Macromolecular crowding in animal component-free, xeno-free and foetal bovine serum media for human bone marrow mesenchymal stromal cell expansion and differentiation.

Background: Cell culture media containing undefined animal-derived components and prolonged in vitro culture periods in the absence of native extracellular matrix result in phenotypic drift of human bone marrow stromal cells (hBMSCs). Methods: Herein, we assessed whether animal component-free (ACF) or xeno-free (XF) media formulations maintain hBMSC phenotypic characteristics more effectively than foetal bovine serum (FBS)-based media. In addition, we assessed whether tissue-specific extracellular matrix, induced via macromolecular crowding (MMC) during expansion and/or differentiation, can more tightly control hBMSC fate. Results: Cells expanded in animal component-free media showed overall the highest phenotype maintenance, as judged by cluster of differentiation expression analysis. Contrary to FBS media, ACF and XF media increased cellularity over time in culture, as measured by total DNA concentration. While MMC with Ficoll™ increased collagen deposition of cells in FBS media, FBS media induced significantly lower collagen synthesis and/or deposition than the ACF and XF media. Cells expanded in FBS media showed higher adipogenic differentiation than ACF and XF media, which was augmented by MMC with Ficoll™ during expansion. Similarly, Ficoll™ crowding also increased chondrogenic differentiation. Of note, donor-to-donor variability was observed for collagen type I deposition and trilineage differentiation capacity of hBMSCs. Conclusion: Collectively, our data indicate that appropriate screening of donors, media and supplements, in this case MMC agent, should be conducted for the development of clinically relevant hBMSC medicines.

Development of three-layer collagen scaffolds to spatially direct tissue-specific cell differentiation for enthesis repair.

Enthesis repair remains a challenging clinical indication. Herein, a three-layer scaffold composed of a tendon-like layer of collagen type I, a fibrocartilage-like layer of collagen type II and a bone-like layer of collagen type I and hydroxyapatite, was designed to recapitulate the matrix composition of the enthesis. To aid tenogenic and fibrochondrogenic differentiation, bioactive molecules were loaded in the tendon-like layer or the fibrocartilage-like layer and their effect was assessed in in vitro setting using human bone marrow derived mesenchymal stromal cells and in an ex vivo model. Seeded human bone marrow mesenchymal stromal cells infiltrated and homogeneously spread throughout the scaffold. As a response to the composition of the scaffold, cells differentiated in a localised manner towards the osteogenic lineage and, in combination with differentiation medium, towards the fibrocartilage lineage. Whilst functionalisation of the tendon-like layer did not improve tenogenic cell commitment within the time frame of this work, relevant fibrochondrogenic markers were detected in the fibrocartilage-like layer when scaffolds were functionalised with bone morphogenetic protein 2 or non-functionalised at all, in vitro and ex vivo, respectively. Altogether, our data advocate the use of compartmentalised scaffolds for the repair and regeneration of interfacial tissues, such as enthesis.

Assessing the combined effect of surface topography and substrate rigidity in human bone marrow stem cell cultures.

The combined effect of surface topography and substrate rigidity in stem cell cultures is still under-investigated, especially when biodegradable polymers are used. Herein, we assessed human bone marrow stem cell response on aliphatic polyester substrates as a function of anisotropic grooved topography and rigidity (7 and 12 kPa). Planar tissue culture plastic (TCP, 3 GPa) and aliphatic polyester substrates were used as controls. Cell morphology analysis revealed that grooved substrates caused nuclei orientation/alignment in the direction of the grooves. After 21 days in osteogenic and chondrogenic media, the 3 GPa TCP and the grooved 12 kPa substrate induced significantly higher calcium deposition and alkaline phosphatase (ALP) activity and glycosaminoglycan (GAG) deposition, respectively, than the other groups. After 14 days in tenogenic media, the 3 GPa TCP upregulated four and downregulated four genes; the planar 7 kPa substrate upregulated seven genes and downregulated one gene; and the grooved 12 kPa substrate upregulated seven genes and downregulated one gene. After 21 days in adipogenic media, the softest (7 kPa) substrates induced significantly higher oil droplet deposition than the other substrates and the grooved substrate induced significantly higher droplet deposition than the planar. Our data pave the way for more rational design of bioinspired constructs.

Craniofacial tendon development-Characterization of extracellular matrix morphology and spatiotemporal protein distribution.

