Background Implanted biomaterials are subject to a significant reaction from the host, known as the foreign body response (FBR). We quantified the FBR to five materials following subcutaneous implantation in mice. Materials and methods Polyvinyl alcohol (PVA) and silicone sheets are considered highly biocompatible biomaterials and were cut into 8mm-diameter disks. Expanded PTFE (ePTFE)and polypropylene are also widely used biocompatible biomaterials and were cut into 2cm-long cylinders. Cotton was selected as a negative control material that would invoke an intense FBR, was cut into disks and implanted. The implants were inserted subcutaneously into female C57BL/6 mice. On post-implantation days 14, 30, 60, 90 and 180, implants were retrieved. Cellularity was assessed with DAPI stain, collagen with Masson’s trichrome stain. mast cells with toluidine-blue, macrophages with F4/80 immunohistochemical-stain, and capsular thickness and foreign body giant cells with hematoxylin & eosin. Results DAPI revealed a significantly increased cellularity in both PVA andsilicone, and ePTFE had the lowest cell density. Silicone showed the lowest cellularity at d14 and d90 whereas ePTFE showed the lowest cellularity at days 30, 60, and 180. Masson’s trichrome staining demonstrated no apparent difference in collagen. Toluidine blue showed no differences in mast cells. There were, however, fewer macrophages associated with ePTFE. On d14, PVA had highest number of macrophages, whereas polypropylene had the highest number at all time points after d14. Giant cells increased earlier and gradually decreased later. On d90, PVA exhibited a significantly increased number of giant cells compared to polypropylene and silicone. Silicone consistently formed the thinnest capsule throughout all time points. On d14, cotton had formed the thickest capsule. On d30 polypropylenehas formed thickest capsule and on days 60, 90 and 180, PVA had formed thickest capsule. Conclusion These data reveal differences in capsule thickness and cellular response in an implant-related manor, indicating that fibrotic reactions to biomaterials are implant specific and should be carefully considered when performing studies on fibrosis when biomaterials are being used.
Hypertrophic scar contraction (HSc) is caused by granulation tissue contraction propagated by myofibroblast and fibroblast migration and contractility. Identifying the stimulants that promote migration and contractility are key to mitigating HSc. AngiotensinII (AngII) promotes migration and contractility of heart, liver, and lung fibroblasts; thus, we investigated the mechanisms of AngII in HSc. Human scar and unwounded dermis were immunostained for AngII receptors AT1-receptor and AT2-receptor, and analyzed for AT1-receptor expression using western blot. In-vitro assays of fibroblast contraction and migration under AngII stimulation were conducted with AT1-receptor, AT2-receptor, p38, JNK, MEK, and ALK5 antagonism. Excisional wounds were created on AT1-receptor KO and WT mice treated with AngII ± Losartan, and ALK5 and JNK inhibitors SB-431542 and SP-600125 respectively. Granulation tissue contraction was quantified and wounds analyzed by immunohistochemistry. AT1-receptor expression was increased in scar, but not unwounded tissue. AngII induced fibroblast contraction and migration through AT1-receptor. Cell migration was inhibited by ALK5 and JNK, but not p38 or MEK blockade. In-vivo experiments determined that absence of AT1-receptor and chemical AT1-receptor antagonism diminished granulation tissue contraction while AngII stimulated wound contraction. AngII granulation tissue contraction was diminished by ALK5 inhibition, but not JNK. AngII, promotes granulation tissue contraction through AT1-receptor and downstream canonical TGFβ signaling pathway; ALK5. Further understanding the pathogenesis of HSc as an integrated signaling mechanism could improve our approach to establishing effective therapeutic interventions.
Hypertrophic scar (HSc) contraction following burn injury causes contractures. Contractures are painful and disfiguring. Current therapies are marginally effective. To study pathogenesis and develop new therapies, a murine model is needed. We have created a validated immune-competent murine HSc model. A third-degree burn was created on dorsum of C57BL/6 mice. Three days postburn, tissue was excised and grafted with ear skin. Graft contraction was analyzed and tissue harvested on different time points. Outcomes were compared with human condition to validate the model. To confirm graft survival, green fluorescent protein (GFP) mice were used, and histologic analysis was performed to differentiate between ear and back skin. Role of panniculus carnosus in contraction was analyzed. Cellularity was assessed with 4′,6-diamidino-2-phenylindole. Collagen maturation was assessed with Picro-sirius red. Mast cells were stained with Toluidine blue. Macrophages were detected with F4/80 immune. Vascularity was assessed with CD31 immune. RNA for contractile proteins was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Elastic moduli of skin and scar tissue were analyzed using a microstrain analyzer. Grafts contracted to ∼45% of their original size by day 14 and maintained their size. Grafting of GFP mouse skin onto wild-type mice, and analysis of dermal thickness and hair follicle density, confirmed graft survival. Interestingly, hair follicles disappeared after grafting and regenerated in ear skin configuration by day 30. Radiological analysis revealed that panniculus carnosus doesn't contribute to contraction. Microscopic analyses showed that grafts show increase in cellularity. Granulation tissue formed after day 3. Collagen analysis revealed increases in collagen maturation over time. CD31 stain revealed increased vascularity. Macrophages and mast cells were increased. qRT-PCR showed up-regulation of transforming growth factor beta, alpha smooth muscle actin, and rho-associated protein kinase 2 in HSc. Tensile testing revealed that human skin and scar tissues are tougher than mouse skin and scar tissues.
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