The synergistic effect of physicochemical in vitro microenvironment modulators in human bone marrow stem cell cultures.

Modern bioengineering utilises biomimetic cell culture approaches to control cell fate during in vitro expansion. In this spirit, herein we assessed the influence of bidirectional surface topography, substrate rigidity, collagen type I coating and macromolecular crowding (MMC) in human bone marrow stem cell cultures. In the absence of MMC, surface topography was a strong modulator of cell morphology. MMC significantly increased extracellular matrix deposition, albeit in a globular manner, independently of the surface topography, substrate rigidity and collagen type I coating. Collagen type I coating significantly increased cell metabolic activity and none of the assessed parameters affected cell viability. At day 14, in the absence of MMC, none of the assessed genes was affected by surface topography, substrate rigidity and collagen type I coating, whilst in the presence of MMC, in general, collagen type I α1 chain, tenascin C, osteonectin, bone sialoprotein, aggrecan, cartilage oligomeric protein and runt-related transcription factor were downregulated. Interestingly, in the presence of the MMC, the 1000 kPa grooved substrate without collagen type I coating upregulated aggrecan, cartilage oligomeric protein, scleraxis homolog A, tenomodulin and thrombospondin 4, indicative of tenogenic differentiation. This study further supports the notion for multifactorial bioengineering to control cell fate in culture.

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.

Scaffold-based delivery of mesenchymal stromal cells to diabetic wounds.

Foot ulceration is a major complication of diabetes mellitus, which results in significant human suffering and a major burden on healthcare systems. The cause of impaired wound healing in diabetic patients is multifactorial with contributions from hyperglycaemia, impaired vascularization and neuropathy. Patients with non-healing diabetic ulcers may require amputation, creating an urgent need for new reparative treatments. Delivery of stem cells may be a promising approach to enhance wound healing because of their paracrine properties, including the secretion of angiogenic, immunomodulatory and anti-inflammatory factors. While a number of different cell types have been studied, the therapeutic use of mesenchymal stromal cells (MSCs) has been widely reported to improve delayed wound healing. However, topical administration of MSCs via direct injection has several disadvantages, including low cell viability and poor cell localization at the wound bed. To this end, various biomaterial conformations have emerged as MSC delivery vehicles to enhance cell viability and persistence at the site of implantation. This paper discusses biomaterial-based MSCs therapies in diabetic wound healing and highlights the low conversion rate to clinical trials and commercially available therapeutic products.

Macromolecular crowding in the development of a three-dimensional organotypic human breast cancer model.

Although cell-derived matrices are at the forefront of scientific research and technological innovation for the development of in vitro tumour models, their two-dimensional structure and low extracellular matrix composition restrict their capacity to accurately predict toxicity of candidate molecules. Herein, we assessed the potential of macromolecular crowding (a biophysical phenomenon that significantly enhances and accelerates extracellular matrix deposition, resulting in three-dimensional tissue surrogates) in improving cell-derived matrices in vitro tumour models. Among the various decellularisation protocols assessed (NH4OH, DOC, SDS/EDTA, NP40), the NP40 appeared to be the most effective in removing cellular matter and the least destructive to the deposited matrix. Among the various cell types (mammary, skin, lung fibroblasts) used to produce the cell-derived matrices, the mammary fibroblast derived matrices produced under macromolecular crowding conditions and decellularised with NP40 resulted in significant increase in focal adhesion molecules, matrix metalloproteinases and proinflammatory cytokines, when seeded with MDA-MB-231 cells. Further, macromolecular crowding derived matrices significantly increased doxorubicin resistance and reduced the impact of intracellular reactive oxygen species mediated cell death. Collectively our data clearly illustrate the potential of macromolecular crowding in the development of cell-derived matrices-based in vitro tumour models that more accurately resemble the tumour microenvironment.

Allogeneic Serum and Macromolecular Crowding Maintain Native Equine Tenocyte Function in Culture.

