Garry P. Duffy

The healing of bony defects by cell-free collagen-based scaffolds compared to stem cell-seeded tissue engineered constructs

One of the key challenges in tissue engineering is to understand the host response to scaffolds and engineered constructs. We present a study in which two collagen-based scaffolds developed for bone repair: a collagen-glycosaminoglycan (CG) and biomimetic collagen-calcium phosphate (CCP) scaffold, are evaluated in rat cranial defects, both cell-free and when cultured with MSCs prior to implantation. The results demonstrate that both cell-free scaffolds showed excellent healing relative to the empty defect controls and somewhat surprisingly, to the tissue engineered (MSC-seeded) constructs. Immunological analysis of the healing response showed higher M1 macrophage activity in the cell-seeded scaffolds. However, when the M2 macrophage response was analysed, both groups (MSC-seeded and non-seeded scaffolds) showed significant activity of these cells which are associated with an immunomodulatory and tissue remodelling response. Interestingly, the location of this response was confined to the construct periphery, where a capsule had formed, in the MSC-seeded groups as opposed to areas of new bone formation in the non-seeded groups. This suggests that matrix deposited by MSCs during in vitro culture may adversely affect healing by acting as a barrier to macrophage-led remodelling when implanted in vivo. This study thus improves our understanding of host response in bone tissue engineering.

Innovative Collagen Nano‐Hydroxyapatite Scaffolds Offer a Highly Efficient Non‐Viral Gene Delivery Platform for Stem Cell‐Mediated Bone Formation

The ability of nano-hydroxyapatite (nHA) particles developed in-house to act as non-viral delivery vectors is assessed. These nHA particles are combined with collagen to yield bioactive, biodegradable collagen nano-hydroxyapatite (coll-nHA) scaffolds. Their ability to act as gene-activated matrices for BMP2 delivery is demonstrated with successful transfection of mesenchymal stem cells (MSCs) resulting in high calcium production.

Drug and cell delivery for cardiac regeneration

The spectrum of ischaemic cardiomyopathy, encompassing acute myocardial infarction to congestive heart failure is a significant clinical issue in the modern era. This group of diseases is an enormous source of morbidity and mortality and underlies significant healthcare costs worldwide. Cardiac regenerative therapy, whereby pro-regenerative cells, drugs or growth factors are administered to damaged and ischaemic myocardium has demonstrated significant potential, especially preclinically. While some of these strategies have demonstrated a measure of success in clinical trials, tangible clinical translation has been slow. To date, the majority of clinical studies and a significant number of preclinical studies have utilised relatively simple delivery methods for regenerative therapeutics, such as simple systemic administration or local injection in saline carrier vehicles. Here, we review cardiac regenerative strategies with a particular focus on advanced delivery concepts as a potential means to enhance treatment efficacy and tolerability and ultimately, clinical translation. These include (i) delivery of therapeutic agents in biomaterial carriers, (ii) nanoparticulate encapsulation, (iii) multimodal therapeutic strategies and (iv) localised, minimally invasive delivery via percutaneous transcatheter systems.

The delayed addition of human mesenchymal stem cells to pre-formed endothelial cell networks results in functional vascularization of a collagen–glycosaminoglycan scaffold in vivo

This paper demonstrates a method to engineer, in vitro, a nascent microvasculature within a collagen-glycosaminoglycan scaffold with a view to overcoming the major issue of graft failure due to avascular necrosis of tissue-engineered constructs. Human umbilical vein endothelial cells (ECs) were cultured alone and in various co-culture combinations with human mesenchymal stem cells (MSCs) to determine their vasculogenic abilities in vitro. Results demonstrated that the delayed addition of MSCs to pre-formed EC networks, whereby MSCs act as pericytes to the nascent vessels, resulted in the best developed vasculature. The results also demonstrate that the crosstalk between ECs and MSCs during microvessel formation occurs in a highly regulated, spatio-temporal fashion, whereby the initial seeding of ECs results in platelet derived growth factor (PDGF) release; the subsequent addition of MSCs 3 days later leads to a cessation in PDGF production, coinciding with increased vascular endothelial cell growth factor expression and enhanced vessel formation. Functional assessment of these pre-engineered constructs in a subcutaneous rat implant model demonstrated anastomosis between the in vitro engineered vessels and the host vasculature, with significantly increased vascularization occurring in the co-culture group. This study has thus provided new information on the process of in vitro vasculogenesis within a three-dimensional porous scaffold for tissue engineering and demonstrates the potential for using these vascularized scaffolds in the repair of critical sized bone defects.

Comparison of biomaterial delivery vehicles for improving acute retention of stem cells in the infarcted heart

Cell delivery to the infarcted heart has emerged as a promising therapy, but is limited by very low acute retention and engraftment of cells. The objective of this study was to compare a panel of biomaterials to evaluate if acute retention can be improved with a biomaterial carrier. Cells were quantified post-implantation in a rat myocardial infarct model in five groups (n = 7-8); saline injection (current clinical standard), two injectable hydrogels (alginate, chitosan/β-glycerophosphate (chitosan/ß-GP)) and two epicardial patches (alginate, collagen). Human mesenchymal stem cells (hMSCs) were delivered to the infarct border zone with each biomaterial. At 24 h, retained cells were quantified by fluorescence. All biomaterials produced superior fluorescence to saline control, with approximately 8- and 14-fold increases with alginate and chitosan/β-GP injectables, and 47 and 59-fold increases achieved with collagen and alginate patches, respectively. Immunohistochemical analysis qualitatively confirmed these findings. All four biomaterials retained 50-60% of cells that were present immediately following transplantation, compared to 10% for the saline control. In conclusion, all four biomaterials were demonstrated to more efficiently deliver and retain cells when compared to a saline control. Biomaterial-based delivery approaches show promise for future development of efficient in vivo delivery techniques.

