Gene-activated Matrix Research Articles

Recent advances in polymeric biomaterials-based gene delivery for cartilage repair

Untreated articular cartilage damage normally results in osteoarthritis and even disability that affects millions of people. However, both the existing surgical treatment and tissue engineering approaches are unable to regenerate the original structures of articular cartilage durably, and new strategies for integrative cartilage repair are needed. Gene therapy provides local production of therapeutic factors, especially guided by biomaterials can minimize the diffusion and loss of the genes or gene complexes, achieve accurate spatiotemporally release of gene products, thus provideing long-term treatment for cartilage repair. The widespread application of gene therapy requires the development of safe and effective gene delivery vectors and supportive gene-activated matrices. Among them, polymeric biomaterials are particularly attractive due to their tunable physiochemical properties, as well as excellent adaptive performance. This paper reviews the recent advances in polymeric biomaterial-guided gene delivery for cartilage repair, with an emphasis on the important role of polymeric biomaterials in delivery systems.

Extracellular matrix mechanics regulate transfection and SOX9-directed differentiation of mesenchymal stem cells

Gene delivery within hydrogel matrices can potentially direct mesenchymal stem cells (MSCs) towards a chondrogenic fate to promote regeneration of cartilage. Here, we investigated whether the mechanical properties of the hydrogel containing the gene delivery systems could enhance transfection and chondrogenic programming of primary human bone marrow-derived MSCs. We developed collagen-I-alginate interpenetrating polymer network hydrogels with tunable stiffness and adhesion properties. The hydrogels were activated with nanocomplexed SOX9 polynucleotides to direct chondrogenic differentiation of MSCs. MSCs transfected within the hydrogels showed higher expression of chondrogenic markers compared to MSCs transfected in 2D prior to encapsulation. The nanocomplex uptake and resulting expression of transfected SOX9 were jointly enhanced by increased stiffness and cell-adhesion ligand density in the hydrogels. Further, transfection of SOX9 effectively induced MSCs chondrogenesis and reduced markers of hypertrophy compared to control matrices. These findings highlight the importance of matrix stiffness and adhesion as design parameters in gene-activated matrices for regenerative medicine. Statement of SignificanceGene-activated matrices (GAMs) are biodegradable polymer networks integrating gene therapies, and they are promising technologies for supporting tissue regeneration. Despite this interest, there is still limited information on how to rationally design these systems. Here, we provide a systematic study of the effect of matrix stiffness and cell adhesion ligands on gene transfer efficiency. We show that high stiffness and the presence of cell-binding sites promote transfection efficiency and that this result is related to more efficient internalization and trafficking of the gene therapies. GAMs with optimized mechanical properties can induce cartilage formation and result in tissues with better characteristics for articular cartilage tissue engineering as compared to previously described standard methods.

MRNA-activated matrices encoding transcription factors as primers of cell differentiation in tissue engineering

Gene-activated matrices (GAMs) encoding pivotal transcription factors (TFs) represent a powerful tool to direct stem cell specification for tissue engineering applications. However, current TF-based GAMs activated with pDNA, are challenged by their low transfection efficiency and delayed transgene expression. Here, we report a GAM technology activated with mRNAs encoding TFs SOX9 (cartilage) and MYOD (muscle). We find that these mRNA-GAMs induce a higher and faster TF expression compared to pDNA-GAMs, especially in the case of RNase resistant mRNA sequences. This potent TF expression was translated into a high synthesis of cartilage- and muscle-specific markers, and ultimately, into successful tissue specification in vitro. Additionally, we show that the expression of tissue-specific markers can be further modulated by altering the properties of the mRNA-GAM environment. These results highlight the value of this GAM technology for priming cell lineage specification, a key centerpiece for future tissue engineering devices.

