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 \u3e45% 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

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

Highly versatile cell-penetrating peptide loaded scaffold for efficient and localised gene delivery to multiple cell types: From development to application in tissue engineering

Gene therapy has recently come of age with seven viral vector-based therapies gaining regulatory approval in recent years. In tissue engineering, non-viral vectors are preferred over viral vectors, however, lower transfection efficiencies and difficulties with delivery remain major limitations hampering clinical translation. This study describes the development of a novel multi-domain cell-penetrating peptide, GET, designed to enhance cell interaction and intracellular translocation of nucleic acids; combined with a series of porous collagen-based scaffolds with proven regenerative potential for different indications. GET was capable of transfecting cell types from all three germ layers, including stem cells, with an efficiency comparable to Lipofectamine® 3000, without inducing cytotoxicity. When implanted in vivo, GET gene-activated scaffolds allowed for host cell infiltration, transfection localized to the implantation site and sustained, but transient, changes in gene expression – demonstrating both the efficacy and safety of the approach. Finally, GET carrying osteogenic (pBMP-2) and angiogenic (pVEGF) genes were incorporated into collagen-hydroxyapatite scaffolds and with a single 2μg dose of therapeutic pDNA, induced complete repair of critical-sized bone defects within 4 weeks. GET represents an exciting development in gene therapy and by combining it with a scaffold-based delivery system offers tissue engineering solutions for a myriad of regenerative indications

Multifunctional biomaterials from the sea: Assessing the effects of chitosan incorporation into collagen scaffolds on mechanical and biological functionality

Natural biomaterials such as collagen show promise in tissue engineering applications due to their inherent bioactivity. The main limitation of collagen is its low mechanical strength and somewhat unpredictable and rapid degradation rate; however, combining collagen with another material, such as chitosan, can reinforce the scaffold mechanically and may improve the rate of degradation. Additionally, the high cost and the risk of prion transmission associated with mammal-derived collagen has prompted research into alternative sources such as marine-origin collagen. In this context, the overall goal of this study was to determine if the incorporation of chitosan into collagen scaffolds could improve the mechanical and biological properties of the scaffold. In addition the study assessed if collagen, derived from salmon skin (marine), can provide an alternative to collagen derived from bovine tendon (mammal) for tissue engineering applications. Scaffold architecture and mechanical properties were assessed as well as their ability to support mesenchymal stem cell growth and differentiation. Overall, the addition of chitosan to bovine and salmon skin-derived collagen scaffolds improved the mechanical properties, increasing the compressive strength, swelling ratio and prolonged the degradation rate. Mesenchymal stem cell (MSC) attachment and proliferation was most improved on the bovine-derived collagen scaffold containing a 75:25 ratio of collagen:chitosan, and when MSC osteogenic and chondrogenic potential on the scaffold was assessed, a significant increase in calcium production (p < 0.001) and sulfated glycosaminoglycan (sGAG) production (p < 0.001) was observed respectively. Regardless of chitosan content, the bovine-derived collagen scaffolds out-performed the salmon skin-derived collagen scaffolds, displaying a larger pore size and higher percentage porosity, more regular architecture, higher compressive modulus, a greater capacity for water uptake and allowed for more MSC proliferation and differentiation. This versatile scaffold incorporating the marine biomaterial chitosan show great potential as appropriate platforms for promoting orthopaedic tissue repair while the use of salmon skin-derived collagen may be more suitable in the repair of soft tissues such as skin.This work was funded by Science Foundation Ireland (SFI) through the Research Frontiers Programme (Grant No. 11/RFP/ENM/3063) and by the European Regional Development Fund (ERDF) through INTERREG 2007-2013 Program (POCTEP project 0687_NOVOMAR_1_P). Bovine collagen materials were provided by Integra Life Sciences, Inc. through a Material Transfer Agreement. Salmon skins were kindly offered by Pingo Doce, Braga (Portugal)

In vitro efficacy of a gene-activated nerve guidance conduit incorporating non-viral PEI-pDNA nanoparticles carrying genes encoding for NGF, GDNF and c-Jun.

