Growth factor analysis in children with non-syndromic orofacial clefts: a systematic review

Abstract

Background

Orofacial clefts (OFCs) are one of the most prevalent congenital abnormalities that affect the lip and/or palate and can cause significant growth retardation in newborns. Several studies have revealed that children with congenital defects or genetic syndromes have their own growth pattern, which may differ from that of normal children. However, during infancy and until around age two, these babies usually show a period of catch-up growth in length, weight, and head circumference for both boys and girls. It was noted out that both genders had smaller physical dimensions than normal children. In order to partially elucidate the biological mechanism affecting children with non-syndromic OFCs, this systematic review aims to assess the relative expression and localization of growth factors and their receptors in craniofacial tissues.

Method

A comprehensive literature search was carried out on May 1, 2025, using three important databases: Web of Science Core collection, PubMed, and Scopus. The search was limited to only English-language studies involving human subjects, but it was not limited by publication date. To find potentially relevant publications, specific keywords and database-specific search techniques were used.

Results

Based on predefined inclusion criteria, 20 studies were selected from a total of 191 articles following a thorough screening process. Growth factors and susceptibility to OFCs were found to be significantly correlated in the analysis of the reports of the chosen studies. Particularly, OFCs and their risk were consistently associated with fibroblast growth factor receptor (FGFR1) and transforming growth factor (TGFβ3). More angiogenesis in compromised tissues is suggested by the elevated levels of two angiogenesis-related proteins, vascular endothelial growth factor (VEGF) and Cluster of differentiation (CD34). Similarly, the studies that used unilateral samples showed relatively high levels of TGFβ3, FGFR1, and bone morphogenetic protein (BMP2/4), which were also higher in bone cells, connective tissues, and epithelia.

Conclusion

The reviewed studies indicate a role of growth-related proteins in the pathophysiology of non-syndromic OFCs. This is demonstrated by the fact that FGFR1 is higher in all studies, TGFβ3 is upregulated in all the studies, and angiogenesis markers (VEGF and CD34) are consistently higher in cases. Additional investigation is needed to determine whether FGFR1 and TGFβ3 can serve as biomarkers or targets for intervention in OFC-related abnormalities.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12903-026-07715-x.

Introduction

Rational and objectives

Orofacial clefts (OFCs) are among the most frequent congenital malformations, affecting around 1 in 700 live births globally, with substantial variation by ancestral origin which are highest in Asian groups, followed by Caucasians and Africans [1]. These defects originate from the Palatal shelves, which are distinct prefabricated viscerocranial structures during embryogenesis, fail to develop, elevate, or combine adequately, resulting in OFCs [2–4]. OFCs have a complex etiology that involves interactions between environmental and genetic variables [5, 6]. This complex interaction leads to non-syndromic OFCs, while syndromic variants are usually associated with chromosomal abnormalities or monogenic disorders [7–9].

Facial development begins with the formation and fusion of five facial prominences frontonasal, paired maxillary, and paired mandibular. This process involves cell migration, proliferation, differentiation, and apoptosis. These primordiae consist of an outer layer made up of ectodermally-derived epithelial cells and a central part mainly formed from neural crest cells [10].

During early embryogenesis, neural crest cells move from the neural folds to the craniofacial region after undergoing epithelial-mesenchymal transition. The neural crest cell-derived ectomesenchymal cells, together ectodermal and mesodermal cells, contribute to the formation of branchial arches and facial processes. The oral cavity is surrounded by the frontonasal, maxillary, and mandibular prominences by the fourth week of human embryonic stages of development. The frontonasal prominence is divided into paired medial and lateral nasal processes by the formation of the nasal placodes. The medial nasal processes combine with the maxillary and lateral nasal processes during the sixth week of the embryonic stage to form the upper lip. The medial nasal processes unite in the midline to form the inter-maxillary segment, which produces the philtrum of the upper lip and the primary palate. While the maxillary and medial nasal processes form the upper lip, the lateral nasal processes eventually form the alae of the nose [11].

