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

Abstract

SummaryBackground/ObjectiveWe 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.MethodsIn 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 ± 0.010 μm vs. 0.114 ± 0.004 μm), fibre orientation (0.51 ± 0.02 vs. 0.52 ± 0.02), and pore size (0.092 ± 0.012 μm vs. 0.087 ± 0.008 μm). 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.ResultsThe results showed that FGF-2 and TGF-β1 in the SIS hydrogel were preserved by 50% (65.9 ± 26.1 ng/g dry SIS) and 90% (4.4 ± 0.6 ng/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.ConclusionOur 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...

The cytoptrotection of small intestinal submucosa‐derived gel in HL‐1 cells during hypoxia/reoxygenation‐induced injury

“…The results indicated that SIS gel had a highly porous and interconnected interior structure (Wang et al, ). In this paper, the results of SEM indicated that SIS gel possessed randomly oriented fibrils and formed porous, interconnected structures similar to other reported ECM gels, such as nerve tissue hydrogel (Lin et al, ) and SIS gel (Liang et al, ). The cause of different structure features of SIS gel may be attributed that typical SEM prepare protocol resulted in gel contracted seriously after dehydration.…”

Extracellular matrix hydrogels from decellularized tissues: Structure and function

“…Gel electrophoresis of UBM hydrogels shows similar bands to SIS hydrogels and both show additional bands corresponding to other ECM proteins [20]. Intact growth factors have also been confirmed in adipose [50], colon [51], liver [52], and SIS [53] ECM hydrogels, although present in reduced amounts compared to native tissue or ECM scaffolds. The impact of solubilization on cryptic peptide and matrix-bound nanovesicle content or activity has yet to be evaluated.…”

“…Various source tissue ECMs showing an average fiber diameter of approximately 100 nm have been reported (e.g. cardiac [42], SIS [53], adipose [67]).…”

“…For example, pore size and fiber diameter are independent of concentration in UBM [23] and liver ECM gels [56], but vary with ECM concentration in dermal ECM gels [23]. UBM hydrogels also show randomly organized fibers, whereas more aligned fiber architecture has been observed in SIS hydrogels [53]. Qualitative analysis of SEM images show easily recognizable differences in structure depending upon the gelation mechanism (temperature- vs. pH-induced) used to create dermal hydrogels [44].…”

Stem cells from the dental apical papilla in extracellular matrix hydrogels mitigate inflammation of microglial cells

“…Hydrogels, in addition to serving as a vehicle for local delivery of SCAP, may also actively participate in immunoregulation of the local environment. Extracellular matrix (ECM) hydrogels can be generated from almost any tissue 21 and have been shown to contain growth factors 22 , matrix-bound nanovesicles 23 and exposed cryptic peptides and bioactive motifs 24,25 . ECM hydrogels provide general and tissue specific cues for regeneration in vitro and in vivo 26,27 .…”