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Stem cells are an essential consideration in the fields of tissue engineering and regenerative medicine. Understanding how nanoengineered biomaterials and mesenchymal stem cells (MSCs) interact is crucial for their role in bone regeneration. Taking advantage of the structural stability of selenium nanoparticles (Se-NPs) and biological properties of natural polymers, Se-NPs-functionalized, injectable, thermoresponsive hydrogels with an interconnected molecular structure were prepared to identify their role in the osteogenic differentiation of different types of mesenchymal stem cells. Further, comprehensive characterization of their structural and biological properties was performed. The results showed that the hydrogels undergo a sol to gel transition with the help of β-glycerophosphate, while functionalization with Se-NPs significantly enhances their biological response through stabilizing their polymeric structure by forming Se-O covalent bonds. Further results suggest that Se-NPs enhance the differentiation of MSCs toward osteogenic lineage in both the 2D as well as 3D. We demonstrated that the Se-NPs-functionalized hydrogels could enhance the differentiation of osteoporotic bone-derived MSCs. We also focused on specific cell surface marker expression (CD105, CD90, CD73, CD45, CD34) based on the exposure of healthy rats' bone marrow-derived stem cells (BMSCs) to the Se-NP-functionalized hydrogels. This study provides essential evidence for pre-clinical/clinical applications, highlighting the potential of the nanoengineered biocompatible elastic hydrogels for bone regeneration in diseased bone.

The properties of the extracellular matrix (ECM) have profound impact upon cell behaviour. As an abundant protein in mammals, collagen is a desirable base material to engineer an ECM tissue scaffold, but its structural weakness generally requires molecular crosslinking or incorporation of additional ECM-based macromolecules such as glycosaminoglycans. We have performed microscopic indentation to test collagen films under dry and aqueous conditions prepared with different levels of physical and chemical crosslinking. Our technique isolates intrinsic properties of the poro-viscoelastic matrix in a regime minimizing the influence of drainage hydrodynamics and allows direct measurement of the effect of hydrating a specific sample. A doubling of the effective stress-strain stiffness under crosslinking could be directly correlated to structural changes in X-ray diffraction spectra, while electron microscopy revealed possible fibril bridging mechanisms explaining observed toughness. Overall, an intrinsic viscoelastic stress-strain response of collagen under various conditions of cross-linking was observed for both dry and wet conditions, with the latter most affected by indentation rate. Under creep testing, a three order of magnitude increase in dynamic compliance and factor three reduction in relaxation time was found going from the dry to hydrated state. When fitted to a simple viscoelastic model, crosslinking showed a tendency to decrease relaxation time in both states, but reduced dynamic compliance only in the hydrated case. This suggests a reduced role of virtual crosslinks under hydration. This is the first study reporting consistent mechanical testing of dry and hydrated ECM-derived biomaterials, accessing the intrinsic material mechanics under in vivo-like conditions. STATEMENT OF SIGNIFICANCE: This manuscript presents new insights into the effect of crosslinking on mechanical properties of dry and hydrated collagen intended for tissue scaffolding applications. A novel microscopic indentation technique allowed testing of the poro-viscoelastic matrix isolated in a regime minimizing the influence of drainage hydrodynamics, so direct comparison of the effect of hydration on the intrinsic material behaviour to could be made. A variety of experimental techniques including X-ray diffraction, infrared spectroscopy, and scanning electron and atomic force microscopy were used to augment the mechanical testing. The results of creep testing were numerically analysed using a four-component viscoelastic model. This is the first mechanical testing of dry and hydrated ECM-derived biomaterials, accessing the intrinsic material mechanics under in vivo-like conditions., Competing Interests: Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The healing of large, critically sized bone defects remains an unmet clinical need in modern orthopaedic medicine. The tissue engineering field is increasingly using biomaterial scaffolds as 3D templates to guide the regenerative process, which can be further augmented via the incorporation of recombinant growth factors. Typically, this necessitates supraphysiological doses of growth factor to facilitate an adequate therapeutic response. Herein, we describe a cell-free, biomaterial implant which is functionalised with a low dose, combinatorial growth factor therapy that is capable of rapidly regenerating vascularised bone tissue within a critical-sized rodent calvarial defect. Specifically, we demonstrate that the dual delivery of the growth factors bone morphogenetic protein-2 (osteogenic) and vascular endothelial growth factor (angiogenic) at a low dose (5 μg/scaffold) on an osteoconductive collagen-hydroxyapatite scaffold is highly effective in healing these critical-sized bone defects. The high affinity between the hydroxyapatite component of this biomimetic scaffold and the growth factors functions to sequester them locally at the defect site. Using this growth factor-loaded scaffold, we show complete bridging of a critical-sized calvarial defect in all specimens at a very early time point of 4 weeks, with a 28-fold increase in new bone volume and seven-fold increase in new bone area compared with a growth factor-free scaffold. Overall, this study demonstrates that a collagen-hydroxyapatite scaffold can be used to locally harness the synergistic relationship between osteogenic and angiogenic growth factors to rapidly regenerate bone tissue without the need for more complex controlled delivery vehicles or high total growth factor doses., (© 2019 John Wiley & Sons, Ltd.)

