Investigators tested the preclinical safety of the innovative implant ARTiCAR for the treatment of osteoarticular lesions. Thanks to the combination of two advanced therapy medicinal products, a polymeric nanofibrous bone wound dressing and bone marrow-derived mesenchymal stem cells, the ARTiCAR promoted both subchondral bone and cartilage regeneration. [Nat Commun]

The extended life expectancy and the raise of accidental trauma call for an increase of osteoarticular surgical procedures. Arthroplasty, the main clinical option to treat osteoarticular lesions, has limitations and drawbacks. In this manuscript, we test the preclinical safety of the innovative implant ARTiCAR for the treatment of osteoarticular lesions. Thanks to the combination of two advanced therapy medicinal products, a polymeric nanofibrous bone wound dressing and bone marrow-derived mesenchymal stem cells, the ARTiCAR promotes both subchondral bone and cartilage regeneration. In this work, the ARTiCAR shows 1) the feasibility in treating osteochondral defects in a large animal model, 2) the possibility to monitor non-invasively the healing process and 3) the overall safety in two animal models under GLP preclinical standards. Our data indicate the preclinical safety of ARTiCAR according to the international regulatory guidelines; the ARTiCAR could therefore undergo phase I clinical trial.

Regeneration of osteochondral defects represents a major challenge, especially considering the aging of the population. The surgical procedures currently applied (bone graft, mosaicplasty, micro-fracture, articular prosthesis, therapeutic implant), are invasive and/or painful for the patient, with limited efficacy and side effects. Lesions of the femoral condyles, for example, are a common side effect that could have serious consequences. A 2002 study found that ≥60% of patients undergoing arthroscopy showed osteochondral defects; in more than half of the cases, such a lesion was classified as grade 3 or higher, according to the International Cartilage Repair Society (ICRS) scale. Osteochondral defects do not heal properly and, even when treated (e.g. by Pridie’s marrow stimulation or by mosaicplasty treatment) consistently led to osteoarthritis (OA). This inevitably has a high impact on the public health system, with the direct costs of the treatment, but it also has repercussions on the general economy (social costs and loss of economic production), setting the overall costs of the disease between 0.25 and 1% of a country’s GDP. The unique properties of the cartilage (multilayered cell structure, different extracellular matrix composition and fibril orientation) make it difficult to repair. Surgical techniques like micro-fracture, mosaicplasty, osteoarticular transplantation or autologous chondrocytes implant may allow a partial functional recovery, but are mostly aimed to relieve the pain and prevent the lesion to spread. Besides their variable outcome and intrinsic limitations none of the afore-mentioned techniques was shown to restore the hyaline articular surface, justifying the search for alternatives to promote osteoarticular regeneration (OAR). Recently, autologous chondrocytes pre-cultured on a membrane of mammalian collagen, were used to fill articular focal lesions and promote cartilage regeneration. However, when performed on subchondral bone, they showed site morbidity and fibrocartilage formation, leading to a dysfunctional repair. To overcome these limitations, mesenchymal stem cell (MSCs)-based therapies emerged, which employ autologous bone marrow-derived MSCs to increase the efficiency of OAR. A combination of biomaterials, stem cells and active molecules are therefore needed to promote an effective tissue repair and to achieve a functional recovery of the articulation.

Recently, we proposed the ARTiCAR (ARTicular CArtilage and subchondRal bone implant) combined Advanced Therapy Medicinal Products (ATMPs) for personalized OAR (Fig. 1a, b). The implant is made of a nanofibrous wound dressing (FDA-approved resorbable polymeric Poly-ε-caprolactone) and autologous bone marrowderived MSCs (Fig. 1a). The wound dressing is nanofunctionalized with nanoreservoirs, for cell contact-dependent delivery of physiological concentrations of bone morphogenetic factor 2 (BMP2). The nanoreservoirs technology enabled to reduce the dose of BMP2 to physiological levels, making it locally and sustainably available, and reducing the adverse effects of its massive release, e.g. from soaked collagen sponges currently used in the clinic. In this work, we test the safety of the ARTiCAR combined ATMPs in two different animal models. Moreover, we assess the feasibility of non-invasive monitoring of the healing process in sheep, via MRI. The results of the toxicity and biodistribution tests, run accordingly to the international regulatory guidelines for cell therapies and medical devices and Good Laboratory Practice (GLP), prove the biosafety of the ARTiCAR combined ATMPs, which can therefore be used in phase I clinical trials as a ready-to-use, flexible implant to address both cartilage and subchondral bone regeneration in OA patients.

The global cartilage repair/regeneration market is valued at USD 4.2 billion in 2016 and is expected to grow at a CAGR of 5.4% during the 2014-2025 period, owing to the increased life expectancy. Currently, the international standard treatment for OA is total knee arthroplasty (TKA). Despite its rapidly increasing utilization (77,000 patients/year younger than 55 years, in the US), TKA is prone to complications, like a higher risk of infection, persistent knee pain, patellar resurfacing problems and prosthetic fracture. Once complications developed, the consequences for the patients are severe, which is why 20% of the patients who underwent TKA were unsatisfied abut the surgery outcome. In most cases, revisions were required within 2-5 years after the primary implant. Regenerative nanomedicine combines the use of biomaterials, nanotechnologies and cells to offer better solutions to issues like OAR, where a complex interface regeneration is required. In this work, we assessed the feasibility, non-invasive monitoring and safety of the ARTiCAR combined ATMPs. Similarly to other smart implantable scaffolds that promote osteochondral differentiation, the ARTiCAR releases a bone promoting factor. However, thanks to our patented nanoreservoir technology that provide cell contactdependent gradual release, the total amount of BMP2 used in the ARTiCAR is 10.000 times lower than that of BMP2-soaked collagen membranes used in the clinic, reducing both potential inflammatory side effects and the overall costs of the procedure. Differently to other approaches where a poor subchondral bone regeneration was achieved, the ARTiCAR address simultaneous regeneration of both the subchondral bone and the cartilage, representing an innovative technology for promoting OAR in a localized osteochondral defect. For cartilage regeneration, the ARTiCAR incorporates MSCs. Human MSCs are currently used in clinical trials for promoting OAR, because of their transdifferentiation potential coupled to immunomodulatory effect. In future clinical trials, the MSCs could be directly harvested from patients, allowing for autologous transplantation. In order to be transplanted in human, MSCs will undergo an implemented quality control and will be used only if the minimum release criteria for this type of ATMP are satisfied. However, since tumorigenicity of MSCs is still debated, the biodistribution of hMSCs is a critical concern of preclinical safety. After the implant of ARTiCAR, traces of hMSC DNA were found in the testes of one male nude rat, out of 40 implanted animals. Also, no tumor formation was observed in the transplanted rats and sheep, neither at the implant site, nor in the rest of the body. Altogether, these data pinpoint the safety of the ARTiCAR implant in respect to the tumorigenicity of the transplanted MSCs. In summary, we showed the feasibility of the ARTiCAR implant in a large animal model and the possibility to follow OAR non-invasively, by mean of MRI. More importantly, we showed the safety of the ARTiCAR, as no acute or long term toxicity was detected, neither in nude rats nor in sheep. Therefore, the ARTiCAR can enter phase I clinical trials as a treatment for osteochondral defects, with the potential to be used also for other conditions, like tendon degeneration and age-related degenerative musculoskeletal issues. As such, the ARTiCAR could replace more invasive current treatments, with the potential to impact 300.000 to 450.000 patients/year only in the US ($4-5 billion global market).

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