Quantitative assessment and comparison of the heterogeneous chondrogenic progenitors resident in various tissues around osteoarthritic knee for cartilage repair

Purpose: Cell-based therapy for cartilage repair is becoming an established technique for modern healthcare. Unfortunately, there is no consensus on an optimal cell source yet. Connective tissue progenitors (CTPs), are a heterogeneous population of colony-founding cells, resident in connective tissue, whose progeny can differentiate into a chondrogenic phenotype to contribute to the formation of new cartilage tissue. CTPs are generally low in number, and their verification is not easy. Colony-forming-unit assay has been used extensively to perform quantitative and functional measurements of the CTPs from different cell sources. In the absence of definitive surface markers that can be used to distinguish between cells with and without CTP potential in freshly isolated cells, it is necessary to directly measure function (self-renewal capacity demonstrated by colony formation and their chondrogenic differentiation expression) in order to identify chondrogenic progenitor cells. The recently adopted ASTM standard test method F2944-12 defines reproducible methods that enable quantitative, automated colony analysis and eliminate the challenges of subjective manual counting that have diminished the rigor (accuracy, repeatability, reproducibility, and documentation) of colony analysis in the past. Methods: Ten patients with varus deformity undergoing total knee arthroplasty (TKA) were recruited (mean age: 59years, range: 37-76years, male=4, female=6) under an IRB approved protocol. During TKA, Outerbridge grade 1-2 (G1-2) cartilage was obtained from the lateral femoral condyle, Outerbridge grade 3-4 (G3-4) cartilage was obtained from medical femoral condyle, synovial membrane was harvested from the medial suprapatellar area, fat was harvested from the infrapatellar fat pad after removal of overlying synovium, periosteum was harvested from the anterior femoral region, proximal to the trochlear grove, and bone marrow was aspirated from iliac crest. Wet weight of tissues was recorded and cells were isolated by enzymatic digestion. The total number of nucleated cells per cc of tissue were counted using hemocytometer (cell concentration, [Cells]). This cell pellet was then suspended in chondrogenic medium and plated at a density of 100,000/well (24,000cells/cm2). For bone marrow, buffy-coat was prepared and cell were counted to plate at a density of 500,000cells/well (120,000/cm2). Cells were cultured at 37oC, 98% humidity, 20% O2, and 5% CO2 for 6 days. On day 6, cultures were harvested, fixed and stained for nuclei with bis-benzimide (BB), and for glycosaminoglycan-containing extracellular matrix (GAG-ECM) with acridine orange (AO). Automated quantitative colony forming unit (CFU) analysis was performed using ColonyzeTM image analysis software to assess CTP-derived colonies. A significance level of 0.05 was used overall, if the overall test for cell source was significant at the 0.05 level, Tukey’s multiple comparison method was used to exam all pairwise comparisons between cell sources. Results: Statistical significant differences were found between A) Cell concentration (cells/mg) were significantly higher in G3-4 (p=0.002) and bone marrow (p=0.001) than in other sources , B) CTP-C prevalence (CTP-Cs per million cells plated) was higher in fat (p=0.001), synovium (p=0.003) and G1-2 cartilage (p=0.02) than in bone marrow, C) Proliferation potential of CTP-Cs obtained from synovium was significantly higher than other tissue sources (p<0.001), and fat, periosteum and bone marrow derived CTP-C had greater proliferation potential than G3-4 cartilage, D) Colony density was significantly higher in both G1-2 and G3-4 cartilage than other tissue sources (p<0.001), suggesting lower migration potential for cartilage derived CTP-Cs, and E) AO per cell expression was also significantly higher in both G1-2 and G3-4 cartilage than other tissue sources (p<0.001), suggesting higher GAG-ECM production by cartilage derived CTP-Cs (Table 1). Conclusions: Our data suggests that both G1-2 and G3-4 cartilage can be used as a source of CTPs and synovium was identified as the most promising non-cartilage tissue source for generation of chondrogenic cells for clinical applications. To our knowledge, this study is the first to quantitatively compare and characterize the cell and CTP-C population in Outerbridge grade 1-2 and grade 3-4 cartilage, infrapatellar fatpad, synovium, periosteum and bone marrow using systematic and reproducible methods for automated assay of colony-forming units in accordance with ASTM standard test method F2944-12. These results provides quantitative assessment and comparison between potential tissues that have been used as sources of progenitors for cartilage repair for long time now without much information on the standard measurements of the quantity and quality of chondrogenic progenitors resident in these tissues. Currently, there is lack of evidence behind the translation of stem cell therapies from basic science to the clinical practice of cartilage repair. These data contribute to the rational development of cell therapy procedures to treat OA. However, the diversity of CTP-C performance at the level of individual colonies, suggests the potential value of selecting CTP-C clones with preferred attributes for vitro expansion.