Comparison of growth factors isolated from exosomes present in maternal blood of cleft and non-cleft infants for identification and quantification of growth factors involved in the growth and development of cartilage.

Background and rationale

Cleft lip and palate (C.L.P.) constitutes one of the common congenital disabilities associated with inherited genetic conditions affecting the orofacial region of a child. This condition can be present in numerous variations and combinations.¹ The prevalence of C.L.P. is very high in the Asian and American populations compared to the African population.¹ According to a study in 2009, India, with an estimated 1.1 billion population, yields 24.5 million births per year, with a birth prevalence of around 27,000-33,000 clefts per year. The incidence of C.L.P. is higher in males than in females.¹ This congenital disability creates medical, psychological, and social problems affecting individuals and their families. Genetic and environmental variables play a role in the complex multifactorial aetiology of C.L.P.¹ The genes responsible for non-syndromic C.L.P. are FGFR2, IRF6, FGF8, and BMP4² and shared environmental factors are maternal smoking, alcohol consumption, consanguineous marriage consumption of certain medications such as topiramate, valproic acid during the first trimester, nutritional deficiency like folic acid deficiency, vitamin A deficiency, chemical exposure like cosmetics, pesticides and psychological factors such as stress and depression.¹

The anatomy of C.L.P. includes the abnormal shape and position of the nasal septum and nasal tip cartilage, which creates an uneven appearance of the nostril, nasal tip, and nasal dome/contour. The deviated nasal cartilage is moulded and corrected with the help of various P.S.I.O. appliances to mould the nasal cartilage pre-surgically.³ The nasal cartilage is a structure within the nose that provides form and support to the nasal cavity and is made of hyaline cartilage.⁴ As the cartilage is avascular connective tissue with no direct blood supply, it receives its nutrition by diffusion from the surrounding enviroment, and this increases when there is an increase in compressive forces acting on it. The structure and organisation of cartilage’s extra-cellular matrix are crucial for the proper functioning of cartilage because cartilage contains particular, differentiated cells whose main job is to maintain the stability of matrix elements. When wounded, its capacity for self-repair is constrained.⁵

The growth and development of cartilages are regulated by various growth factors that signal the extracellular matrix and other components.⁶

Even after a prosperous cleft palate and lip surgery in a C.L.P. child, there are chances for relapse, possibly due to something lacking on a cellular level. Intercellular communication is an essential function for maintaining homeostasis, or direct cell-to-cell interaction.

Since the previous decade, extra-cellular vesicles, particularly exosomes, have drawn more attention for their potential applications.⁶ Recent research has shown that exosomes can serve as a portal for cell-free drug delivery systems that retain their original characteristics from the parent cell. Extra-cellular vesicles, or exosomes, are found in nearly all cells, tissues, and bodily secretions.⁷ They support intercellular communication and preserve tissue homeostasis in pathological physiology. Nine thousand seven hundred sixty-nine proteins, 2838 miRNAs, 3408 mRNAs, and 1116 lipids are found in exosomes.

These exosomes can act as a drug delivery tool as their characteristics are derived from the parent cell. Almost all the cells, blood, tissue, saliva, tear, breast milk, urine, and G.I.T. secretion secrete exosomes. Exosomes can cross the blood-brain barrier and the lipid bilayer guards against immune cells and enzymes. They can regenerate new blood vessels and cellular proliferation.⁸

Exosomes are secreted as extra-cellular vesicles and contain growth factors, amongst many other essential cellular components. These exosomes contain signalling growth factors that may have therapeutic potential in the growth and development of nasal cartilage in C.L.P. infants.⁹

There is a need to access various factors, illustrated in Table 1 (genes, D.N.A., R.N.A., protein, enzyme, growth factor), responsible for the growth and development of cartilage. It was thought that accessing the quality and quantity of growth factors in the exosomes would play a role in the cartilage’s growth and development.

The following study is one of a kind. The growth factors present in the mother are genetically transferred to the child to produce a type of cartilage with specific characteristics. They thought to assess the quality and quantity of growth factors in the mother of a child with C.L.P. and a child with non-cleft. The current conventional treatment regime for C.L.P. management does not address the cellular aspect, especially the cellular signalling for cartilage moulding. This rate-limiting factor for C.L.P. management may be overcome by providing cartilage growth and development-triggering growth factors.

The following study is designed with the hypothesis that there may be a difference in the quality and quantity of growth factors in the blood of lactating mothers of cleft and non-cleft infants.

