Promotion Of Hearing Recovery From Traumatic Tympanic Membrane Perforation By Implanting Collagen Membranes Integrated With Collagen-Binding Basic Fibroblast Growth Factor

Background: Collagen is a major structural protein of extracellular matrix of the human body that has been studied as scaffold for tympanic membrane(TM) regeneration.Basic fibroblast growth factor(bFGF) is a potent growth factor that can promote the differentiation and proliferation of cells and tissues. BFGF has been studied to accelerate the healing of tympanic membrane perforation. In order to find a more appropriate method of repairing tympanic membrane perforation that take advantages of both collagen and bFGF, we use a kind of artificial modified bFGF that add a collagen binding domain(CBD)with the end, and make it combine with collagen membrane(CM) specially. This functional membrane was explored to repair traumatic tympanic membrane perforation in rats.Methods:(1) In vitro: compared with commercialized bFGF, CCK 8 method was adopted to detect effects of CBD-bFGF on promoting the proliferation of human skin fibroblasts(HS-865). Then we combined both them to the collagen membranes and used BCA kit(Total protein quantification test kit) to test concentration of cytokine and get a binding rate.(2) In vivo: Seventy-two SD rats underwent perforation of the bilateral TM and were divided into three experimental groups(n=24). The perforations were repaired with CM-CBD-bFGF and CM using on-lay myringoplasty, or they were allowed to heal spontaneously(control). An additional group of 8 rats without perforation or scaffold was allocated as a sham group. Rats in each experimental group(n=8) were evaluated at 7, 14 and 28 days after surgery. TM structural healing was evaluated by otomicroscopy,histology, immunohistochemistry and transmission electron microscopy(TEM) and functional hearing was analyzed by auditory brainstem responses(ABR).Results:(1) In vitro: compared with commercialized bFGF, CBD-bFGF significantly promoted the proliferation of human skin fibroblasts(HS-865). The binding rate of CBD-bFGF and collagen was 81.9±11.64%, higher than bFGF(72.3±12.01%).(2) In vivo:Microscopy revealed healing rate of CM-CBD-bFGF group was obviously higher than thatof CM group and control group at the eary stage. The appearance of regenerative TMs were similar to the sham TMs. TMs from CS-CBD-bFGF group at all time points showed well-organized tissue layers, including outer epidermal layer, middle fibrous layer and inner mucosal layer. Especially, at 28 days, the middle fibrous layers were more compact and organized, which were similar to the sham TMs. In the CM group, the healed TMs showed loose and immature fibrous layers. In contrast, the control group was thinnest, due to unconspicuous middle layer. According to ABR, CM-CBD-bFGF group got earlier and better hearing recovery than CM group and control group.Conclusion: With the help of collagen binding domain(CBD), CBD-bFGF can bind on the collagen membrane efficiently. CM-CBD-bFGF can notably retrieved the structure and function of TM at early. This functional membrane has a potential clinical application for traumatic TM regeneration.