Localized Nanoparticle-Mediated Delivery of miR-29b Normalizes the Dysregulation of Bone Homeostasis Caused by Osteosarcoma whilst Simultaneously Inhibiting Tumor Growth.

Patients diagnosed with osteosarcoma undergo extensive surgical intervention and chemotherapy resulting in dismal prognosis and compromised quality of life owing to poor bone regeneration, which is further compromised with chemotherapy delivery. This study aims to investigate if localized delivery of miR-29b-which is shown to promote bone formation by inducing osteoblast differentiation and also to suppress prostate and cervical tumor growth-can suppress osteosarcoma tumors whilst simultaneously normalizing the dysregulation of bone homeostasis caused by osteosarcoma. Thus, the therapeutic potential of microRNA (miR)-29b is studied to promote bone remodeling in an orthotopic model of osteosarcoma (rather than in bone defect models using healthy mice), and in the context of chemotherapy, that is clinically relevant. A formulation of miR-29b:nanoparticles are developed that are delivered via a hyaluronic-based hydrogel to enable local and sustained release of the therapy and to study the potential of attenuating tumor growth whilst normalizing bone homeostasis. It is found that when miR-29b is delivered along with systemic chemotherapy, compared to chemotherapy alone, the therapy provided a significant decrease in tumor burden, an increase in mouse survival, and a significant decrease in osteolysis thereby normalizing the dysregulation of bone lysis activity caused by the tumor.

Macrophage metabolic profile is altered by hydroxyapatite particle size.

Since the recent observation that immune cells undergo metabolic reprogramming upon activation, there has been immense research in this area to not only understand the basis of such changes, but also to exploit metabolic rewiring for therapeutic benefit. In a resting state, macrophages preferentially utilise oxidative phosphorylation to generate energy; however, in the presence of immune cell activators, glycolytic genes are upregulated, and energy is generated through glycolysis. This facilitates the rapid production of biosynthetic intermediates and a pro-inflammatory macrophage phenotype. While this is essential to mount responses to infectious agents, more evidence is accumulating linking dysregulated metabolism to inappropriate immune responses. Given that certain biomaterials are known to promote an inflammatory macrophage phenotype, this prompted us to investigate if biomaterial particulates can impact on macrophage metabolism. Using micron and nano sized hydroxyapatite (HA), we demonstrate for the first time that these biomaterials can indeed drive changes in metabolism, and that this occurs in a size-dependent manner. We show that micronHA, but not nanoHA, particles upregulate surrogate markets of glycolysis including the glucose transporter (GLUT1), hexokinase 2 (HK2), GAPDH, and PKM2. Furthermore, we demonstrate that micronHA alters mitochondrial morphology and promotes a bioenergetic shift to favour glycolysis. Finally, we demonstrate that glycolytic gene expression is dependent on particle uptake and that targeting glycolysis attenuates the pro-inflammatory profile of micronHA-treated macrophages. These results not only further our understanding of biomaterial-based macrophage activation, but also implicate immunometabolism as a new area for consideration in intelligent biomaterial design and therapeutic targeting. STATEMENT OF SIGNIFICANCE: Several recent studies have reported that immune cell activation occurs concurrently with metabolic reprogramming. Furthermore, metabolic reprogramming of innate immune cells plays a prominent role in determining cellular phenotype and function. In this study we demonstrate that hydroxyapatite particle size alters macrophage metabolism, in turn driving their functional phenotype. Specifically, the pro-inflammatory phenotype promoted by micron-sized HA-particles is accompanied by changes in mitochondrial dynamics and a bioenergetic shift favouring glycolysis. This effect is not seen with nano-HA particles and can be attenuated upon inhibition of glycolysis. This study therefore not only identifies immunometabolism as a useful tool for characterising the immune response to biomaterials, but also highlights immunometabolism as a targetable aspect of the host response for therapeutic benefit.

A Spheroid Model of Early and Late-Stage Osteosarcoma Mimicking the Divergent Relationship between Tumor Elimination and Bone Regeneration.

