Development of a dual-component infection-resistant arterial replacement for small-caliber reconstructions: A proof-of-concept study.

Introduction: Synthetic vascular grafts perform poorly in small-caliber (<6mm) anastomoses, due to intimal hyperplasia and thrombosis, whereas homografts are associated with limited availability and immunogenicity, and bioprostheses are prone to aneurysmal degeneration and calcification. Infection is another important limitation with vascular grafting. This study developed a dual-component graft for small-caliber reconstructions, comprising a decellularized tibial artery scaffold and an antibiotic-releasing, electrospun polycaprolactone (PCL)/polyethylene glycol (PEG) blend sleeve. Methods: The study investigated the effect of nucleases, as part of the decellularization technique, and two sterilization methods (peracetic acid and γ-irradiation), on the scaffold's biological and biomechanical integrity. It also investigated the effect of different PCL/PEG ratios on the antimicrobial, biological and biomechanical properties of the sleeves. Tibial arteries were decellularized using Triton X-100 and sodium-dodecyl-sulfate. Results: The scaffolds retained the general native histoarchitecture and biomechanics but were depleted of glycosaminoglycans. Sterilization with peracetic acid depleted collagen IV and produced ultrastructural changes in the collagen and elastic fibers. The two PCL/PEG ratios used (150:50 and 100:50) demonstrated differences in the structural, biomechanical and antimicrobial properties of the sleeves. Differences in the antimicrobial activity were also found between sleeves fabricated with antibiotics supplemented in the electrospinning solution, and sleeves soaked in antibiotics. Discussion: The study demonstrated the feasibility of fabricating a dual-component small-caliber graft, comprising a scaffold with sufficient biological and biomechanical functionality, and an electrospun PCL/PEG sleeve with tailored biomechanics and antibiotic release.

Towards the development of osteochondral allografts with reduced immunogenicity.

Nowadays, repair and replacement of hyaline articular cartilage still challenges orthopedic surgery. Using a graft of decellularized articular cartilage as a structural scaffold is considered as a promising therapy. So far, successful cell removal has only been possible for small samples with destruction of the macrostructure or loss of biomechanics. Our aim was to develop a mild, enzyme-free chemical decellularization procedure while preserving the biomechanical properties of cartilage. Porcine osteochondral cylinders (diameter: 12 mm; height: 10 mm) were divided into four groups: Native plugs (NA), decellularized plugs treated with PBS, Triton-X-100 and SDS (DC), and plugs additionally treated with freeze-thaw-cycles of -20 °C, -80 °C or shock freezing in nitrogen (N2) before decellularization. In a non-decalcified HE stain the decellularization efficiency (cell removal, cell size, depth of decellularization) was calculated. For biomechanics the elastic and compression modulus, transition and failure strain as well as transition and failure stress were evaluated. The -20 °C, -80 °C, and N2 groups showed a complete decellularization of the superficial and middle zone. In the deep zone cells could not be removed in any experimental group. The biomechanical analysis showed only a reduced elastic modulus in all decellularized samples. No significant differences were found for the other biomechanical parameters.

Assessment of Migration of Human MSCs through Fibrin Hydrogels as a Tool for Formulation Optimisation.

Control of cell migration is fundamental to the performance of materials for cell delivery, as for cells to provide any therapeutic effect, they must migrate out from the delivery material. Here the influence of fibrinogen concentration on the migration of encapsulated human mesenchymal stem cells (hMSCs) from a cell spheroid through fibrin hydrogels is tracked over time. Fibrin was chosen as a model material as it is routinely employed as a haemostatic agent and more recently has been applied as a localised delivery vehicle for potential therapeutic cell populations. The hydrogels consisted of 5 U/mL thrombin and between 5 and 50 mg/mL fibrinogen. Microstructural and viscoelastic properties of different compositions were evaluated using SEM and rheometry. Increasing the fibrinogen concentration resulted in a visibly denser matrix with smaller pores and higher stiffness. hMSCs dispersed within the fibrin gels maintained cell viability post-encapsulation, however, the migration of cells from an encapsulated spheroid revealed that denser fibrin matrices inhibit cell migration. This study provides the first quantitative study on the influence of fibrinogen concentration on 3D hMSC migration within fibrin gels, which can be used to guide material selection for scaffold design in tissue engineering and for the clinical application of fibrin sealants.

Stiffening by Osmotic Swelling Constraint in Cartilage-Like Cell Culture Scaffolds.

