1,721,074 research outputs found
Highlights from TERMIS EU 2019
No full textThis eBook is a collection of articles from a Frontiers Research Topic. Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office: frontiersin.org/about/contac
Highlights from TERMIS EU 2019
No full textThis eBook is a collection of articles from a Frontiers Research Topic. Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office: frontiersin.org/about/contac
Macromolecular crowding for chondrogenic phenotype maintenance and stem cell differentiation
Get PDFChondrocyte-based tissue engineering therapies require in vitro cell expansion, which is associated with loss of phenotype, decrease in synthesis of collagen type II and increase in synthesis of collagen type I. Another major obstacle in clinical translation of chondrocyte-based therapies is the lack of extracellular matrix (ECM) in the cartilage substitutes. Macromolecular crowding (MMC) is a biophysical phenomenon, based on the excluded volume effect, known to dramatically increase tissue-specific ECM deposition during in vitro culture and to modulate cell phenotype and stem cell differentiation. The effect of MMC in chondrogenesis has not been well investigated as yet. Herein, we hypothesized that MMC will increase hyaline ECM deposition in chondrocytes and favour chondrogenic, as opposed to osteogenic and adipogenic, differentiation of bone marrow mesenchymal stem cells (BMSCs).
In early passage (3) human chondrocytes, MMC increased, albeit not significantly, both collagen type I and collagen type II deposition. In late passage (7) chondrocytes, re-differentiated using a commercial medium, MMC significantly increased collagen type I deposition, whilst almost no collagen type II was detected. These data suggested that the commercial medium used was not able to correctly restore cell phenotype and that the optimal medium for expansion of adult chondrocytes is still elusive.
MMC was then assessed in the pre-condition and/or differentiation media of human BMSCs. A moderate increase in collagen type II and chondroitin sulphate, a lower increase in collagen type I and collagen type X and an unaffected Sox-9 expression were detected, suggesting that carrageenan, the highly sulphated crowder used, enhanced chondrogenesis. Osteogenesis was also enhanced, as evidenced by increased mineralisation, collagen type I deposition and osteopontin expression. Adipogenesis was not affected.
Collectively, these data provide further knowledge on the use of MMC for the modulation of chondrogenic phenotype maintenance and stem cell differentiation.2020-01-3
Development of anisotropic polymeric substrates for tendon tissue engineering
No full textTendon injuries and degenerative conditions constitute an unmet clinical need with
pharmacological strategies and tissue grafts failing to recapitulate native tendon
function. Advancements in bioengineering have enabled the development of various
scaffold fabrication technologies, using natural or synthetic in origin polymers that
closely imitate the native tendon anisotropic architecture. Anisotropic collagen
sponges, extruded collagen fibres, isoelectric focused collagen fibres, electro-spun
polymeric fibres and imprinted polymeric substrates are at the forefront of scientific
and technological research and innovation. Herein, we ventured to assess whether onestep
functionalisation of electro-spun fibres with nano / micro particles is possible and
whether anisotropic imprinted substrates can maintain tenogenic phenotype in vitro
and promote functional neotissue formation in vivo.
Starting with electro-spinning, mechanical evaluation demonstrated that aligned
orientated electro-spun fibres exhibited significant higher stress at break values than
their random aligned counterparts and random orientated electro-spun fibres exhibited
significant higher strain at break values than the aligned orientated scaffolds. While
maintaining fibre structure, a co-deposition method of spraying and electro-spinning
was developed that enabled the incorporation of microspheres within the threedimensional
structure of the scaffold. Of significant importance is that bovine
tenocytes aligned perpendicular to the fibre orientation, possibly due to the absence of
mechanical tension.
With respect to imprinting, it is still unclear whether surface topography can be
translated into a clinically functional response in vivo at the tissue / device interface.
Herein, we demonstrated that anisotropic substrates with groove depth of ~317 nm
and ~1988 nm promoted human tenocyte alignment parallel to underlined topography
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in vitro. However, none of the topographies assessed (~37 nm, ~317 nm and ~1988
nm groove depth) induced parallel to the substrate cellular orientation in a
subcutaneous model and none of the topographies promoted directional tenogenesis
in vivo. Further, the rigid poly(lactic-co-glycolic acid) substrate used induced transdifferentiation
towards chondrogenic / osteogenic lineage, as evidenced by gene
analysis. Collectively, these data indicate that two-dimensional imprinting
technologies are useful tools for in vitro cell phenotype maintenance, rather than for
directional neotissue formation, should multifactorial approaches that consider both
surface topography and substrate rigidity are established.