The upper lip and palate are formed by the fusion of frontonasal and maxillary processes; failure of these instances throughout development results in the formation of the OFCs of the lip with or without palate [9, 12].

The secondary palate forms as bilateral outgrowths from the maxillary processes, with the palatal shelves rising vertically behind the primary palate and laterally to the developing tongue. The palatal shelves reorient over the tongue during the eighth week of pregnancy, beginning in the anterior region and moving posteriorly. These palatal shelves then enlarge and come into contact with one another as they approach the midline. A midline epithelial seam (MES) is formed when the medial edge epithelia (MEE) at the palatal shelf tips fuse and adhere. Contact and fusion begin in the anterior third of the palate and progress anteriorly and posteriorly throughout the ninth week of pregnancy. Then, the anterior area of the secondary palate fused with the primary palate. The MES degeneration causes mesenchymal continuity over the palate. The palatal mesenchyme divides into bone and muscle, forming the hard and soft palates, respectively [13, 14].

The formation of the secondary palate is one of the most complicated processes. The secondary palate is mostly composed of two shelf-like extensions from the maxillary prominences. The MEE encompasses the palatal primordiae that have been described following the fusion of palatal shelves the MEE attaches to form the epithelial midline edge [15].

In particular, several growth factors and their receptors play an important role in regulating and managing all of the activities outlined above in a growing embryonic tissue. The Transforming growth factor-beta (TGFβ) contains the most researched secreted proteins in palatal development. These growth and differentiation factors, which include bone morphogenetic proteins (BMP) and TGFβs, are structurally similar. Numerous biological processes, including cell proliferation, apoptosis, extracellular matrix (ECM) production and deposition, cell migration, epithelial mesenchymal transition, and basement membrane disintegration, are influenced by the growth factors discussed and also take place during palatogenesis [16–18]. Similarly, it has been identified that fibroblast growth factors (FGFs) and their receptors are crucial for development of the palate and upper lip as they regulate cell motility, differentiation, and proliferation [19].

The FGF signalling pathways plays an important role in craniofacial growth, including the development of the lip and palate. Extracellular FGF ligands activate FGF receptors, which then regulate cellular processes such as migration and morphogenesis by guiding the activation of certain target genes. The interaction of mesodermal and neural crest-derived mesenchyme with ecto - and endodermal-derived epithelium is a significant factor in the development of orofacial structures. Patients with FGFR1 and FGFR2 mutations are most frequently affected, and cleft palate is part of a broader craniofacial phenotype that also includes craniosynostosis. Similarly, FGF8 mutations have been reported in patients with idiopathic hypogonadotropic hypogonadism, with some additionally having OFCs [20].

It has been suggested that palatogenesis, which initiates the regulatory activities by stimulating the FGF receptor tyrosine kinase, depends on the FGF family. OFC involving palate is linked to mutations in either the FGF or FGFr gene. Although Fgfr1 is expressed in the cranial neural crest (CNC)-derived palate mesenchyme and associated with palate defects, its role in palatogenesis remains unclear [21].

OFC patients often have feeding difficulties due to issues with suction while nursing and milk entering into their nasal cavity [22]. Therefore, a number of measures should be taken into account when feeding these infants, such as evaluating their capacity for sucking, teaching the correct position for nursing, providing them with specialized feeding supplies (such as specific bottles and teats), and educating families on child nutrition [23–25].

As a consequence, OFCs can cause significant growth retardation during infancy, with compensatory growth occurring in the first two years of life. Some studies have underlined the need for precise diagnosis of the specific type of OFCs for growth evaluation in a homogeneous population. The researchers believed that a child with OFCs without concurrent genetic disorders would have the same growth. A few studies have suggested children with congenital abnormalities or genetic syndromes have their own growth pattern, which may differ from that of normal children, and constitute small body dimensions due to disease-specific growth patterns. A research investigation revealed catch up growth in terms of length, weight, and head circumference for girls and boys. The authors have pointed out that children of both genders had decreased physical dimensions compared to normal children. Even though the boys did not show catch up growth in length but weight was the most compromised parameter for both genders followed by head circumference [26–28].