Despite the success of tissue engineered nerve guidance conduits (NGCs) for the treatment of small peripheral nerve injuries, autografts remain the clinical gold standard for larger injuries. The delivery of neurotrophic factors from conduits might enhance repair for more effective treatment of larger injuries but the efficacy of such systems is dependent on a safe, effective platform for controlled and localised therapeutic delivery. Gene therapy might offer an innovative approach to control the timing, release and level of neurotrophic factor production by directing cells to transiently sustain therapeutic protein production in situ. In this study, a gene-activated NGC was developed by incorporating non-viral polyethyleneimine-plasmid DNA (PEI-pDNA) nanoparticles (N/P 7 ratio, 2 μg dose) with the pDNA encoding for nerve growth factor (NGF), glial derived neurotrophic factor (GDNF) or the transcription factor c-Jun. The physicochemical properties of PEI-pDNA nanoparticles, morphology, size and charge, were shown to be suitable for gene delivery and demonstrated high Schwann cell transfection efficiency (60 ± 13%) in vitro. While all three genes showed therapeutic potential in terms of enhancing neurotrophic cytokine production while promoting neurite outgrowth, delivery of the gene encoding for c-Jun showed the greatest capacity to enhance regenerative cellular processes in vitro. Ultimately, this gene-activated NGC construct was shown to be capable of transfecting both Schwann cells (S42 cells) and neuronal cells (PC12 and dorsal root ganglia) in vitro, demonstrating potential for future therapeutic applications in vivo. STATEMENT OF SIGNIFICANCE: The basic requirements of biomaterial-based nerve guidance conduits have now been well established and include being able to bridge a nerve injury to support macroscopic guidance between nerve stumps, while being strong enough to withstand longitudinal tension and circumferential compression, in addition to being mechanically sound to facilitate surgical handling and implantation. While meeting these criteria, conduits are still limited to the treatment of small defects clinically and might benefit from additional biochemical stimuli to enhance repair for the effective treatment of larger injuries. In this study, a gene activated conduit was successfully developed by incorporating non-viral nanoparticles capable of efficient Schwann cell and neuronal cell transfection with therapeutic genes in vitro, which showed potential to enhance repair in future applications particularly when taking advantage of the transcription factor c-Jun. This innovative approach may provide an alternative to conduits used as platforms for the delivery neurotrophic factors or genetically modified cells (viral gene therapy), and a potential solution for the unmet clinical need to repair large peripheral nerve injury effectively.</p

Activation of the SOX-5, SOX-6, and SOX-9 trio of transcription factors using a gene-activated scaffold stimulates mesenchymal stromal cell chondrogenesis and inhibits endochondral ossification

Current treatments for articular cartilage defects relieve symptoms but often only delay cartilage degeneration. Mesenchymal stem cells (MSCs) have shown chondrogenic potential but tend to undergo endochondral ossification when implanted in vivo. Harnessing factors governing joint development to functionalize biomaterial scaffolds, termed developmental engineering, might allow to prime host MSCs to regenerate mature articular cartilage in situ without requiring cell isolation or ex vivo expansion. Therefore, the aim of this study is to develop a gene-activated scaffold capable of delivering developmental cues to host MSCs, thus priming MSCs for articular cartilage differentiation and inhibiting endochondral ossification. It is shown that delivery of the SOX-Trio induced MSCs to over-express COL2A1 and ACAN and deposit a sulfated and collagen type II rich extracellular matrix while hypertrophic gene expression and collagen type X deposition is inhibited. When cell-free SOX-Trio-activated scaffolds are implanted ectopically in vivo, they induced spontaneous chondrogenesis without evidence of hypertrophy. MSCs pre-cultured on SOX-Trio-activated scaffolds prior to implantation differentiate into phenotypically stable chondrocytes as evidenced by a lack of collagen X expression or vascular invasion. This SOX-trio-activated scaffold represents a potent, single treatment, developmentally inspired strategy to prime MSCs in situ for articular cartilage defect repair. </p

Lineage-specific differences and regulatory networks governing human chondrocyte development

To address large gaps in our understanding of the molecular regulation of articular and growth plate cartilage development in humans, we used our directed differentiation approach to generate these distinct cartilage tissues from human embryonic stem cells. The resulting transcriptomic profiles of hESC-derived articular and growth plate chondrocytes were similar to fetal epiphyseal and growth plate chondrocytes, with respect to genes both known and previously unknown to cartilage biology. With the goal to characterize the regulatory landscapes accompanying these respective transcriptomes, we mapped chromatin accessibility in hESC-derived chondrocyte lineages, and mouse embryonic chondrocytes, using ATAC-sequencing. Integration of the expression dataset with the differentially accessible genomic regions revealed lineage-specific gene regulatory networks. We validated functional interactions of two transcription factors (TFs) (RUNX2 in growth plate chondrocytes and RELA in articular chondrocytes) with their predicted genomic targets. The maps we provide thus represent a framework for probing regulatory interactions governing chondrocyte differentiation. This work constitutes a substantial step towards comprehensive and comparative molecular characterizations of distinct chondrogenic lineages and sheds new light on human cartilage development and biology

