Scaffold-free Three-dimensional Graft From Autologous Adipose-derived Stem Cells for Large Bone Defect Reconstruction: Clinical Proof of Concept.

Long bone nonunion in the context of congenital pseudarthrosis or carcinologic resection (with intercalary bone allograft implantation) is one of the most challenging pathologies in pediatric orthopedics. Autologous cancellous bone remains the gold standard in this context of long bone nonunion reconstruction, but with several clinical limitations. We then assessed the feasibility and safety of human autologous scaffold-free osteogenic 3-dimensional (3D) graft (derived from autologous adipose-derived stem cells [ASCs]) to cure a bone nonunion in extreme clinical and pathophysiological conditions. Human ASCs (obtained from subcutaneous adipose tissue of 6 patients and expanded up to passage 4) were incubated in osteogenic media and supplemented with demineralized bone matrix to obtain the scaffold-free 3D osteogenic structure as confirmed in vitro by histomorphometry for osteogenesis and mineralization. The 3D "bone-like" structure was finally transplanted for 3 patients with bone tumor and 3 patients with bone pseudarthrosis (2 congenital, 1 acquired) to assess the clinical feasibility, safety, and efficacy. Although minor clones with structural aberrations (aneuploidies, such as tri or tetraploidies or clonal trisomy 7 in 6%-20% of cells) were detected in the undifferentiated ASCs at passage 4, the osteogenic differentiation significantly reduced these clonal anomalies. The final osteogenic product was stable, did not rupture with forceps manipulation, did not induce donor site morbidity, and was easily implanted directly into the bone defect. No acute (<3 mo) side effects, such as impaired wound healing, pain, inflammatory reaction, and infection, or long-term side effects, such as tumor development, were associated with the graft up to 4 years after transplantation. We report for the first time that autologous ASC can be fully differentiated into a 3D osteogenic-like implant without any scaffold. We demonstrated that this engineered tissue can safely promote osteogenesis in extreme conditions of bone nonunions with minor donor site morbidity and no oncological side effects.

Characterization of Abcc4 gene amplification in stepwise-selected mouse J774 macrophages resistant to the topoisomerase II inhibitor ciprofloxacin.

Exposure of J774 mouse macrophages to stepwise increasing concentrations of ciprofloxacin, an antibiotic inhibiting bacterial topoisomerases, selects for resistant cells that overexpress the efflux transporter Abcc4 (Marquez et al. [2009] Antimicrob. Agents Chemother. 53: 2410-2416), encoded by the Abcc4 gene located on Chromosome 14qE4. In this study, we report the genomic alterations occurring along the selection process. Abcc4 expression progressively increased upon selection rounds, with exponential changes observed between cells exposed to 150 and 200 µM of ciprofloxacin, accompanied by a commensurate decrease in ciprofloxacin accumulation. Molecular cytogenetics experiments showed that this overexpression is linked to Abcc4 gene overrepresentation, grading from a partial trisomy of Chr 14 at the first step of selection (cells exposed to 100 µM ciprofloxacin), to low-level amplifications (around three copies) of Abcc4 locus on 1 or 2 Chr 14 (cells exposed to 150 µM ciprofloxacin), followed by high-level amplification of Abcc4 as homogeneous staining region (hsr), inserted on 3 different derivative Chromosomes (cells exposed to 200 µM ciprofloxacin). In revertant cells obtained after more than 60 passages of culture without drug, the Abcc4 hsr amplification was lost in approx. 70% of the population. These data suggest that exposing cells to sufficient concentrations of an antibiotic with low affinity for eukaryotic topoisomerases can cause major genomic alterations that may lead to the overexpression of the transporter responsible for its efflux. Gene amplification appears therefore as a mechanism of resistance that can be triggered by non-anticancer agents but contribute to cross-resistance, and is partially and slowly reversible.