Assessment of Cell Cytotoxicity in 3D Biomaterial Scaffolds Following miRNA Transfection.

Assessment of cell cytotoxicity following transfection of cells with microRNA (miRNA) is an essential step in the evaluation of basic miRNA functional effects within cells in both 2D and 3D microenvironments. The lactate dehydrogenase (LDH) assay is a colorimetric assay that provides a basic, dependable method for determining cellular cytotoxicity through assessment of the level of plasma membrane damage in a cell population. Here, we describe the overexpression of miRNA in breast cancer cells when cultured in 3D collagen-based biomaterial scaffolds, achieved by Lipofectamine transfection, with subsequent examination of cell cytotoxicity using the LDH assay.

Development of a Gene-Activated Scaffold Incorporating Multifunctional Cell-Penetrating Peptides for pSDF-1α Delivery for Enhanced Angiogenesis in Tissue Engineering Applications.

Non-viral gene delivery has become a popular approach in tissue engineering, as it permits the transient delivery of a therapeutic gene, in order to stimulate tissue repair. However, the efficacy of non-viral delivery vectors remains an issue. Our lab has created gene-activated scaffolds by incorporating various non-viral delivery vectors, including the glycosaminoglycan-binding enhanced transduction (GET) peptide into collagen-based scaffolds with proven osteogenic potential. A modification to the GET peptide (FLR) by substitution of arginine residues with histidine (FLH) has been designed to enhance plasmid DNA (pDNA) delivery. In this study, we complexed pDNA with combinations of FLR and FLH peptides, termed GET* nanoparticles. We sought to enhance our gene-activated scaffold platform by incorporating GET* nanoparticles into collagen-nanohydroxyapatite scaffolds with proven osteogenic capacity. GET* N/P 8 was shown to be the most effective formulation for delivery to MSCs in 2D. Furthermore, GET* N/P 8 nanoparticles incorporated into collagen-nanohydroxyapatite (coll-nHA) scaffolds at a 1:1 ratio of collagen:nanohydroxyapatite was shown to be the optimal gene-activated scaffold. pDNA encoding stromal-derived factor 1α (pSDF-1α), an angiogenic chemokine which plays a role in BMP mediated differentiation of MSCs, was then delivered to MSCs using our optimised gene-activated scaffold platform, with the aim of significantly increasing angiogenesis as an important precursor to bone repair. The GET* N/P 8 coll-nHA scaffolds successfully delivered pSDF-1α to MSCs, resulting in a significant, sustained increase in SDF-1α protein production and an enhanced angiogenic effect, a key precursor in the early stages of bone repair.

The Effect of Fluid Flow Shear Stress and Substrate Stiffness on Yes-Associated Protein (YAP) Activity and Osteogenesis in Murine Osteosarcoma Cells.

Osteosarcoma (OS) is an aggressive bone cancer originating in the mesenchymal lineage. Prognosis for metastatic disease is poor, with a mortality rate of approximately 40%; OS is an aggressive disease for which new treatments are needed. All bone cells are sensitive to their mechanical/physical surroundings and changes in these surroundings can affect their behavior. However, it is not well understood how OS cells specifically respond to fluid movement, or substrate stiffness-two stimuli of relevance in the tumor microenvironment. We used cells from spontaneous OS tumors in a mouse engineered to have a bone-specific knockout of pRb-1 and p53 in the osteoblast lineage. We silenced Sox2 (which regulates YAP) and tested the effect of fluid flow shear stress (FFSS) and substrate stiffness on YAP expression/activity-which was significantly reduced by loss of Sox2, but that effect was reversed by FFSS but not by substrate stiffness. Osteogenic gene expression was also reduced in the absence of Sox2 but again this was reversed by FFSS and remained largely unaffected by substrate stiffness. Thus we described the effect of two distinct stimuli on the mechanosensory and osteogenic profiles of OS cells. Taken together, these data suggest that modulation of fluid movement through, or stiffness levels within, OS tumors could represent a novel consideration in the development of new treatments to prevent their progression.