Osteosarcoma is the most diagnosed bone tumor in children. The use of tissue engineering strategies after malignant tumor resection remains a subject of scientific controversy. As a result, there is limited research that focuses on bone regeneration postresection, which is further compromised following chemotherapy. This study aims to develop the first co-culture spheroid model for osteosarcoma, to understand the divergent relationship between tumor elimination and bone regeneration. By manipulating the ratio of stromal to osteosarcoma cells the modelled cancer state (early/late) is modified, as is evident by the increased tumor growth rates and an upregulation of a panel of well-established osteosarcoma prognostic genes. Validation of the authors' model is conducted by analyzing its ability to mimic the cytotoxic effects of the FDA-approved chemotherapeutic Doxorubicin. Next, the model is used to investigate what effect osteogenic supplements have, if any, on tumor growth. When their model is treated with osteogenic supplements, there is a stimulatory effect on the surrounding stromal cells. However, when treated with chemotherapeutics this stimulatory effect is significantly diminished. Together, the results of this study present a novel multicellular model of osteosarcoma and provide a unique platform for screening potential therapeutic options for osteosarcoma before conducting in vivo experiments.

Harnessing the innate and adaptive immune system for tissue repair and regeneration: Considering more than macrophages.

Tissue healing and regeneration is a complex, choreographed, spatiotemporal process involving a plethora of cell types, the activity of which is stringently regulated in order for effective tissue repair to ensue post injury. A number of globally prevalent conditions such as heart disease, organ failure, and severe musculoskeletal disorders require new therapeutic strategies to repair damaged or diseased tissue, particularly given an ageing population in which obesity, diabetes, and consequent tissue defects have reached epidemic proportions. This is further compounded by the lack of intrinsic healing and poor regenerative capacity of certain adult tissues. While vast progress has been made in the last decade regarding tissue regenerative strategies to direct self-healing, for example, through implantation of tissue engineered scaffolds, several challenges have hampered the clinical application of these technologies. Control of the immune response is growing as an attractive approach in regenerative medicine and it is becoming increasingly apparent that an in depth understanding of the interplay between cells of the immune system and tissue specific progenitor cells is of paramount importance. Furthermore, the integration of immunology and bioengineering promises to elevate the efficacy of biomaterial-based tissue repair and regeneration. In this review, we highlight the role played by individual immune cell subsets in tissue repair processes and describe new approaches that are being taken to direct appropriate healing outcomes via biomaterial mediated targeting of immune cell activity. STATEMENT OF SIGNIFICANCE: It is becoming increasingly apparent that controlling the immune response is as an attractive approach in regenerative medicine. Here, we propose that an in-depth understanding of immune system and tissue specific progenitor cell interactions may reveal mechanisms by which tissue healing and regeneration takes place, in addition to identifying novel therapeutic targets that could be used to enhance the tissue repair process. To date, most reviews have focused solely on macrophage subsets. This manuscript details the role of other innate and adaptive immune cells such as innate lymphoid cells (ILCs), natural killer (NK) cells and γδT cells (in addition to macrophages) in tissue healing. We also describe new approaches that are being taken to direct appropriate healing outcomes via biomaterial mediated cytokine and drug delivery.

Nano-particle mediated M2 macrophage polarization enhances bone formation and MSC osteogenesis in an IL-10 dependent manner.

Engineering a pro-regenerative immune response following scaffold implantation is integral to functional tissue regeneration. The immune response to implanted biomaterials is determined by multiple factors, including biophysical cues such as material stiffness, topography and particle size. In this study we developed an immune modulating scaffold for bone defect healing containing bone mimetic nano hydroxyapatite particles (BMnP). We first demonstrate that, in contrast to commercially available micron-sized hydroxyapatite particles, in-house generated BMnP preferentially polarize human macrophages towards an M2 phenotype, activate the transcription factor cMaf and specifically enhance production of the anti-inflammatory cytokine, IL-10. Furthermore, nano-particle treated macrophages enhance mesenchymal stem cell (MSC) osteogenesis in vitro and this occurs in an IL-10 dependent manner, demonstrating a direct pro-osteogenic role for this cytokine. BMnPs were also capable of driving pro-angiogenic responses in human macrophages and HUVECs. Characterization of immune cell subsets following incorporation of functionalized scaffolds into a rat femoral defect model revealed a similar profile, with micron-sized hydroxyapatite functionalized scaffolds eliciting pro-inflammatory responses characterized by infiltrating T cells and elevated expression of M1 macrophages markers compared to BMnP functionalized scaffolds which promoted M2 macrophage polarization, tissue vascularization and increased bone volume. Taken together these results demonstrate that nano-sized Hydroxyapatite has immunomodulatory potential and is capable of directing anti-inflammatory innate immune-mediated responses that are associated with tissue repair and regeneration.