Despite several investigations the distribution and localization of different growth factors and growth factor receptors in tissues impacted by human clefts remain unclear. The etiopathogenesis of OFCs is still debated. Current genetic study on numerous growth factors and investigations suggests that OFC is produced by a combination of variables. This leads to a preference for a multifactorial model of inheritance, where environmental variables may interact with genetic risk factors that have a minor individual influence. Combining these elements makes it more difficult to analyze non-syndromic variants of OFCs genetically. Despite the recent identification of genes likely to influence the risk of non-syndromic OFCs, these results yet to have any direct impact on genetic counselling or clinical management. Improved epidemiologic information does, however, allow for better point estimates for familial recurrence risks [29, 30].

Hence, understanding the pathogenesis of OFCs is essential as most of the studies conducted on animal models only, and the availability of data on humans is limited due to ethical constraints and lack of embryonic tissues. Also, knowledge of the tissue localization of growth factors will aid in better surgical planning, targeted therapy, and enhanced regeneration in cleft patients. Thus, the main objective of this review was to determine the expression patterns (location and intensity) of growth factors and the associated growth factor receptors in the local tissues of children with OFCs.

Research questions

1. The expression patterns of growth factors in the local tissues of children with OFCs?

2. The associated growth factor receptors responsible for causing OFCs in children?

Materials and methods

Research design

This review was successfully registered with the INPLASY number (INPLASY202550054) and followed the 2020 checklist of preferred reporting items for systematic review and meta-analysis (PRISMA). However, it summarized the literature through narrative-based research studies.

Eligibility criteria

• Inclusion criteria

  • Retrospective cross-sectional studies, case-control studies, original research articles, longitudinal observational studies, systematic reviews, and literature reviews that involves identification of growth factors and their receptors in non-syndromic cleft affected tissues of children.
• Exclusion criteria

  • Articles containing only abstracts without full text.
  • Unpublished papers with only manuscripts.
  • Articles that are published in languages other than English.
  • Case studies, case series, blog posts, editorials, magazine articles, and book chapters.

Information sources

The initial search was conducted on May 1st, 2025. Although the search was limited to English-language articles and studies involving human subjects, there was no time constraint on publication. An electronic search was conducted utilizing databases such as PubMed, Scopus, and the Web of Science Core Collection. Two reviewers searched the databases both independently and concurrently.

Search strategy

The search criteria for each database were accompanied with Medical Subject Heading (MeSH) terminology and free keyword searches. The set of words and syntax of terms have been altered in each database. Table 1 display the search strings and keywords utilized in their respective databases.

Table 1.

Keywords & search strings used with their respective databases

Nos	Databases Keyword strings
1	PubMed	((Facial tissue [MeSH Terms]) AND (((Cleft lip and palate [MeSH Terms]) OR (rhGFs [MeSH Terms])) OR (Transforming growth factor [MeSH Terms]))) AND (Nestin [MeSH Terms]) AND (Fibroblast [MeSH Terms])) OR (((Oral clefts [MeSH Terms]) AND (Bone morphogenic protein [MeSH Terms]))])) OR (Nerve growth factor [MeSH Terms]))) AND (Polymorphisms [MeSH Terms]))) OR (Endothelial growth factor [MeSH Terms])))
2	Scopus	(TITLE-ABS-KEY (Facial tissue)) AND ((TITLE-ABS-KEY (Cleft lip and palate)) OR (TITLE-ABS-KEY (rhGFs)) OR (TITLE-ABS-KEY (Transforming growth factor)) OR (TITLE-ABS-KEY (Nestin))) AND (TITLE-ABS-KEY (Fibroblast)) OR (TITLE-ABS-KEY (Oral clefts))) AND (TITLE-ABS-KEY (Bone morphogenic protein)) OR (TITLE-ABS-KEY (Nerve growth factor))) AND (TITLE-ABS-KEY (Polymorphisms)) OR (TITLE-ABS-KEY (Endothelial growth factor))
3	Web of Science	(TITLE- Facial tissue AND Cleft lip and palate AND Transforming growth factor AND Nestin OR Fibroblast AND Bone morphogenic protein AND Matrix metalloproteinases AND Nerve growth factor OR rhGFs OR Oral clefts AND polymorphisms OR Endothelial growth factor)