It is increasingly being recognised within the field of tissue engineering that the regenerative capacity of biomaterial scaffolds can be augmented via the incorporation of gene therapeutics. However, the field still lacks a biocompatible gene delivery vector which is capable of functionalizing scaffolds for tailored nucleic acid delivery. Herein, we describe a versatile, collagen based, gene-activated scaffold platform which can transfect autologous host cells in vivo via incorporation of star-shaped poly(˪-lysine) polypeptides (star-PLLs) and a plasmid DNA (pDNA) cargo. Two star-PLL vectors with varying number and length of poly(˪-lysine) arms were assessed. In vitro, the functionalization of a range of collagen based scaffolds containing either glycosaminoglycans (chondroitin sulfate or hyaluronic acid) or ceramics (hydroxyapatite or nano-hydroxyapatite) with star-PLL-pDNA nanomedicines facilitated prolonged, non-toxic transgene expression by mesenchymal stem cells (MSCs). We demonstrate that the star-PLL structure confers enhanced spatiotemporal control of nanomedicine release from functionalized scaffolds over a 28-day period compared to naked pDNA. Furthermore, we identify a star-PLL composition with 64 poly(˪-lysine) arms and 5 (˪-lysine) subunits per arm as a particularly effective vector, capable of facilitating a 2-fold increase in reporter transgene expression compared to the widely used vector polyethylenimine (PEI), a 44-fold increase compared to a 32 poly(˪-lysine) armed star-PLL and a 130-fold increase compared to its linear analogue, linear poly(˪-lysine) (L-PLL) from a collagen-chondroitin sulfate gene activated scaffold. In an in vivo subcutaneous implant model, star-PLL-pDNA gene activated scaffolds which were implanted cell-free exhibited extensive infiltration of autologous host cells, nanomedicine retention within the implanted construct and successful host cell transfection at the very early time point of just seven days. Overall, this article illustrates for the first time the significant ability of the star-PLL polymeric structure to transfect autologous host cells in vivo from an implanted biomaterial scaffold thereby forming a versatile platform with potential in numerous tissue engineering applications., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The bone infection osteomyelitis (typically by Staphylococcus aureus) usually requires a multistep procedure of surgical debridement, long-term systemic high-dose antibiotics, and - for larger defects - bone grafting. This, combined with the alarming rise in antibiotic resistance, necessitates development of alternative approaches. Herein, we describe a one-step treatment for osteomyelitis that combines local, controlled release of non-antibiotic antibacterials with a regenerative collagen-based scaffold. To maximise efficacy, we utilised bioactive glass, an established osteoconductive material with immense capacity for bone repair, as a delivery platform for copper ions (proven antibacterial, angiogenic, and osteogenic properties). Multifunctional collagen-copper-doped bioactive glass scaffolds (CuBG-CS) were fabricated with favourable microarchitectural and mechanical properties (up to 1.9-fold increase in compressive modulus over CS) within the ideal range for bone tissue engineering. Scaffolds demonstrated antibacterial activity against Staphylococcus aureus (up to 66% inhibition) whilst also enhancing osteogenesis (up to 3.6-fold increase in calcium deposition) and angiogenesis in vitro. Most significantly, when assessed in a chick embryo in vivo model, CuBG-CS not only demonstrated biocompatibility, but also a significant angiogenic and osteogenic response, consistent with in vitro studies. Collectively, these results indicate that the CuBG-CS developed here show potential as a one-step osteomyelitis treatment: reducing infection, whilst enhancing bone healing., (Copyright © 2019. Published by Elsevier Ltd.)