Exosome-mimetics as an engineered gene-activated matrix induces in-situ vascularized osteogenesis

Exosome has been considered as an instructive supplement between complicated cell therapy and single gene/protein drug treatment in the field of regenerative medicine due to its excellent biocompatibility, efficient cellular internalization and large loading capacity. Nevertheless, one major issue that extremely restricts the potential application as gene/drug vehicles is the low yield of nanoscale exosome. Moreover, the intravenous injection of targeted exosomes may cause the obstruction of blood-rich organs. Thus, herein we fabricated a specific exosome-mimetics (EMs) that could come true mass and fast production exhibited the similar size, morphology and membrane protein markers in comparison with conventional exosomes. To bypass the risk of intravenous injection and improve the efficiency of topical therapy, we simultaneously applied the engineered EMs to design a gene-activated matrix (GAM) that could be locally released by encapsulating the plasmid of vascular endothelial growth factor (VEGF) and flexibly binding onto a core-shell nanofiber film. Our findings showed that the well-designed engineered EMs-mediated GAM was able to sustainably deliver VEGF gene and significantly enhance the vascularized osteogenesis in vivo. The current work can not only consolidate the applied foundation of EMs through the breakthrough of high yield, but also provide a local and effective delivery of engineered EMs for the in-situ therapy.

Poly lactic-co-glycolic acid scaffold loaded with plasmid DNA encoding fibroblast growth factor-2 promotes periodontal ligament regeneration of replanted teeth.

This study investigated the effects of poly lactic-co-glycolic acid (PLGA) loaded with plasmid DNA encoding fibroblast growth factor-2 (pFGF-2) on human periodontal ligament cells (hPDLCs) in vitro and evaluated the ability of the PLGA/pFGF-2 scaffold to promote periodontal ligament (PDL) regeneration in a beagle dog teeth avulsion animal model. Growth factor and scaffold play important roles in PDL regeneration. PLGA is a kind of biodegradable and biocompatible polymer that can be used as a carrier to deliver growth factors or genes. FGF-2 can induce potent proliferative responses, promote cell migration and regulate the production of extracellular matrix. Therefore, a gene-activated matrix composed of scaffold and genes is supposed to be a superior approach for promoting tissue regeneration. In this study, PLGA and PLGA/pFGF-2 scaffolds were fabricated using electrospinning. The characterization of scaffolds was shown by scanning electron microscope (SEM) and transmission electron microscope (TEM). dsDNA HS was used to test the plasmid release of PLGA/pFGF-2 scaffold. The viability and proliferation of hPDLCs on the two kinds of scaffolds were evaluated by the CCK-8 assay, and the expression of collagen I and scleraxis were analysed by RT-qPCR. The roots of avulsed teeth were covered by the two types of scaffolds and replanted into the alveolar pockets in beagles. Haematoxylin-eosin and Masson staining were used to evaluate the effects of PLGA/pFGF-2 scaffold on promoting PDL regeneration. The smooth and uniform fibres can be observed in both scaffolds, and the plasmids were randomly distributed in the PLGA/pFGF-2 scaffold. dsDNA HS analysis demonstrated that the PLGA/pFGF-2 scaffold released up to 123ng pFGF-2 over 21days in a sustained manner without any obvious burst release. The PLGA/pFGF-2 scaffold promoted the proliferation of hPDLCs and increased the expression levels of collagen I and scleraxis compared with PLGA scaffold. Animal experiments showed that more regular PDL-like tissues and less root surface resorption occurred in the PLGA/pFGF-2 scaffold group compared with the PLGA scaffold group. The PLGA/pFGF-2 scaffold promoted hPDLCs proliferation and facilitated periodontal ligament-related differentiation. The PLGA/pFGF-2 scaffold possesses excellent biological characteristics and could be used as a promising biomaterial for improving the treatment prognosis of replanted tooth.

