Bone Regeneration Using Gene-Activated Matrices.

Gene transfer approaches to the healing of bone and cartilage.

Minicircle DNA-based Gene Therapy Coupled With Immune Modulation Permits Long-term Expression of α-L-Iduronidase in Mice With Mucopolysaccharidosis Type I

Ex vivo regional gene therapy is a promising tissue-engineering strategy for bone regeneration: osteogenic mesenchymal stem cells (MSCs) can be genetically modified to express an osteoinductive stimulus (e.g., bone morphogenetic protein-2), seeded onto an osteoconductive scaffold, and then implanted into a bone defect to exert a therapeutic effect. Compared to recombinant human BMP-2 (rhBMP-2), which is approved for clinical use, regional gene therapy may have unique benefits related to the addition of MSCs and the sustained release of BMP-2. However, the cellular and transcriptional mechanisms regulating the response to these two strategies for BMP-2 mediated bone regeneration are largely unknown. Here, for the first time, we performed single-cell RNA sequencing (10x Genomics) of hematoma tissue in six rats with critical-sized femoral defects that were treated with either regional gene therapy or rhBMP-2. Our unbiased bioinformatic analysis of 2393 filtered cells in each group revealed treatment-specific differences in their cellular composition, transcriptional profiles, and cellular communication patterns. Gene therapy treatment induced a more robust chondrogenic response, as well as a decrease in the proportion of fibroblasts and the expression of profibrotic pathways. Additionally, gene therapy was associated with an anti-inflammatory microenvironment; macrophages expressing canonical anti-inflammatory markers were more common in the gene therapy group. In contrast, pro-inflammatory markers were more highly expressed in the rhBMP-2 group. Collectively, the results of our study may offer insights into the unique pathways through which ex vivo regional gene therapy can augment bone regeneration compared to rhBMP-2. Furthermore, an improved understanding of the cellular pathways involved in segmental bone defect healing may allow for the further optimization of regional gene therapy or other bone repair strategies.

piggyBac Transposon-mediated Long-term Gene Expression in Mice

Event Abstract Back to Event Bone agumentation by gene activated matrix composed of plasmid DNA, BMP4 or Runx2, embedded in atelocollagen Mayumi Umebayashi1, Yoshinori Sumita1 and Izumi Asahina1 1 Nagasaki University, Oral surgery, Japan Background: To date, therapeutic method for in vivo gene delivery has not been established on bone engineering though their potential usefulness has been suggested. For clinical applications, an effective condition should be developed to transfer the genes in vivo without any of transfection-reagents or virus-vectors. Objective: The aim of this study is to have a new understanding on the usefulness of delivering non-viral GAM without cell transplantation or any transfection reagents/kits for facilitating clinical setting of bone engineering. Methods: In this study, to facilitate the clinical setting of this strategy, we simply investigated whether manufactured gene activated-matrix (GAM) with atelocollagen containing a certain amount of plasmid (p) DNA encoding osteogenic-proteins could augment the cranial bone in rat. GAMs were manufactured by mixing 0.02, 0.1, 0.5 or 1 mg of AcGFP plasmid-vectors harboring cDNA of BMP4 (pBMP4) or Runx2 (pRunx2) with 2% bovine-atelocollagen and β-TCP granules. Before manufactured GAMs, to determine the biological activity of generated pDNAs, we confirmed GFP-expression and increased-level of ALP activities in MC3T3-E1 cells transfected with pBMP4 or pRunx2 during culture. Then, GAMs were lyophilized and transplanted to onlay placement on the cranium. Results: At 2 weeks of transplantation, GFP-expressing cells could be detectable in only GAMs containing 0.5 and 1 mg of AcGFP plasmid vectors. Then, at 4 weeks, significant bone formation was recognized in GAMs containing 0.5 and 1 mg of pDNAs encoding BMP4 or Runx2 but not in 0.02 or 0.1 mg of GAMs. These newly formed bone tissues surrounded by osteocalcin-stained area were augmented markedly until 8 weeks after transplantation. In contrast, minimal bone formation was observed in GAMs without harboring cDNA of osteogenic-proteins. Meanwhile, when GAMs were transplanted to the cranial bone defect, bone formation was detectable in specimens containing 1 mg of pBmp4 or pRunx2 at 8 weeks as well. Conclusion: Thus, atelocollagen-based GAM reliably could form the engineered-bone even for the vertical augmentation when contained a certain amount of plasmid-vectors encoding osteogenic-proteins. This study supports facilitating the clinical application of GAM for bone engineering. Keywords: Bone Regeneration, Tissue Engineering, biomaterial, gene delivery Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Biomimetic materials Citation: Umebayashi M, Sumita Y and Asahina I (2016). Bone agumentation by gene activated matrix composed of plasmid DNA, BMP4 or Runx2, embedded in atelocollagen. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.00373 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Mayumi Umebayashi Yoshinori Sumita Izumi Asahina Google Mayumi Umebayashi Yoshinori Sumita Izumi Asahina Google Scholar Mayumi Umebayashi Yoshinori Sumita Izumi Asahina PubMed Mayumi Umebayashi Yoshinori Sumita Izumi Asahina Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Nonviral Gene Delivery Embedded in Biomimetically Mineralized Matrices for Bone Tissue Engineering

