Biomaterial based modulation of macrophage polarization: a review and suggested design principles

Macrophages have long been known for their phagocytic capabilities and immune defence; however, their role in healing is being increasingly recognized in recent years due to their ability to polarize into pro-inflammatory and anti-inflammatory phenotypes. Historically, biomaterials were designed to be inert to minimize the host response. More recently, the emergence of tissue engineering and regenerative medicine has led to the design of biomaterials that interact with the host through tailored mechanical, chemical and temporal characteristics. Due to such advances in biomaterial functionality and an improved understanding of macrophage responses to implanted materials, it is now possible to identify biomaterial design characteristics that dictate the host response and contribute to successful tissue integration. Herein, we begin by briefly reviewing macrophage cell origin and the key cytokine/chemokine markers of macrophage polarization and then describe which responses are favorable for both replacement and regenerative biomaterials. The body of the review focuses on macrophage polarization in response to inherent cues directly provided by biomaterials and the consequent cuesthat result from events related to biomaterial implantation. To conclude, a section on potential design principles for both replacement and regenerative biomaterials is presented. An in depth understanding of biomaterial cues to selectively polarize macrophages may prove beneficial in the design of a new generation of ‘immuno-informed’ biomaterials that can positively interact with the immune system to dictate a favorable macrophage response following implantation

Freeze dried, acellular nerve scaffolds with longitudinal channels for Peripheral Nerve Regeneration

Nerve injuries can occur due to automobile accidents, gunshot injuries, thermal or electric shock and lacerations caused by sharp instruments. At least 2 million people worldwide suffer from peripheral nerve injuries annually, with estimated costs of $7 billion incurred due to paralysis alone. The current "gold standard" is the autograft?sensory nerves (eg. Sural nerve) harvested from other parts of the patient. However, this poses disadvantages such as loss of sensation at donor site, insufficient length of graft and high cost in addition to a low success rate of 50$7 billion incurred due to paralysis alone. The current "gold standard" is the autograft'sensory nerves (eg. Sural nerve) harvested from other parts of the patient. However, this poses disadvantages such as loss of sensation at donor site, insufficient length of graft and high cost in addition to a low success rate of 50%. While several clinical alternatives composed of natural and synthetic biomaterials have been developed, none of them match the regenerative levels of the autograft. Allografts are nerves harvested from other human donors and may provide a viable alternative. However, patients who receive allograftsneed to undergo intense immunosuppression treatments due to host rejection of grafts. For this reason, it is of interest to remove cellular materialfrom the allografts. These acellular nerve grafts perform better than other clinically available nerve grafts but not as well as autografts. Current research on acellular nerve grafts focuses on the incorporation of additional components such as growth factors and cells that provide chemical guidance to regenerating axons.However, the pore size of acellular nerve grafts is ~10?m which does not allow effective penetration of cells or axons into the graft. In this study, we developed a decellularization protocol that sufficiently removed immunogenic cellular components without damaging the extracellular matrix. We then induced the formation of longitudinal channels of 20-60?m (diameter) inthese acellular grafts using a novel freeze drying process. These highly porous scaffolds exhibited similar tensileproperties asnative nerve tissue. Furthermore, when compared to acellular scaffoldsthat were not freeze dried, enhanced cell penetration and neurite outgrowth was observed with PC12 cells after 14 days of culture. The results of this study emphasize the importance of pore size on cellular infiltration. We therefore conclude that our novel freeze dried acellular scaffolds with longitudinal channelsserve as a basis for future peripheral nerve regenerative strategies using acellular allografts

Investigating biomaterial-based biophysical cues for modulating macrophage polarization towards bone regeneration applications

