In vitro and preclinical characterisation of compressed, macro-porous and collagen coated poly-ε-caprolactone electro-spun scaffolds.

Low in macro-porosity electro-spun scaffolds are often associated with foreign body response, whilst macro-porous electro-spun scaffolds have low mechanical integrity. Herein, compressed, macro-porous and collagen (bovine Achilles tendon and human recombinant) coated electro-spun poly-ε-caprolactone scaffolds were developed and their biomechanical, in vitro and in vivo properties were assessed. Collagen coating, independently of the source, did not significantly affect the biomechanical properties of the scaffolds. Although no significant difference in cell viability was observed between the groups, collagen coated scaffolds induced significantly higher DNA concentration. In vivo, no signs of adverse tissue effect were observed in any of the groups and all groups appeared to equally integrate into the subcutaneous tissue. It is evidenced that macro-porous poly-ε-caprolactone electro-spun meshes with adequate mechanical properties and acceptable host response can be developed for biomedical applications.

Polydispersity and negative charge are key modulators of extracellular matrix deposition under macromolecular crowding conditions.

Macromolecular crowding is a biophysical phenomenon that stems from the volume excluded by macromolecules, as they undergo steric repulsion and electrostatic interactions. The excluded volume depends on the shape, size, charge and polydispersity of the molecules. Although theoretical/computational models have been used to assess the influence of macromolecular crowding in biological media, real-time experiments are scarce. Herein, we evaluated 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. The use of crowding cocktails with different molecular weight/concentrations of Ficoll™ or dextran sulphate molecules led to increased polydispersity and enhanced collagen type I deposition in comparison to their mono-domain counterparts. Carrageenan, however, induced the highest deposition of collagen type I due to its negative charge and inherent polydispersity. Our data contribute to a better understanding of the influence of the biophysical properties of the crowders on extracellular matrix deposition in vitro. STATEMENT OF SIGNIFICANCE: Macromolecular crowding is a biophysical phenomenon that accelerates and enhances extracellular matrix deposition in cell culture systems. Herein, we demonstrate that negatively charged and polydispersed macromolecules or cocktails of macromolecules, as opposed to neutral and monodomain macromolecules, induce highest extracellular matrix deposition in human dermal fibroblast cultures.

Development macro-porous electro-spun meshes with clinically relevant mechanical properties-a technical note.

The nano-fibrous architecture of electro-spun meshes favours their use in biomedicine, but their low mechanical properties prohibit their wide use in clinical practice. Introduction of porosity, essential of tissue integration, decreases further mechanical integrity. Herein, we hypothesised that macro-porous electro-spun meshes with adequate mechanical properties can be fabricated through layering and subsequent compression. Two and three layers electro-spun poly-ε-caprolactone scaffolds were fabricated, compressed and subsequently 30% circular porosity was introduced through laser cutting. Three-layered porous electro-spun meshes exhibited mechanical properties similar to commercially available scaffolds without any structural or cytotoxic effect. This study brings electro-spun materials closer to clinical translation and commercialisation.

Influence of Cross-Linking Method and Disinfection/Sterilization Treatment on the Structural, Biophysical, Biochemical, and Biological Properties of Collagen-Based Devices.

