Positive effects of an extracellular matrix hydrogel on rat anterior cruciate ligament fibroblast proliferation and collagen mRNA expression

Background/Objective: We have previously shown that an extracellular matrix (ECM) bioscaffold derived from porcine small intestine submucosa (SIS) enhanced the healing of a gap injury of the medial collateral ligament as well as the central third defect of the patellar tendon. With the addition of a hydrogel form of SIS, we found that a transected goat anterior cruciate ligament (ACL) could also be healed. The result begs the research question of whether SIS hydrogel has positive effects on ACL fibroblasts (ACLFs) and thus facilitates ACL healing. Methods: In the study, ECM-SIS hydrogel was fabricated from the digestion of decellularised and sterilised sheets of SIS derived from αGal-deficient (GalSafe) pigs. As a comparison, a pure collagen hydrogel was also fabricated from commercial collagen type I solution. The morphometrics of hydrogels was assessed with scanning electron microscopy. The ECM-SIS and collagen hydrogels had similar fibre diameters (0.105±0.010μm vs. 0.114±0.004μm), fibre orientation (0.51±0.02 vs. 0.52±0.02), and pore size (0.092±0.012μm vs. 0.087±0.008μm). The preservation of bioactive properties of SIS hydrogel was assessed by detecting bioactive molecules sensitive to processing and enzyme digestion, such as growth factors fibroblast growth factor-2 (FGF-2) and transforming growth factor-beta 1 (TGF-β1), with enzyme-linked immunosorbent assay. ACLFs were isolated and expanded in culture from explants of rat ACLs (n=3). The cells were then seeded on the hydrogels and cultured with 0%, 1%, and 10% foetal bovine serum (FBS) for 3 days and 7 days. Cell attachment was observed using a light microscope and scanning electron microscopy, whereas cell proliferation and matrix production (collagen types I and III) were examined with bromodeoxyuridine assays and reverse transcription-polymerase chain reaction, respectively. Results: The results showed that FGF-2 and TGF-β1 in the SIS hydrogel were preserved by 50% (65.9±26.1ng/g dry SIS) and 90% (4.4±0.6ng/g dry SIS) relative to their contents in ECM-SIS sheets, respectively. At Day 3 of culture, ACLFs on the SIS hydrogel were found to proliferate 39%, 31%, and 22% more than those on the pure collagen hydrogel at 0%, 1%, and 10% FBS, respectively (p<0.05). Collagen type I mRNA expression was increased by 150%, 207%, and 100%, respectively, compared to collagen hydrogel (p<0.05), whereas collagen type III mRNA expression was increased by 123% and 132% at 0% and 1% FBS, respectively (all p<0.05) but not at 10% FBS. By Day 7, collagen type I mRNA expression was still elevated by 137% and 100% compared to collagen hydrogel at 1% and 10% FBS, respectively (p<0.05). Yet, collagen type III mRNA levels were not significantly different between the two groups at any FBS concentrations. Conclusion: Our data showed that the ECM-SIS hydrogel not only supported the growth of ACLFs, but also promoted their proliferation and matrix production relative to a pure collagen hydrogel. As such, ECM-SIS hydrogel has potential therapeutic value to facilitate ACL healing at the early stage after injury.