As one of the first cells to respond to biomaterial implantation, macrophages, through polarization into pro- (M1) and anti-inflammatory (M2) states, secrete cytokines and chemokines that determine the subsequent immune response and eventual success of an implanted biomaterial. Little is known about how biomaterial properties, especially biophysical cues, modulate the macrophage response and their interaction with mesenchymal stem cells (MSCs), another important cell type in the implant environment. The overall objective of the research presented in this thesis was to understand the role of biophysical cues presented by biomaterials in directing macrophage polarization, function, migration and interaction with MSCs, and to investigate the role of these biomaterial-based immune responses in promoting bone regeneration. The objective of Chapter 2 was to investigate the role of substrate stiffness (of collagen-coated 2D polyacrylamide hydrogels) in modulating macrophage morphology and polarization. Substrate stiffness ‘primed’ macrophages to a rounded cell shape and a predominantly anti-inflammatory M2-like phenotype on soft (11kPa) and medium (88kPa) stiffness gels and a spread shape and a pro-inflammatory M1-like phenotype on stiff (323kPa) gels. Soft gels also promoted a pro-inflammatory response in addition to an anti-inflammatory response from macrophages, resulting in a ‘hybrid’ phenotype. Building on these results, a comprehensive assessment of the role of substrate stiffness in modulating macrophage function and migration mode was undertaken in Chapter 3, with the aim of elucidating the mechanism of macrophage mechanosensing. It was shown that macrophage functions (phagocytosis and migration) were enhanced on softer gels, and impaired on stiff gels. Moreover, macrophages migrated using the RhoA Kinase (ROCK)-dependent amoeboid migration mode on softer gels and a proteolytic podosome-dependent mesenchymal migration mode on stiff gels. ROCK signalling was also shown to be involved in both macrophage polarization and migration, providing a mechanistic understanding of the role of the cytoskeleton in macrophage mechanosensing. Chapter 4 aimed to assess the role of substrate stiffness in MSC immunomodulation and their crosstalk with macrophages, in addition to evaluating the role of biomaterial-based macrophage responses in driving downstream events involved in bone regeneration. A stiffness-dependent cross-talk between MSCs and macrophages was established, with macrophages on softer gels producing decreased pro-inflammatory and increased anti-inflammatory factors in the presence of MSCs. It was also shown that factors secreted by the ‘hybrid’ macrophage phenotype on soft gels played an important role in mediating MSC osteogenesis. An alternative approach to investigate the immune response to biomaterial-based biophysical cues was undertaken in Chapter 5, which aimed to evaluate the role of material properties of collagen-based scaffolds such as stiffness, crosslinking and incorporated particle size in directing macrophage polarization and macrophage-mediated MSC osteogenesis. Macrophage response to 3D porous scaffold stiffness was shown to be dependent on the crosslinking method employed to modulate the stiffness, with no evident stiffness-related responses. Needle-shaped 5μm particles embedded in collagen-hydroxyapatite (Coll-HA) scaffolds promoted a ‘hybrid’ phenotype in macrophages, with upregulation of pro- and anti-inflammatory markers, which was not observed in scaffolds with spherical particles (5μm and 30μm). MSC osteogenesis was significantly increased when co-cultured with macrophages, with scaffolds with needle-shaped particles further enhancing this response through a pro-osteogenic macrophage phenotype. The differences between scaffold groups and between MSC mono-culture and co-culture with macrophages highlights the importance of analysing the macrophage response (in addition to MSCs alone) to biomaterial physical properties in directing subsequent osteogenic responses. In summary, this thesis has definitively demonstrated that biophysical properties of biomaterials like stiffness and particle size can be tailored to modulate macrophage polarization, function, migration and their interaction with MSCs, and has highlighted the role of biomaterial-based macrophage responses in mediating bone regeneration outcomes.</p

The shape and size of hydroxyapatite particles dictate inflammatory responses following implantation.

The extent of regeneration following biomaterial implantation is dependent on the microenvironment surrounding the implant. Since implant composition can have a profound effect on inflammation, it is essential to understand this process as a non-resolving inflammatory response can lead to fibrous encapsulation and insufficient integration. Incorporation of particulates into implants confers structural and functional benefits, thus optimizing particulate characteristics to enhance immune mediated efficacy is important. We investigated the relationship between the nature of hydroxyapatite (HA) particles and the innate immune response, focusing on how particle size (0.1 µm, 5 µm, 20 µm, 100 µm) and morphology (needle-shaped/spherical; smooth/rough surface) modulates inflammatory responses. We observed a shape and size-dependent activation of the NLRP3 inflammasome and IL-1β secretion; while needle-shaped and smaller HA particles significantly enhanced cytokine secretion, larger particles did not. Moreover, HA particle characteristics profoundly influenced patterns of innate immune cell recruitment and cytokine production following injection. While small, needle-shaped particles induced a strong inflammatory response, this was not observed with smooth, spherical particles of comparable size or with larger particles. These findings indicate that hydroxyapatite particle characteristics dictate immune cell recruitment and the ensuing inflammatory response, providing an opportunity to tailor HA particle characteristics to regulate immune responses induced after biomaterial implantation