Influences of the 3D microenvironment on cancer cell behaviour and treatment responsiveness: A recent update on lung, breast and prostate cancer models.

The majority of in vitro studies assessing cancer treatments are performed in two-dimensional (2D) monolayers and are subsequently validated in in vivo animal models. However, 2D models fail to accurately model the tumour microenvironment. Furthermore, animal models are not directly applicable to mimic the human scenario. Three-dimensional (3D) culture models may help to address the discrepancies of 2D and animal models. When cancer cells escape the primary tumour, they can invade at distant organs building secondary tumours, called metastasis. The development of metastasis leads to a dramatic decrease in the life expectancy of patients. Therefore, 3D systems to model the microenvironment of metastasis have also been developed. Several studies have demonstrated changes in cell behaviour and gene expression when cells are cultured in 3D compared to 2D and concluded a better comparability to cells in vivo. Of special importance is the effect seen in response to anti-cancer treatments as models are built primarily to serve as drug-testing platforms. This review highlights these changes between cancer cells grown in 2D and 3D models for some of the most common cancers including lung, breast and prostate tumours. In addition to models aiming to mimic the primary tumour site, the effects of 3D cell culturing in bone metastasis models are also described. STATEMENT OF SIGNIFICANCE: Most in vitro studies in cancer research are performed in 2D and are subsequently validated in in vivo animal models. However, both models possess numerous limitations: 2D models fail to accurately model the tumour microenvironment while animal models are expensive, time-consuming and can differ considerably from humans. It is accepted that the cancer microenvironment plays a critical role in the disease, thus, 3D models have been proposed as a potential solution to address the discrepancies of 2D and animal models. This review highlights changes in cell behaviour, including proliferation, gene expression and chemosensitivity, between cancer cells grown in 2D and 3D models for some of the most common cancers including lung, breast and prostate cancer as well as bone metastasis.

Rapid bone repair with the recruitment of CD206+M2-like macrophages using non-viral scaffold-mediated miR-133a inhibition of host cells.

microRNAs offer vast therapeutic potential for multiple disciplines. From a bone perspective, inhibition of miR-133a may offer potential to enhance Runx2 activity and increase bone repair. This study aims to assess the therapeutic capability of antagomiR-133a delivery from collagen-nanohydroxyapatite (coll-nHA) scaffolds following cell-free implantation in rat calvarial defects (7 mm diameter). This is, to the best of our knowledge, the first report of successful in vivo antagomiR uptake in host cells of fully immunocompetent animals without distribution to other off-target tissues. Our results demonstrate the localized release of antagomiR-133a to the implant site at 1 week post-implantation with increased calcium deposits already evident in the antagomiR-133a loaded scaffolds at this early timepoint. This was followed by an approximate 2-fold increase in bone volume versus antagomiR-free scaffolds and a significant 10-fold increase over the empty defect controls, after just 4 weeks. An increase in host CD206+ cells suggests an accelerated pro-remodeling response by M2-like macrophages accompanying bone repair with this treatment. Overall, this non-viral scaffold-mediated antagomiR-133a delivery platform demonstrates capability to accelerate bone repair in vivo - without the addition of exogenous cells - and underlines the role of M2 macrophage-like cells in directing accelerated bone repair. Expanding the repertoire of this platform to deliver alternative miRNAs offers exciting possibilities for a variety of therapeutic indications. STATEMENT OF SIGNIFICANCE: microRNAs, small non-coding RNA molecules involved in gene regulation, may have potential as a new class of bone healing therapeutics as they can enhance the regenerative capacity of bone-forming cells. We developed a collagen-nanohydroxyapatite-microRNA scaffold system to investigate whether miR133a inhibition can enhance osteogenesis in rat MSCs and ultimately accelerate endogenous bone repair by host cells in vivo without pre-seeding cells prior to implantation. Overall, this off-the-shelf, non-viral scaffold-mediated antagomiR-133a delivery platform demonstrates capability to accelerate bone repair in vivo - without the requirement of exogenous cells - and highlights the role of CD206+M2 macrophage-like cells in guiding accelerated bone repair. Translating the repertoire of this platform to deliver alternative miRNAs offers exciting possibilities for a vast myriad of therapeutic indications.