Cartilage wounds result in chronic pain and degradation of the quality of life for millions of people. A synthetic cellular scaffold able to heal the damage by substituting the natural tissue is of great potential value. Here, it is shown for the first time that the unique interplay between the molecular components of cartilage can be reproduced in composite materials made of a polyelectrolyte hydrogel embedding a collagen scaffold. These composites possess a mechanical response determined by osmotic and electrostatic effects, comparable to articular cartilage in terms of elastic modulus, time-dependent response, and permeability to interstitial fluid flow. Made entirely from biocompatible materials, the cartilage-like composite materials developed permit 3D culture of chondrocyte-like cells through their microporosity. The biomimetic materials presented here constitute an entirely new class of osmotically stiffened composites, which may find use outside of biomedical applications.

Osseointegration of porous apatite-wollastonite and poly(lactic acid) composite structures created using 3D printing techniques.

A novel apatite-wollastonite/poly(lactic acid) (AW/PLA) composite structure, which matches cortical and cancellous bone properties has been produced and evaluated in vitro and in vivo. The composites structure has been produced using an innovative combination of 3D printed polymer and ceramic macrostructures, thermally bonded to create a hybrid composite structure. In vitro cell assays demonstrated that the AW structure alone, PLA structure alone, and AW/PLA composite were all biocompatible, with the AW structure supporting the proliferation and osteogenic differentiation of rat bone marrow stromal cells. Within a rat calvarial defect model the AW material showed excellent osseointegration with the formation of new bone, and vascularisation of the porous AW structure, both when the AW was implanted alone and when it was part of the AW/PLA composite structure. However, the AW/PLA structure showed the largest amount of the newly formed bone in vivo, an effect which is considered to be a result of the presence of the osteoinductive AW structure stimulating bone growth in the larger pores of the adjacent PLA structure. The layered AW/PLA structure showed no signs of delamination in any of the in vitro or in vivo studies, a result which is attributed to good initial bonding between polymer and ceramic, slow resorption rates of the two materials, and excellent osseointegration. It is concluded that macro-scale composites offer an alternative route to the fabrication of bioactive bone implants which can provide a match to both cortical and cancellous bone properties over millimetre length scales.

Impact of Collagen/Heparin Multilayers for Regulating Bone Cellular Functions.

Bone cell interaction with extracellular matrix (ECM) microenvironment is of critical importance when engineering surface interfaces for bone regeneration. In this work layer-by-layer films of type I collagen (coll), the major constituent of bone ECM, and heparin (hep), a glycosaminoglycan, were assembled on poly(l-lactic acid) (PLLA) substrates to evaluate the impact of the biomacromolecular coating on cell activity. The surface modification of PLLA demonstrated that the hep/coll multilayer is stable after 10 bilayers (confirmed by contact angle, infrared spectroscopy, and morphological analysis). This simple approach provided novel information on the effect of heparin on type I collagen hierarchical organization and subsequent cell response of osteoblast-like (MC3T3-E1) and human bone marrow-derived mesenchymal stem cells (hMSCs). Interestingly, the number of deposited heparin layers (1 or 10) appeared to play an important role in the self-assembly of collagen into fibrils, stabilizing the fibrous collagen layer, and potentially impacting hMSCs activity.

A novel technique for the production of electrospun scaffolds with tailored three-dimensional micro-patterns employing additive manufacturing.

Electrospinning is a common technique used to fabricate fibrous scaffolds for tissue engineering applications. There is now growing interest in assessing the ability of collector plate design to influence the patterning of the fibres during the electrospinning process. In this study, we investigate a novel method to generate hybrid electrospun scaffolds consisting of both random fibres and a defined three-dimensional (3D) micro-topography at the surface, using patterned resin formers produced by rapid prototyping (RP). Poly(D,L-lactide-co-glycolide) was electrospun onto the engineered RP surfaces and the ability of these formers to influence microfibre patterning in the resulting scaffolds visualized by scanning electron microscopy. Electrospun scaffolds with patterns mirroring the microstructures of the formers were successfully fabricated. The effect of the resulting fibre patterns and 3D geometries on mammalian cell adhesion and proliferation was investigated by seeding enhanced green fluorescent protein labelled 3T3 fibroblasts onto the scaffolds. Following 24 h and four days of culture, the seeded scaffolds were visually assessed by confocal macro- and microscopy. The patterning of the fibres guided initial cell adhesion to the scaffold with subsequent proliferation over the geometry resulting in the cells being held in a 3D micro-topography. Such patterning could be designed to replicate a specific in vivo structure; we use the dermal papillae as an exemplar here. In conclusion, a novel, versatile and scalable method to produce hybrid electrospun scaffolds has been developed. The 3D directional cues of the patterned fibres have been shown to influence cell behaviour and could be used to culture cells within a similar 3D micro-topography as experienced in vivo.