Secretion of Angiogenic Proteins by Human Multipotent Mesenchymal Stromal Cells (MSC) under Hypoxic Culture Conditions and Clinical Application of MSC in Steroid Induced Osteonecrosis in Children.
Authors: Müller Ingo , Vaegler Martin , Holzwarth Christina , Schütt Burkhardt , Pfister Stefan M. , Greil Johann , Rudert Maximilian , Handgretinger Rupert 
Published: April 2020

Abstract
Osteonecrosis is a frequent complication after treatment for childhood leukemia.17% of adolescents above the age of 15 years treated in the ALL-BFM 95 trial protocol suffered from avascular necrosis of the bone (AVN). In addition, children receiving steroid-based treatment, e. g. autoimmune diseases, are frequently affected as well. If conservative measures fail, core decompression surgery is required. The success rate of surgery varies depending on the stage from 29% to 84% (Castro et al., 2000). Evidently, improved intervention strategies are warranted. Therefore, we evaluated the safety of implanting autologous MSC during surgical intervention in order to improve bone regeneration. AVN represent an area of absent blood perfusion and consequently low supplies of nutrients and oxygen. Therefore, we cultivated MSC under oxygen tensions of 3% or lower. These cells showed slowed proliferation and differentiation into bone. However, MSC grown under these low-oxygen conditions secreted significant amounts of vascular endothelial derived factor and selected insulin-like growth factors and their binding proteins. These factors might facilitate reperfusion and regeneration of bone at the site of implantation during core decompression surgery. 10 ml of heparinized bone marrow was drawn from the iliac crest of the patient. MSC were expanded under GMP conditions without the use of animal proteins employing platelet lysate and human fresh frozen plasma as source for growth factors (Müller et al., 2006). Recombinant trypsin was used for detachment and harvest of the cells. MSC could be expanded from 5 out of 5 juvenile bone marrow aspiates. Within three weeks of culture, sufficient numbers of cells, i. e. 112·106 ± 67·106, were generated in a certified GMP lab. At each of the three passages diagnostic cultures for aerobic bacteria were established and remained sterile throughout the culture period. MSC grown under these conditions were able to differentiate into osteoblasts. No chromosomal aberrations were detected by matrix-based comparative genomic hybridization using DNA microarrays containing &gt;6,400 genomic DNA fragments. At the day of core decompression surgery, cells were harvested and resuspended to a final volume of 3 ml in saline. As core decompression implies drilling a hole into the necrotic area, the MSC were implanted through the same channel, which was sealed thereafter. Hence, instillation of the MSC did not require additional surgical procedures. Application of MSC was feasible and successful during core decompression surgery in all of the 5 patients. Follow up of the patients is between 14 and 8 months. None of the patients experienced adverse side effects, most importantly, there were no infectious complications. Clinically, no patient deteriorated and all patients reported less or no pain two months after surgery. Although the number of patients in this pilot trial is small, initial data for safety and feasibility of employing MSC in juvenile steroid-induced osteonecrosis is very promising.