The absence of a native extracellular matrix and the use of xenogeneic sera are often associated with rapid tenocyte function losses during in vitro culture. Herein, we assessed the influence of different sera (equine serum and foetal bovine serum) on equine tenocyte morphology, viability, metabolic activity, proliferation and protein synthesis as a function of tissue-specific extracellular matrix deposition (induced via macromolecular crowding), aging (passages 3, 6, 9) and time in culture (days 3, 5, 7). In comparison to cells at passage 3, at day 3, in foetal bovine serum and without macromolecular crowding (traditional equine tenocyte culture), the highest number of significantly decreased readouts were observed for cells in foetal bovine serum, at passage 3, at day 5 and day 7 and without macromolecular crowding. Again, in comparison to traditional equine tenocyte culture, the highest number of significantly increased readouts were observed for cells in equine serum, at passage 3 and passage 6, at day 7 and with macromolecular crowding. Our data advocate the use of an allogeneic serum and tissue-specific extracellular matrix for effective expansion of equine tenocytes.

Influence of the Thermodynamic and Kinetic Control of Self-Assembly on the Microstructure Evolution of Silk-Elastin-Like Recombinamer Hydrogels.

Complex recombinant biomaterials that merge the self-assembling properties of different (poly)peptides provide a powerful tool for the achievement of specific structures, such as hydrogel networks, by tuning the thermodynamics and kinetics of the system through a tailored molecular design. In this work, elastin-like (EL) and silk-like (SL) polypeptides are combined to obtain a silk-elastin-like recombinamer (SELR) with dual self-assembly. First, EL domains force the molecule to undergo a phase transition above a precise temperature, which is driven by entropy and occurs very fast. Then, SL motifs interact through the slow formation of ß-sheets, stabilized by H-bonds, creating an energy barrier that opposes phase separation. Both events lead to the development of a dynamic microstructure that evolves over time (until a pore size of 49.9 ± 12.7 µm) and to a delayed hydrogel formation (obtained after 2.6 h). Eventually, the network is arrested due to an increase in ß-sheet secondary structures (up to 71.8 ± 0.8%) within SL motifs. This gives a high bond strength that prevents the complete segregation of the SELR from water, which results in a fixed metastable microarchitecture. These porous hydrogels are preliminarily tested as biomimetic niches for the isolation of cells in 3D cultures.

Local pharmacological induction of angiogenesis: Drugs for cells and cells as drugs.

The past decades have seen significant advances in pro-angiogenic strategies based on delivery of molecules and cells for conditions such as coronary artery disease, critical limb ischemia and stroke. Currently, three major strategies are evolving. Firstly, various pharmacological agents (growth factors, interleukins, small molecules, DNA/RNA) are locally applied at the ischemic region. Secondly, preparations of living cells with considerable bandwidth of tissue origin, differentiation state and preconditioning are delivered locally, rarely systemically. Thirdly, based on the notion, that cellular effects can be attributed mostly to factors secreted in situ, the cellular secretome (conditioned media, exosomes) has come into the spotlight. We review these three strategies to achieve (neo)angiogenesis in ischemic tissue with focus on the angiogenic mechanisms they tackle, such as transcription cascades, specific signalling steps and cellular gases. We also include cancer-therapy relevant lymphangiogenesis, and shall seek to explain why there are often conflicting data between in vitro and in vivo. The lion's share of data encompassing all three approaches comes from experimental animal work and we shall highlight common technical obstacles in the delivery of therapeutic molecules, cells, and secretome. This plethora of preclinical data contrasts with a dearth of clinical studies. A lack of adequate delivery vehicles and standardised assessment of clinical outcomes might play a role here, as well as regulatory, IP, and manufacturing constraints of candidate compounds; in addition, completed clinical trials have yet to reveal a successful and efficacious strategy. As the biology of angiogenesis is understood well enough for clinical purposes, it will be a matter of time to achieve success for well-stratified patients, and most probably with a combination of compounds.