The study presents a challenging scenario in managing cleft lip and palate (C.L.P.) due to the complex process of nasoalveolar cartilage moulding. The importance of time in the initial 2-3 months of a child’s life for cartilage moulding is emphasised, but C.L.P. infants are deprived of essential nutrients in the mother’s milk. While some developed countries provide fortified milk for C.L.P. infants, developing countries like India rely heavily on animal-derived milk, which has limited nutrients. This lack of essential nutrients can lead to a decline in the crucial 2–3-month period to less than one month, failing mechanical appliances like nasal stunts.

Objective

To identify growth factors present in the exosomes of lactating maternal blood of cleft and non-cleft infants.

To quantify the identified growth factors in the exosomes of lactating maternal blood of cleft and non-cleft infants.

To compare the identified growth factors present in the exosomes of lactating maternal blood of cleft and non-cleft infants.

Study design

Analytical observational study.

Study setting

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

Study participants

The lactating mothers visiting the Department of Orthodontics and Dentofacial Orthopaedics, Department of Gynaecology and Obstetrics, and the Department of Paediatrics and fulfilling the inclusion criteria will be included in the study.

Inclusion criteria

Lactating mothers of non-syndromic isolated cleft lip and palate infants less than six months of age.

Age of mothers: <35 years.

No systemic conditions such as diabetes and hypertension.

Exclusion criteria

Age of mother: >35 years (due to hormonal changes).

Lactating mother with systemic disease.

Lactating mother with developmental and congenital disease.

Lactating mother of an infant of more than six months of age.

Non-lactating mothers

Protocol

Control Group: Mothers of healthy infants (non-cleft infants).

Study Group: Mothers of an infant with a cleft lip and palate.

Methodology

Each lactating mother included in the sample will explain the need for the study and the entire procedure in their language of understanding, and informed consent will be obtained from them. The blood sample collection will be done in the Department of Oral Pathology/Oral Surgery, Sharad Pawar Dental College, in collaboration with the Department of Gynecology. 2 ml of the blood sample will be collected in EDTA collection tubes. Once sample collection is done, it will be taken to the laboratory in the R&D house (D.M.I.H.E.R.) for evaluation. First, the plasma will be separated through a centrifugation process and stored at -80 degrees Celsius until further experimentation. The isolation of exosomes from the plasma and the subsequent isolation of total proteins from the exosome will be done following standard protocol, and followed by an analysis of growth factors from the isolated total proteins (from both the 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 host antibodies identify a target protein via an antigen-antibody reaction as it identifies the target protein among the number of unrelated proteins. Proteins are electrophoretically separated and then moved to nitrocellulose membranes. This method makes use of the following three components:

Examples of equipment used for each component mentioned above are listed here:

Variables and data measurements

OUTCOME: Following are the outcomes with their descriptions for the measurements mentioned below:

The purpose of the study is to assess the growth factors, such as TGF-β and B.M.P., found in the exosomes of lactating mothers' blood. These exosomes can be used to identify, quantify, and compare growth factors that have been isolated from the maternal blood of mothers of infants with and without clefts.

This means that separated mother exosomes should have a good number of growth factors that are important for cartilage growth and development. These growth factors can be used as a therapy in addition to common CLP management procedures.

BIAS: Bias will be minimised as patients are random and will be selected from the O.P.D. of the Department of Orthodontics and Dentofacial Orthopaedics of Sharad Pawar Dental College and Hospital, Wardha.

Study sample

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

Where,

Z∝2 = 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 = 30

Statistical analysis

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

Descriptive statistics for continuous variables will be used to offer data interpretations tested for normality using the KALMOGOROV-SMIRNOV TEST for continuous data to test the outcome variable. The R software version of SPSS software 3.2 will be used to analyse the results.

Growth factors are categorised according to the range that will be distributed for analyzing the data into the normal range and not in the normal range. The chi-square test can be used to find the result of an association of growth factors with the blood of mothers of cleft and non-cleft infants.

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

An independent t-test will be used to find the results in two groups for outcome variables if the data falls under the normal distribution. A non-parametric test will find a significant difference if the data does not come under a normal distribution.

Dissemination

This study aims to evaluate the growth factors present in the exosomes of lactating maternal blood for their potential use in cleft lip and palate (C.L.P.) management. The study hypothesises that growth factors enhance cartilage growth and development, crucial during the first 2-3 months of a child’s life. However, C.L.P. infants are often deprived of essential nutrients in the mother’s milk, leading to a decline in the duration of cartilage moulding. Exosomes in lactating maternal blood can carry essential growth factors, hormones, and estrogen, which can be transported to the required site for therapeutic potential in C.L.P. management.

Study status

The study has yet to start.