Enhancement of in vitro proliferation and bioactivity of human anterior cruciate ligament fibroblasts using an in situ tissue isolation method and basic fibroblast growth factor culture conditions

Abstract

Background:Previous studies have reported poor proliferation and bioactivity of human anterior cruciate ligament fibroblasts (hACLFs) after injury. As hACLFs are one of the most significant and indispensable source of seed cells in constructing tissue-engineered ligament, enhancing hACLF proliferation would offer favorable cellular-biological ability and induce the extracellular matrix secretion of hACLFs after loading on multiple types of scaffolds. Enhancing the bioactivity of hACLFs would improve tissue repair and functional recovery after tissue-engineered ligament transplantation. This study compared cells prepared by collagenase digestion and the in situ culture of tissue pieces and investigated the effect of basic fibroblast growth factor (bFGF) on hACLFs.Methods:Six adult patients participated in this study. Of these patients, tissues from three were compared after culture establishment through collagenase digestion or in situ tissue isolation. hACLF phenotypic characteristics were assessed, and the effect of bFGF on hACLF cultures was observed. hACLFs cultured with and without bFGF served as the experimental and control groups, respectively. Cell Counting Kit-8 was used to detect proliferation. The expression of ligament-related genes and proteins was evaluated by immunofluorescence staining, real-time polymerase chain reaction (PCR) assays, and Western blot assays.Results:The morphology of hACLFs isolated using the two methods differed after the 2nd passage. The proliferation of cells obtained by in situ culture was higher than that of cells obtained by collagenase digestion. hACLFs cultured with bFGF after the 3rd passage exhibited a higher proliferation rate than the controls. Immunofluorescence staining, real-time PCR, and Western blot analysis showed a significant increase in ligament-related gene and protein expression in the hACLFs cultured with bFGF.Conclusions:The in situ isolation of tissue pieces enhanced hACLF proliferation in vitro, and the hACLFs exhibited phenotypic characteristics of fibroblasts. hACLFs cultured with bFGF exhibited increased hACLF bioactivity.