Plasmid BMP-2–embedded gelatin sponge as a gene-activated matrix for preosteoblast differentiation

Gene-activated matrices (GAM), a combination of scaffolds and plasmid delivery systems, are widely used in regenerative medicine, but few compared plasmid BMP-2 in hemostatic gelatin sponge. We compared the osteogenic differentiation ability of bone morphogenetic protein-2 (BMP-2)–transfected preosteoblasts cultured into a hemostatic gelatin sponge with that of non-transfected cells cultured into BMP-2–activated hemostatic gelatin sponge. GAM was prepared from hemostatic gelatin sponge embedded with complexes (plasmid BMP-2 mixed with TransIT-2020® transfection reagent). The standard transfection method using mouse preosteoblasts (MC3T3-E1) mixed with the complex loaded onto the scaffold was compared with the GAM group. Non-transfected cells were loaded onto the scaffold as the control group. Transmission and scanning electron microscopy results revealed that TransIT-2020®/plasmid BMP-2 complexes formed homogeneous non-aggregated structures, which were round in shape and 200–250 nm in size. Alkaline phosphatase activity, the marker of bone differentiation, was significantly higher in GAM group compared to that in standard and control groups on differentiation days 14, 21, and 28. The concentration of released BMP-2 was significantly higher in GAM group than that in standard group on days 3 and 7, consistent with confocal microscopy results.

Immobilization of plasmids in bacterial nanocellulose as gene activated matrix

The synergy of the local delivery of nucleic acids using a hydrogel-based gene activated matrix (GAM) might support regenerative processes on a genetic level by concurrently providing a cell-friendly microenvironment. To investigate bacterial nanocellulose (BNC) as GAM, two plasmids (pSV-β-Gal and pGL3) were incorporated by reswelling and injection techniques forming matrix and core-shell systems as determined by SEM and staining experiments. The release was found to be dependent on the type of BNC, the plasmid and the loading technique, and lasted over at least 20 days. No morphological or mechanical changes of the BNC due to the presence of plasmids were observed. Immobilized plasmids especially in the matrix systems were protected against enzymatic degradation by maintaining the high biocompatibility of BNC and transfection efficacy of the plasmids. These results indicate that BNC can be used as a promising and renewable carrier for the application as local gene delivery system.

Polyethylenimine-alginate nanocomposites based bone morphogenetic protein 2 gene-activated matrix for alveolar bone regeneration.

The repair and treatment of lost or damaged alveolar bone is of great significance in dentistry. Gene-activated matrix (GAM) technology provides a new way for bone regeneration. It is a local gene delivery system, which can not only recruit cells, but also influence their fate. For this purpose, we fabricated a bone morphogenetic protein 2 (BMP-2) gene-loaded absorbable gelatin sponge (AGS) and studied its effect on promoting alveolar bone formation and preventing resorption following tooth extraction in rats. In order to obtain better transfection efficiency, polyethylenimine-alginate (PEI-al) nanocomposites were synthesized and used as gene vectors to deliver BMP-2 cDNA plasmids (PEI-al/pBMP-2). The transfection efficiency, BMP-2 protein expression and osteogenic differentiation of the cells were investigated in vitro. In vivo, we established an alveolar bone regeneration model by extracting the rats' left mandibular incisors. The rats were randomly assigned into 3 groups: control group, unfilled sockets; AGS group, sockets filled with PEI-al solution-loaded gelatin sponges; AGS/BMP group, sockets filled with PEI-al/pBMP-2 solution-loaded gelatin sponge. Radiological and histological assays were performed at 4 and 8 weeks later. In vitro transfection assays indicated that PEI-al/pBMP-2 complexes could effectively transfect MC3T3-E1 cells, promoting the secretion of BMP-2 protein for at least 14 days, as well as increasing the expression of osteogenesis-related gene, ALP activity and calcium deposition. In vivo, western blot analysis showed BMP-2 protein was expressed in bone tissues of AGS/BMP group. The relative height of the residual alveolar ridge and bone mineral density (BMD) of the AGS/BMP group were significantly greater than those in the AGS and control groups at 4 and 8 weeks, respectively. Histological examination showed that, at 4 weeks, osteoblasts had grown in a cubic shape around the new bone in the AGS/BMP group, suggesting new bone formation. In conclusion, the combination of PEI-al/pBMP-2 complexes and gelatin sponge could promote alveolar bone regeneration, which may provide an easy and valuable method for alveolar ridge preservation and augmentation.

