Gene-activated hyaluronic acid-based cryogels for cartilage tissue engineering.

Articular cartilage injuries are very frequent lesions that if left untreated may degenerate into osteoarthritis. Gene transfer to mesenchymal stem cells (MSCs) provides a powerful approach to treat these lesions by promoting their chondrogenic differentiation into the appropriate cartilage phenotype. Non-viral vectors constitute the safest gene transfer tools, as they avoid important concerns of viral systems including immunogenicity and insertional mutagenesis.

Non-viral gene delivery to human mesenchymal stem cells: a practical guide towards cell engineering.

In recent decades, human mesenchymal stem cells (hMSCs) have gained momentum in the field of cell therapy for treating cartilage and bone injuries. Despite the tri-lineage multipotency, proliferative properties, and potent immunomodulatory effects of hMSCs, their clinical potential is hindered by donor variations, limiting their use in medical settings. To address this challenge, gene delivery technologies have emerged as a promising approach to modulate the phenotype and commitment of hMSCs towards specific cell lineages, thereby enhancing osteochondral repair strategies.

Development of new non-viral systems for genetic modification of senescent cells.

Senescence is a process characterized by a prolonged irreversible cell-cycle arrest. The accumulation of senescent cells in tissues is related to aging and to the development of age-related diseases. Recently, gene therapy has emerged as a powerful tool for treating age-associated diseases by the transference of specific genes into the target cell population.

Chondrogenic Differentiation of Human Mesenchymal Stem Cells via SOX9 Delivery in Cationic Niosomes.

Gene transfer to mesenchymal stem cells constitutes a powerful approach to promote their differentiation into the appropriate cartilage phenotype. Although viral vectors represent gold standard vehicles, because of their high efficiency, their use is precluded by important concerns including an elevated immunogenicity and the possibility of insertional mutagenesis. Therefore, the development of new and efficient non-viral vectors is under active investigation.

"Vermellogens" and the Development of CB[8]-Based Supramolecular Switches Using pH-Responsive and Non-Toxic Viologen Analogues.

We present herein the "vermellogens", a new class of pH-responsive viologen analogues, which replace the direct linking between -substituted pyridinium moieties within those by a hydrazone functional group. A series of such compounds have been efficiently synthesized in aqueous media by hydrazone exchange reactions, displaying a marked pH-responsivity. Furthermore, the parent ,'-dimethylated "vermellogen": the "red thread", an analogue of the herbicide paraquat and used herein as a representative model of the series, showed anion-recognition abilities, non-reversible electrochemical behavior, and non-toxicity of the modified bis-pyridinium core.

Niosomes-based gene delivery systems for effective transfection of human mesenchymal stem cells.

Gene transfer to mesenchymal stem cells (MSCs) has arisen as a powerful approach to increase the therapeutic potential of this effective cell population. Over recent years, niosomes have emerged as self-assembled carriers with promising performance for gene delivery. The aim of our work was to develop effective niosomes-based DNA delivery platforms for targeting MSCs.

Recent Progress on Polysaccharide-Based Hydrogels for Controlled Delivery of Therapeutic Biomolecules.

A plethora of applications using polysaccharides have been developed in recent years due to their availability as well as their frequent nontoxicity and biodegradability. These polymers are usually obtained from renewable sources or are byproducts of industrial processes, thus, their use is collaborative in waste management and shows promise for an enhanced sustainable circular economy. Regarding the development of novel delivery systems for biotherapeutics, the potential of polysaccharides is attractive for the previously mentioned properties and also for the possibility of chemical modification of their structures, their ability to form matrixes of diverse architectures and mechanical properties, as well as for their ability to maintain bioactivity following incorporation of the biomolecules into the matrix.

Hydrogel-Guided, rAAV-Mediated IGF-I Overexpression Enables Long-Term Cartilage Repair and Protection against Perifocal Osteoarthritis in a Large-Animal Full-Thickness Chondral Defect Model at One Year In Vivo.

The regeneration of focal articular cartilage defects is complicated by the reduced quality of the repair tissue and the potential development of perifocal osteoarthritis (OA). Biomaterial-guided gene therapy may enhance cartilage repair by controlling the release of therapeutic sequences in a spatiotemporal manner. Here, the benefits of delivering a recombinant adeno-associated virus (rAAV) vector coding for the human insulin-like growth factor I (IGF-I) via an alginate hydrogel (IGF-I/AlgPH155) to enhance repair of full-thickness chondral defects following microfracture surgery after one year in minipigs versus control (lacZ/AlgPH155) treatment are reported.

pNaSS-Grafted PCL Film-Guided rAAV TGF-β Gene Therapy Activates the Chondrogenic Activities in Human Bone Marrow Aspirates.