Study selection and data collection process

Two reviewers screened the titles and abstracts of studies discovered by database searches, manual searches, and reference lists to identify papers that matched the inclusion criteria. To make an informed decision, one reviewer reviewed the abstracts and entire texts of each of these qualifying papers. A second author validated the decisions. The duplicate articles were deleted using the Mendeley reference manager software’s (version 2.132.1) check for duplication feature.

Data extraction

The following information was obtained from the methodology and results section of the papers that were selected. The details pertaining to the growth factor expression patterns in the local tissues of children who have OFCs and their associated growth factor receptors that cause OFCs.

Data items

Data was gathered using the following criteria:

1. Population – Non-syndromic OFCs or.

2. Intervention – Growth factors and their receptors in children with OFCs or.

3. Comparison – Non-cleft (NC) children or.

4. Outcomes – Long term assessment of growth factors and their receptors responsible for causing OFCs.

Quality assessment

Two reviewers evaluated the studies individually and then discussed their quality. In case, if there is any discrepancy, a third author was assigned for further evaluation. Newcastle - Ottawa scale (NOS) adapted version was used for quality assessment of cross-sectional studies [31]. Risk Of Bias in Non-randomised Studies of Interventions (ROBINS-I) tool was applied for case-control studies [32]. Risk of Bias in systematic review (ROBIS) assessment tool was utilized as per four phases of domains for systematic reviews included [33]. The in-vitro studies were evaluated using a quality assessment tool (QUIN) that includes 12 points with grading and scoring options [34]. Inter-rater reliability for quality assessment of the studies was done using Kohen’s Kappa score. Three raters reviewed the quality of each study included and percentage of agreement was decided based on the criteria of scoring 0–1 [35].

Additional synthesis

Due to the marked differences in biological functions and the degree of heterogeneity among the growth factors studied, performing a meta-analysis was not considered methodologically appropriate, as it could compromise the validity and reliability of the synthesized findings. Hence, we preferred a narrative synthesis over a meta-analysis because the included studies had different designs like cross-sectional, in-vitro, case-control, longitudinal, and systematic reviews. The outcomes were measured in different ways (e.g., gene expression and location of protein using immunohistochemistry). This is because standardized effect sizes, such as odds ratios (ORs) and confidence intervals (CI's) were not frequently reported in all the study findings.

Results

Study selection

Using specified keyword searches and database-specific terms, 191 studies were found in Scopus, PubMed, and Web of Science. The PRISMA 2020 flowchart (Fig. 1) displays the initial article screening and full-text review process. After an extensive review of the full texts, 20 studies were included and 12 were excluded. Table 2 shows possible reasons for exclusion.

Fig. 1.

PRISMA flow diagram for database searches and included studies

Table 2.