Scaffold-Based Delivery of Nucleic Acid Therapeutics for Enhanced Bone and Cartilage Repair.

Recent advances in tissue engineering have made progress toward the development of biomaterials capable of the delivery of growth factors, such as bone morphogenetic proteins, in order to promote enhanced tissue repair. However, controlling the release of these growth factors on demand and within the desired localized area is a significant challenge and the associated high costs and side effects of uncontrolled delivery have proven increasingly problematic in clinical orthopedics. Gene therapy may be a valuable tool to avoid the limitations of local delivery of growth factors. Following a series of setbacks in the 1990s, the field of gene therapy is now seeing improvements in safety and efficacy resulting in substantial clinical progress and a resurgence in confidence. Biomaterial scaffold-mediated gene therapy provides a template for cell infiltration and tissue formation while promoting transfection of cells to engineer therapeutic proteins in a sustained but ultimately transient fashion. Additionally, scaffold-mediated delivery of RNA-based therapeutics can silence specific genes associated with orthopedic pathological states. This review will provide an overview of the current state-of-the-art in the field of gene-activated scaffolds and their use within orthopedic tissue engineering applications. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1671-1680, 2019.

Harnessing an Inhibitory Role of miR-16 in Osteogenesis by Human Mesenchymal Stem Cells for Advanced Scaffold-Based Bone Tissue Engineering.

MicroRNA (miRNA) therapeutics is increasingly being developed to either target bone-related diseases such as osteoporosis and osteoarthritis or as the basis for novel bone tissue engineering strategies. A number of miRNAs have been reported as potential osteo-therapeutics but no consensus has yet been established on the optimal target. miR-16 has been studied extensively in nonosteogenic functions and used as functionality reporter target in the development of nonviral miRNA delivery platforms. This study hypothesized that miR-16 may also play an inhibitory role in osteogenesis due to its ability to directly target Smad5 and AcvR2a. This study thus aimed to assess the potential of miR-16 inhibition to increase osteogenesis in human mesenchymal stem cells (hMSCs) using a previously established miRNA delivery platform composed of nanohydroxyapatite (nHA) particles as nonviral vectors in combination with collagen-nHA scaffolds designed specifically for bone repair. Initial results showed that antagomiR-16 delivery efficiently increased the relative levels of both putative targets and Runx2, the key transcription factor for osteogenesis, while also increasing osteocalcin levels. Furthermore, significant increases in mineral calcium deposition by hMSCs were found in both monolayer and most importantly in scaffold-based osteodifferentiation studies, ultimately demonstrating that miR-16 inhibition further enhances the therapeutic potential of a scaffold with known potential for bone repair applications and thus holds significant therapeutic potential as a novel bone tissue engineering strategy. Furthermore, we suggest that harnessing the additional functions known to miR-16 by incorporating either its enhancers or inhibitors to tissue-specific tailored scaffolds provides exciting opportunities for a diverse range of therapeutic indications.

Scaffold-Based microRNA Therapies in Regenerative Medicine and Cancer.

microRNA-based therapies are an advantageous strategy with applications in both regenerative medicine (RM) and cancer treatments. microRNAs (miRNAs) are an evolutionary conserved class of small RNA molecules that modulate up to one third of the human nonprotein coding genome. Thus, synthetic miRNA activators and inhibitors hold immense potential to finely balance gene expression and reestablish tissue health. Ongoing industry-sponsored clinical trials inspire a new miRNA therapeutics era, but progress largely relies on the development of safe and efficient delivery systems. The emerging application of biomaterial scaffolds for this purpose offers spatiotemporal control and circumvents biological and mechanical barriers that impede successful miRNA delivery. The nascent research in scaffold-mediated miRNA therapies translates know-how learnt from studies in antitumoral and genetic disorders as well as work on plasmid (p)DNA/siRNA delivery to expand the miRNA therapies arena. In this progress report, the state of the art methods of regulating miRNAs are reviewed. Relevant miRNA delivery vectors and scaffold systems applied to-date for RM and cancer treatment applications are discussed, as well as the challenges involved in their design. Overall, this progress report demonstrates the opportunity that exists for the application of miRNA-activated scaffolds in the future of RM and cancer treatments.

