Preclinical safety study of a combined therapeutic bone wound dressing for osteoarticular regeneration

Abstract

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.

Fig. 1

Composite advanced therapy medicinal product (ATMP) developed for the osteoarticular regeneration (OAR) of cartilage focal lesions. a The ARTiCAR is a composite ATMP that combines an FDA-approved synthetic wound dressing and a living therapeutic made of autologous mesenchymal stem cells (MSCs), embedded in alginate/hyaluronic acid hydrogel. The wound dressing, named NanoM1-BMP2 is made of nanofibrous poly-ε-caprolactone nano-functionalized with Bone morphogenetic protein 2 and aims at subchondral bone regeneration. The cellular hydrogel aims at articular cartilage regeneration. The NanoM1-BMP2 was tested for cytotoxicity in vitro (step 1); the whole ARTiCAR was tested for acute toxicity, biodistribution and persistence in an osteochondral defect nude rat model (step 2) and for feasibility, safety, and non-invasive monitoring of the procedure (step 3) in a sheep intra-articular model. b The ARTiCAR aims at the simultaneous regeneration of both the articular cartilage and the subchondral bone in a one-step surgical procedure. After harvesting MSCs from patient’s bone marrow, the NanoM1-BMP2 is applied in contact with the injured subchondral bone; afterwards, the MSCs mixed with the hydrogel is applied to fill the defect

Fig. 2

In vitro evaluation of the NanoM1-BMP2 wound dressing cytotoxicity. MRC-5 cell line was cultured in the presence of Polyurethane film, (RM-A; a–e), NanoM1-BMP2 wound dressing (f–j) or high density polyethylene film (RM-C; k–q). Five sizes (20, 16, 9, 4, 1 mm²) of membranes were used. Cells were inspected for morphological changes over a 3 day period. Proliferation/viability was assessed via WST-1 assay (p–n) at 24 h (C.Ris Pharma, CRO, France). Error bars represent standard deviation. Scale bar: 100 µm

Fig. 3

clinical, hematological, biochemical and histopathology evaluation of the ARTiCAR safety in an osteochondral defect nude rat model. a Individual body weight curves of five male (M1-M5) and five female (F1-F5) rats, after implant of the ARTiCAR ATMP. b, c Seven days after implantation, ventricular blood of implanted rats was collected (group 1: ARTiCAR, group 2: hydrogel without hMSCs; (n = 20) and analyzed for hematological and biochemical parameters. WBCs: white blood cells; RBCs: red blood cells; HGB: haemoglobin; HCT: hematocrits; MCV: mean corpuscular volume; PLT: platelets. Data are presented as the mean ± SD. One-way ANOVA followed by the Bonferroni post-hoc test was performed for significance. A p value ≤ 0.05 was considered significant. (C.Ris Pharma, CRO, France). d, e Histological sections of the intra-articular implant site in nude rats with femoral bone/cartilage defect, 7 days after surgery (n = 20) for group 1 (d) and 2 (e), at ×5 magnification. Comparable inflammatory responses involved in bone healing process and no systemic changes were observed in both group. Asterisks represent the limits of the defect. Scale bar: 1 mm

Fig. 4

Feasibility, non-invasive monitoring and safety evaluation of the ARTiCAR combined ATMPs implanted in sheep intra-articular defect model. a–e the OAR process after ARTiCAR implant was monitored non-invasively by means of MRI immediately after surgery (a) and at 12 (b) and 26 (c) weeks post implant. The extension of the bone defect (white asterisks) and the ongoing OAR (white arrowheads) are visible. Micro CT-scan (d) followed by 3D surface rendering (e) were performed on freshly explanted joints. f The level of cartilage regeneration was macroscopically assessed based on ICSR score system. g, h Whole explants were sectioned and stained with safranin o - fast green (blue: bone; red: cartilage), imaged at ×4 and stitched together (g) to evaluate the OAR according to the ICRS II score system (g). Scale bar: 1 mm. ICRS II parameters were examined for ARTiCAR and the control groups considered and analyzed by mean of two-way ANOVA followed by Bonferroni post hoc test (h). * = p ≤ 0.1; ** = p ≤ 0.05. Dots represent individual scores, bars represent mean scores and error bars represent standard errors. (i–n) Zones of osteochondral remodeling within the treated defects in animals from AG (I,L), NT (j, m) and ARTiCAR (k, n) groups, either at 12 or 26 weeks post implant. Bone (white asterisks), cartilage (black asterisk), fibrocartilage (black arrow) and subchondral bone (yellow arrow) are shown. Scale bar: 500 µm