Commitment of human mesenchymal stromal cells towards ACL fibroblast differentiation upon rAAV-mediated FGF-2 and TGF-ß overexpression using pNaSS-grafted PCL films.

Despite various clinical options, human anterior cruciate ligament (ACL) lesions do not fully heal. Biomaterial-guided gene therapy using recombinant adeno-associated virus (rAAV) vectors may improve the intrinsic mechanisms of ACL repair. Here, we examined whether poly(sodium styrene sulfonate)-grafted poly(ε-caprolactone) (pNaSS-grafted PCL) films can deliver rAAV vectors coding for the reparative basic fibroblast growth factor (FGF-2) and transforming growth factor beta (TGF-ß) in human mesenchymal stromal cells (hMSCs) as a source of implantable cells in ACL lesions. Efficient and sustained rAAV-mediated reporter (red fluorescent protein) and therapeutic (FGF-2 and TGF-ß) gene overexpression was achieved in the cells for at least 21 days in particular with pNaSS-grafted PCL films relative to all other conditions (up to 5.2-fold difference). Expression of FGF-2 and TGF-ß mediated by rAAV using PCL films increased the levels of cell proliferation, the DNA contents, and the deposition of proteoglycans and of type-I and -III collagen (up to 2.9-fold difference) over time in the cells with higher levels of transcription factor expression (Mohawk, Scleraxis) (up to 1.9-fold difference), without activation of inflammatory tumor necrosis alpha especially when using pNaSS-grafted PCL films compared with the controls. Overall, the effects mediated by TGF-ß were higher than those promoted by FGF-2, possibly due to higher levels of gene expression achieved upon rAAV gene transfer. This study shows the potential of using functionalized PCL films to apply rAAV vectors for ACL repair.

rAAV TGF-ß and FGF-2 Overexpression via pNaSS-Grafted PCL Films Stimulates the Reparative Activities of Human ACL Fibroblasts.

Lesions in the human anterior cruciate ligament (ACL) are frequent, unsolved clinical issues due to the limited self-healing ability of the ACL and lack of treatments supporting full, durable ACL repair. Gene therapy guided through the use of biomaterials may steadily activate the processes of repair in sites of ACL injury. The goal of the present study was to test the hypothesis that functionalized poly(sodium styrene sulfonate)-grafted poly(ε-caprolactone) (pNaSS-grafted PCL) films can effectively deliver recombinant adeno-associated virus (rAAV) vectors as a means of overexpressing two reparative factors (transforming growth factor beta-TGF-ß and basic fibroblast growth factor-FGF-2) in primary human ACL fibroblasts. Effective, durable rAAV reporter red fluorescent protein and candidate TGF-ß and FGF-2 gene overexpression was achieved in the cells for at least 21 days, especially when pNaSS-grafted PCL films were used versus control conditions, such as ungrafted films and systems lacking vectors or films (between 1.8- and 5.2-fold differences), showing interactive regulation of growth factor production. The expression of TGF-ß and FGF-2 from rAAV via PCL films safely enhanced extracellular matrix depositions of type-I/-III collagen, proteoglycans/decorin, and tenascin-C (between 1.4- and 4.5-fold differences) in the cells over time with increased levels of expression of the specific transcription factors Mohawk and scleraxis (between 1.7- and 3.7-fold differences) and without the activation of the inflammatory mediators IL-1ß and TNF-α, most particularly with pNaSS-grafted PCL films relative to the controls. This work shows the value of combining rAAV gene therapy with functionalized PCL films to enhance ACL repair.

Advanced Gene Therapy Strategies for the Repair of ACL Injuries.

The anterior cruciate ligament (ACL), the principal ligament for stabilization of the knee, is highly predisposed to injury in the human population. As a result of its poor intrinsic healing capacities, surgical intervention is generally necessary to repair ACL lesions, yet the outcomes are never fully satisfactory in terms of long-lasting, complete, and safe repair. Gene therapy, based on the transfer of therapeutic genetic sequences via a gene vector, is a potent tool to durably and adeptly enhance the processes of ACL repair and has been reported for its workability in various experimental models relevant to ACL injuries in vitro, in situ, and in vivo. As critical hurdles to the effective and safe translation of gene therapy for clinical applications still remain, including physiological barriers and host immune responses, biomaterial-guided gene therapy inspired by drug delivery systems has been further developed to protect and improve the classical procedures of gene transfer in the future treatment of ACL injuries in patients, as critically presented here.

pNaSS-Grafted PCL Film-Guided rAAV TGF-ß 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-ß), 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-ß 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. The production of rAAV-mediated TGF-ß by pNaSS-grafted and ungrafted PCL films significantly triggered the biological activities and chondrogenic processes in the samples (proteoglycan and type-II collagen deposition and cell proliferation), while containing premature mineralization and hypertrophy relative to the other conditions, with overall superior effects supported by the pNaSS-grafted films. These observations demonstrate the potential of PCL film-assisted rAAV TGF-ß gene transfer as a convenient, off-the-shelf technique to enhance the reparative potential of the bone marrow in patients in future approaches for improved cartilage repair.