Translating the role of osteogenic-angiogenic coupling in bone formation: Highly efficient chitosan-pDNA activated scaffolds can accelerate bone regeneration in critical-sized bone defects.

The clinical translation of bioactive scaffolds for the treatment of large segmental bone defects has remained a challenge due to safety and efficacy concerns as well as prohibitive costs. The design of an implantable, biocompatible and resorbable device, which can fill the defect space, allow for cell infiltration, differentiation and neovascularisation, while also recapitulating the natural repair process and inducing cells to lay down new bone tissue, would alleviate the problems with existing treatments. We have developed a gene-activated scaffold platform using a bone-mimicking collagen hydroxyapatite scaffold loaded with chitosan nanoparticles carrying genes encoding osteogenic (BMP-2) and angiogenic (VEGF) proteins. With a single treatment, protein expression by mesenchymal stem cells (MSCs) seeded onto the scaffold is sustained for up to 28 days and is functional in inducing MSC osteogenesis. The in vivo safety and efficacy of this gene-activated scaffold platform was demonstrated resulting in the successful transfection of host cells, abrogating the requirement for multiple procedures to isolate cells or ex vivo cell culture. Furthermore, the level of bone formation at the exceptionally early time-point of 28 days was comparable to that achieved following recombinant BMP-2 protein delivery after 8 weeks in vivo, without the adverse side effects and at a fraction of the cost. This naturally derived cell-free gene-activated scaffold thus represents a new 'off-the-shelf' product capable of accelerating bone repair in critical-sized bone defects.

Formulation and Evaluation of Anisamide-Targeted Amphiphilic Cyclodextrin Nanoparticles To Promote Therapeutic Gene Silencing in a 3D Prostate Cancer Bone Metastases Model.

In recent years, RNA interference (RNAi) has emerged as a potential therapeutic offering the opportunity to treat a wide range of diseases, including prostate cancer. Modified cyclodextrins have emerged as effective gene delivery vectors in a range of disease models. The main objective of the current study was to formulate anisamide-targeted cyclodextrin nanoparticles to interact with the sigma receptor (overexpressed on the surface of prostate cancer cells). The inclusion of octaarginine in the nanoparticle optimized uptake and endosomal release of siRNA in two different prostate cancer cell lines (PC3 and DU145 cells). Resulting nanoparticles were less than 200 nm in size with a cationic surface charge (∼+20 mV). In sigma receptor-positive cell lines, the uptake of anisamide-targeted nanoparticles was reduced in the presence of the sigma receptor competitive ligand, haloperidol. When cells were transfected in 2D, the levels of PLK1 mRNA knockdown elicited by targeted versus untargeted nanoparticles tended to be greater but the differences were not statistically different. In contrast, when cells were grown on 3D scaffolds, recapitulating bone metastasis, targeted formulations showed significantly higher levels of PLK1 mRNA knockdown (46% for PC3 and 37% for DU145, p < 0.05). To our knowledge, this is the first time that a targeted cyclodextrin has been used to transfect prostate cancer cells in a 3D model of bone metastasis.

Nanoparticle-mediated siRNA delivery assessed in a 3D co-culture model simulating prostate cancer bone metastasis.