Scaffold-guided viral gene therapy is a novel, powerful tool to enhance the processes of tissue repair in articular cartilage lesions by the delivery and overexpression of therapeutic genes in a noninvasive, controlled release manner based on a procedure that may protect the gene vehicles from undesirable host immune responses. In this study, we examined the potential of transferring a recombinant adeno-associated virus (rAAV) vector carrying a sequence for the highly chondroregenerative transforming growth factor beta (TGF-β), using poly(ɛ-caprolactone) (PCL) films functionalized by the grafting of poly(sodium styrene sulfonate) (pNaSS) in chondrogenically competent bone marrow aspirates as future targets for therapy in cartilage lesions. Effective overexpression of TGF-β in the aspirates by rAAV was achieved upon delivery using pNaSS-grafted and ungrafted PCL films for up to 21 days (the longest time point evaluated), with superior levels using the grafted films, compared with respective conditions without vector coating.

Scaffold-Mediated Gene Delivery for Osteochondral Repair.

Osteochondral defects involve both the articular cartilage and the underlying subchondral bone. If left untreated, they may lead to osteoarthritis. Advanced biomaterial-guided delivery of gene vectors has recently emerged as an attractive therapeutic concept for osteochondral repair.

Hydrogel-Based Localized Nonviral Gene Delivery in Regenerative Medicine Approaches-An Overview.

Hydrogel-based nonviral gene delivery constitutes a powerful strategy in various regenerative medicine scenarios, as those concerning the treatment of musculoskeletal, cardiovascular, or neural tissues disorders as well as wound healing. By a minimally invasive administration, these systems can provide a spatially and temporarily defined supply of specific gene sequences into the target tissue cells that are overexpressing or silencing the original gene, which can promote natural repairing mechanisms to achieve the desired effect. In the present work, we provide an overview of the most avant-garde approaches using various hydrogels systems for controlled delivery of therapeutic nucleic acid molecules in different regenerative medicine approaches.

Therapeutic Delivery of rAAV via Polymeric Micelles Counteracts the Effects of Osteoarthritis-Associated Inflammatory Cytokines in Human Articular Chondrocytes.

Osteoarthritis (OA) is a prevalent joint disease linked to the irreversible degradation of key extracellular cartilage matrix (ECM) components (proteoglycans, type-II collagen) by proteolytic enzymes due to an impaired tissue homeostasis, with the critical involvement of OA-associated pro-inflammatory cytokines (interleukin 1 beta, i.e., IL-1β, and tumor necrosis factor alpha, i.

rAAV-Mediated Overexpression of SOX9 and TGF-β via Carbon Dot-Guided Vector Delivery Enhances the Biological Activities in Human Bone Marrow-Derived Mesenchymal Stromal Cells.

Scaffold-assisted gene therapy is a highly promising tool to treat articular cartilage lesions upon direct delivery of chondrogenic candidate sequences. The goal of this study was to examine the feasibility and benefits of providing highly chondroreparative agents, the cartilage-specific sex-determining region Y-type high-mobility group 9 (SOX9) transcription factor or the transforming growth factor beta (TGF-β), to human bone marrow-derived mesenchymal stromal cells (hMSCs) via clinically adapted, independent recombinant adeno-associated virus (rAAV) vectors formulated with carbon dots (CDs), a novel class of carbon-dominated nanomaterials. Effective complexation and release of a reporter rAAV- vector was achieved using four different CDs elaborated from 1-citric acid and pentaethylenehexamine (CD-1); 2-citric acid, poly(ethylene glycol) monomethyl ether (MW 550 Da), and ,-dimethylethylenediamine (CD-2); 3-citric acid, branched poly(ethylenimine) (MW 600 Da), and poly(ethylene glycol) monomethyl ether (MW 2 kDa) (CD-3); and 4-citric acid and branched poly(ethylenimine) (MW 600 Da) (CD-4), allowing for the genetic modification of hMSCs.

Controlled Release of rAAV Vectors from APMA-Functionalized Contact Lenses for Corneal Gene Therapy.

As an alternative to eye drops and ocular injections for gene therapy, the aim of this work was to design for the first time hydrogel contact lenses that can act as platforms for the controlled delivery of viral vectors (recombinant adeno-associated virus, rAAV) to the eye in an effective way with improved patient compliance. Hydrogels of hydroxyethyl methacrylate (HEMA) with aminopropyl methacrylamide (APMA) (H: 40, and H: 80 mM) or without (H: 0 mM) were synthesized, sterilized by steam heat (121 °C, 20 min), and then tested for gene therapy using rAAV vectors to deliver the genes to the cornea. The hydrogels showed adequate light transparency, oxygen permeability, and swelling for use as contact lenses.

Enhanced Chondrogenic Differentiation Activities in Human Bone Marrow Aspirates via Overexpression Mediated by pNaSS-Grafted PCL Film-Guided rAAV Gene Transfer.

