Macromolecular crowding meets oxygen tension in human mesenchymal stem cell culture - A step closer to physiologically relevant in vitro organogenesis

Abstract

Modular tissue engineering is based on the cells’ innate ability to create bottom-up supramolecular assemblies with efficiency and efficacy still unmatched by man-made devices. Although the regenerative potential of such tissue substitutes has been documented in preclinical and clinical setting, the prolonged culture time required to develop an implantable device is associated with phenotypic drift and/or cell senescence. Herein, we demonstrate that macromolecular crowding significantly enhances extracellular matrix deposition in human bone marrow mesenchymal stem cell culture at both 20% and 2% oxygen tension. Although hypoxia inducible factor - 1α was activated at 2% oxygen tension, increased extracellular matrix synthesis was not observed. The expression of surface markers and transcription factors was not affected as a function of oxygen tension and macromolecular crowding. The multilineage potential was also maintained, albeit adipogenic differentiation was significantly reduced in low oxygen tension cultures, chondrogenic differentiation was significantly increased in macromolecularly crowded cultures and osteogenic differentiation was not affected as a function of oxygen tension and macromolecular crowding. Collectively, these data pave the way for the development of bottom-up tissue equivalents based on physiologically relevant developmental processes.

Multifactorial bottom‐up bioengineering approaches for the development of living tissue substitutes

“…Enhanced deposition of COL1A1 that was due to MMC has been described repeatedly in the literature for various crowders (42, 50–52, 86). Among them, Cr has shown superior performance in terms of accelerated and enhanced ECM deposition by TCs; osteoblasts; BMSCs; and dermal, lung, and corneal fibroblasts (41, 42, 47). The confined environment created by the high natural polydispersity of Cr in the culture medium accelerates cleavage of the N‐ and C ‐ propeptides of the procollagen molecules by the N‐ and C ‐ proteinases, thereby creating the insoluble collagen molecule (40, 41).…”

“…COL1A1 was not increased at 10% strain, which contradicts previous studies in which mechanical stimulation led to increased COL1A1 synthesis in TCs (82, 87–89) and BMSCs (30, 90, 91), a process mediated by TGF‐β1 (92). Increased deposition of collagen types III, IV, V, and VI has been observed in fibroblasts (41), and increased deposition of collagen III has been described in BMSCs (47) and corneal fibroblasts (42) when using Cr as a crowder. Furthermore, deposition of collagen type IV by BMSCs has also been increased by a Ficoll 70 and 400 kDa cocktail, though this was performed in adipogenic differentiation conditions (93).…”

“…Human TCs, human ADFs, and human NDFs were purchased from DV Biologics (Yorba Linda, CA, USA) and LG Standards (Teddington, United Kingdom), respectively. Human BMSCs were isolated from fresh bone marrow (Lonza Group, Basel, Switzerland), as previously described (47). TCs, ADFs, and NDFs were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.…”

“…It is worth noting that in the deposited under MMC conditions collagen exhibits the characteristic d ‐periodicity (41, 42), a strong indicator for a regular self‐assembly of collagen molecules (53). Although MMC has been previously combined with other microenvironmental modulators, such as hypoxia (47, 54), it is yet to be used simultaneously with mechanical stimulation. Herein, we ventured to assess the synergistic effect of uniaxial stretching and MMC on cell [human adult TCs, human neonatal dermal fibroblasts (NDFs), human adult dermal fibroblasts (ADFs), and human BMSCs] morphology, deposited ECM composition and cell phenotype maintenance (TCs), differentiation (BMSCs), and transdifferentiation (NDFs and ADFs) for the fabrication of tendon‐like tissue substitutes.…”

Harnessing mesenchymal stem cell secretome: Effect of extracellular matrices on proangiogenic signaling

“…Ascorbic acid, also known as vitamin C, has been demonstrated to influence ECM synthesis and remodeling (Cigognini et al, 2016; Kumar, Satyam, Cigognini, Pandit, & Zeugolis, 2018; Tagler et al, 2014). To explore the effect of media supplementation on deposition/synthesis of various ECM components, the decellularized matrices generated by MSCs (P4) were stained for collagen, fibronectin, and laminin.…”