Collagen/GAG scaffolds activated by RALA-siMMP-9 complexes with potential for improved diabetic foot ulcer healing

Impaired wound healing of diabetic foot ulcers has been linked to high MMP-9 levels at the wound site. Strategies aimed at the simultaneous downregulation of the MMP-9 level in situ and the regeneration of impaired tissue are critical for improved diabetic foot ulcer (DFU) healing. To fulfil this aim, collagen/GAG (Col/GAG) scaffolds activated by MMP-9-targeting siRNA (siMMP-9) were developed in this study. The siMMP-9 complexes were successfully formed by mixing the RALA cell penetrating peptide with siMMP-9. The complexes formulated at N:P ratios of 6 to 15 had a diameter around 100 nm and a positive zeta potential about 40 mV, making them ideal for cellular uptake. In 2 dimensional (2D) culture of human fibroblasts, the cellular uptake of the complexes surpassed 60% and corresponded to a 60% reduction in MMP-9 gene expression in low glucose culture. In high glucose culture, which induces over-expression of MMP-9 and therefore serves as an in vitro model mimicking conditions in DFU, the MMP-9 gene could be downregulated by around 90%. In the 3D culture of fibroblasts, the siMMP-9 activated Col/GAG scaffolds displayed excellent cytocompatibility and ~60% and 40% MMP-9 gene downregulation in low and high glucose culture, respectively. When the siMMP-9 complexes were applied to THP-1 macrophages, the primary cell type producing MMP-9 in DFU, MMP-9 gene expression was significantly reduced by 70% and 50% for M0 and M1 subsets, in 2D culture. In the scaffolds, the MMP-9 gene and protein level of M1 macrophages decreased by around 50% and 30% respectively. Taken together, this study demonstrates that the RALA-siMMP-9 activated Col/GAG scaffolds possess high potential as a promising regenerative platform for improved DFU healing

Functionalising Collagen-Based Scaffolds With Platelet-Rich Plasma for Enhanced Skin Wound Healing Potential

Porous collagen-glycosaminoglycan (collagen-GAG) scaffolds have shown promising clinical results for wound healing; however, these scaffolds do not replace the dermal and epidermal layer simultaneously and rely on local endogenous signaling to direct healing. Functionalizing collagen-GAG scaffolds with signaling factors, and/or additional matrix molecules, could help overcome these challenges. An ideal candidate for this is platelet-rich plasma (PRP) as it is a natural reservoir of growth factors, can be activated to form a fibrin gel, and is available intraoperatively. We tested the factors released from PRP (PRPr) and found that at specific concentrations, PRPr enhanced cell proliferation and migration and induced angiogenesis to a greater extent than fetal bovine serum (FBS) controls. This motivated us to develop a strategy to successfully incorporate PRP homogeneously within the pores of the collagen-GAG scaffolds. The composite scaffold released key growth factors for wound healing (FGF, TGFβ) and vascularization (VEGF, PDGF) for up to 14 days. In addition, the composite scaffold had enhanced mechanical properties (when compared to PRP gel alone), while providing a continuous upper surface of extracellular matrix (ECM) for keratinocyte seeding. The levels of the factors released from the composite scaffold were sufficient to sustain proliferation of key cells involved in wound healing, including human endothelial cells, mesenchymal stromal cells, fibroblasts, and keratinocytes; even in the absence of FBS supplementation. In functional in vitro and in vivo vascularization assays, our composite scaffold demonstrated increased angiogenic and vascularization potential, which is known to lead to enhanced wound healing. Upon pro-inflammatory induction, macrophages released lower levels of the pro-inflammatory marker MIP-1α when treated with PRPr; and released higher levels of the anti-inflammatory marker IL1-ra upon both pro- and anti-inflammatory induction when treated with the composite scaffold. Finally, our composite scaffold supported a co-culture system of human fibroblasts and keratinocytes that resulted in an epidermal-like layer, with keratinocytes constrained to the surface of the scaffold; by contrast, keratinocytes were observed infiltrating the PRP-free scaffold. This novel composite scaffold has the potential for rapid translation to the clinic by isolating PRP from a patient intraoperatively and combining it with regulatory approved scaffolds to enhance wound repair