Glyoxal cross-linking of solubilized extracellular matrix to produce highly porous, elastic, and chondro-permissive scaffolds for orthopedic tissue engineering.

Extracellular matrix (ECM)-derived implants hold great promise for tissue repair, but new strategies are required to produce efficiently decellularized scaffolds with the necessary porosity and mechanical properties to facilitate regeneration. In this study, we demonstrate that it is possible to produce highly porous, elastic, articular cartilage (AC) ECM-derived scaffolds that are efficiently decellularized, nonimmunogenic, and chondro-permissive. Pepsin solubilized porcine AC was cross-linked with glyoxal, lyophilized and then subjected to dehydrothermal treatment. The resulting scaffolds were predominantly collagenous in nature, with the majority of sulphated glycosaminoglycan (sGAG) and DNA removed during scaffold fabrication. Four scaffold variants were produced to examine the effect of both ECM (10 or 20 mg/mL) and glyoxal (5 or 10 mM) concentration on the mechanical and biological properties of the resulting construct. When seeded with human infrapatellar fat pad-derived stromal cells, the scaffolds with the lowest concentration of both ECM and glyoxal were found to promote the development of a more hyaline-like cartilage tissue, as evident by increased sGAG and type II collagen deposition. Furthermore, when cultured in the presence of human macrophages, it was found that these ECM-derived scaffolds did not induce the production of key proinflammatory cytokines, which is critical to success of an implantable biomaterial. Together these findings demonstrate that the novel combination of solubilized AC ECM and glyoxal crosslinking can be used to produce highly porous scaffolds that are sufficiently decellularized, highly elastic, chondro-permissive and do not illicit a detrimental immune response when cultured in the presence of human macrophages.

S100A8 acts as an autocrine priming signal for heme-induced human Mϕ pro-inflammatory responses in hemolytic inflammation.

Intravascular hemolysis, in addition to reducing red cell counts, incurs extensive vascular inflammation and oxidative stress. One product of hemolysis, heme, is a potent danger associated molecular pattern (DAMP), activating leukocytes and inducing cytokine expression and processing, among other pro-inflammatory effects. We explored pathways by which heme-induced inflammation may be amplified under sterile conditions. Incubation of human Mϕs, differentiated from CD14+ cells, with heme induced time- and concentration-dependent gene and protein expression of S100A8, a myeloid cell-derived alarmin. Human Mϕ stimulation with recombinant S100A8, in turn, induced robust pro-IL-1ß expression that was dependent upon NF-κB activation, gene transcription, and partially dependent upon TLR4-mediated signaling. Moreover, heme itself stimulated significant Mϕ pro-IL-1ß gene and protein expression via an S100A8-mediated mechanism and greatly amplified S100A8-driven NLRP3 inflammasome-mediated IL-1ß secretion. In vivo, induction of acute intravascular hemolysis in mice induced a rapid elevation of plasma S100A8 that could be abolished by hemopexin, a heme scavenger. Finally, plasma S100A8 levels were found to be significantly elevated in patients with the inherited hemolytic anemia, sickle cell anemia, when compared with levels in healthy individuals. In conclusion, we demonstrate that hemolytic processes are associated with S100A8 generation and that some of the inflammatory effects of heme may be amplified by autocrine S100A8 production. Findings suggest a mechanism by which hemolytic inflammation could be propagated via leukocyte priming by endogenous proteins, even in sterile inflammatory environments such as those that occur in the hemolytic diseases. S100A8 may represent a therapeutic target for reducing inflammation in hemolytic disorders.

Tissue-specific extracellular matrix scaffolds for the regeneration of spatially complex musculoskeletal tissues.