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. +

Gene activated scaffolds incorporating star-shaped polypeptide-pDNA nanomedicines accelerate bone tissue regeneration in vivo

Increasingly, tissue engineering strategies such as the use of biomaterial scaffolds augmented with specific biological cues are being investigated to accelerate the regenerative process. For example, significant clinical challenges still exist in efficiently healing large bone defects which are above a critical size. Herein, we describe a cell-free, biocompatible and bioresorbable scaffold incorporating a novel star-polypeptide biomaterial as a gene vector. This gene-loaded scaffold can accelerate bone tissue repair in vivo in comparison to a scaffold alone at just four weeks post implantation in a critical sized bone defect. This is achieved via the in situ transfection of autologous host cells which migrate into the implanted collagen-based scaffold via gene-loaded, star-shaped poly(l-lysine) polypeptides (star-PLLs). In vitro, we demonstrate that star-PLL nanomaterials designed with 64 short poly(l-lysine) arms can be used to functionalise a range of collagen based scaffolds with a dual therapeutic cargo (pDual) of the bone-morphogenetic protein-2 plasmid (pBMP-2) and vascular endothelial growth factor plasmid (pVEGF). The versatility of this polymeric vector is highlighted in its ability to transfect Mesenchymal Stem Cells (MSCs) with both osteogenic and angiogenic transgenes in a 3D environment from a range of scaffolds with various macromolecular compositions. In vivo, we demonstrate that a bone-mimetic, collagen-hydroxyapatite scaffold functionalized with star-PLLs containing either 32-or 64-poly(l-lysine) arms can be used to successfully deliver this pDual cargo to autologous host cells. At the very early timepoint of just 4 weeks, we demonstrate the 64-star-PLL-pDual functionalised scaffold as a particularly efficient platform to accelerate bone tissue regeneration, with a 6-fold increase in new bone formation compared to a scaffold alone. Overall, this article describes for the first time the incorporation of novel star-polypeptide biomaterials carrying two therapeutic genes into a cell free scaffold which supports accelerated bone tissue formation in vivo

In vitro vascularization of tissue engineered constructs by non-viral delivery of pro-angiogenic genes

Vascularization is still one of the major challenges in tissue engineering. In the context of tissue regeneration, the formation of capillary-like structures is often triggered by the addition of growth factors which are associated with high cost, bolus release and short half-life. As an alternative to growth factors, we hypothesized that delivering genes-encoding angiogenic growth factors to cells in a scaffold microenvironment would lead to a controlled release of angiogenic proteins promoting vascularization, simultaneously offering structural support for new matrix deposition. Two non-viral vectors, chitosan (Ch) and polyethyleneimine (PEI), were tested to deliver plasmids encoding for vascular endothelial growth factor (pVEGF) and fibroblast growth factor-2 (pFGF2) to human dermal fibroblasts (hDFbs). hDFbs were successfully transfected with both Ch and PEI, without compromising the metabolic activity. Despite low transfection efficiency, superior VEGF and FGF-2 transgene expression was attained when pVEGF was delivered with PEI and when pFGF2 was delivered with Ch, impacting the formation of capillary-like structures by primary human dermal microvascular endothelial cells (hDMECs). Moreover, in a 3D microenvironment, when PEI-pVEGF and Ch-FGF2 were delivered to hDFbs, cells produced functional pro-angiogenic proteins which induced faster formation of capillary-like structures that were retained in vitro for longer time in a Matrigel assay. The dual combination of the plasmids resulted in a downregulation of the production of VEGF and an upregulation of FGF-2. The number of capillary-like segments obtained with this system was inferior to the delivery of plasmids individually but superior to what was observed with the non-transfected cells. This work confirmed that cell-laden scaffolds containing transfected cells offer a novel, selective and alternative approach to impact the vascularization during tissue regeneration. Moreover, this work provides a new platform for pathophysiology studies, models of disease, culture systems and drug screening