Tissue engineering offers new treatments for disorders such as temporomandibular joint disorder which among others frequently afflicts patients. In this thesis, tissue engineering strategies are explored with an emphasis on addressing bone tissue repair. The primary technique used was gene therapy in conjunction with a matrix to provide therapeutic growth factors and a foundation for new bone to form on, respectively. The first chapter describes the previous research of tissue engineering to address TMD; the next four chapters then describe optimizations and combination of gene therapy to improve current bone tissue engineering outcomes. The final chapter looks at the future of tissue engineering in the field of bone tissue repair with a focus on TMD.Damage or disease in the TMJ adversely affects masticatory function and speaking, reducing patients' quality of life. Effective treatment options for patients suffering from severe temporomandibular joint disorders are in high demand because surgical options are restricted to removal of damaged tissue or complete replacement of the joint with prosthetics. Tissue engineering approaches for the temporomandibular joint are a promising alternative to the limited clinical treatment options. However, tissue engineering is still a developing field and only in its formative years for the temporomandibular joint. This chapter outlines the anatomical and physiological characteristics of the temporomandibular joint, clinical management of temporomandibular joint disorder, and current perspectives in the tissue engineering approach for the temporomandibular joint disorder. In the subsequent chapters, the goal was to investigate different bone tissue engineering approaches with gene therapy. The first was to identify if calcium ion (Ca2+) concentration affects the transfection of bone marrow stromal cells because these cells play a major role in bone healing and can infiltrate gene‐activated matrices designed to promote bone growth. The results indicate that Ca2+ levels between 8 and 12 mM positively impacted transfection of BMSCs with PEI‐pDNA complexes in terms of cell viability and transfection efficiency, and a Ca2+ concentration of 10 mM also increased the expression of an osteogenic gene, osteocalcin, when the cells were transfected with plasmid DNA encoding bone morphogenetic protein 2 (BMP‐2). In the third chapter, gene-activated matrices (GAMs) mineralized with a simulated body fluid (SBF) were prepared and implanted in rat calvarial defects. The optimal GAM consisted of a collagen sponge with PEI-pDNA complexes embedded in a calcium phosphate coating produced by SBF, which increased total bone formation by 39% compared to 19% for control samples. A follow up in vivo study was performed to optimize the ratio of growth factors included in the GAM. The optimal ratio for supporting bone formation after 6 weeks of implantation was 5 parts of pBMP-2 to 3 parts pFGF-2. These studies demonstrated that collagen matrices biomimetically mineralized and activated with plasmids encoding FGF-2 and BMP-2 can optimally improve bone regeneration outcomes. The next chapter focuses on the development of a system to control the formation of bone to complement developments that have enabled potent regeneration of bony tissue. Scaffolds were fabricated with chemically modified RNA encoding for bone morphogenetic protein-9 (cmBMP9) and capped with salicylic acid (SA)-containing polymer (SAPAE) with the goal to determine if SAPAE could inhibit the formation of bone in a pilot animal study since cmBMP9 has been demonstrated to be highly effective in regenerating bone in a rat calvarial defect model. The results indicated that cmBMP9 increased bone formation (30% increase in area covered compared to control) and that SAPAE trended toward reducing the bone formation. These results suggest SAPAE could be useful as a chemical agent in reducing unwanted bone formation in implants loaded with cmBMP9. The final chapter investigates synthesis of chemically modified RNA and the application of these cmRNAs to bone defects. EGFP cmRNA was first synthesized to measure transfection efficiency and viability of BMSCs transfected with PEI-cmRNA complexes. Then BMP-2 and FGF-2 cmRNAs were created from plasmid DNA. These cmRNAs were demonstrated to increase protein expression in vitro. The cmRNAs were then loaded into GAMs and used to treat rat calvarial defects. In conclusion, gene therapy is a promising method for improving outcomes related to bone tissue engineering, and the techniques and data described in this thesis are paramount in furthering this field. The main takeaways from this thesis are calcium can improve gene therapy efficiency, and chemically modified is a more potent form of gene therapy as compared to plasmid DNA when target bone tissue.