Overall, both electro-spinning and imprinting technologies show great promise for
tendon repair and regeneration. Imprinting could be the ideal technology for cell
phenotype maintenance in vitro, as we can closely control architectural features.
Although it was not investigated here, electro-spinning is the ideal technology for in
vivo positive outcomes, as the three-dimensional architecture would allow directional
tissue formation within the fibrous construct
Biophysical and biochemical tools for cell phenotype maintenance, differentiation and trans-differentiation
No full textTendon injuries constitute an unmet clinical need, with 3 to 5 million new incidents occurring annually worldwide. Tissue grafting and biomaterial-based approaches fail to provide environments that are conducive to regeneration; instead they lead to nonspecific cell adhesion and scar tissue formation, which collectively impair functionality. Cell based therapies may potentially recover native tendon function, if tenocyte trans-differentiation can be evaded and cell differentiation / transdifferentiation towards tenogenic lineage is attained. To this end, recreating an artificial in vivo tendon niche by engineering functional in vitro microenvironments is a research priority. In this work, the effect of biophysical (macromolecular crowding, mechanical stimulation) and biochemical (oxygen tension) modulators on the behaviour of permanently differentiated cell sources (human adult and neonatal dermal fibroblasts and tenocytes) and stem cells (bone marrow derived mesenchymal stem cells) was assessed. Firstly, the influence of hydrodynamic radius, charge and polydispersity of (a) various concentrations of different crowders (carrageenan, Ficoll™ and dextran sulphate); (b) various molecular weights of different crowders (70, 400 and 100 kDa of Ficoll™ and 10, 100 and 500 kDa of dextran sulphate) and (c) various cocktails of the same crowders (cocktails of various concentrations of different molecular weights Ficoll™ and dextran sulphate) on extracellular matrix deposition in human dermal fibroblast culture was analysed. The different crowders were tested individually and carrageenan was found to induce the highest extracellular matrix deposition due to its natural polydispersity and negative charge, conducive to more efficient volume exclusion. Cocktails of different molecular weight / concentrations of Ficoll™ or dextran sulphate, although presented increased polydispersity than their mono-domain counterparts, did not match carrageenan’s performance. Carrageenan was utilised to accelerate extracellular matrix deposition in human bone marrow mesenchymal stem cells and it was found to outperform a Ficoll™ cocktail (70 kDa and 400 kDa). Additionally, under low oxygen conditions (2 %), increased matrix deposition was also observed. The multi-differentiation potential was maintained under both macromolecular crowding and hypoxia, though adipogenesis was significantly reduced after pre-treatment with 2 % oxygen tensionand chondrogenesis was significantly increased after pre-conditioning with macromolecular crowding. Finally, the combined effect of macromolecular crowding and mechanical stimulation on morphology, deposition of extracellular matrix and phenotype of human tenocytes, adult and neonatal dermal fibroblasts and bone marrow stem cells was assessed. Mechanical stimulation induced alignment perpendicular to the load of the permanently differentiated cell sources. Macromolecular crowding accelerated deposition of collagen type I. All cells deposited collagen types I, III, V and VI and bone marrow stem cells also deposited collagen type IV. Extracellular matrix synthesis was not increased by mechanical stimulation. Gene expression analysis revealed upregulation of scleraxis by tenocytes, potentially indicating phenotypic maintenance. Both neonatal and adult dermal fibroblasts presented upregulation of thrombospondin 4 and alkaline phosphatase or cartilage oligomeric protein, respectively, whilst the expression profile of bone marrow stem cells was unchanged. Taken together, these results provide further knowledge on the use of biophysical and biochemical in vitro microenvironment modulators for control of cell phenotype and development of tissue-like supramolecular assemblies.2019-02-2
Biophysical and biochemical tools for cell phenotype maintenance, differentiation and trans-differentiation