siRNA has emerged as a potential therapeutic for the treatment of prostate cancer but effective delivery remains a major barrier to its clinical application. This study aimed to develop and characterise a 3D in vitro co-culture model to simulate prostate cancer bone metastasis and to assess the ability of the model to investigate nanoparticle-mediated siRNA delivery and gene knockdown. PC3 or LNCaP prostate cancer cells were co-cultured with hFOB 1.19 osteoblast cells in 2D on plastic tissue culture plates and in 3D on collagen scaffolds mimicking the bone microenvironment. To characterise the co-culture model, cell proliferation, enzyme secretion and the utility of two different gene delivery vectors to mediate siRNA uptake and gene knockdown were assessed. Cell proliferation was reduced by∼50% by day 7 in the co-culture system relative to monoculture (PC3 and LNCaP co-cultures, in 2D and 3D) and an enhanced level of MMP9 (a marker of bone metastasis) was secreted into the media (1.2-4-fold increase depending on the co-culture system). A cationic cyclodextrin gene delivery vector proved significantly less toxic in the co-culture system relative to the commercially available vector Lipofectamine 2000(®). In addition, knockdown of both the GAPDH gene (minimum 15%) and RelA subunit of the NF-κB transcription factor (minimum 20%) was achieved in 2D and 3D cell co-cultures. Results indicate that the prostate cancer-osteoblast in vitro co-culture model was more physiologically relevant vs the monoculture. This model has the potential to help improve the design and efficacy of gene delivery formulations, to more accurately predict in vivo performance and, therefore, to reduce the risk of product failure in late-stage clinical development.

Next generation bone tissue engineering: non-viral miR-133a inhibition using collagen-nanohydroxyapatite scaffolds rapidly enhances osteogenesis.

Bone grafts are the second most transplanted materials worldwide at a global cost to healthcare systems valued over $30 billion every year. The influence of microRNAs in the regenerative capacity of stem cells offers vast therapeutic potential towards bone grafting; however their efficient delivery to the target site remains a major challenge. This study describes how the functionalisation of porous collagen-nanohydroxyapatite (nHA) scaffolds with miR-133a inhibiting complexes, delivered using non-viral nHA particles, enhanced human mesenchymal stem cell-mediated osteogenesis through the novel focus on a key activator of osteogenesis, Runx2. This study showed enhanced Runx2 and osteocalcin expression, as well as increased alkaline phosphatase activity and calcium deposition, thus demonstrating a further enhanced therapeutic potential of a biomaterial previously optimised for bone repair applications. The promising features of this platform offer potential for a myriad of applications beyond bone repair and tissue engineering, thus presenting a new paradigm for microRNA-based therapeutics.

Life in 3D is never flat: 3D models to optimise drug delivery.

The development of safe, effective and patient-acceptable drug products is an expensive and lengthy process and the risk of failure at different stages of the development life-cycle is high. Improved biopharmaceutical tools which are robust, easy to use and accurately predict the in vivo response are urgently required to help address these issues. In this review the advantages and challenges of in vitro 3D versus 2D cell culture models will be discussed in terms of evaluating new drug products at the pre-clinical development stage. Examples of models with a 3D architecture including scaffolds, cell-derived matrices, multicellular spheroids and biochips will be described. The ability to simulate the microenvironment of tumours and vital organs including the liver, kidney, heart and intestine which have major impact on drug absorption, distribution, metabolism and toxicity will be evaluated. Examples of the application of 3D models including a role in formulation development, pharmacokinetic profiling and toxicity testing will be critically assessed. Although utilisation of 3D cell culture models in the field of drug delivery is still in its infancy, the area is attracting high levels of interest and is likely to become a significant in vitro tool to assist in drug product development thus reducing the requirement for unnecessary animal studies.

The use of collagen-based scaffolds to simulate prostate cancer bone metastases with potential for evaluating delivery of nanoparticulate gene therapeutics.