Parathyroid hormone gene-activated matrix with DFDBA/collagen composite matrix enhances bone regeneration in rat calvarial bone defects

BackgroundGene-activated matrix (GAM) induces sustained local production of growth factors to promote tissue regeneration. GAM contains a plasmid DNA (pDNA) encoding target proteins that is physically entrapped within a biodegradable matrix carrier. GAM with a pDNA encoding the first 34 amino acids of parathyroid hormone (PTH 1–34) and a collagen matrix enhances bone regeneration in long bone defects. Demineralized freeze-dried bone allograft (DFDBA) is a widely used osteoinductive bone graft. The present study determined the osteogenic effects of PTH-GAM with a collagen or DFDBA/collagen composite (D/C) matrix for treating craniofacial bone defects. MethodsWe constructed a pDNA encoding human PTH 1–34 and performed cyclic AMP ELISA to verify the bioactivity of PTH 1–34. Next, we generated a D/C matrix and PTH-GAMs containing a collagen matrix (PTH-C-GAM) or D/C matrix (PTH-D/C-GAM). Rats with critical-sized calvarial bone defects were divided into four groups, namely, untreated rats (sham group) and rats grafted with D/C matrix, PTH-C-GAM, or PTH-D/C-GAM (D/C, PTH-C-GAM, or PTH-D/C-GAM groups, respectively). PTH expression was determined by performing immunohistochemical staining after 4 and 8 weeks. New bone formation was evaluated by performing radiography, dual-energy X-ray absorptiometry, microcomputed tomography, and histological examination. ResultsPTH pDNA-transfected cells secreted bioactive PTH 1–34. Moreover, PTH was expressed at 4 and 8 weeks after the surgery in rats in the PTH-C-GAM group but not in rats in the D/C group. New bone formation in the calvarial bone defects, from more to less, was in the order of PTH-D/C-GAM, PTH-C-GAM, D/C, and sham groups. ConclusionOur results indicate that PTH-GAM with a collagen matrix promotes local PTH production for at least 8 weeks and bone regeneration in craniofacial bone defect. Moreover, our results indicate that replacement of the collagen matrix with the D/C matrix improves the osteogenic effects of PTH-GAM.

Projection Stereolithographic Fabrication of BMP-2 Gene-activated Matrix for Bone Tissue Engineering

Currently, sustained in vivo delivery of active bone morphogenetic protein-2 (BMP-2) protein to responsive target cells, such as bone marrow-derived mesenchymal stem cells (BMSCs), remains challenging. Ex vivo gene transfer method, while efficient, requires additional operation for cell culture and therefore, is not compatible with point-of-care treatment. In this study, two lentiviral gene constructs – (1) Lv-BMP/GFP, containing human BMP-2 and green fluorescent protein (GFP) gene (BMP group); or (2) Lv-GFP, containing GFP gene (GFP group) – were incorporated with human BMSCs into a solution of photocrosslinkable gelatin, which was then subjected to visible light-based projection stereolithographic printing to form a scaffold with desired architectures. Upon in vitro culture, compared to the GFP group, cells from BMP group showed >1,000-fold higher BMP-2 release, and the majority of them stained intensely for alkaline phosphatase activity. Real-time RT-PCR also showed dramatically increased expression of osteogenesis marker genes only in the BMP group. 3.5 months post-implantation into SCID mice, the micro-computed tomography imaging showed detectable mineralized areas only in the BMP group, which was restricted within the scaffolds. Alizarin red staining and immunohistochemistry of GFP and osteocalcin further indicated that the grafted hBMSCs, not host cells, contributed primarily to the newly formed bone.

Enzyme-crosslinked gene-activated matrix for the induction of mesenchymal stem cells in osteochondral tissue regeneration.

