Although surgery is needed to fuse the lip and palate, there are other conditions that children with CLP face after surgery, such as otitis media (OM), speech defects, and difficulty breathing. This thesis is a research proposal to evaluate whether γ-PGA can be used in nasal rinses to safely prevent nasopharyngeal infections such as OM from occurring among children with CLP. A double-blind randomized clinical study will also be conducted to analyze the effects of γ-PGA among 400 children with CLP (6 - 12 years old).
By creating a nasal wash that is more effective at killing bacteria compared to saline, but also non-toxic unlike chlorhexidine and listerine, γ-PGA can prevent harmful nasopharyngeal infections in people with CLP.
What is a Cleft Lip and Palate?
Given that CLP is a relatively common birth condition, raising awareness about the challenges people with CLP face is critical. Because many of the direct causes of cleft lip and palate are still unknown, it is currently not possible to prevent the condition worldwide. While surgeries are considered expensive for many, surgeries often save people with CLP from other health problems and treatments that are even more expensive when children with CLP grow up with an illness.
Dedicated programs have helped children with CLP in poor communities receive safe surgery to fuse the lip and palate free of charge. However, many children with CLP still need several more surgeries and additional treatments that may not be prevented by lip and palate repair alone.
Management of CLP beyond Surgical Treatment
Many children with CLP may have difficulty feeding, especially when the lip and palate are not successfully surgically fused. In addition, abnormal salivary glands are formed with CLP due to the presence of the mutated IRF6 gene. Orthodontic treatment is often required, which can improve the structure and alignment of the teeth and jaws.
The opening and closing of the Eustachian tube also equalizes pressure and drains various bacteria and fluids from the middle ear (Schilder et al., 2015; Zambonato et al., 2009).
Anatomy and Genetics of Cleft Lip and Palate
The palatal shelves first grow vertically along the sides of the developing tongue, but eventually grow horizontally as the tongue begins to flatten. The nasal and oral cavities are well separated as long as this fusion of the secondary palate is successful (Leslie and Marazita, 2013) (Figure 1.1). The mandibular processes fuse together to form the lower jaw. d) the sixth week of development: the secondary palate develops from the maxillary processes, which grow vertically down the side of the tongue. e) the palatal shelves settle into a horizontal position above the tongue and eventually begin to fuse together. f) Palatal shelf fusion splits the oronasal space into separate oral and nasal cavities (Dixon et al., 2011).
Advances in molecular biology have successfully evaluated the genetic basis for the development of CLP.
Clinical Perspectives and Proposed Therapeutic Non-Invasive Approaches for
A better understanding of CLP was not promoted until Fabricius ab Aquapendente proposed the embryology of a condition where the upper lip of the fetus fuses only along the midline in the late stages of development. Ambroise Paré, one of the best surgeons in the 16th century, conducted studies on the anatomy of the lip and palate and improved suturing techniques (Bhattacharya et al., 2009). For example, nose correction such as rhinoplasty was not introduced until the 1970s due to fears that the surgeries could affect the growth of the nose.
Specifically, the tensor veli palatini aponeurosis attaches to the edges of the cleft palate rather than the posterior border of the hard palate (Sharma and Nanda, 2009). A study also reveals that skull dimensions such as the small size of the spheno-occipital bone and the upward position of the maxilla are other possible reasons why children with CLP are more prone to OM (Sharma and Nanda, 2009). Fluid accumulation in the middle ear prevents the bones from moving fully (Mayo Clinic, 2022).
Eustachian tube connection between nasopharynx and middle ear in infants and adults without CLP. Note that the Eustachian tube in adults is more oblique to allow easier drainage of the middle ear (Cleveland Nasal Sinus and Sleep Center, 2022). Improper Eustachian tube drainage allows bacteria and viruses to lodge in the middle ear, causing otitis media (Schilder et al., 2016).
Children with ear tubes often have chronic ear discharge that drains from the ear. Tympanic membrane affected by OM. d) An ear tube surgically implanted in the eardrum to drain fluid from the middle ear (Schilder et al., 2016).