Craniofacial (CF) tendons are often affected by traumatic injuries and painful disorders that can severely compromise critical jaw functions, such as mastication and talking. Unfortunately, tendons lack the ability to regenerate, and there are no solutions to restore their native properties or function. An understanding of jaw tendon development could inform tendon regeneration strategies to restore jaw function, however CF tendon development has been relatively unexplored. Using the chick embryo, we identified the jaw-closing Tendon of the musculus Adductor Mandibulae Externus (TmAM) and the jaw-opening Tendon of the musculus Depressor Mandibulae (TmDM) that have similar functions to the masticatory tendons in humans. Using histological and immunohistochemical (IHC) analyses, we characterized the TmAM and TmDM on the basis of cell and extracellular matrix (ECM) morphology and spatiotemporal protein distribution from early to late embryonic development. The TmAM and TmDM were detectable as early as embryonic day (d) 9 based on histological staining and tenascin-C (TNC) protein distribution. Collagen content increased and became more organized, cell density decreased, and cell nuclei elongated over time during development in both the TmAM and TmDM. The TmAM and TmDM exhibited similar spatiotemporal patterns for collagen type III (COL3), but differential spatiotemporal patterns for TNC, lysyl oxidase (LOX), and matrix metalloproteinases (MMPs). Our results demonstrate markers that play a role in limb tendon formation are also present in jaw tendons during embryonic development, implicate COL3, TNC, LOX, MMP2, and MMP9 in jaw tendon development, and suggest TmAM and TmDM possess different developmental programs. Taken together, our study suggests the chick embryo may be used as a model with which to study CF tendon extracellular matrix development, the results of which could ultimately inform therapeutic approaches for CF tendon injuries and disorders.

Macromolecular crowding transforms regenerative medicine by enabling the accelerated development of functional and truly three-dimensional cell assembled micro tissues.

Scaffold-free in vitro organogenesis exploits the innate ability of cells to synthesise and deposit their own extracellular matrix to fabricate tissue-like assemblies. Unfortunately, cell-assembled tissue engineered concepts require prolonged ex vivo culture periods of very high cell numbers for the development of a borderline three-dimensional implantable device, which are associated with phenotypic drift and high manufacturing costs, thus, hindering their clinical translation and commercialisation. Herein, we report the accelerated (10 days) development of a truly three-dimensional (338.1 ± 42.9 µm) scaffold-free tissue equivalent that promotes fast wound healing and induces formation of neotissue composed of mature collagen fibres, using human adipose derived stem cells seeded at only 50,000 cells/cm2 on an poly (N-isopropylacrylamide-co-N-tert-butylacrylamide (PNIPAM86-NTBA14) temperature-responsive electrospun scaffold and grown under macromolecular crowding conditions (50 µg/ml carrageenan). Our data pave the path for a new era in scaffold-free regenerative medicine.

The Influence of Bloom Index, Endotoxin Levels and Polyethylene Glycol Succinimidyl Glutarate Crosslinking on the Physicochemical and Biological Properties of Gelatin Biomaterials.

In the medical device sector, bloom index and residual endotoxins should be controlled, as they are crucial regulators of the device's physicochemical and biological properties. It is also imperative to identify a suitable crosslinking method to increase mechanical integrity, without jeopardising cellular functions of gelatin-based devices. Herein, gelatin preparations with variable bloom index and endotoxin levels were used to fabricate non-crosslinked and polyethylene glycol succinimidyl glutarate crosslinked gelatin scaffolds, the physicochemical and biological properties of which were subsequently assessed. Gelatin preparations with low bloom index resulted in hydrogels with significantly (p < 0.05) lower compression stress, elastic modulus and resistance to enzymatic degradation, and significantly higher (p < 0.05) free amine content than gelatin preparations with high bloom index. Gelatin preparations with high endotoxin levels resulted in films that induced significantly (p < 0.05) higher macrophage clusters than gelatin preparations with low endotoxin level. Our data suggest that the bloom index modulates the physicochemical properties, and the endotoxin content regulates the biological response of gelatin biomaterials. Although polyethylene glycol succinimidyl glutarate crosslinking significantly (p < 0.05) increased compression stress, elastic modulus and resistance to enzymatic degradation, and significantly (p < 0.05) decreased free amine content, at the concentration used, it did not provide sufficient structural integrity to support cell culture. Therefore, the quest for the optimal gelatin crosslinker continues.

Hyaluronic Acid as Macromolecular Crowder in Equine Adipose-Derived Stem Cell Cultures.

The use of macromolecular crowding in the development of extracellular matrix-rich cell-assembled tissue equivalents is continuously gaining pace in regenerative engineering. Despite the significant advancements in the field, the optimal macromolecular crowder still remains elusive. Herein, the physicochemical properties of different concentrations of different molecular weights hyaluronic acid (HA) and their influence on equine adipose-derived stem cell cultures were assessed. Within the different concentrations and molecular weight HAs, the 10 mg/mL 100 kDa and 500 kDa HAs exhibited the highest negative charge and hydrodynamic radius, and the 10 mg/mL 100 kDa HA exhibited the lowest polydispersity index and the highest % fraction volume occupancy. Although HA had the potential to act as a macromolecular crowding agent, it did not outperform carrageenan and Ficoll®, the most widely used macromolecular crowding molecules, in enhanced and accelerated collagen I, collagen III and collagen IV deposition.