No full textTendon injuries constitute an unmet clinical need, with 3 to 5 million new incidents occurring annually worldwide. Tissue grafting and biomaterial-based approaches fail to provide environments that are conducive to regeneration; instead they lead to nonspecific cell adhesion and scar tissue formation, which collectively impair functionality. Cell based therapies may potentially recover native tendon function, if tenocyte trans-differentiation can be evaded and cell differentiation / transdifferentiation towards tenogenic lineage is attained. To this end, recreating an artificial in vivo tendon niche by engineering functional in vitro microenvironments is a research priority. In this work, the effect of biophysical (macromolecular crowding, mechanical stimulation) and biochemical (oxygen tension) modulators on the behaviour of permanently differentiated cell sources (human adult and neonatal dermal fibroblasts and tenocytes) and stem cells (bone marrow derived mesenchymal stem cells) was assessed. Firstly, the influence of hydrodynamic radius, charge and polydispersity of (a) various concentrations of different crowders (carrageenan, Ficoll™ and dextran sulphate); (b) various molecular weights of different crowders (70, 400 and 100 kDa of Ficoll™ and 10, 100 and 500 kDa of dextran sulphate) and (c) various cocktails of the same crowders (cocktails of various concentrations of different molecular weights Ficoll™ and dextran sulphate) on extracellular matrix deposition in human dermal fibroblast culture was analysed. The different crowders were tested individually and carrageenan was found to induce the highest extracellular matrix deposition due to its natural polydispersity and negative charge, conducive to more efficient volume exclusion. Cocktails of different molecular weight / concentrations of Ficoll™ or dextran sulphate, although presented increased polydispersity than their mono-domain counterparts, did not match carrageenan’s performance. Carrageenan was utilised to accelerate extracellular matrix deposition in human bone marrow mesenchymal stem cells and it was found to outperform a Ficoll™ cocktail (70 kDa and 400 kDa). Additionally, under low oxygen conditions (2 %), increased matrix deposition was also observed. The multi-differentiation potential was maintained under both macromolecular crowding and hypoxia, though adipogenesis was significantly reduced after pre-treatment with 2 % oxygen tensionand chondrogenesis was significantly increased after pre-conditioning with macromolecular crowding. Finally, the combined effect of macromolecular crowding and mechanical stimulation on morphology, deposition of extracellular matrix and phenotype of human tenocytes, adult and neonatal dermal fibroblasts and bone marrow stem cells was assessed. Mechanical stimulation induced alignment perpendicular to the load of the permanently differentiated cell sources. Macromolecular crowding accelerated deposition of collagen type I. All cells deposited collagen types I, III, V and VI and bone marrow stem cells also deposited collagen type IV. Extracellular matrix synthesis was not increased by mechanical stimulation. Gene expression analysis revealed upregulation of scleraxis by tenocytes, potentially indicating phenotypic maintenance. Both neonatal and adult dermal fibroblasts presented upregulation of thrombospondin 4 and alkaline phosphatase or cartilage oligomeric protein, respectively, whilst the expression profile of bone marrow stem cells was unchanged. Taken together, these results provide further knowledge on the use of biophysical and biochemical in vitro microenvironment modulators for control of cell phenotype and development of tissue-like supramolecular assemblies.2019-02-2
Macromolecular crowding transforms regenerative medicine by enabling the accelerated development of functional and truly three-dimensional tissue moduli
No full textScaffold-free in vitro organogenesis exploits the innate ability of cells to synthesise and deposit their own extracellular matrix to fabricate tissue-like assemblies. Unfortunately, traditional 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. Macromolecular crowding, a biophysical phenomenon based on the principle of excluded-volume effect, dramatically accelerates and increases extracellular matrix deposition during in vitro culture. However, the optimal macromolecular crowder is still elusive and the therapeutic potential of macromolecular crowding has yet to be evaluated.
With respect to the optimal macromolecular crowder, the biophysical properties of various concentrations of different seaweed in origin sulphated polysaccharides (carrageenan, fucoidan, galactofucan, arabinogalactan, ulvan) and their effect on human adipose derived stem cell cultures were assessed. Carrageenan, possibly due to its high sulphation degree, exhibited the highest negative charge values. No correlation was observed between the different concentrations of the crowders and charge, polydispersity index, hydrodynamic radius and fraction volume occupancy across all crowders. None of the crowders, but arabinogalactan, negatively affected cell viability. Carrageenan, fucoidan, galactofucan and ulvan increased extracellular matrix (especially collagen type I and collagen type V) deposition. Carrageenan induced the highest osteogenic effect and galactofucan and fucoidan demonstrated the highest chondrogenic effect.