Poly-Gamma-Glutamic Acid (γ-PGA) and its Usefulness
The ability to inhibit four different types of highly pathogenic bacteria in the nasopharynx of children with CLP will be tested. All measurements and tests will be performed 8 times (in eight times) to ensure the accuracy and precision of the experiments. The mixture on the microplate will be incubated for an hour and a half at 37 ºC without light.
Then, with a spectrophotometer plate reader (ELISA), the optical density (OD) value will be measured at 450 nm. The bacterial strains will be added to a centrifuge tube containing 10 ml of nutrient broth in the laminar flow hood. After that, the control treatment for each bacterial strain will include 1 mL of the bacterial culture and mixed with 9 mL of saline.
Results will be presented as mean and standard error of the mean (SEM). The effect of four different concentrations of γ-PGA on cell survival and toxicity will be evaluated in L929 mouse embryonic fibroblasts (Figures 6.1 and 6.2). I expect that in the LDH assays, γ-PGA will be associated with high viability, reaching nearly 100% cell viability at each of the four concentrations.
Given the expected results, I would conclude that γ-PGA will have strong antibacterial activity and the optimal concentration will be 10,000 ppm of γ-PGA. To assess the safety and effects of γ-PGA in preventing infections that particularly affect those with CLP, this thesis proposes that clinical trials be conducted in children with CLP aged 6-12 years. The study protocol will be approved by local ethics committees and Institutional Review Boards (IRBs).
Children in both the treatment and control groups will be given instructions on using nasal rinses. Baseline characteristics (made on the first day of the study after 0 months) will be compared between the two groups. The effectiveness of γ-PGA will be assessed by examining the rhinology scores and the use of medications, which include antipyretics, nasal decongestants, mucolytics, and antibiotics.
In-Vitro Studies of γ-PGA
The Evaluation of the Effectiveness of γ-PGA in Saline Application to Prevent Upper
The aim of the study is to evaluate this hypothesis by testing the efficacy of γ-PGA in reducing these infections and symptoms in children with CLP. The double-blind, placebo-controlled, randomized phase 3 clinical study will be conducted at various craniofacial clinics in San Francisco; every child will be evaluated equally. The number of bottles used by each patient will be recorded and each patient will be required to return any empty bottles.
Patients will be observed for 9 months; their symptoms, health status, presence of ear infections and medication use will be assessed during 4 visits (at 0, 3, 6 and 9 months) throughout the trial. Baseline parameters will be assessed at initial visits, including gender, age, type of cleft condition, health status, and health history. The parameters to be used are those commonly used by otolaryngologists in the clinical management of upper respiratory tract and otolaryngological infections in children and have been used to evaluate other products in clinical studies (Tomooka et al., 2000; Šlapak et al. , 2008; Ramalingam et al., 2019).
Quantitative data will be presented as mean, standard deviation and median, and qualitative data will be presented as frequency and absolute percentage. For data measured on a single scale (including rhinologic system scores, nasal secretion scores, and nasal breathing scores), the hypothesis of no difference in mean values will be evaluated through a Student's t test. A total of 400 patients will be examined; 200 patients will be assigned to each of the saline (placebo) and γ-PGA (treatment) groups through double-blind randomization.
Symptom scores that differed significantly between the two groups are scores for sore throat, nasal secretion type, nasal secretion, and nasal breathing scores (p < 0.05; Tables 7.2, 7.3, and 7.4). This is a proposed double-blind, placebo-controlled phase 3 clinical trial to evaluate the potential of γ-PGA in preventing nasopharyngeal infections in children with CLP.
Last Thoughts on this Proposed Study
I am grateful that they have given me the wonderful opportunity to write this thesis about my passion to help fellow cleft lip and palate individuals. Cleft lip and palate: Parental experiences of stigma, discrimination and social/structural inequalities. Annals of Oral Surgery. Use of stem cells in bone regeneration in patients with cleft palate: Review and recommendations Journal of the Korean Society of Oral and Maxillofacial Surgeons.
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