Identification and quantification of growth factors involved in growth and development of cartilage present in exosomes of lactating mother’s milk of cleft infant as compared to non-cleft infant – a protocol for an analytical observational study

Rationale

Even after successful cleft palate and lip surgeries in CLP children, there are chances of relapse as there may be a lack something on a cellular level. Direct cell-to-cell interaction and the maintenance of homeostasis depend on intercellular communication. Since the last decade, there has been an increased interest in the role of extracellular vesicles, especially exosomes. Recent studies have illustrated that exosomes act as a potential portal for a cell-free drug delivery system with the native characteristic of the parent cell of origin. Exosomes, called extracellular vesicles, are present in almost all cells, tissue and body fluids. It helps in intercellular signaling and maintaining tissue homeostasis in diseased physiology.⁴ It consists of 9769 proteins, 2838 miRNAs, 3408 mRNAs & 1116 lipids.

These exosomes can act as a key player in treating a cleft patient. It acts as the drug delivery tool as its characteristics are derived from the parent cell. Almost all cells, blood, tissue, saliva, tear, breast milk, urine, and the gastrointestinal tract (GIT) secrete exosomes. Exosomes have the potential to cross the blood-brain barrier.⁴ Its lipid-bilayer-coated with extracellular vesicles protect against immune cells and enzymes. It can promote cellular growth and the regeneration of new blood vessels.⁴

Recently many studies have been done which state that exosome contains many factors:

Mengna Duan et al. in 2020 studied the effects of exosomes derived from epidermal stem cells on the rate of wound healing in rat skin. When epidermal stem cells were combined with exosomes, scar formation was found to be reduced and wound healing increased.⁵ Kan Yin et al. in 2019 found that exosomes derived from mesenchymal cells have many growth factors like TGFβ1, VEGF, HGF, cytokines, and proteins.⁶

Extracellular vesicles that are secreted are known as exosomes, and they are vital components of cells that also contain growth factors. Exosomes contain signalling growth factors that are responsible for the growth and development of cartilage, and this has therapeutic potential in the management of CLP.

A variety of molecules, including proteins, enzymes, growth hormones, genes, DNA, and RNA, are found in exosomes. Exosomes are believed to include growth factors that are crucial for the growth and development of cartilage, thus it was expected to evaluate their quantity and quality. It was necessary to determine the elements influencing cartilage development in a patient with CLP since the exosome can express the growth of cartilage in a newborn with CLP.

The following study is one of a kind. It was thereby thought to assess the quality and quantity of growth factors in the mother of a child with CLP and a child with non-cleft and to evaluate the mother’s growth factors, which are genetically passed on to the child and produce a particular type of cartilage with a particular set of properties.

This literature highlights the current conventional treatment regime for CLP management which is not addressing the cellular aspect and especially the cellular signaling for cartilage molding. This is a rate-limiting factor towards CLP management, which may be overcome by providing cartilage growth and development-triggering growth factors.

The following study was thereby designed with a hypothesis to assess the quality and quantity of growth factors in lactating mothers of cleft children and non-cleft children.

Objectives

Ethical considerations

Ethical approval for the study was obtained from the institutional ethics committee of Datta

Meghe Institute of Higher Education and Research (Deemed to be University) (ref. no: DMIHER (DU)/IEC/2023/571) Date of approval: 06-02-2023. A written participant information sheet will be given regarding the details of the study, and it will be explained to participants and their parents before enrolment to the study. Their involvement benefits and harm will be explained to the participants. Written informed consent from the participants will be obtained before involving them in study.

Trial design

This will be a parallel group, analytical observational study.

Study setting

The following study will be conducted in the Department of Orthodontics and Dentofacial Orthopedics at Sharad Pawar Dental College in collaboration with Central Research Laboratory (Center of Translation Sciences), Sawangi, Wardha.

Eligibility criteria

Inclusion criteria

Exclusion criteria

Intervention

Lactating mothers reporting in the Department of Orthodontics and Dentofacial Orthopedics and Department of Gynecology & Obstetrics fulfilling the inclusion criteria will be included in the study.