In this study, a gene-activated matrix (GAM) to promote the growth of both cartilage and subchondral bone within a single integrated construct is developed. It has the capacity to promote transfection of human mesenchymal stem cells (hMSCs) and the functional osteochondral tissues formation. The results show that the sustained release of plasmids including TGF-beta and BMP-2 from GAM could promote prolonged transgene expression and stimulate hMSCs differentiation into the osteogenic and chondrogenic lineages by spatial control manner. This improved delivery method should enhance the functionalized composite graft to accelerate healing process in vitro and in vivo for osteochondral tissue regeneration.

Biopolymers/poly(ε-caprolactone)/polyethylenimine functionalized nano-hydroxyapatite hybrid cryogel: Synthesis, characterization and application in gene delivery

Nano-hydroxyapatite (nHAp), surface functionalized with linear polyethylenimine (LPEI), was used for the preparation of biocomposites in combination with biopolymers and poly(ε-caprolactone) (PCL), by cryogelation technique, to yield biomimetic scaffolds with controlled interconnected macroporosity, mechanical stability, and predictable degradation behavior. The structural characteristics, swelling and degradation behavior of hydroxyapatite and hydroxyapatite/β-tricalcium phosphate (β-TCP) filled matrices were investigated as compared to the corresponding naked polymer 3D system. It was found that the homogeneity and cohesivity of the composite are significantly dependent on the size and amount of the included inorganic particles, which are thus determining the structural parameters. Surface modification with LPEI and nanodimensions favored the nHAp integration in the organic matrix, with preferential location along protein fibers, while β-TCP microparticles induced an increased disorder in the hybrid system. The biocomposite including nHAp only was further investigated targeting biomedical uses, and proved to be non-cytotoxic and capable of acting as gene-activated matrix (GAM). It allowed sustained delivery over time (until 22days) of embedded PEI25-pDNA polyplexes at high levels of transgene expression, while insuring a decrease in cytotoxicity as compared to polyplexes alone. Experimental data recommend such biocomposite as an attractive material for regenerative medicine.

Ultrasound-responsive gene-activated matrices for osteogenic gene therapy using matrix-assisted sonoporation

Gene-activated matrix (GAM)-based therapeutics for tissue regeneration are limited by efficacy, the lack of spatiotemporal control and availability of target cells, all of which impact negatively on their translation to the clinic. Here, an advanced ultrasound-responsive GAM is described containing target cells that facilitates matrix-assisted sonoporation (MAS) to induce osteogenic differentiation. Ultrasound-responsive GAMs consisting of fibrin/collagen hybrid-matrices containing microbubbles, bone morphogenetic protein BMP2/7 coexpression plasmids together with C2C12 cells were treated with ultrasound either in vitro or following parenteral intramuscular implantation in vivo. Using direct measurement for alkaline phosphatase activity, von Kossa staining and immunohistochemical analysis for osteocalcin expression, MAS-stimulated osteogenic differentiation was confirmed in the GAMs in vitro 7days after treatment with ultrasound. At day 30 post-treatment with ultrasound, ectopic osteogenic differentiation was confirmed in vivo using X-ray microcomputed tomography and histological analysis. Osteogenic differentiation was indicated by the presence of ectopic bone structures in all animals treated with MAS. In addition, bone volumes in this group were statistically greater than those in the control groups. This novel approach of incorporating a MAS capability into GAMs could be exploited to facilitate ex vivo gene transfer with subsequent surgical implantation or alternatively provide a minimally invasive means of stimulating in situ transgene delivery for osteoinductive gene-based therapies. Copyright © 2017 John Wiley & Sons, Ltd.