Supramolecular Hydrogels with Reverse Thermal Gelation Properties from (Oligo)tyrosine Containing Block Copolymers

Novel block copolymers comprising poly(ethylene glycol) (PEG) and an oligo(tyrosine) block were synthesized in different compositions by N-carboxyanhydride (NCA) polymerization. It was shown that PEG2000-Tyr(6) undergoes thermoresponsive hydrogelation at a low concentration range of 0.25-3.0 wt % within a temperature range of 25-50 °C. Cryogenic transmission electron microscopy (Cryo-TEM) revealed a continuous network of fibers throughout the hydrogel sample, even at concentrations as low as 0.25 wt %. Circular dichroism (CD) results suggest that better packing of the β-sheet tyrosine block at increasing temperature induces the reverse thermogelation. A preliminary assessment of the potential of the hydrogel for in vitro application confirmed the hydrogel is not cytotoxic, is biodegradable, and produced a sustained release of a small-molecule drug.

Combinatorial Gene Therapy Accelerates Bone Regeneration: Non‐Viral Dual Delivery of VEGF and BMP2 in a Collagen‐Nanohydroxyapatite Scaffold

Vascularization and bone repair are accelerated by a series of gene-activated scaffolds delivering both an angiogenic and an osteogenic gene. Stem cell-mediated osteogenesis in vitro, in addition to increased vascularization and bone repair by host cells in vivo, is enhanced using all systems while the use of the nanohydroxyapatite vector to deliver both genes markedly enhances bone healing.

Mechanical characterization of a customized decellularized scaffold for vascular tissue engineering.

Several challenges persist when attempting to utilize decellularized tissue as a scaffold for vascular tissue engineering. Namely: poor cell infiltration/migration, excessive culture times associated with repopulating the scaffolds, and the achievement of a quiescent medial layer. In an attempt to create an optimum vascular scaffold, we customized the properties of decellularized porcine carotid arteries by: (i) creating cavities within the medial layer to allow direct injection of cells, and (ii) controlling the amount of collagen digestion to increase the porosity. Histological examination of our customized scaffold revealed a highly porous tissue structure containing consistent medial cavities running longitudinally through the porous scaffold wall. Mechanical testing of the customized scaffold showed that our minimal localized disruption to the ECM does not have a detrimental effect on the bulk mechanical response of the tissue. The results demonstrate that an increased stiffness and reduced distensibility occurs after decellularization when compared to the native tissue, however post scaffold customization we can revert the scaffold tensile properties back to that of the native tissue. This most noteworthy result occurs in the elastin dominant phase of the tensile response of the scaffold, indicating that no disruption has occurred to the elastin network by our decellularization and customization techniques. Additionally, the bulk seeding potential of the customized scaffold was demonstrated by direct injection of human smooth muscle cells through the medial cavities. The optimum cell dispersion was observed in the highest porosity scaffold, with large cell numbers retained within the medial layer after 24 h static culture. In summary, this study presents a novel customized decellularized vascular scaffold that has the capability of bulk seeding the media, and in tandem to this method, the porosity of the scaffold has been increased without compromising the mechanical integrity.

RANKL and OPG activity is regulated by injury size in networks of osteocyte-like cells.

Bone remodelling is an intricate process encompassing numerous paracrine and autocrine biochemical pathways and mechanical mechanisms. It is responsible for maintaining bone homeostasis, structural integrity and function. The RANKL-RANK-OPG cytokine system is one of the principal mediators in the maintenance of bone cell function and activation of bone remodelling by the Basic Multicellular Unit (BMU) which carries out remodelling. Theories surrounding the initiation of bone remodelling include mechanical loading, fluid flow and microdamage as potential stimuli. This study focused on microdamage. In an in vitro simulated bone environment, gel embedded MLO-Y4 cell networks were subjected to damage in the form of planar, crack-like defects of constant area and varying thickness. The biochemical response was determined by ELISA and luciferase assay. The results showed that RANKL release increased and OPG decreased in a manner which depended on injury size (i.e. thickness) and time following application of injury. The effect of microdamage on cell viability and apoptosis was also evaluated. This work demonstrates that injury alone, in the absence of imposed strain or fluid flow, is sufficient to initiate changes in cytokine concentrations of the type which are known to stimulate bone remodeling.

The development of non-viral gene-activated matrices for bone regeneration using polyethyleneimine (PEI) and collagen-based scaffolds.

The healing potential of scaffolds for tissue engineering can be enhanced by combining them with genes to produce gene-activated matrices (GAMs) for tissue regeneration. We examined the potential of using polyethyleneimine (PEI) as a vector for transfection of mesenchymal stem cells (MSCs) in monolayer culture and in 3D collagen-based GAMs. PEI-pDNA polyplexes were fabricated at a range of N/P ratios and their optimal transfection parameters (N/P 7 ratio, 2μg dose) and transfection efficiencies (30±8%) determined in monolayer culture. The polyplexes were then loaded onto collagen, collagen-glycosaminoglycan and collagen-nanohydroxyapatite scaffolds where gene expression was observed up to 21 days with a polyplex dose as low as 2μg. Transient expression profiles indicated that the GAMs act as a polyplex depot system whereby infiltrating cells become transfected over time as they migrate throughout the scaffold. The collagen-nHa GAM exhibited the most prolonged and elevated levels of transgene expression. This research has thus demonstrated that PEI is a highly efficient pDNA transfection agent for both MSC monolayer cultures and in the 3D GAM environment. By combining therapeutic gene therapy with highly engineered scaffolds, it is proposed that these GAMs might have immense capability to promote tissue regeneration.