Biological scaffolds generated from tissue-derived extracellular matrix (ECM) are commonly used clinically for soft tissue regeneration. Such biomaterials can enhance tissue-specific differentiation of adult stem cells, suggesting that structuring different ECMs into multi-layered scaffolds can form the basis of new strategies for regenerating damaged interfacial tissues such as the osteochondral unit. In this study, mass spectrometry is used to demonstrate that growth plate (GP) and articular cartilage (AC) ECMs contain a unique array of regulatory proteins that may be particularly suited to bone and cartilage repair respectively. Applying a novel iterative freeze-drying method, porous bi-phasic scaffolds composed of GP ECM overlaid by AC ECM are fabricated, which are capable of spatially directing stem cell differentiation in vitro, promoting the development of graded tissues transitioning from calcified cartilage to hyaline-like cartilage. Evaluating repair 12-months post-implantation into critically-sized caprine osteochondral defects reveals that these scaffolds promote regeneration in a manner distinct to commercial control-scaffolds. The GP layer supports endochondral bone formation, while the AC layer stimulates the formation of an overlying layer of hyaline cartilage with a collagen fiber architecture better recapitulating the native tissue. These findings support the use of a bi-layered, tissue-specific ECM derived scaffolds for regenerating spatially complex musculoskeletal tissues.

Disease-Associated Particulates and Joint Inflammation; Mechanistic Insights and Potential Therapeutic Targets.

It is now well established that intra-articular deposition of endogenous particulates, such as osteoarthritis-associated basic calcium phosphate crystals, gout-associated monosodium urate crystals, and calcium deposition disease-associated calcium pyrophosphate crystals, contributes to joint destruction through the production of cartilage-degrading enzymes and pro-inflammatory cytokines. Furthermore, exogenous wear-debris particles, generated from prosthetic implants, drive periprosthetic osteolysis which impacts on the longevity of total joint replacements. Over the last few years, significant insight has been gained into the mechanisms through which these particulates exert their effects. Not only has this increased our understanding of the pathological processes associated with crystal deposition but it has also led to the identification of a number of therapeutic targets to treat particulate-associated disease. In this review, we discuss recent developments regarding the cellular events triggered by joint-associated particulates, as well as future directions in therapy for particulate-related arthropathies.

Orthopaedic implant materials drive M1 macrophage polarization in a spleen tyrosine kinase- and mitogen-activated protein kinase-dependent manner.

Total joint replacements (TJR) are costly procedures required to relieve pain and restore function in patients suffering from end-stage arthritis. Despite great progress in the development and durability of TJRs, the generation of prosthesis-associated wear particles over time leads to an inflammatory cascade which culminates in periprosthetic osteolysis. Studies suggest that wear particles drive the polarization/differentiation of immature macrophages towards a pro-inflammatory M1 phenotype rather than an anti-inflammatory M2 phenotype associated with normal bone and wound healing. This, in turn, contributes to the initiation of peri-implant inflammation. As a result, modulating M1 macrophage cytokine production has been recognised as a viable therapeutic option. The aim of this study was to examine the impact of hydroxyapatite (HA) and poly(methyl methacrylate) (PMMA) particles on human macrophage polarization by comparing their effect on M1/M2-associated gene expression using real-time PCR. Furthermore, using immunoblotting to assess kinase activation, we sought to identify the intracellular signalling molecules activated by PMMA/HA particles and to determine whether pharmacological blockade of these molecules impacts on macrophage phenotype and cytokine production as measured by ELISA. We report that wear particles preferentially polarize macrophages towards an M1 phenotype, an effect that is dependent on activation of the membrane proximal kinase, Syk and members of the mitogen-activated protein kinase (MAPK) family of signalling molecules. Pre-treatment of macrophages with Syk inhibitors (R788/piceatannol) or MAPK inhibitors (SB203580 and PD98059), not only prevents M1 polarization, but also attenuates production of key pro-inflammatory mediators that have been specifically implicated in periprosthetic osteolysis and osteoclast differentiation. STATEMENT OF SIGNIFICANCE: It is now well established that wear-debris particles from implanted materials drive deleterious inflammatory responses which can eventually lead to implant loosening. In this study, we provide further insight into the specific cellular pathways activated by wear particles in primary human immune cells. We demonstrate that PMMA bone cement and hydroxyapatite, a commonly used biomaterial, drive the polarization of macrophages towards an inflammatory phenotype and identify the specific signalling molecules that are activated in this process. Pre-treatment of macrophages with pharmacological inhibitors of these molecules in turn prevents macrophage polarization and dampens inflammatory cytokine production. Hence these signalling molecules represent potential therapeutic targets to treat or possibly prevent particulate induced osteolysis.