason. Although macromolecular crowding has been available since the 1980s, only a handful of studies have assessed its potential in cell culture context. Standardization may be the fifth reason. Rarely will one find published papers reporting that authors extracted the cells in the same fashion, used the same media, applied the same preconditioning conditions, and conducted the same analysis. Regulatory issues may be the sixth reason. Most of the scaffold-based surface topography/substrate rigidity experiments are performed using non-FDA approved polymers. Undeniably, the cell culture market is growing exponentially; it is expected to worth $18.63 billion by 202053 and $37 billion by 2022.54 Unless a disruptive innovation comes along, it is likely that functional reparative therapies will involve the delivery of a relevant cell population that has been expanded in vitro. It is therefore imperative to direct our efforts toward the creation of physiologically/clinically relevant, industrially scalable, and regulatory compliant in vitro microenvironments in order to develop in the years to come remedial patient bedside cell-based therapies.

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