Long-term hydrolytic degradation study of polycaprolactone films and fibers grafted with poly(sodium styrene sulfonate): Mechanism study and cell response.

Polycaprolactone (PCL) is a widely used biodegradable polyester for tissue engineering applications when long-term degradation is preferred. In this article, we focused on the analysis of the hydrolytic degradation of virgin and bioactive poly(sodium styrene sulfonate) (pNaSS) functionalized PCL surfaces under simulated physiological conditions (phosphate buffer saline at 25 and 37 °C) for up to 120 weeks with the aim of applying bioactive PCL for ligament tissue engineering. Techniques used to characterize the bulk and surface degradation indicated that PCL was hydrolyzed by a bulk degradation mode with an accelerated degradation-three times increased rate constant-for pNaSS grafted PCL at 37 °C when compared to virgin PCL at 25 °C. The observed degradation mechanism is due to the pNaSS grafting process (oxidation and radical polymerization), which accelerated the degradation until 48 weeks, when a steady state is reached. The PCL surface was altered by pNaSS grafting, introducing hydrophilic sulfonate groups that increase the swelling and smoothing of the surface, which facilitated the degradation. After 48 weeks, pNaSS was largely removed from the surface, and the degradation of virgin and pNaSS grafted surfaces was similar. The cell response of primary fibroblast cells from sheep ligament was consistent with the surface analysis results: a better initial spreading of cells on pNaSS surfaces when compared to virgin surfaces and a tendency to become similar with degradation time. It is worthy to note that during the extended degradation process the surfaces were able to continue inducing better cell spreading and preserve their cell phenotype as shown by collagen gene expressions.

Enhanced Chondrogenic Differentiation Activities in Human Bone Marrow Aspirates via sox9 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 sox9 overexpression was observed in aspirates treated with pNaSS-grafted or ungrafted PCL films coated with the candidate rAAV-FLAG-hsox9 (FLAG-tagged rAAV vector carrying a human sox9 gene sequence) vector for at least 21 days relative to other conditions (pNaSS-grafted and ungrafted PCL films without vector coating). Overexpression of sox9 via rAAV sox9/pNaSS-grafted or ungrafted PCL films led to increased biological and chondrogenic differentiation activities (matrix deposition) in the aspirates while containing premature osteogenesis and hypertrophy without impacting cell proliferation, with more potent effects noted when using pNaSS-grafted films. CONCLUSIONS: These findings show the benefits of targeting patients' bone marrow via PCL film-guided therapeutic rAAV (sox9) delivery as an off-the-shelf system for future strategies to enhance cartilage repair in translational applications.

Biomaterial-Guided Recombinant Adeno-associated Virus Delivery from Poly(Sodium Styrene Sulfonate)-Grafted Poly(ɛ-Caprolactone) Films to Target Human Bone Marrow Aspirates.

Scaffold-guided gene transfer offers strong systems to develop noninvasive convenient therapeutic options for the treatment of articular cartilage defects, especially when targeting bone marrow aspirates from patients containing chondroregenerative mesenchymal stromal cells in a native microenvironment. In this study, we examined the feasibility of delivering reporter (red fluorescent protein [RFP], lacZ) recombinant adeno-associated virus (rAAV) vectors over time to such samples through biocompatible mechanically stable poly(ɛ-caprolactone) (PCL) films grafted with poly(sodium styrene sulfonate) (pNaSS) for improved biological responses as clinically adapted tools for cartilage repair. Effective transgene expression (RFP, lacZ) was noted over time in human bone marrow aspirates using pNaSS-grafted films (up to 90% efficiency for at least 21 days) versus control conditions (ungrafted films, absence of vector coating on the films, free or no vector treatment), without displaying cytotoxic nor detrimental effects on the osteochondrogenic or hypertrophic potential of the samples. These findings demonstrate the potential of directly modifying therapeutic bone marrow from patients by controlled delivery of rAAV using biomaterial-guided procedures as a future noninvasive strategy for clinical cartilage repair. Impact statement Injured articular cartilage does not fully regenerate on itself and none of the currently available clinical and experimental therapeutic procedures are capable of restoring an original hyaline cartilage in sites of injury. Biomaterial-guided gene delivery has a strong potential to enhance the processes of cartilage repair. The system presented here based on the FDA-approved biocompatible poly(ɛ-caprolactone) material provides a functional scaffold for the controlled delivery of clinically adapted recombinant adeno-associated virus vectors as an off-the-shelf compound that could be applicable in a minimally invasive manner in patients.