POSSIBILITIES OF CURRENT CELLULAR TECHNOLOGIES FOR ARTICULAR CARTILAGE REPAIR (ANALYTICAL REVIEW)

Despite a wide variety of surgical procedures utilized in clinical practice for treatment of articular cartilage lesions, the search for other options of articular reconstruction remains a relevant and open issue at the current stage of medicine and biotechnologies development. The recent years demonstrated a strong belief in cellular methods of hyaline cartilage repair such as implantation of autologous chondrocytes (ACI) or cultures of mesenchymal stem cells (MSC) including techniques for genetic modification of cells.The purpose of presented review is to summarize the published scientific data on up to date results of perspective cellular technologies for articular cartilage repair that are being developed. Autologous chondrocyte transplantation originally performed by Swedish researchers in 1987 is considered the first clinically applied technique for restoration of hyaline cartilage using cellular technologies. However, the transplanted cell culture featured low proliferative capacity and inability to form a regenerate resistant to high physical activity. Another generation of methods originated at the turn of the century utilized mesenchymal stem cells instead of autologous chondrocytes. Preparation of MSCs is a less invasive procedure compared to chondrocytes harvesting and the culture is featured by a higher proliferative ability. Researchers use various biodegradable carriers (matrices) to secure cell fixation. Despite good clinical mid-term outcomes the transplanted tissue-engineering structures deteriorate with time due to cellular de-differentiation. Next generation of techniques being currently under pre-clinical studies is featured by the preliminary chondrogenic modification of transplanted cell culture. Usage of various growth factors, modified cell product and gene-activated matrices allow to gain a stable regulatory and key proteins synthesis and achieve a focused influence on regenerate's chondrogenic proliferation and in result to form a good hyaline cartilage resistant to high physical load in long term period.Thus, development of methods for articular cartilage repair has long ago went beyond the interests of clinical physicians, and only the close interdisciplinary cooperation of clinicians and specialists for cytology, molecular genetics and, probably, virology would enable replacement of a defect with a rigorous hyaline cartilage.

Bone Regeneration Using Gene-Activated Matrices.

Gene delivery to bone is a potential therapeutic strategy for directed, sustained, and regulated protein expression. Tissue engineering strategies for bone regeneration include delivery of proteins, genes (viral and non-viral-mediated delivery), and/or cells to the bone defect site. In addition, biomimetic scaffolds and scaffolds incorporating bone anabolic agents greatly enhance the bone repair process. Regional gene therapy has the potential of enhancing bone defect healing and bone regeneration by delivering osteogenic genes locally to the osseous lesions, thereby reducing systemic toxicity and the need for using supraphysiological dosages of therapeutic proteins. By implanting gene-activated matrices (GAMs), sustained gene expression and continuous osteogenic protein production in situ can be achieved in a way that stimulates osteogenesis and bone repair within osseous defects. Critical parameters substantially affecting the therapeutic efficacy of gene therapy include the choice of osteogenic transgene(s), selection of non-viral or viral vectors, the wound environment, and the selection of ex vivo and in vivo gene delivery strategies, such as GAMs. It is critical for gene therapy applications that clinically beneficial amounts of proteins are synthesized endogenously within and around the lesion in a sustained manner. It is therefore necessary that reliable and reproducible methods of gene delivery be developed and tested for their efficacy and safety before translating into clinical practice. Practical considerations such as the age, gender, and systemic health of patients and the nature of the disease process also need to be taken into account in order to personalize the treatments and progress towards developing a clinically applicable gene therapy for healing bone defects. This review discusses tissue engineering strategies to regenerate bone with specific focus on non-viral gene delivery systems.

Non-viral gene activated matrices for mesenchymal stem cells based tissue engineering of bone and cartilage.

Recent regenerative medicine and tissue engineering strategies for bone and cartilage repair have led to fascinating progress of translation from basic research to clinical applications. In this context, the use of gene therapy is increasingly being considered as an important therapeutic modality and regenerative technique. Indeed, in the last 20 years, nucleic acids (plasmid DNA, interferent RNA) have emerged as credible alternative or complement to proteins, which exhibited major issues including short half-life, loss of bioactivity in pathologic environment leading to high dose requirement and therefore high production costs. The relevance of gene therapy strategies in combination with a scaffold, following a so-called "Gene-Activated Matrix (GAM)" approach, is to achieve a direct, local and sustained delivery of nucleic acids from a scaffold to ensure efficient and durable cell transfection. Among interesting cells sources, Mesenchymal Stem Cells (MSC) are promising for a rational use in gene/cell therapy with more than 1700 clinical trials approved during the last decade. The aim of the present review article is to provide a comprehensive overview of recent and ongoing work in non-viral genetic engineering of MSC combined with scaffolds. More specifically, we will show how this inductive strategy can be applied to orient stem cells fate for bone and cartilage repair.