Background: The delivery of therapeutic genes in sites of articular cartilage lesions using non-invasive, scaffold-guided gene therapy procedures is a promising approach to stimulate cartilage repair while protecting the cargos from detrimental immune responses, particularly when targeting chondroreparative bone marrow-derived mesenchymal stromal cells in a natural microenvironment like marrow aspirates.

Methods: Here, we evaluated the benefits of providing a sequence for the cartilage-specific sex-determining region Y-type high-mobility group box 9 (SOX9) transcription factor to human marrow aspirates via recombinant adeno-associated virus (rAAV) vectors delivered by poly(ε-caprolactone) (PCL) films functionalized via grafting with poly(sodium styrene sulfonate) (pNaSS) to enhance the marrow chondrogenic potential over time.

Results: Effective overexpression was observed in aspirates treated with pNaSS-grafted or ungrafted PCL films coated with the candidate rAAV-FLAG-h (FLAG-tagged rAAV vector carrying a human gene sequence) vector for at least 21 days relative to other conditions (pNaSS-grafted and ungrafted PCL films without vector coating).

Thermosensitive Hydrogel Based on PEO-PPO-PEO Poloxamers for a Controlled In Situ Release of Recombinant Adeno-Associated Viral Vectors for Effective Gene Therapy of Cartilage Defects.

Advanced biomaterial-guided delivery of gene vectors is an emerging and highly attractive therapeutic solution for targeted articular cartilage repair, allowing for a controlled and minimally invasive delivery of gene vectors in a spatiotemporally precise manner, reducing intra-articular vector spread and possible loss of the therapeutic gene product. As far as it is known, the very first successful in vivo application of such a biomaterial-guided delivery of a potent gene vector in an orthotopic large animal model of cartilage damage is reported here. In detail, an injectable and thermosensitive hydrogel based on poly(ethylene oxide) (PEO)-poly(propylene oxide) (PPO)-PEO poloxamers, capable of controlled release of a therapeutic recombinant adeno-associated virus (rAAV) vector overexpressing the chondrogenic sox9 transcription factor in full-thickness chondral defects, is applied in a clinically relevant minipig model in vivo.

Effects of rAAV-Mediated Overexpression on the Biological Activities of Human Osteoarthritic Articular Chondrocytes in Their Intrinsic Three-Dimensional Environment.

Gene therapy for osteoarthritis offers powerful, long-lasting tools that are well adapted to treat such a slow, progressive disorder, especially those therapies based on the clinically adapted recombinant adeno-associated viral (rAAV) vectors. Here, we examined the ability of an rAAV construct carrying a therapeutic sequence for the cartilage-specific SOX9 transcription factor to modulate the phenotype of human osteoarthritic articular chondrocytes compared with normal chondrocytes in a three-dimensional environment where the cells are embedded in their extracellular matrix. Successful overexpression via rAAV was noted for at least 21 days, leading to the significant production of major matrix components (proteoglycans, type-II collagen) without affecting the proliferation of the cells, while the cells contained premature hypertrophic processes relative to control conditions (reporter rAAV- application, absence of vector treatment).

Remodeling of Human Osteochondral Defects via rAAV-Mediated Co-Overexpression of TGF-β and IGF-I from Implanted Human Bone Marrow-Derived Mesenchymal Stromal Cells.

The application of chondrogenic gene sequences to human bone marrow-derived mesenchymal stromal cells (hMSCs) is an attractive strategy to activate the reparative activities of these cells as a means to enhance the processes of cartilage repair using indirect cell transplantation procedures that may improve the repopulation of cartilage lesions. In the present study, we examined the feasibility of co-delivering the highly competent transforming growth factor beta (TGF-β) with the insulin-like growth factor I (IGF-I) in hMSCs via recombinant adeno-associated virus (rAAV) vector-mediated gene transfer prior to implantation in a human model of osteochondral defect (OCD) ex vivo that provides a microenvironment similar to that of focal cartilage lesions. The successful co-overexpression of rAAV TGF-β/IGF-I in implanted hMSCs promoted the durable remodeling of tissue injury in human OCDs over a prolonged period of time (21 days) relative to individual gene transfer and the control (reporter Z gene) treatment, with enhanced levels of cell proliferation and matrix deposition (proteoglycans, type-II collagen) both in the lesions and at a distance, while hypertrophic, osteogenic, and catabolic processes could be advantageously delayed.

Current Trends in Viral Gene Therapy for Human Orthopaedic Regenerative Medicine.

Background: Viral vector-based therapeutic gene therapy is a potent strategy to enhance the intrinsic reparative abilities of human orthopaedic tissues. However, clinical application of viral gene transfer remains hindered by detrimental responses in the host against such vectors (immunogenic responses, vector dissemination to nontarget locations). Combining viral gene therapy techniques with tissue engineering procedures may offer strong tools to improve the current systems for applications .