Collagen-glycosaminoglycan (CG) scaffolds have been widely developed for a range of regenerative medicine applications. To enhance their efficacy, CG scaffolds have previously been prevascularised in vitro using human umbilical vein endothelial cells and human mesenchymal stromal cells (hMSCs); however, at later timepoints, a regression of vascularisation is observed. This is undesirable for longer preculture periods (e.g., for partial/full organ regeneration) and for in vitro vascularised tissue model systems (e.g., for drug testing/modelling). We hypothesised that delayed platelet-derived growth factor (PDGF)-BB addition could stabilise vessels, preventing their regression. In 2D, we identified 25 ng/ml as a suitable dose that enhanced hMSC metabolic activity and proliferation, without affecting endothelial cells, or migration in either cell type. In our 3D model of CG scaffold vascularisation, early addition of PDGF (Day 3) behaved similarly to no PDGF controls. However, PDGF addition at later timepoints (i.e., Days 4 and 5), with a second addition on Day 10, prevented vascular regression. In quantifying our observations, we identified a need for a tool to measure in vitro vascularisation in porous scaffolds. This was a second key objective of this work. A novel ImageJ macro was developed, which allowed us to analyse vessel-like structures, evaluating their number and morphology, and confirmed our qualitative observations. Finally, upregulation of angiogenic genes (ANG1, KDR, and TEK2) involved in vessel maturation illustrated how PDGF addition contributed to vascular stability. Taken together, the results suggest that addition of PDGF at specific timepoints can be used to stabilise vasculature in CG scaffolds., (© 2018 John Wiley & Sons, Ltd.)

Cardiac stem cells (CSCs) represent a logical cell type to exploit as a regenerative treatment option for tissue damage accrued as a result of a myocardial infarction. However, the isolation and expansion of CSCs prior to cell transplantation is time consuming, costly and invasive, and the reliability of cell expansion may also prove to be a major obstacle in the clinical application of CSC-based transplantation therapy after a myocardial infarction. In order to overcome this, we propose the incorporation of growth factor-eluting alginate microparticles into collagen-based scaffolds as an implantable biomaterial to promote the recruitment and expansion of CSCs in the myocardium. In order to obtain scaffolds able to enhance the motogenic and proliferative potential of CSCs, the aim of this work was to achieve a sustained delivery of both hepatocyte growth factor and insulin-like growth factor-1. Both proteins were initially encapsulated in alginate microparticles by spray drying and subsequently incorporated into a collagen scaffold. Microparticles were seen to homogeneously distribute through the interconnected scaffold pore structure. The resulting scaffolds were capable of extending the release of both proteins up to 15 days, a three-fold increase over non-encapsulated proteins embedded in the scaffolds. In vitro assays with isolated CSCs demonstrated that the sustained release of both bioactive proteins resulted in an increased motogenic and proliferative effect. As presently practiced, the isolation and expansion of CSCs for autologous cell transplantation is slow, expensive and difficult to attain. Thus, there is a need for strategies to specifically activate in situ the intrinsic cardiac regenerative potential represented by the CSCs using combinations of growth factors obviating the need for cell transplantation. By favouring the natural regenerative capability of CSCs, it is hypothesized that the cardiac patch presented here will result in positive therapeutic outcomes in MI and heart failure patients in the future. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Keratoconus can behave more aggressively in pediatric than in adult patients. We systematically reviewed the literature to determine the effectiveness of corneal collagen cross-linking (CXL) in children. For this study, MEDLINE ® and Cochrane databases were searched for all studies examining the effects of standard, trans-epithelial or accelerated CXL protocols in patients age 18 years or younger. Primary outcomes were; uncorrected visual acuity (UCVA) and maximum keratometry (Kmax) and secondary outcomes were; best-corrected visual acuity (BCVA), mean refractive spherical equivalent (MRSE), central corneal thickness (CCT) and endothelial cell density (ECD). Standardized mean differences (SMD) and 95% confidence