Disinfection/sterilization is an essential step during the manufacturing process of any implantable medical device. Cross-linking is also required for biopolymers to control resistance to degradation and enhance mechanical integrity. To date, there is still no single disinfection/sterilization treatment and cross-linking method that can be used universally for collagen-based devices. Herein, we assessed the influence of ethylene oxide, ethanol, gamma irradiation, and gas plasma disinfection/sterilization on the structural, biophysical, biochemical, and biological properties of self-assembled collagen films cross-linked with 4-arms polyethylene glycol succinimidyl glutarate and genipin. Microscopy analysis revealed that gas plasma treatment induced the most profound differences in the non-cross-linked and 4-arms polyethylene glycol succinimidyl glutarate cross-linked collagen films. Gas plasma also significantly increased the swelling ratio of the non-cross-linked and the 4-arms polyethylene glycol succinimidyl glutarate cross-linked films. Non-cross-linked and gas plasma treated 4-arms polyethylene glycol succinimidyl glutarate collagen films exhibited the lowest resistance to collagenase degradation and denaturation temperature. Between the non-cross-linked groups, the gas plasma treatment resulted in the collagen films with the lowest stress at break, strain at break, force at break, and Young's modulus values. Within the 4-arms polyethylene glycol succinimidyl glutarate groups, the ethylene oxide treatment resulted in collagen films with the lowest stress at break, strain at break, force at break, and Young's modulus values. Within the genipin groups, the gas plasma treatment resulted in collagen films with the lowest stress at break, strain at break, force at break, and Young's modulus values. Proliferation, metabolic activity, and viability of human skin fibroblasts were not affected as a function of cross-linking method and disinfection/sterilization treatment. However, proliferation, metabolic activity, and viability of THP1 cells were significantly reduced as a function of the cross-linking method, but they were not affected as a function of the disinfection/sterilization treatment. Overall, our data illustrate that the cross-linking method and the disinfection/sterilization treatment differentially affect the structural, biophysical, biochemical, and biological properties of collagen-based devices, and thus, they should be optimized according to the clinical indication.

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.

Influence of porosity and pore shape on structural, mechanical and biological properties of poly ϵ-caprolactone electro-spun fibrous scaffolds.

BACKGROUND: Electro-spun scaffolds are utilized in a diverse spectrum of clinical targets, with an ever-increasing quantity of work progressing to clinical studies and commercialization. The limited number of conformations in which the scaffolds can be fabricated hampers their wide acceptance in clinical practice. MATERIALS & METHODS: Herein, we assessed a single-strep fabrication process for predesigned electro-spun scaffold preparation and the ramifications of the introduction of porosity (0, 30, 50, 70%) and pore shape (circle, rhomboid, square) on structural, mechanical (tensile and ball burst) and biological (dermal fibroblast and THP-1) properties. RESULTS: The collector design did not affect the fibrous nature of the scaffold. Modulation of the porosity and pore shape offered control over the mechanical properties of the scaffolds. Neither the porosity nor the pore shape affected cellular (dermal fibroblast and THP-1) response. CONCLUSION: Overall, herein we provide evidence that electro-spun scaffolds of controlled architecture can be fabricated with fibrous fidelity, adequate mechanical properties and acceptable cytocompatibility for a diverse range of clinical targets.

Harnessing Hierarchical Nano- and Micro-Fabrication Technologies for Musculoskeletal Tissue Engineering.

Cells within a tissue are able to perceive, interpret and respond to the biophysical, biomechanical, and biochemical properties of the 3D extracellular matrix environment in which they reside. Such stimuli regulate cell adhesion, metabolic state, proliferation, migration, fate and lineage commitment, and ultimately, tissue morphogenesis and function. Current scaffold fabrication strategies in musculoskeletal tissue engineering seek to mimic the sophistication and comprehensiveness of nature to develop hierarchically assembled 3D implantable devices of different geometric dimensions (nano- to macrometric scales) that will offer control over cellular functions and ultimately achieve functional regeneration. Herein, advances and shortfalls of bottom-up (self-assembly, freeze-drying, rapid prototype, electrospinning) and top-down (imprinting) scaffold fabrication approaches, specific to musculoskeletal tissue engineering, are discussed and critically assessed.

An academic, clinical and industrial update on electrospun, additive manufactured and imprinted medical devices.

Electrospinning, additive manufacturing and imprint lithography scaffold fabrication technologies have attracted great attention in biomedicine, as they allow production of two- and three- dimensional constructs with tuneable topographical and geometrical features. In vitro data demonstrate that electrospun and imprinted substrates offer control over permanently differentiated and stem cell function. Advancements in functionalisation strategies have further enhanced the bioactivity and reparative capacity of electrospun and additive manufactured devices, as has been evidenced in several preclinical models. Despite this overwhelming success in academic setting, only a few technologies have reached the clinic and only a fraction of them have become commercially available products.