Prostate cancer bone metastases are a leading cause of cancer-related death in men with current treatments offering only marginally improved rates of survival. Advances in the understanding of the genetic basis of prostate cancer provide the opportunity to develop gene-based medicines capable of treating metastatic disease. The aim of this work was to establish a 3D cell culture model of prostate cancer bone metastasis using collagen-based scaffolds, to characterise this model, and to assess the potential of the model to evaluate delivery of gene therapeutics designed to target bone metastases. Two prostate cancer cell lines (PC3 and LNCaP) were cultured in 2D standard culture and compared to 3D cell growth on three different collagen-based scaffolds (collagen and composites of collagen containing either glycosaminoglycan or nanohydroxyapatite). The 3D model was characterised for cell proliferation, viability and for matrix metalloproteinase (MMP) enzyme and Prostate Specific Antigen (PSA) secretion. Chemosensitivity to docetaxel treatment was assessed in 2D in comparison to 3D. Nanoparticles (NPs) containing siRNA formulated using a modified cyclodextrin were delivered to the cells on the scaffolds and gene silencing was quantified. Both prostate cancer cell lines actively infiltrated and proliferated on the scaffolds. Cell culture in 3D resulted in reduced levels of MMP1 and MMP9 secretion in PC3 cells. In contrast, LNCaP cells grown in 3D secreted elevated levels of PSA, particularly on the scaffold composed of collagen and glycosaminoglycans. Both cell lines grown in 3D displayed increased resistance to docetaxel treatment. The cyclodextrin.siRNA nanoparticles achieved cellular uptake and knocked down the endogenous GAPDH gene in the 3D model. In conclusion, development of a novel 3D cell culture model of prostate cancer bone metastasis has been initiated resulting, for the first time, in the successful delivery of gene therapeutics in a 3D in vitro model. Further enhancement of this model will help elucidate the pathogenesis of prostate cancer and also accelerate the design of effective therapies which can penetrate into the bone microenvironment for prostate cancer therapy.

Development of a gene-activated scaffold platform for tissue engineering applications using chitosan-pDNA nanoparticles on collagen-based scaffolds.

Biomaterial scaffolds that support cell infiltration and tissue formation can also function as platforms for the delivery of therapeutics such as drugs, proteins, and genes. As burst release of supraphysiological quantities of recombinant proteins can result in adverse side effects, the objective of this study was to explore the potential of a series of collagen-based scaffolds, developed in our laboratory, as gene-activated scaffold platforms with potential in a range of tissue engineering applications. The potential of chitosan, a biocompatible material derived from the shells of crustaceans, as a gene delivery vector was assessed using mesenchymal stem cells (MSCs). A transfection efficiency of >45% is reported which is similar to what is achieved with polyethyleneimine (PEI), a non-viral gold standard vector, without causing cytotoxic side effects. When the optimised chitosan nanoparticles were incorporated into a series of collagen-based scaffolds, sustained transgene expression from MSCs seeded on the scaffolds was maintained for up to 28days and interestingly the composition of the scaffold had an effect on transfection efficiency. These results demonstrate that by simply varying the scaffold composition and the gene (or combinations thereof) chosen; the system has potential for a myriad of therapeutic applications.

A novel collagen-nanohydroxyapatite microRNA-activated scaffold for tissue engineering applications capable of efficient delivery of both miR-mimics and antagomiRs to human mesenchymal stem cells.

Manipulation of gene expression through the use of microRNAs (miRNAs) offers tremendous potential for the field of tissue engineering. However, the lack of sufficient site-specific and bioactive delivery systems has severely hampered the clinical translation of miRNA-based therapies. In this study, we developed a novel non-viral bioactive delivery platform for miRNA mimics and antagomiRs to allow for a vast range of therapeutic applications. By combining nanohydroxyapatite (nHA) particles with reporter miRNAs (nanomiRs) and collagen-nanohydroxyapatite scaffolds, this work introduces the first non-viral, non-lipid platform to date, capable of efficient delivery of mature miRNA molecules to human mesenchymal stem cells (hMSCs), a particularly difficult cell type to transfect effectively, with minimal treatment-associated cytotoxicity. Firstly, miRNAs were successfully delivered to hMSCs in monolayer, with internalisation efficiencies of 17.4 and 39.6% for nanomiR-mimics and nanoantagomiRs respectively, and both nanomiR-mimics and nanoantagomiRs yielded sustained interfering activity of greater than 90% in monolayer over 7 days. When applied to 3D scaffolds, significant RNA interference of 20% for nanomiR-mimics and 88.4% for nanoantagomiRs was achieved with no cytotoxicity issues over a 7 day period. In summary, in-house synthesised non-viral nHA particles efficiently delivered reporter miRNAs both in monolayer and on scaffolds demonstrating the immense potential of this innovative miRNA-activated scaffold system for tissue engineering applications.

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.

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.