Decellularised porcine peritoneum as a tendon protector sheet.

Tissue grafts achieve high levels of compositional and mechanical integrity biomimicry and are often considered as the gold standard in clinical practice. Herein, we assessed the potential of decellularised porcine peritoneum (XenoMEM) as a tendon protector sheet and correlated its properties to a commercially available product (TenoGlide®). XenoMEM presented lower cross-linking ratio (p < 0.05), higher mechanical properties (p < 0.01), lower coefficient of friction (p < 0.01) and higher (p < 0.05) cytocompatibility with human tenocytes than TenoGlide®. In addition, XenoMEM exhibited lower (p < 0.05) immune response than TenoGlide® with macrophages. Collectively, these data support the use of XenoMEM in tendon tissue engineering.

Production and Characterization of Chemically Cross-Linked Collagen Scaffolds.

Chemical cross-linking of collagen-based devices is used as a means of increasing the mechanical stability and control the degradation rate upon implantation. Herein, we describe techniques to produce cross-linked with glutaraldehyde (GTA; amine terminal cross-linker), 4-arm polyethylene glycol succinimidyl glutarate (4SP; amine terminal cross-linker), diphenyl phosphoryl azide (DPPA; carboxyl terminal cross-linker), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; carboxyl terminal cross-linker) collagen films. In addition, we provide protocols to characterize the biophysical (swelling), biomechanical (tensile), and biological (metabolic activity, proliferation and viability using human dermal fibroblasts and THP-1 macrophages) properties of the cross-linked collagen scaffolds.

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

Bottom-up bioengineering utilizes the inherent capacity of cells to build highly sophisticated structures with high levels of biomimicry. Despite the significant advancements in the field, monodomain approaches require prolonged culture time to develop an implantable device, usually associated with cell phenotypic drift in culture. Herein, we assessed the simultaneous effect of macromolecular crowding (MMC) and mechanical loading in enhancing extracellular matrix (ECM) deposition while maintaining tenocyte (TC) phenotype and differentiating bone marrow stem cells (BMSCs) or transdifferentiating neonatal and adult dermal fibroblasts toward tenogenic lineage. At d 7, all cell types presented cytoskeleton alignment perpendicular to the applied load independently of the use of MMC. MMC enhanced ECM deposition in all cell types. Gene expression analysis indicated that MMC and mechanical loading maintained TC phenotype, whereas tenogenic differentiation of BMSCs or transdifferentiation of dermal fibroblasts was not achieved. Our data suggest that multifactorial bottom-up bioengineering approaches significantly accelerate the development of biomimetic tissue equivalents.-Gaspar, D., Ryan, C. N. M., Zeugolis, D. I. Multifactorial bottom-up bioengineering approaches for the development of living tissue substitutes.

Battling adhesions: from understanding to prevention.

Adhesions represent a major burden in clinical practice, particularly following abdominal, intrauterine, pericardial and tendon surgical procedures. Adhesions are initiated by a disruption in the epithelial or mesothelial layer of tissue, which leads to fibrin adhesion sites due to the downregulation of fibrinolytic activity and an increase in fibrin deposition. Hence, the metabolic events involved in tissue healing, coagulation, inflammation, fibrinolysis and angiogenesis play a pivotal role in adhesion formation. Understanding these events, their interactions and their influence on the development of post-surgical adhesion is crucial for the development of effective therapies to prevent them. Mechanical barriers, antiadhesive agents and combination thereof are customarily used in the battle against adhesions. Although these systems seem to be effective at reducing adhesions in clinical procedures, their prevention remains still elusive, imposing the need for new antiadhesive strategies.

State of art and limitations in genetic engineering to induce stable chondrogenic phenotype.