Excluded Article with possible reasons for exclusion

Study and year of publication	Study title	Reason for exclusion
Wang et al. [21]	Type 1 Fgf Receptor in Cranial Neural Crest Cell-derived Mesenchyme Is Required for Palatogenesis	Study involves the assessment of growth factors on mice.
Marques et al. [27]	Longitudinal study of growth of children with unilateral cleft-lip palate from birth to two years of age	Study was done to assess different parameters such as length, weight, and head circumference in OFC children.
Li et al. [36]	The Fibroblast Growth Factor 9 (Fgf9) Participates in Palatogenesis by Promoting Palatal Growth and Elevation	Study was conducted on Mice.
Li et al. [37]	Efficacy of Recombinant Human BMP2 and PDGF-BB in Orofacial Bone Regeneration: A Systematic Review and Meta-analysis	Target condition was regeneration of orofacial bones.
Hao et al. [38]	Roles of FGF8 subfamily in embryogenesis and oral‑maxillofacial diseases (Review)	Target conditions were oro-maxillofacial diseases.
Sun et al. [39]	Repair of alveolar cleft bone defects by bone collagen particles combined with human umbilical cord mesenchymal stem cells in rabbit	Study was conducted on rabbit with different target condition.
Hammoudeh et al. [40]	A Comparative Analysis of Recombinant Human Bone Morphogenetic Protein-2 with a Demineralized Bone Matrix versus Iliac Crest Bone Graft for Secondary Alveolar Bone Grafts in Patients with OFCs: Review of 501 Cases.	It’s a comparative study which is done for different indication.
Palumbo et al. [41]	A novel dominant-negative FGFR1 variant causes Hartsfield syndrome by deregulating RAS/ERK1/2 pathway	The study discusses about FGFR1 variant that causes Hartsfield syndrome.
Brajadenta et al. [42]	Molecular analysis of exon 7 of the fibroblast growth factor receptor 2 (FGFR2) gene in an Indonesian patient with Apert syndrome: a case report	It’s a case report which discusses about growth factor in patient with apert syndrome.
Liu et al. [43]	Distinct functions for Bmp signaling in lip and palate fusion in mice.	Study was conducted on mice.
Boyne, [44]	Application of bone morphogenetic proteins in the treatment of clinical oral and maxillofacial osseous defects.	The research involves treatment of osseous defects.
Courage et al. [45]	Novel synonymous and missense variants in FGFR1 causing Hartsfield syndrome	The study involves the FGFR variants that causes Hartsfield syndrome.

Characteristics of the included studies

This review included 20 relevant papers in accordance with the predefined inclusion criteria [6, 9, 18, 46–62]. Table 3 shows the detailed characteristics of these research studies, such as the study design, sample population, and key findings. These studies determine a variety of growth factors and their associated receptors that are linked to the development of oral clefts in children.

Table 3.