Bone repair strategies continue to be developed for alternatives to autografting, allogeneic implants of banked bone, and other bone substitutes. Efforts have included the delivery of potent growth and/or differentiation factors and the use of gene therapy. For bone regeneration, gene therapy is the delivery, uptake and expression of DNA that has been localized to a wound bed. The objective of the current study is to investigate methods to enhance non-viral-mediated means of gene uptake and expression for use in bone regeneration. Several types of DNA-polymer complexes, either applied directly to baby hamster kidney (BHK) cells, or released from a porous, resorbable gene-activated matrix (GAM), were evaluated in vitro for their ability to transfect cells with a circular plasmid DNA construct expressing green fluorescent protein. Complexes included conjugates containing a lipophilic reagent, liposomes, poly-ethyl-oxazoline, and poly-ethyleneimine (PEI). Data were subjected to analysis of variance and Fisher's protected least significant difference for multiple comparisons with significance established at p < 0.05. Transfection efficiencies of the liposome and PEI complexes improved in vitro when released from resorbable GAMs. The lipophilic reagent FuGene 6 demonstrated abundant uptake and expression in the initial 1- and 2-day evaluation periods. In contrast, the DNA-liposome and PEI GAM complexes demonstrated a sustained release, uptake and expression by the BHK cells at the 2-, 4-, and 7-day, and 4- and 7-day evaluation intervals, respectively. GAM technology appears to improve the functional stability and release duration of incorporated DNA-polymer complexes in the present in vitro studies. The ongoing objective of our research is to develop a localized treatment to improve the uptake and expression of plasmid DNA by non-viral-mediated gene therapy.

In the context of producing enhanced therapeutics for regenerative medicine, our laboratory develops gene-activated matrices (GAMs) using non-viral gene therapy (GT) in combination with collagen-based scaffolds engineered specifically for tissue repair. Non-viral vectors have been referred to as a minority pursuit in GT but considering the concerns associated with viral vectors and as transient gene expression is such a key consideration, further research is clearly warranted for tissue engineering (TE) applications. Mesenchymal stem cells (MSCs) are well regarded for their capability in bone regeneration but as primary cells, they are difficult to transfect. We have recently optimised the non-viral vector, polyethyleneimine (PEI), to achieve high transfection efficiencies in MSCs. Subsequently, a series of PEI-based GAMs were developed using collagen, collagen-glycosaminoglycan and collagen-nanohydroxyapatite (collagen-nHa) scaffolds whereby transgene expression was detected up to 21 d with the collagen-nHa scaffold providing the most prolonged expression. Moreover, all PEI-based GAMs contained a low plasmid DNA dose of 2 µg which is far below doses often required in previous GAMs. Having successfully developed these GAMs, the ephrinB2 gene has recently been incorporated to produce a novel therapeutic GAM for bone repair. Herein, we discuss our recent investigations in the development and application of non-viral GAMs.

Lentivirus Immobilization to Nanoparticles for Enhanced and Localized Delivery From Hydrogels

Gene therapy is one of the most promising approaches in regenerative medicine. Gene-activated matrices provide stable gene expression and the production of osteogenic proteins in situ to stimulate osteogenesis and bone repair. In this study, we developed new gene-activated matrices based on polylactide granules (PLA) impregnated with BMP2 polyplexes and included in chitosan hydrogel or PRP-based fibrin hydrogel. The matrices showed high biocompatibility both in vitro with mesenchymal stem cells and in vivo when implanted intramuscularly in rats. The use of porous PLA granules allowed the inclusion of a high concentration of polyplexes, and the introduction of the granules into hydrogel provided the gradual release of the plasmid constructs. All gene-activated matrices showed transfecting ability and ensured long-term gene expression and the production of target proteins in vitro. At the same time, the achieved concentration of BMP-2 was sufficient to induce osteogenic differentiation of MSCs. When implanted into critical-size calvarial defects in rats, all matrices with BMP2 polyplexes led to new bone formation. The most significant effect on osteoinduction was observed for the PLA/PRP matrices. Thus, the developed gene-activated matrices were shown to be safe and effective osteoplastic materials. PLA granules and PRP-based fibrin hydrogel containing BMP2 polyplexes were shown to be the most promising for future applications in bone regeneration.

Bringing gene therapy to regenerative medicine

Regional Gene Therapy with Transduced Human Cells: The Influence of “Cell Dose” on Bone Repair

Controlled pVEGF delivery via a gene-activated matrix comprised of a peptide-modified non-viral vector and a nanofibrous scaffold for skin wound healing

Fibrin microbeads (FMB) as a 3D platform for kidney gene and cell therapy