Page 30 - ISAKOS 2020 Newsletter Volume 2

CURRENT CONCEPTS
Stem Cell-Based Treatment for Rotator Cuff Repair: Are We Talking About the Same Thing?
Preparations of highly concentrated primary cells from lipoaspirates, also referred to as stromal vascular fraction (SVF), are also obtained by means of purely physical methods. Collagenase-digested lipoaspirates are filtered, and the SVF is concentrated by centrifugation. These cell concentrates also include cells of the hematopoietic line such as granulocytes, monocytes, and lymphocytes in addition to endothelial cells, pericytes, and stromal cells. Although less rich in platelets and growth factors than bone-marrow aspirates, the SVF of lipoaspirates is also promising for regeneration and biological augmentation1.
In order to compare the results from different studies involving bone marrow-derived “stem cells,” a common terminology is essential3. The term mesenchymal stem cell (MSC) is often used interchangeably with the term connective-tissue progenitor cell (CTP). The number of nucleated cells found in concentrated bone marrow is counted and used as a predictor of the number of MSCs present. The nucleated cell population in bone marrow consists not only of MSCs but also macrophages, lymphocytes, and granulocytes. However, MSCs represent a very small fraction (0.001% – 0.01%) of the total population of nucleated cells in bone marrow and are defined as cells that (1) are able to proliferate and form colony-forming units (CFUs) (consisting of ≥8 cells, with all cells being clones from one stem cell), (2) have the ability to adhere to plastic, and (3) express surface markers. These three characteristics set MSCs apart from the other nucleated cells found in bone marrow.
01 Standard method of counting CFUs after cultivation a sample on a culture dish. Only 25% of each culture dish area is counted microscopically, usually by one trained individual. (Courtesy
of Mary Beth McCarthy, UConn Musculoskeletal Institute, Farmington, CT, USA)
It should be mentioned that the nucleated cell count is obtained via an automatic cell-counter machine, which counts every single cell. When counting CFUs, only 25% of each culture dish is counted microscopically, usually by one specially trained examiner. This number is then used to calculate the total number of CFUs in the culture plate and assumes an equal distribution of cells (Fig. 1)5.
Although this technique is the gold standard for counting colonies, its results may vary and are dependent on the examiner’s experience, the CFU distribution, and the time point of counting. Factors such as the patient’s age may influence the number of CFUs, as laboratory studies have shown that younger patients have a more rapid appearance of CFUs compared with older patients.
In a culture dish in which CFUs have been established and multiple stem cells are communicating, it becomes impractical to know whether this group of cells comes from a single CFU or from multiple CFUs. Because of the increased age of the patient population with rotator cuff tears, a normal time point to count CFUs in clinical application would be 7-10 days after harvesting. In addition, the CTP prevalence (CFUs or CTPs / million nucleated cells) is an important marker for clinicians as it is a reference for the potential number of stem cells for biological augmentation.
02 Photograph showing CFUs in a specimen from a young patient after 48 hours of cultivation in a culture dish. The photograph was taken at 10x magnification, at which determination of 2 single CFUs is almost impossible. (Note that, because of formatting, there might be some scale differences in the image.) (Courtesy of Mary Beth McCarthy, UConn Musculoskeletal Institute, Farmington, CT, USA.)
Two different strategies for MSC application are currently used. The first technique is a simple local injection of a cell suspension that has been previously isolated or even purified ex vivo prior to reinjection. The aim of this strategy is for these cells to replace damaged cells within a tissue in order to reconstitute integrity and function4.
28 ISAKOS NEWSLETTER 2020: VOLUME II