Donor-matched comparison of chondrogenic progenitors resident in human infrapatellar fat pad, synovium and periosteum – Implications for cartilage repair

Abstract

Purpose: Over the past decades, multiple tissue sources have been used as a source of cells in clinical trials for cartilage repair, including infrapatellar fat pad, synovium, bone marrow, peripheral blood, adipose-stromal vascular tissue, and periosteum. Despite the increased clinical use of bone marrow/peripheral blood cells and adipose tissue obtained subcutaneously or through liposuction, there are reports suggesting higher concentration and chondrogenic potential of progenitors derived from synovium and infrapatellar fat pad. Such tissues around the knee are easily accessible and can be harvested via arthroscopy or open surgery using minimally invasive techniques, with minimal complications at the donor site. However, to date, there is no consensus on the optimal cell source for cartilage repair. Furthermore, no in-vitro or in-vivo study has systematically compared tissue sources used in clinical practice. Therefore, the aims of this study were to quantitatively define and compare the progenitor populations in infrapatellar fat pad, synovium and periosteum in donor-matched osteoarthritic (OA) knee joints with respect to 1) cell concentration, 2) chondrogenic connective tissue progenitor (CTP-Cs) prevalence and concentration, and 3) biological potential of the CTP-Cs progenitors, and 4) to determine the effect of demographic factors (age and gender) on these variables. Methods: Twenty-eight patients with varus deformity undergoing total knee arthroplasty (TKA) were recruited (mean age:63.1years, range:37-82years, male=12, female=16) under an IRB approved protocol. During TKA, 0.5-1.5cc of synovial membrane was harvested from the medial suprapatellar area, assuring that fat tissue was not present. 2.5cc of fat was harvested from the infrapatellar fat pad after removal of overlying synovium. 0.5-1.5cc of periosteum was harvested from the anterior femoral region, proximal to the trochlear grove. Wet weight of tissues was recorded and cells were isolated by enzymatic digestion. The total number of nucleated cells per mg of tissue was 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) 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 from fat, synovium, and periosteum (Fig 1). A significance level of 0.05 was used overall, with Holm’s method used to control the family-wise error rate. Pearson’s correlation coefficient (r) was used to estimate the within-patient association between two colony metrics for each individual patient, while the repeated measures correlation coefficient (rrm) was used to estimate the within-patient association between two colony metrics that was common across all patients. All analyses were performed in R 3.4.1 (R Foundation for Statistical Computing, Vienna, Austria). Results: Cell concentration (nucleated cells/mg) did not differ between fat, synovium, and periosteum across all age groups and gender. Patients <60years did not differ on CTP prevalence (PCTP-C)(CTP-Cs/million nucleated cells) between the three sources. However, in patients >60years, both fat and synovium had a higher PCTP-C than periosteum (p<0.001). For females, PCTP-C was higher in fat (p<0.001) and synovium (p=0.001) than periosteum (after adjusting for age). No differences in PCTP-C were observed in males. Overall CTP-C concentration (CTP-C/mg) did not differ significantly between the three sources (p=0.226), however significant differences were encountered with age (p<0.001). Proliferation potential of CTP-Cs differed significantly between tissue sources (synovium>fat>periosteum), specifically in patients >60years. Cell migration (density) and differentiation potential were comparable (p>0.5) (Table1). Conclusions: This study isolated cells using an established protocol from three potential tissue sources resident in the knee, and cultured them in the same chondrogenic conditions for systematically quantifying and comparing the performance of the progenitors in vitro. To the best of our knowledge, this study is the first to systematically assess these tissue sources by implementing reproducible methods for automated colony-forming unit assay in accordance with ASTM Standard Method F2944-12 to quantitatively compare and characterize the cell and CTP-C population resident in these donor-matched tissues. These results provide insight into potential patient population that may benefit from one or the other tissue as source of progenitors for cartilage repair. Chondrogenic progenitors are available in fat, synovium, and periosteum, with differences encountered based on age and gender. The heterogeneity among the CTP-Cs suggests performance-based selection might be useful to optimize cell-sourcing strategies to improve efficacy of cellular therapies for cartilage repair.View Large Image Figure ViewerDownload Hi-res image Download (PPT)