With respect to therapeutic effect, carrageenan (50 μg/ml) was used as macromolecular crowding agent to produce extracellular matrix-rich tissue equivalents, using only 50,000 human adipose derived stem cells per cm2 on an 85:15 poly-N-isopropylacrylamide-N-tert-butylacrylamide temperature-responsive electrospun scaffold. The combination of macromolecular crowding and the temperature-responsive electrospun scaffold enabled the accelerated (10 days) development of a truly three-dimensional (338.1 ± 42.9 μm) scaffold-free tissue equivalent that promoted fast wound healing and induced neotissue formation composed of mature collagen fibres.
Collectively, data obtained highlight the potential of macromolecular crowding to transform regenerative medicine by enabling the accelerated development of functional and truly three-dimensional tissue modulus and pave the path for a new era in scaffold-free tissue engineering.2024-11-0
Collagen scaffolds with controlled topography and stiffness and mechanical stimulation direct tissue-specific cell phenotype for tendon regeneration
No full textTendon and ligament injuries represent a major global cause of disability, frequently requiring surgical intervention to restore function. While tissue grafts remain the clinical gold standard for tendon augmentation, their use is constrained by risks of disease transmission and suboptimal tissue integration. Emerging tissue engineering strategies aim to overcome these limitations through the ex vivo development of tendon substitutes using biomaterial scaffolds and targeted microenvironmental cues; however, further refinement is required to achieve functional regeneration. Collagen type I, the principal structural component of tendons, holds great potential as a scaffold biomaterial, yet its adoption is hindered by sourcing and processing concerns. On the one hand, traditional sources such as bovine and porcine tissues raise concerns related to zoonosis risks and ethical acceptability. On the other hand, collagen manipulation complexity has traditionally limited the architectural and mechanical properties of collagen scaffolds. In this study, we hypothesised that an appropriate collagen type I scaffold with defined architectural and mechanical properties can maintain the phenotype of human tendon cells and induce the deposition of organised tendon-specific ECM in vitro.
In the first phase of this study, collagen type I was extracted from caprine skin, digital flexor and digital extensor tendons and compared to that extracted from bovine and porcine Achilles tendons. Biochemical analysis confirmed that caprine collagen type I purity was on pair with that of traditional sources. Subsequently, collagen was either non-crosslinked or crosslinked with 4-arm succinimidyl glutarate and processed into films. Caprine scaffolds displayed macroscopic and microscopic features, including fibril diameter ranges, similar to their bovine and porcine counterparts regardless of the tissue source. Mechanical characterisation revealed that caprine scaffolds were intrinsically stiffer and less susceptible to modulus increases upon crosslinking, suggesting underlying species-specific biochemical differences. With respect to cytocompatibility, caprine tendon-derived scaffolds supported the attachment, proliferation and metabolic activity of fibroblast and macrophages at levels consistent with those observed on bovine and porcine scaffolds. Notably, caprine skin-derived collagen enhanced both fibroblast and macrophage attachment relative to tendon-derived collagen from all species, pointing to tissue-specific functional differences. Immunogenicity analysis revealed all caprine scaffolds induced lower pro-inflammatory responses than Escherichia coli lipopolysaccharides on tissue culture plastic and elicited responses comparable to traditional collagen scaffolds. Minor increases in tumour necrosis factor alpha expression were observed in crosslinked groups, likely reflecting the influence of increased surface stiffness on macrophage behaviour rather than a direct effect of the crosslinker agent, as this was not observed in the indirect cultures. Collectively, these results underscore the potential of caprine tissues as an alternative source of collagen type I for the fabrication of medical devices.
In the second phase of this study, collagen scaffolds featuring either planar or grooved (2 x 2 x 2 μm) surface topographies and tuneable mechanical properties were fabricated using soft lithography and chemical crosslinking with different concentrations of succinimidyl glutarate (0.5 mM, 1.0 mM and 1.5 mM). Surface characterisation confirmed the presence of well-defined surface grooves, particularly in crosslinked scaffolds. The crosslinking agent reduced scaffold free amine content and increased Young’s modulus, indicating the formation of covalent bonds. In addition, micro-indentation measurements revealed a concentration-dependent increase in surface stiffness. In vitro experiments using human tendon cells demonstrate that grooved topographies promoted anisotropic cell and extracellular matrix alignment, especially in crosslinked scaffolds, highlighting the importance of collagen crosslinking for pattern stability. Crosslinking also exerted a dose-dependent effect on cell phenotype, with the highest concentration reducing cell proliferation, the lowest concentration inducing the broadest tendon-marker upregulation and all tested concentrations inducing higher tenascin C deposition than the non-crosslinked counterparts, a phenomenon we attributed to crosslinker-mediated surface smoothening and scaffold stiffening, respectively. Scaffolds crosslinked with the lowest crosslinker concentration and subjected to different tensional regimes (no tension, static tension and cyclic tension) showed that, within the parameters utilised in this study (frequency, strain and rest interval), static tension resulted in higher cell proliferation, enhanced cell and extracellular alignment and increased tendon marker upregulation compared to cyclic stimulation in planar, grooved, and both planar and grooved scaffolds, respectively. Collectively, this study advocates the use of combined biophysical cues to maintain physiological cell function.