Current protocols for chondrocyte expansion and chondrogenic differentiation of stem cells fail to reduce phenotypic loss and to mitigate hypertrophic tendency. To this end, cell genetic manipulation is gaining pace as a means of generating cells with stable chondrocyte phenotype. Herein, we provide an overview of candidate genes that either induce cartilage regeneration or inhibit cartilage degeneration. We further discuss in vitro, ex vivo and in vivo viral transduction and non-viral transfection strategies for targeted cells (chondrocytes, mesenchymal stem cells, induced pluripotent stem cells and synovial cells), along with the most representative results obtained in pre-clinical models and in clinical trials. We highlight current challenges and associated risks that slowdown clinical acceptance and commercialisation of gene transfer technologies.

Acetic acid and pepsin result in high yield, high purity and low macrophage response collagen for biomedical applications.

Collagen based devices are frequently associated with foreign body response. Although several pre- (e.g. species, state of animal, tissue) and post- (e.g. cross-linking, scaffold architecture) extraction method factors have a profound effect on foreign body response, little is known about which and how during the extraction process factors mediate foreign body response. In this study, we assessed the influence of acetic acid and hydrochloric acid and the utilisation or not of pepsin or salt precipitation during collagen extraction on the yield, purity, free amines, denaturation temperature, resistance to collagenase degradation and macrophage response. Acetic acid/pepsin extracted collagen exhibited the highest yield, purity and free amine content and the lowest denaturation temperature. No differences in resistance to collagenase digestion were detected between the groups. Although all treatments exhibited similar macrophage morphology comprised of round cells (M1 phenotype), elongated cells (M2 phenotype) and cell aggregates (foreign body response), significantly more elongated cells were observed on HC films. Although no differences in metabolic activity were observed between the groups, the DNA concentration was significantly lower for the hydrochloric acid treatments. Further, cytokine analysis revealed that hydrochloric acid treatments induced significantly higher IL-1ß and TNF-α release with respect to acetic acid treatments. Salt precipitation did not influence the parameters assessed. Collectively, these data suggest that during the collagen extraction process variables should also be monitored as, evidently, they affect the physicochemical and biological properties of collagen preparations.

* Collagen Cross-Linking: Biophysical, Biochemical, and Biological Response Analysis.

Extracted forms of collagen are subjected to chemical cross-linking to enhance their stability. However, traditional cross-linking approaches are associated with toxicity and inflammation. This work investigates the stabilization capacity, cytotoxicity and inflammatory response of collagen scaffolds cross-linked with glutaraldehyde (GTA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, 4-arm polyethylene glycol (PEG) succinimidyl glutarate (4SP), genipin (GEN), and oleuropein. Although all cross-linking methods reduced free amine groups, variable data were obtained with respect to denaturation temperature, resistance to collagenase digestion, and mechanical properties. With respect to biological analysis, fibroblast cultures showed no significant difference between the treatments. Although direct cultures with human-derived leukemic monocyte cells (THP-1) clearly demonstrated the cytotoxic effect of GTA, THP-1 cultures supplemented with conditioned medium from the various groups showed no significant difference between the treatments. With respect to cytokine profile, no significant difference in secretion of proinflammatory (e.g., interleukin [IL]-1ß, IL-8, tumor necrosis factor-α) and anti-inflammatory (e.g., vascular endothelial growth factor) cytokines was observed between the noncross-linked and the 4SP and GEN cross-linked groups, suggesting the suitability of these agents as collagen cross-linkers.

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

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.

Recreating complex pathophysiologies in vitro with extracellular matrix surrogates for anticancer therapeutics screening.

In vitro tumour models utilise various cancer cells and an appropriate extracellular matrix equivalent to recapitulate the in vivo tumour microenvironment. Three-dimensional tissue surrogates (e.g., decellularised tissue grafts, decellularised monolayers, hydrogels, electrospun fibres and sponges) are increasingly used as alternatives to conventional two-dimensional monolayer cultures to model the tissue environment more faithfully for drug development and screening. Herein, we critically assess the advances and shortfalls of these three-dimensional systems as in vitro models of cancer.

Preferential tendon stem cell response to growth factor supplementation.

