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Customers Also Viewed. To detach cells possibly adherent to the tube, they were incubated with 0. RBC lysis was performed by incubating the cells for 25 min. Non-labeled primary antibodies were detected by secondary antibody, goat-anti mouse-PE after incubation on ice for 20 min Table 1. For gating strategy see Additional file 1 : Figure S1. The media was changed every 3 days, and the cultures were evaluated after 10 days microscopically.
Colonies were counted in replicates and subsequently compared as mean data for each condition and donor. The calculated ratios represent the specific fold changes for each tested marker, or marker combination, of BMAC compared to the respective internal control single donor. To get an estimate about the respective cell numbers, percentages of subpopulations related to the recorded viable cell counts are presented in Additional file 2 : Table S1. Presented data for blood cell counts, CFU-F and growth factors were calculated of original non-diluted samples based on analyses of diluted samples considering respective sample dilutions both BMAC: 4.
Mean cell viability after processing was similar for unprocessed controls Cell viability in both BMAC groups and controls. Blood cell counts in both BMAC groups and controls. We observed for both BMAC a donor-depending non-hematopoietic progenitor cells enrichment between 4.
ELISA growth factor analysis of the cell lysates illustrated considerable variability between donors. Points represent mean values for each donor. Both commercial BMAC systems significantly concentrated MSC populations, platelets and growth factors, but not hematopoietic progenitor cells, compared to untreated marrow aspirate from single donors.
Despite enrichment of biologic factors, the question remains as to whether higher MSC content in BMAC leads to improved clinical efficacy. Hernigou et al. In their study of patients with atrophic non-unions of the tibia who received percutaneous BMAC injections, the volume of mineralization of the fracture callus at 4 months was directly related to the number of progenitor cells in the original injection, providing evidence that the efficacy of BM aspirations for fracture healing can be enhanced with BMAC [ 3 ].
Moreover, the CFU-F assay cannot provide timely results as it requires several days ex vivo culture time. BMAC may also promote tissue healing by delivering platelets as well as non-cellular growth factors [ 2 , 5 , 11 , 13 , 14 , 15 ]. Additionally, it up regulates angiogenesis thereby initiating a cascade of bone and soft tissue repair mechanisms in the presence of injury [ 16 ].
As we analyzed the growth factors from the lysates of the mononuclear cell fraction we hypothesize that the higher growth factors concentrations we observed in BMAC were related to the higher numbers of progenitor cells rather than platelets contents. Yet, it may be reasonable to assume that both cell types contribute synergistically as they have been shown to carry such proteins [ 18 ]. Yet, to date, the effects of immune cell subset depletion or concentration by BMAC on tissue regeneration remains unclear.
This specific subpopulation was concentrated in BMAC, providing further proof of concept for its clinical use. This is of particular importance as the frequency of these cells in the BM is very low, i. Previous studies have illustrated that BMAC application resulted in significant bone and cartilage healing in both animal models and human clinical trials [ 2 , 3 , 23 ].
In a prospective non-randomized human clinical trial, patients with large patellofemoral chondral lesions showed significantly improved clinical outcome scores at a minimum 3 year follow-up after treatment with autologous BMAC implanted into the chondral defect which was comparable to patients treated with matrix-induced autologous chondrocyte implantation [ 25 ].
However, there is currently a lack of consensus for or against the use of a scaffold coupled with BMAC for clinical applications [ 27 ]. Our data could contribute to the development of BMAC quality control assays as both commercial BMAC systems significantly concentrate MSC populations, platelets and growth factors, but not hematopoietic progenitor cells, compared to un-concentrated marrow aspirate.
Clinical trials will be necessary to correlate specific MSC subpopulations and growth factors with therapeutic efficacy. Establishing proof of concept: platelet-rich plasma and bone marrow aspirate concentrate may improve cartilage repair following surgical treatment for osteochondral lesions of the talus. World J Orthop. Article Google Scholar. In vivo comparison of the bone regeneration capability of human bone marrow concentrates vs.
Percutaneous autologous bone-marrow grafting for nonunions. Influence of the number and concentration of progenitor cells. J Bone Joint Surg Am. PubMed Google Scholar. Phenotype, donor age and gender affect function of human bone marrow-derived mesenchymal stromal cells. BMC Med. Jones E, Schafer R. Stem Cell Res Ther. Regenerate augmentation with bone marrow concentrate after traumatic bone loss. Orthop Rev Pavia. Cannulated screw delivery of bone marrow aspirate concentrate to a stress fracture nonunion: technique tip.
Foot Ankle Int. Bone marrow concentrate and platelet-rich plasma differ in cell distribution and interleukin 1 receptor antagonist protein concentration. Knee Surg Sports Traumatol Arthrosc. A prospective comparison of 3 approved systems for autologous bone marrow concentration demonstrated nonequivalency in progenitor cell number and concentration. J Orthop Trauma. The use of percutaneous autologous bone marrow transplantation in nonunion and avascular necrosis of bone.
J Bone Joint Surg Br. Mesenchymal stem cell concentration and bone repair: potential pitfalls from bench to bedside. Implant Dent. Therapeutic applications of mesenchymal stromal cells: paracrine effects and potential improvements. Tissue Eng Part B Rev. Engineering principles of clinical cell-based tissue engineering. Curr Pharm Des. Vascular endothelial growth factor receptor structure, function, intracellular signalling and therapeutic inhibition. Cell Signal.
Platelet lysate from whole blood-derived pooled platelet concentrates and apheresis-derived platelet concentrates for the isolation and expansion of human bone marrow mesenchymal stromal cells: production process, content and identification of active components. CD73 and CD39 ectonucleotidases in T cell differentiation: beyond immunosuppression.
FEBS Lett. Myelo-lymphoid lineage restriction occurs in the human haematopoietic stem cell compartment before lymphoid-primed multipotent progenitors. Nat Commun. Arthritis Rheum. Single-platform quality control assay to quantify multipotential stromal cells in bone marrow aspirates prior to bulk manufacture or direct therapeutic use.
Development of an osteogenic bone-marrow preparation.