In conclusion, our findings underscore the viability of caprine tissue as a source of collagen type I for the manufacturing of collagen devices and demonstrate that precise modulation of collagen scaffold architecture and mechanical properties directly influences the maintenance of a tendon cell phenotype in vitro. Together, these insights offer a foundation for the design of collagen-based functional tendon substitutes
Three-layer collagen-based composite scaffolds to spatially direct tissue-specific cell differentiation for enthesis repair
No full textThe enthesis is a specialised interfacial tissue responsible to minimise stress concentrations between tendon and bone. Fibrocartilaginous entheses are composed of four distinct areas, namely tendon, unmineralised fibrocartilage, mineralised fibrocartilage and bone, and they are characterised by spatial gradients of cell phenotype, matrix composition and organisation. Given the intrinsic complexity of this tissue and the inherently poor healing of tendons and ligaments, regeneration of the enthesis is particularly challenging, usually resolving in a scar populated by fibroblasts. Surgical approaches still fail to restore the native fibrocartilaginous transition, while tissue engineering strategies have offered some valid approaches. Among the most promising strategies, multiphasic scaffolds seeded with adult differentiated cell types or adult mesenchymal stromal cells are worth of mentioning. Although the former strategy exploits the heterotypic interactions between different cell phenotypes in proximity to each other, it poses considerable scalability and regulatory challenges. The latter approach benefits from the use of a single cell population (making its applicability far easier) that will differentiate towards the appropriate tissue lineage through scaffold- and local microenvironment- induced signals. Recent advances in biomaterial engineering have inspired the development of multi-cargo delivery vehicles to mimic naturally occurring gradients in composition, signalling cues and other constituents of the enthesis but, despite the very promising preclinical data, none of them has been clinically translated yet. Herein, the potential of a three-layer enthesis-composition inspired collagen - based scaffold to spatially direct differentiation of human bone marrow derived mesenchymal stromal cells in an in vitro model or maintain phenotype of native tendon-derived cells in an ex vivo model, with the aid of a zonal functionalisation with bioactive molecules were assessed. Before approaching the design of a multi-layer collagen - based scaffold, hydrogels and sponges were singularly tested as monolayer scaffolds and their production and crosslinking were optimised. Collagen type I hydrogels were fabricated with in-house extracted porcine collagen and cross-linked with polyethylene glycol succinimidyl succinate molecule, tested at different number of arms (4 and 8), molecular weights (10, 20 and 40 KDa) and concentrations (0, 0.1, 0.5, 1, 2.5 and 5 mM). Although some of the conditions yielded stable hydrogels, they degraded too quickly in cell culture conditions, so they were deemed unsuitable for in vitro studies. The focus was shifted towards more clinically relevant scaffolds for enthesis repair, such as sponges, and the cross-linker of choice was changed to 4-arm 10 KDa polyethylene glycol succinimidyl glutarate. Collagen type I and collagen type II monolayer sponges were produced and their crosslinking density optimised. Afterwards, the 3-L scaffold production was optimised by testing different freezing and freeze-drying process to finally obtain an organised porous network by iterative layering freeze-drying. This process yielded three interconnected, yet distinguishable layers that mimicked the basic extracellular matrix composition of the enthesis: a bone-like layer made of collagen type I and hydroxyapatite, a fibrocartilage-like layer made of collagen type II and a tendon-like layer made of collagen type I. To assess the potential of the scaffold to promote specific cell lineage commitment, human bone marrow derived mesenchymal stromal cells were seeded onto the scaffolds and their differentiation towards the three cell populations of the enthesis tissue was investigated. Cells infiltrated and homogeneously spread throughout the scaffold; as a response to the composition of the scaffold, cells differentiated in a localised manner in the bone-like layer towards the osteogenic lineage and, in combination with differentiation medium, towards the fibrocartilage lineage. To aid tenogenic and fibrochondrogenic differentiation, different bioactive molecules were screened in supplementation to basal medium on cell - seeded tendon-like layer and fibrocartilage-like layer monolayer scaffolds. The best two candidates [for tenogenic differentiation: platelet-derived growth factor bb and transforming growth factor β3; for fibrochondrogenic differentiation: transforming growth factor β3 and bone morphogenetic protein 2] were incorporated in the tendon-like layer or the fibrocartilage-like layer during the fabrication process and their effect was assessed. To better simulate an in vivo implantation scenario, the three-layer scaffolds were placed in proximity of Achilles rat tendons to assess native tendon-derived cell response. In the absence of bioactive molecule functionalisation, the scaffolds were fully populated and a fibrocartilage interface was initiated, as evidenced by collagen type II presence in the fibrocartilage-like layer. Overall, these results indicate that the three-layer composite collagen scaffolds can stimulate osteogenic and fibrochondrogenic differentiations, even in the absence of bioactive molecule functionalisation, which can help to strengthen the attachment between bone and repaired tendon and lay the foundations for an in vivo functional repair of the enthesis.2025-09-2
Macromolecular crowding meets tissue engineering by self-assembly
No full textIntroduction: Advancements in molecular and cell biology have led to the development of tissue engineering by self-assembly. The driving hypothesis of this concept is that replacement, repair and restoration of lost tissue function can be accomplished best by using the cells\u27 inherent capacity to create highly sophisticated structures with precision and efficiency still unmatched by human-made devices. However, the prolonged culture time required to develop an implantable device jeopardises clinical translation and commercialisation. It has been demonstrated that macromolecular crowding enhances the deposition of extracellular matrix. Herein, the influence of crowding molecules on matrix deposition and the potential of this technology in tissue engineering by self-assembly was investigated.
Materials and Methods: Human fibroblasts (lung and skin), tenocytes and osteoblasts were cultured under various MMC conditions (dextran sulphate, Ficoll® & carrageenan) in a range of fetal bovine serum (FBS) and human serum (HS) concentrations (0.0-10%). ECM deposition was verified by SDS-PAGE, immunocytochemistry (ICC), atomic force microscopy (AFM), scanning electron microscopy (SEM) and mass-spectrometry (MS). The MMC molecules were characterized by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). The influence of crowders on cell morphology, cell viability and metabolic activity were evaluated using phase-contrast microscopy, Live/Dead® and AlamarBlue® assays respectively. pNIPAAm and pNTBA based thermo-responsive copolymers were developed to facilitate detachment of ECM-rich cell-sheets.
Results: The SDS-PAGE and densitometry demonstrated that MMC significantly increases the collagen type-I deposition (p<0.0001) at all tested serum concentrations (maximum deposition was in 2 days & 0.5% FBS or HS). ICC, AFM and SEM further confirmed enhanced deposition of fibrillar ECM in presence of MMC. DLS and NTA demonstrated that CR has highest polydispersity among all tested crowders. Phase-contrast microscopy, Live/Dead® and AlamarBlue® assays confirmed that cellular morphology, viability and metabolic activity respectively were not affected by MMC. Thermo-responsive coating with 65% pNIPAAm: 35% pNTBA facilitated detachment of ECM rich cell-sheet from culture. Complementary ICC for MS validation confirmed the enhanced deposition of collagens (III, IV, V, VI) and other ECM molecules (laminin, fibronectin, hyaluronic acid, decorin, lysyl oxidase), without changing collagen-VII, elastin, fibrillin-1, transglutaminase-2, alpha-smooth muscle actin, epithelial keratin, tubulin, chondroitin sulphate, keratin sulphate, heparin sulphate, aggrecan, biglycan, CD248 and IL-10.
Discussion and conclusions: This work reports that the efficacy of macromolecular crowding in enhancing matrix deposition is amplified in human fibroblast, tenocyte and osteoblast cultures in the presence of low serum concentration, due to the low proteolytic activity of serum; in fact, an over 80-fold increase in extracellular matrix deposition is documented within 48 hours. It further identifies that macromolecular polydispersity is key modulator of extracellular matrix deposition, due to the generation of effective volume exclusion effect. Using a custom-made thermal responsive polymer, living substitutes with tissue-specific protein composition and structure were attained. This approach enables modulation of the in vitro microenvironment, without negatively impacting on cellular functions, and therefore opens new avenues for a more rational design in engineering cohesive tissue modules.2016-05-1
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