Analysis of early cellular responses of anterior cruciate ligament fibroblasts seeded on different molecular weight polycaprolactone films functionalized by a bioactive poly(sodium styrene sulfonate) polymer.

With the growing number of anterior cruciate ligament (ACL) ruptures and the increased interest for regenerative medicine procedures, many studies are now concentrated on developing bioactive and biodegradable synthetic ligaments. For this application, the choice of raw materials with appropriate physicochemical characteristics and long-term degradation features is essential. Polycaprolactone (PCL) has the advantage of slow degradation that depends on its molecular weight. This study evaluates two PCL materials: a technical grade (PC60: 60 kDa) versus a medical grade (PC12: 80 kDa), both before and after functionalization with poly(sodium styrene sulfonate) (pNaSS). After determining the grafting process had little to no effect on the PCL physicochemical properties, sheep ACL fibroblast responses were investigated. The PC12 films induced a significantly lower expression of the tumor necrosis factor alpha inflammatory gene compared to the PC60 films. Both film types induced an overproduction of fibroblast growth factor-2 and transforming growth factor beta compared to the controls on day 5 and demonstrated collagen gene expression profiles similar to the controls on day 7. Upon protein adsorption, pNaSS grafting caused a rapid cell adhesion in the first 30 min and an increased adhesion strength (1.5-fold higher). Moreover, after 7 days, an increase in cell density and actin network development were noted on the grafted films.

Impact of chemical and physical treatments on the mechanical properties of poly(ε-caprolactone) fibers bundles for the anterior cruciate ligament reconstruction.

The anterior cruciate ligament rupture is one of the most common sport injuries. Due to ligaments' poor healing capacity, surgical intervention is often required. Nowadays, these injuries are managed using replacement autografts or to a lesser extent using artificial ligaments. With the expansion of tissue engineering, more recent researches focus on the development of biodegradable structures that could allow graft functioning while enhancing host integration. The main challenge is to develop a structure that gradually loses its mechanical properties when at the same time the neo-ligament gains in solidity. Mechanical behavior and reconstruction of natural tissue are the two key points for such a successful device. This article evaluates the mechanical consistency of poly(ε-caprolactone) fibers bundles grafted with sodium polystyrene sulfonate, as a candidate for ligament prosthesis. In order to be medically used, PCL fibers need to cope with multiple steps before implantation including extensive washings, knitting, grafting and sterilization processes. The evolution of mechanical properties at each step of the elaboration process has been investigated. The results show that PCL bundles have the same visco-elastic behavior than the native ACL. Nevertheless, when undergoing physical treatments such as ionizing radiations, like UV or ß-rays, the material endures a hardening, increasing its stiffness but also its fragility. At this opposite, the thermal radical grafting acts like an annealing step, increasing significantly the elasticity of the PCL fibers. With this chemical treatment, the stiffness is decreasing, leading to higher energy dissipation. Added to the observation of the structure of the material, this demonstrates the possibility of the PCL to modulate it microstructure. In case of orthopedic prosthesis, the need of such a construct is strongly required to avoid distension of the future prosthesis and to restore good knee stabilization, showing the promising future of PCL ligament prosthesis.

Controlled release of gene therapy constructs from solid scaffolds for therapeutic applications in orthopedics.

Injuries occurring in orthopedic tissues do not completely heal if left untreated due to their imperfect self abilities for spontaneous repair. As most of the current clinical treatments often fail to fully restore the damaged tissues, there is a critical need to develop potent alternatives to activate the processes of repair in sites of orthopedic lesions. In this regard, combining gene therapy approaches with tissue engineering procedures to generate therapeutic gene vector-guided delivery systems, especially those based on mechanically stable solid scaffolds, is an attractive strategy to provide off-the-shelf compounds for the convenient spatiotemporal expression of candidate agents in orthopedic tissue defects. The goal of this review is to report the most advanced technologies using such scaffolds as tools for the controlled delivery of gene transfer vectors to improve orthopedic tissue repair.