Tendon injuries are increasingly prevalent around the world, accounting for more than 100 000 new clinical cases/year in the USA alone. Cell-based therapies have been proposed as a therapeutic strategy, with recent data advocating the use of tendon stem cells (TSCs) as a potential cell source with clinical relevance for tendon regeneration. However, their in vitro expansion is problematic, as they lose their multipotency and change their protein expression profile in culture. Herein, we ventured to assess the influence of insulin-like growth factor 1 (IGF-1), growth and differentiation factor-5 (GDF-5) and transforming growth factor-ß1 (TGFß1) supplementation in TSC culture. IGF-1 preserved multipotency for up to 28 days. Upregulation of decorin and scleraxis expression was observed as compared to freshly isolated cells. GDF-5 treated cells exhibited reduced differentiation along adipogenic and chondrogenic pathways after 28 days, and decorin, scleraxis and collagen type I expression was increased. After 28 days, TGFß1 supplementation led to increased scleraxis, osteonectin and collagen type II expression. The varied responses to each growth factor may reflect their role in tendon repair, suggesting that: GDF-5 promotes the transition of tendon stem cells towards tenocytes; TGFß1 induces differentiation along several pathways, including a phenotype indicative of fibrocartilage or calcified tendon, common problems in tendon healing; and IGF-1 promotes proliferation and maintenance of TSC phenotypes, thereby creating a population sufficient to have a beneficial effect. Copyright © 2014 John Wiley & Sons, Ltd.

The influence of poly(ethylene glycol) ether tetrasuccinimidyl glutarate on the structural, physical, and biological properties of collagen fibers.

Various chemical, natural, or synthetic in origin, crosslinking methods have been proposed over the years to stabilise collagen fibers. However, an optimal method has yet to be identified. Herein, we ventured to assess the potential of 4-star poly(ethylene glycol) ether tetrasuccinimidyl glutarate, as opposed to glutaraldehyde (GTA), genipin and carbodiimide, on the structural, physical and biological properties of collagen fibers. The 4-star poly(ethylene glycol) ether tetrasuccinimidyl glutarate induced an intermedium surface smoothness, denaturation temperature and swelling. The 4-star poly(ethylene glycol) ether tetrasuccinimidyl glutarate fibers had significantly higher stress at break values than the carbodiimide fibers, but significantly lower than the GTA and genipin fibers. With respect to strain at break, no significant difference was observed among the crosslinking treatments. The 4-star poly(ethylene glycol) ether tetrasuccinimidyl glutarate fibers exhibited significantly higher cell metabolic activity and DNA concentration that all other crosslinking treatments, promoted consistently cellular elongation along the longitudinal fiber axis and by day 7 they were completely covered by cells. Collectively, this work clearly demonstrates the potential of 4-star poly(ethylene glycol) ether tetrasuccinimidyl glutarate as collagen crosslinker. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 914-922, 2016.

A shape-controlled tuneable microgel platform to modulate angiogenic paracrine responses in stem cells.

Development of cell delivery platforms have been driven based on an empirical cytoprotective design. While cell-matrix and cell-cell interactions that influence biochemical effects beyond survival has been limited and overshadowed in an effort to incrementally improve biomimicking properties of the tissue-engineered constructs. Here we demonstrate fabrication of a shape controlled 3D type-I collagen-based microgel platform that can be tuned to modulate angiogenic paracrine- 'angiocrine' responses of human mesenchymal stem cells (hMSCs). Furthermore, these microgels were characterized as a 3D cell culture tool to assess optimal biological response as a function of cell-matrix and cell-cell interactions. Finally, optimised hMSC embedded microgels were shown to induce vascular repair and functional improvement in vivo in a mouse model of hind-limb ischemia. The approach described here in designing a tuneable cell delivery platform using naturally occurring extracellular matrix molecules highlights the need for highly customised matrices with an array of self-assembling proteins that dictate specific cell function resembling the native tissue of interest for repair.