Hydrogels based on natural polymers offer a range of properties to mimic the native extracellular matrix, and provide microenvironments to preserve cellular function and encourage tissue formation. A tri-component hydrogel using collagen, alginate and fibrin (CAF) was developed and investigated at three collagen concentrations for application as a functional extracellular matrix analogue. Physical-chemical characterization of CAF hydrogels demonstrated a thermo-responsive crosslinking capacity at physiological conditions with stiffness similar to native soft tissues. CAF hydrogels were also assessed for cytocompatibility using L929 murine fibroblasts, pancreatic MIN6 β-cells and human mesenchymal stem cells (hMSCs); and demonstrated good cell viability, proliferation and metabolic activity after 7 days of in vitro culture. CAF hydrogels, especially with 2.5% w/v collagen, increased alkaline phosphatase production in hMSCs indicating potential for the promotion of osteogenic activity. Moreover, CAF hydrogels also increased metabolic activity of MIN6 β-cells and promoted the reconstitution of spherical pseudoislets with sizes ranging between 50 and 150 μm at day 7, demonstrating potential in diabetic therapeutic applications.

A novel electrospun biphasic scaffold provides optimal three-dimensional topography for in vitro co-culture of airway epithelial and fibroblast cells G E Morris, J C Bridge, L A Brace et al. AbstractElectrospinning 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.

Spinal fusion is the gold standard surgical procedure for degenerative spinal conditions when conservative therapies have been unsuccessful in rehabilitation of patients. Novel strategies are required to improve biocompatibility and osseointegration of traditionally used materials for lumbar cages. Furthermore, new design and technologies are needed to bridge the gap due to the shortage of optimal implant sizes to fill the intervertebral disc defect. Within this context, additive manufacturing technology presents an excellent opportunity to fabricate ergonomic shape medical implants. The goal of this study is to design and manufacture a 3D-printed lumbar cage for lumbar interbody fusion. Optimisations of the proposed implant design and its printing parameters were achieved via in silico analysis. The final construct was characterised via scanning electron microscopy, contact angle, x-ray micro computed tomography (μCT), atomic force microscopy, and compressive test. Preliminary in vitro cell culture tests such as morphological assessment and metabolic activities were performed to access biocompatibility of 3D-printed constructs. Results of in silico analysis provided a useful platform to test preliminary cage design and to find an optimal value of filling density for 3D printing process. Surface characterisation confirmed a uniform coating of nHAp with nanoscale topography. Mechanical evaluation showed mechanical properties of final cage design similar to that of trabecular bone. Preliminary cell culture results showed promising results in terms of cell growth and activity confirming biocompatibility of constructs. Thus for the first time, design optimisation based on computational and experimental analysis combined with the 3D-printing technique for intervertebral fusion cage has been reported in a single study. 3D-printing is a promising technique for medical applications and this study paves the way for future development of customised implants in spinal surgical applications.

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.

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.

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.

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.

This paper presents an investigation of how different culture media (i.e. basal and osteogenic media) affect the nanomechanical properties and microstructure of the mineralized matrix produced by the human mesenchymal stem cell line Y201, from both an experimental and theoretical approach. A bone nodule (i.e. mineralized matrix) cultured from basal medium shows a more anisotropic microstructure compared to its counterpart cultured from an osteogenic medium. As confirmed by finite element simulations, this anisotropic microstructure explains the bimodal distribution of the corresponding mechanical properties very well. The overall nanomechanical response of the bone nodule from the osteogenic medium is poorer compared to its counterpart from the basal medium. The bone nodules, from both basal and osteogenic media, have shown reverse aging effects in terms of mechanical properties. These are possibly due to the fact that cell proliferation outcompetes the mineralization process.