title: Advances in autologous chondrocyte implantation and related techniques for cartilage repair.
abstract: Articular cartilage is a specialized tissue exhibiting low intrinsic capabilities of regeneration or healing after injury. Autologous chondrocyte implantation (ACI) and scaffold-supported ACI are often used for treatment of larger chondral defects (> 2 cm2). These utilize open surgery re-implantation of ex vivo cultured autologous chondrocytes harvested as a biopsy arthroscopically in a prior surgery. This two-step procedure is an advanced and expensive treatment that despite high expectations have failed to regenerate articular cartilage in a consistent and predictable fashion, and as many as 25% the operated of patients have dissatisfactory outcomes. The objective of the present thesis was to address and investigate methods for optimizing the steps involved in the ACI and scaffold-supported ACI treatment including chondrocyte culture environment, chondrocyte labeling and tracking, improved biomaterials, and cell seeding densities. We hypothesized that these areas were eligible for targeted optimization, which has been addressed in the five papers constituting the work performed in the present thesis. The first two studies address the in vitro cell expansion of chondrocytes before re-implantation. After validation of hypoxia-suitable housekeeping genes for quantitative gene expression analysis using previously validated algorithms (study 1) the effect of combined hypoxic- and 3D culture on human chondrocytes gene expression was investigated (study 2). An in vitro experiment was performed to determine the effect on gene expression of an intracellular superparamagnetic labeling agent for 1.5T MRI-tracking of alginate-embedded human chondrocytes (study 3). We further performed a literature study, reviewing the cell seeding densities of the implanted chondrocytes used in clinically available cell transplantation-based treatments for cartilage repair (study 4). Finally, we tested the addition of dermatan sulfate to a clinically approved methoxy-polyethen-glycol (MPEG) substituted polylactide-co-glycolic acid (PLGA) scaffold by implantation of cell-free scaffolds in an osteochondral rabbit model (study 5). We determined a set of hypoxia-stable reference genes in study 1 that were then used in study 2. We observed that there was a positive effect on chondrogenic gene expression in human chondrocytes when culturing in 3D compared to monolayer and in hypoxia compared to normoxia and that there was an additional positive combined effect of 3D and hypoxia. Using a clinical MRI-system we were able to track labeled chondrocytes for up to 4 weeks, but we found that the labeling agent had significant effects on chondrocyte gene expression, which could potentially confound results when used in vivo. In our review of chondrocyte seeding densities we found large variability between commercial products and a very limited preclinical basis for the applied densities. Lastly, we found that there was no positive effect in vivo of adding dermatan sulfate to MPEG-PLGA scaffold in osteochondral repair. We conclude that while the outcome of ACI-related treatments certainly is multifactorial it may be improved by optimizing the in vitro culture by hypoxic and 3D culture and by adjusting the chondrocyte seeding density. Our studies on biomaterials and potential system for cell tracking in vivo did not show results that justified further studies and clinical trials.
authors: Orthopaedic Research Laboratory, Department of Orthopaedics, Aarhus University Hospital, Nørrebrogade 44, Aarhus C, Denmark. foldager@ki.au.dk
authors: Casper Bindzus
authors: CB
authors: Foldager
mesh_terms: D000818:Animals; D002358:Cartilage, Articular / Q000033:anatomy & histology / Q000293:injuries / Q000502:physiology* / Q000601:surgery*; D018929:Cell Culture Techniques; D017690:Cell Transplantation / Q000379:methods; D019902:Chondrocytes / Q000166:cytology / Q000637:transplantation*; D003871:Dermatan Sulfate / Q000494:pharmacology; D005353:Fibronectins / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D020869:Gene Expression Profiling; D006801:Humans; D011817:Rabbits; D012038:Regeneration; D054457:Tissue Scaffolds; D014182:Transplantation, Autologous
chemical_list: D005353:Fibronectins; D003871:Dermatan Sulfate