Therapeutic Effects of rAAV-Mediated Concomittant Gene Transfer and Overexpression of TGF-β and IGF-I on the Chondrogenesis of Human Bone-Marrow-Derived Mesenchymal Stem Cells.

Application of chondroreparative gene vectors in cartilage defects is a powerful approach to directly stimulate the regenerative activities of bone-marrow-derived mesenchymal stem cells (MSCs) that repopulate such lesions. Here, we investigated the ability of combined recombinant adeno-associated virus (rAAV) vector-mediated delivery of the potent transforming growth factor beta (TGF-β) and insulin-like growth factor I (IGF-I) to enhance the processes of chondrogenic differentiation in human MSCs (hMSCs) relative to individual candidate treatments and to reporter () gene condition. The rAAV-hTGF-β and rAAV-hIGF-I vectors were simultaneously provided to hMSC aggregate cultures (TGF-β/IGF-I condition) in chondrogenic medium over time (21 days) versus TGF-β/, IGF-I/, and treatments at equivalent vector doses.

Supramolecular Cyclodextrin-Based Hydrogels for Controlled Gene Delivery.

Controlled delivery of gene transfer vectors is a powerful strategy to enhance the temporal and spatial presentation of therapeutic agents in a defined target. Hydrogels are adapted biomaterials for gene delivery capable of acting as a localized depot of genes while maintaining the long term local availability of DNA vectors at a specific location. Supramolecular hydrogels based on cyclodextrins (CDs) have attracted considerable attention as potential biomaterials in a broad range of drug delivery applications.

Improved Chondrogenic Differentiation of rAAV SOX9-Modified Human MSCs Seeded in Fibrin-Polyurethane Scaffolds in a Hydrodynamic Environment.

The repair of focal articular cartilage defects remains a problem. Combining gene therapy with tissue engineering approaches using bone marrow-derived mesenchymal stem cells (MSCs) may allow the development of improved options for cartilage repair. Here, we examined whether a three-dimensional fibrin-polyurethane scaffold provides a favorable environment for the effective chondrogenic differentiation of human MSCs (hMSCs) overexpressing the cartilage-specific SOX9 transcription factor via recombinant adeno-associated virus (rAAV) -mediated gene transfer cultured in a hydrodynamic environment .

Effective Remodelling of Human Osteoarthritic Cartilage by sox9 Gene Transfer and Overexpression upon Delivery of rAAV Vectors in Polymeric Micelles.

Recombinant adeno-associated virus (rAAV) vectors are well suited carriers to provide durable treatments for human osteoarthritis (OA). Controlled release of rAAV from polymeric micelles was already shown to increase both the stability and bioactivity of the vectors while overcoming barriers, precluding effective gene transfer. In the present study, we examined the convenience of delivering rAAV vectors via poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) polymeric (PEO-PPO-PEO) micelles to transfer and overexpress the transcription factor SOX9 in monolayers of human OA chondrocytes and in experimentally created human osteochondral defects.

Chondrogenic Differentiation Processes in Human Bone-Marrow Aspirates Seeded in Three-Dimensional-Woven Poly(ɛ-Caprolactone) Scaffolds Enhanced by Recombinant Adeno-Associated Virus-Mediated SOX9 Gene Transfer.

Combining gene therapy approaches with tissue engineering procedures is an active area of translational research for the effective treatment of articular cartilage lesions, especially to target chondrogenic progenitor cells such as those derived from the bone marrow. This study evaluated the effect of genetically modifying concentrated human mesenchymal stem cells from bone marrow to induce chondrogenesis by recombinant adeno-associated virus (rAAV) vector gene transfer of the sex-determining region Y-type high-mobility group box 9 (SOX9) factor upon seeding in three-dimensional-woven poly(ɛ-caprolactone; PCL) scaffolds that provide mechanical properties mimicking those of native articular cartilage. Prolonged, effective SOX9 expression was reported in the constructs for at least 21 days, the longest time point evaluated, leading to enhanced metabolic and chondrogenic activities relative to the control conditions (reporter lacZ gene transfer or absence of vector treatment) but without affecting the proliferative activities in the samples.

Sustained spatiotemporal release of TGF-β1 confers enhanced very early chondrogenic differentiation during osteochondral repair in specific topographic patterns.

The continuous presence of TGF-β is critically important to induce effective chondrogenesis. To investigate chondrogenesis in a cartilage defect, we tested the hypothesis that the implantation of TGF-β1-releasing scaffolds improves very early cartilage repair in vivo. Spatiotemporal controlled release of TGF-β1 was achieved from multiblock scaffolds that were implanted in osteochondral defects in the medial femoral condyles of adult minipigs.