intervals were calculated, comparing baseline values with those at 6, 12 and 24 months. A total of 13 papers, published between May 2011 and December 2014 examining 490 eyes of 401 patients with a mean age of 15.25 (±1.5) years, were included in the qualitative analysis in this review. Nine papers were included in the meta-analysis, showing significant improvement in UCVA and BCVA and stable Kmax at 12 months, and stable UCVA, improved BCVA and improved Kmax at 24 months in the standard protocol group UCVA, BCVA and KMax were stable at 12 months in the trans-epithelial group. Mean refractive spherical equivalent (MRSE), CCT and ECD remained stable in both groups. In conclusion it was found that standard CXL may be effective in halting progression of keratoconus in pediatric patients at 1 year. However, larger, more long-term studies are required to ascertain its effectiveness., (© 2016 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Today, chronic respiratory disease is one of the leading causes of mortality globally. Epithelial dysfunction can play a central role in its pathophysiology. The development of physiologically-representative in vitro model systems using tissue-engineered constructs might improve our understanding of epithelial tissue and disease. This study sought to engineer a bilayered collagen-hyaluronate (CHyA-B) scaffold for the development of a physiologically-representative 3D in vitro tracheobronchial epithelial co-culture model. CHyA-B scaffolds were fabricated by integrating a thin film top-layer into a porous sub-layer with lyophilisation. The film layer firmly connected to the sub-layer with delamination occurring at stresses of 12-15 kPa. Crosslinked scaffolds had a compressive modulus of 1.9 kPa and mean pore diameters of 70 μm and 80 μm, depending on the freezing temperature. Histological analysis showed that the Calu-3 bronchial epithelial cell line attached and grew on CHyA-B with adoption of an epithelial monolayer on the film layer. Immunofluorescence and qRT-PCR studies demonstrated that the CHyA-B scaffolds facilitated Calu-3 cell differentiation, with enhanced mucin expression, increased ciliation and the formation of intercellular tight junctions. Co-culture of Calu-3 cells with Wi38 lung fibroblasts was achieved on the scaffold to create a submucosal tissue analogue of the upper respiratory tract, validating CHyA-B as a platform to support co-culture and cellular organisation reminiscent of in vivo tissue architecture. In summary, this study has demonstrated that CHyA-B is a promising tool for the development of novel 3D tracheobronchial co-culture in vitro models with the potential to unravel new pathways in drug discovery and drug delivery., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

We have previously examined osteoblast behavior on porous collagen-glycosaminoglycan (CG) scaffolds with a range of mean pore sizes demonstrating superior cell attachment and migration in scaffolds with the largest pores (325 μm). Scaffolds provide a framework for construct development; therefore, it is crucial to identify the optimal pore size for augmented tissue formation. Utilizing the same range of scaffolds (85 μm - 325 μm), this study aimed to examine the effects of mean pore size on subsequent osteoblast differentiation and matrix mineralization, and to understand the mechanism by which pore size influences behavior of different cell types. Consequently, primary mesenchymal stem cells (MSCs) were assessed and their behavior compared to osteoblasts. Results demonstrated that scaffolds with the largest pore size (325 μm) facilitated improved osteoblast infiltration, earlier expression of mature bone markers osteopontin (OPN) and osteocalcin (OCN), and increased mineralization. MSCs responded similarly to osteoblasts whereby cell attachment and scaffold infiltration improved with increasing pore size. However, MSCs showed reduced cell motility, proliferation, and scaffold infiltration compared to osteoblasts. This was associated with differences in the profile of integrin subunits (α2) and collagen receptors (CD44), indicating that osteoblasts have a stronger affinity for CG scaffolds compared to MSCs. In summary, these results reveal how larger pores promote improved cell infiltration, essential for construct development, however the optimal scaffold pore size can be cell type specific. As such, this study highlights a necessity to tailor both scaffold micro-architecture and cell-type when designing constructs for successful bone tissue engineering applications., (© 2015 Wiley Periodicals, Inc.)