The study will include a total sample size of 30. There will be two groups with 15 patients in each group:

The patient will be selected from the smile train outpatient department (OPD) of the Orthodontic Department, while the non-cleft patient will be selected from the Department of Gynaecology. Mothers with a CLP child and a non-cleft child between the age of 0–6 months will be selected for the study. The purpose of the study will be explained to mothers, and those who are willing to participate will be given the consent form in a language they can understand, after which their signature will be obtained. The milk sample from the mother with a CLP child will be collected in the breastfeeding room of the smile train unit in the Department of Orthodontic and Dentofacial Orthopaedic, Sharad Pawar Dental College, and from the mother with the non-cleft child it will be collected from the Department of Gynecology. The milk sample of 2 ml will be collected in a falcon tube and transported with cold storage facilities to a laboratory in R&D house (DMIHER) for evaluation. It will be stored at a temperature of -80°C until the experiment is performed. Initially, the isolation of exosomes from the milk sample will be done from which total proteins will be isolated under the centrifugation process. This will be followed by an analysis of growth factors from the isolated total proteins (from both exosomes) through immunoblotting. Once the analysis is done, the quality and quantity of growth factors responsible for cartilage growth and development will be compared for both groups.

Immunoblotting is a technique in which we use host antibodies to identify a target protein via antigen-antibody reaction as it identifies the target protein among the number of unrelated proteins. Proteins are separated by electrophoresis and transferred onto the nitrocellulose membrane. This technique uses three elements which are:

Outcomes

Primary outcome

The proposed study is intended to highlight the therapeutic potential of maternal exosomes which can be genetically identified for their quality and quantity. Results will be highlighting the growth factors in infants with CLP. Likewise, further modalities for the management of the nasal cartilage can be done. In CLP subjects, the availability of maternal growth factors is limiting owing to its deficiency.

Secondary outcome

Because of this, isolated maternal exosomes are expected to contain ample growth factors involved in cartilage growth and development, highlighting its potential for use as a therapy alongside prevalent procedures in CLP.

Sample size

The sample size was calculated by using Daniel’s formula for sample size:

Where,

$Z_{\raisebox{1ex}{$\propto $}\!\left/ \!\raisebox{-1ex}{$2$}\right.}$ is the level of significance at 5% i.e. 95% confidence interval = 1.96

P = Prevalence of cleft lip and palate = 1% = 0.01

d = Desired error of margin = 6% = 0.06

n is the population size

Total sample size is 30

There is a total two groups which means 15 patients in each group.

Statistical method

All the demographic and outcome data will be presented using descriptive statistics for continuous variables can be categorized using mean, standard deviation, and median for discrete variables, and frequency and proportion for continuous variables.

The outcome variable will be tested for normality using the Kolmogorov-Smirnov test for continuous data. Results will be analysed using SPSS version 27.0.

Growth factors will be categorized according to the range that will be distributed for analysing the data into the normal range and not in the normal range. The chi-squared test will be used for finding the result of the association of growth factors with milk from the mother of cleft and non-cleft infants.

An odds ratio will be used to find the risk involved multiple times.

An independent t-test will be used to find the results of the two groups for outcome variables if data comes under the normal distribution, or a non-parametric test will be used to find the significant difference if data doesn’t come under the normal distribution.

Dissemination

In this study, we expect that maternal exosomes act as carriers of factors that can have therapeutic significance to the natural compensation of the cartilage to maintain nasal symmetry by naso-alveolar molding. Because of growth factors, which in the first 2–3 months help/enhance cartilage development and growth. However, due to a lack of these growth factors in children, this period declines to 1 month, which can result in the failure of many mechanical appliances like stunts in the 3- to 4-month range getting no results due to a lack of certain factors.

Identifying the factors like growth factors, estrogen or hormones can enhance the growth and development of cartilage and, by highlighting their therapeutic significance in CLP management as isolated maternal exosomes are expected to contain ample amount of growth factors involved in cartilage growth and development, highlighting its potential for use as a therapy alongside prevalent procedures in CLP.

Study status

Not started yet.