Bone agumentation by gene activated matrix composed of plasmid DNA, BMP4 or Runx2, embedded in atelocollagen

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.

Gene-activated matrix for in situ regeneration of skin: from structure to function

Gene-activated matrix for in situ regeneration of skin: from structure to function

FGF2 gene activated matrices promote proliferation of bone marrow stromal cells

BackgroundIn this study, we report on the results from the development and early in vitro testing of a gene activated matrix encoding basic human fibroblast growth factor 2 (FGF2) in bone marrow stromal cells (BMSCs). MethodsPolyethylenimine (PEI), a cationic polymer, was utilized as a gene delivery vector and collagen scaffolds were used as the carrier to deliver the PEI-pDNA nano-sized complexes (nanoplexes) encoding the FGF2 protein. Initially, the BMSCs were transfected in vitro with the PEI-pFGF2 nanoplexes, prepared at a N/P ratio of 10, with cells alone and naked DNA as controls. This was followed by transfection experiments using collagen scaffold containing complexes, with the scaffold alone as a control. The transfection efficacy of the nanoplexes was assessed using ELISA for the determination of FGF2 protein expressed by the transfected cells. The functionality of transfection was assessed by evaluating cellular recruitment, attachment, and proliferation of BMSCs on the scaffold using imaging techniques. ResultsBMSCs transfected with the PEI-pFGF2 nanoplexes (either alone or within the scaffold) led to higher expression of FGF2, compared to controls. Scanning electron microscopy and confocal imaging confirmed the recruitment and attachment of BMSCs to scaffolds containing the PEI-pFGF2 nanoplexes. Confocal microscopy showed a significantly higher number of proliferating cells within PEI-pFGF2 nanoplex-loaded scaffolds than with empty scaffolds. ConclusionsThis first in vitro evaluation in BMSCs provides evidence that gene activated matrices (GAMs) encoding the FGF2 protein may have strong translational potential for clinical applications that require enhanced osseous and periodontal tissue regeneration.

Gene-Activated Matrix Comprised of Atelocollagen and Plasmid DNA Encoding BMP4 or Runx2 Promotes Rat Cranial Bone Augmentation.

To date, therapeutic method for in vivo gene delivery has not been established on bone engineering though its potential usefulness has been suggested. For clinical applications, an effective condition should be developed to transfer the genes in vivo without any transfection reagents or virus vectors. In this study, to facilitate the clinical setting of this strategy, particularly aimed at atrophic bone repair, 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, or 1 mg of AcGFP plasmid vectors harboring cDNA of BMP4 (pBMP4) or Runx2 (pRunx2) with 2% bovine atelocollagen and β-tricalcium phosphate granules. Before manufacturing GAMs, to determine the biological activity of generated pDNAs, we confirmed GFP expression and increased level of alkaline phosphatase activities in MC3T3-E1 cells transfected with pBMP4 or pRunx2 during culture. Then, GAMs were lyophilized and transplanted to onlay placement on the cranium. At 2 weeks of transplantation, GFP-expressing cells could be detectable in only GAMs containing 1 mg of AcGFP plasmid vectors. Then, at 4 weeks, significant bone formation was recognized in GAMs containing 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. Thus, atelocollagen-based GAM reliably could form the engineered bone even for the vertical augmentation when containing a certain amount of plasmid vectors encoding osteogenic proteins. This study supports facilitating the clinical application of GAM for bone engineering.