SUMMARY Cleft palate formation is one of the most common craniofacial anomalies in children and occurs in 1:1000 live births. Patients undergo multiple surgeries during their lifetime and, for many, the first surgery is the repair of their cleft palate. However, 60% of cleft palate patients suffer poor wound healing following surgery which leads to the development of an oro-nasal fistula (ONF) characterized by a continued communication between the oral and nasal cavities. The primary approach to repair an ONF is by using human donor tissue, which acts only as a physical barrier and carries the risks of disease associated with human donor tissue, such as prions or HIV infection. Therefore, there is an urgent need to develop new therapeutic strategies to improve wound healing after cleft palate surgical repair, and to reduce the cases of ONF. Wound healing has been studied extensively on the skin and particularly following intestinal injury. Intestinal wound healing efficiency is highly sensitive to environmental factors, especially the microbiome, where specific microbial community structures or supplementation with probiotic bacteria enhances the wound healing process. However, little is known about how the oral microbiome affects ONF wound healing. We directly address this gap in the knowledge and show preliminary data that creating an ONF in a murine model results in marked changes in the microbiome composition, with the complete disappearance of certain microbes, and blooms of other bacterial taxa. Based on this premise, in Aim 1, we will create ONF wounds in germ-free mice, or in conventionally raised mice treated with antibiotics, and assess healing rates and changes in the oral microbiome composition. We expect to show the influence of disrupting the oral microbiome on healing processes and ONF formation. In Aim 2, we will use a hydrogel to re-introduce commensal oral microbes lost following surgery to the site of the oral wound and assess healing rates. We expect to show that repletion of oral commensal bacteria to the wound site can promote wound healing processes. In addition, by assessing transcript enrichment by RNA-seq within ONFs in germ-free, antibiotic treated mice, and after the repletion of oral commensal bacteria, we expect to identify gene networks that function in healing within ONFs that are sensitive to the commensal oral microbiome. Thus, our overall hypothesis is that the oral commensal microbiome exerts positive modulatory influences on oral wound healing and may limit ONF formation. Discovering specific bacterial taxa within the oral cavity or wound tissue that positively influence healing will be essential information for the characterization of an eubiotic oral microbiome for wound healing. Our experiments will also generate critical information for clinicians about the prudent use of antibiotics after cleft palate surgery and the effects of depleting the oral microbiome during healing. We will also substantiate the ap...