Detailed characteristics of the research studies included

Study	Sample population	Key findings
Letra et al. [6]	907 Brazilian individuals, 494 non-syndromic OFC patients, 413 NC children	Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases TIMPs. A strong correlation of MMP3 and TIMP2 was found with all OFC phenotypes.
Quizhpi-Quito et al. [9]	A total of 6 articles (4 for TGFB and 2 for FGF) were included	Both TGFB3 gene alterations are thought to play a role in the genesis of non-syndromic OFCs. The findings revealed that There is not much scientific evidence to link TGFB and FGF polymorphisms to OFCs.
Krivicka et al. [18]	Study sample involved 3 groups from Latvian individuals Group 1: 14 OFC patients involving palate from 8 months to 18.2 years. Group 2: 12 patients with or without OFC from 4 months to 15.4 years. Group 3: 11 NC children	The defective tissues of the OFC patients involving palate have revealed more prominent expression of BMP 2/4 and TGFβ. While expression of TGFBR3 was found to be significantly higher in connective tissue. FGFR1 expression was significantly higher both in general and in the epithelium.
Balode and Pilmane [46]	15 patients from Latvia (11 boys and 4 girls) among them 10 were unilateral OFC, 5 were bilateral OFC patients and 7 were NC children	Positive structures were found in connective tissue (TGFb1, MMP2, MMP9, and TIMP2). CD34 was detected in blood vessel endothelial cells, particularly in tiny vessels. The cell count ranged from zero to plentiful.
Beyeler et al. [47]	Invitro study involved primary lip fibroblasts from 16 OFC patients from Switzerland and 9 foreskin fibroblasts for comparison	The study investigated fibroblasts from a distinct group of OFC patients has shown increased rate of migration into wound and also linked with higher TGFα expression.
Blaha et al. [48]	Tissue samples of 26 boys and 8 girls were collected. Study sample involved children from Czech Republic	Increased concentration of MMP9 was found in dermis and epidermis before 2–4 months of cleft surgery. Whereas TIMP-1 levels in dermis were significantly decreased in early surgery patients when compared with samples collected from 2–4 months surgery.
Derelli Tufekci et al. [49]	70 non-syndromic OFC patients and 85 unrelated NC subjects from Turkey	As per the findings, the TGFα Hinfl gene polymorphism may be linked to non-syndromic OFC susceptibility, resulting in the occurrence of non-syndromic OFCs in Turkish patients.
De aquino et al. [50]	300 non-syndromic OFC patients and 385 unaffected NC children from Brazil	The findings indicate that FGF12, VCL, and CX43 polymorphisms do not contribute to the development of non-syndromic OFCs in Brazilian patients.
Leslie et al. [51]	1,498 Asian and European ancestry from Europe, the United States, China, and the Philippines	FGFR2 plays an important role in craniofacial development. Mutations in FGFR2 result in two craniosynostosis disorders, including OFCs. FGFR2 was found to be the most promising candidate which has been shown to disrupt the activity of a neural crest enhancer.
Letra et al. [52]	Human fibrosarcoma cells (HT-1080) Collected from the American Type Culture (Gaithersburg, MD, USA)	The findings suggest that MMP3 and TIMP2 promoter polymorphisms can impact gene transcription and craniofacial development, potentially contributing to non-syndromic OFCs.
Li et al. [53]	602 non-syndromic OFCs patients and 605 NC subjects as controls from China	Three single nucleotide polymorphisms (FGF2/rs1048201, FGF5/rs3733336, FGF9/rs546782) out of the nine were revealed evidence of a correlation with non-syndromic OFCs in the Chinese Han population.
Souza et al. [54]	127 Brazilian children with non-syndromic OFCs and 116 children without non-syndromic OFCs.	The EGF rs2237051 polymorphism was found to be associated with OFCs. A borderline association was detected with rs1800470 in TGFB1 when tooth agenesis and non-syndromic OFCs were assessed together.
Smane et al. [55]	19 complete bilateral OFC patients and 11 unaffected Latvian individuals as controls	In soft tissue impacted by clefts, MMP-2 expression is prominent, suggesting a potential increase in tissue remodelling. The growing presence of TIMP-2 positive connective tissue cells in the oral mucosa lamina propria indicates that local apoptosis has decreased in OFCs patients.
Smane et al. [56]	10 children with complete unilateral OFC and 19 children with complete bilateral OFC form Latvia.	Nestin immunoreactive structures were seen in the oral mucosa in moderate to abundant amounts. CD34 antibodies were found in all microvessels in the lamina propria of Cleft affected tissues. There were few TGFβ3 positive cells dispersed throughout the connective tissue.
Smane and Pilmane [57]	10 children had complete bilateral OFC, and 12 children had complete unilateral OFC from Latvia.	MMP2-positive cells were seen in all complete unilateral OFC cases, with MMP2-positive osteocytes ranging from infrequent to common. While MMP2-positive cells were detected in all bilateral OFC affected bone tissue samples. TIMP2-positive osteocytes ranged from relatively few to numerous in all complete unilateral and bilateral OFC instances. BMP2/4-positive bone cells ranged from moderate to numerous in all complete unilateral OFC instances. However, positive osteocytes in the complete bilateral OFC group were rare to abundant. TGFβ3-positive osteocyte cells were seen in all complete unilateral and bilateral OFC cases ranged from moderately to abundant.
Rahimov et al. [58]	Genetic study of OFCs. Gene samples from Iowa city, USA	The FGF signalling system facilitates craniofacial development by inducing and migrating cranial neural crest cells and regulating epithelial-mesenchymal interactions during facial prominence fusion. TGFA was one of the first genes linked to non-syndromic OFCS. In the orofacial primordia, conditional deletion of the type 1 BMP receptor gene (BMPR 1a) and its ligand Bmp4 leads in isolated OFC alone, while conditional inactivation of the gene causes bilateral OFCs with tooth agenesis. A variety of aspects of embryonic development and craniofacial morphogenesis are influenced by the sonic hedgehog (SHH) signaling system, as evidenced by the diverse range of craniofacial abnormalities brought on by disruptions.
Souza et al. [59]	96 complete case-triads, consisting of the mother, father, and proband; and 79 incomplete case-parents, consisting of the mother, father, or proband from Brazil	The authors revealed no causal relationship between TGFA/Taq I polymorphisms and OFCs examined in the case parent triad of the Brazilians.
Wang et al. [60]	OFC involving lip with or without palate case-parent trios through treatment centres in Maryland, Taiwan, Singapore, and South Korea	Several genes in the FGF/FGFR family may contribute to OFC involving lip with or without palate risk through different biological pathways, according to the findings.
Ge et al. [61]	6 non-syndromic OFC of lip patients, 8 non-syndromic OFC of palate patients, and 2 traumatized lip tissues patients as controls for Chinese Han population	Findings suggest that FGFR2 plays a significant role in non-syndromic OFC involving lip patients than other FGF/FGFRs in Han Chinese.
Alghonemy and Ashmay, [62]	6 articles were included for review	As per the findings, the MTHFR c.677 C > T and MTHFD1 1958G > polymorphisms have been found to be associated with non-syndromic OFC risk.

Results of risk of bias studies

As per the ROBINS-I assessment, 11 case control studies were assessed except the systematic reviews, In-vitro studies and cross-sectional studies. Among 11 studies included, 5 were rated as moderate and 6 were rated as low risk. The scores are displayed in (Fig. 2) and summarized in (Fig. 3). ROBIS assessment tool was utilized for two systematic reviews included. The tool is utilized in 4 different domains to identify the review process concerns, and determine the risk of bias in four domain categories in systematic reviews which displayed lowest risk of bias as presented in (Fig. 4).

Fig. 2.

Risk of bias assessment of case-control studies as per ROBINS-I assessment criteria

Fig. 3.

ROBINS-I assessment summary of case-control studies

Fig. 4.

Risk of bias in systematic reviews (ROBIS) assessment of included studies

The QUIN quality assessment tool was used for 4 in-vitro studies included as shown in Table 4 and it was graded as low risk of bias. The quality of three cross-sectional studies was assessed using NOS (adapted version), depicted in Table 5, and the overall rating was 7–8, indicating that high-quality studies were included. The inter-rater reliability as per Cohen’s Kappa score was 0.66–1.00.66.00 as shown in Table 6 which demonstrate substantial to nearly perfect agreement between the raters.

Table 4.

QUIN tool of assessment for in vitro studies

Study	Criteria and Assessment Score
	Clearly Stated Aims/objectives	Detailed Explanation Of Sample Size Calculation	Detailed Explanation of Sampling technique	Details Of Comparison Group	Detailed Explanation Of Methodology	Operator details	Randomization	Method of measurement of outcome	Outcome assessor details	Blinding	Statistical analysis	Presentation of results
Beyeler et al. [47]	2	2	2	2	2	1	NA	1	1	NA	2	2
Blaha et al. [48]	2	2	2	2	2	1	NA	2	2	NA	NA	2
Letra et al. [52]	2	1	2	2	2	1	NA	1	1	NA	NA	1
Rahimov et al. [58]	2	1	0	1	1	0	NA	0	1	NA	NA	1

Table 5.

Quality assessment of cross-sectional studies by Newcastle - Ottawa scale (Adapted version)

Author and year of Study	Selection				Comparability	Outcome		Overall Rating
	Representativeness of the cases	Sample size	Non-response rate	Ascertainment of the screening	Potential confounders Investigation	Assessment of outcome	Statistical analysis
Leslie et al. [51]	1	1	0	1	1	2	2	8
Smane et al. [56]	1	1	1	0	1	1	2 -	7
Smane and Pilmane [57]	1	1	1	0	1	2	2	8

Table 6.

Inter-rater agreement as per Cohen’s Kappa score

Study	Rater 1	Rater 2	Rater 3	Agreement %
Letra et al. [6]	1	1	0	0.66
Quizhpi-Quito et al. [9]	1	1	1	1.0
Krivicka et al. [18]	1	1	1	1.0
Balode and Pilmane, [46]	1	1	1	1.0
Beyeler et al. [47]	1	1	1	1.0
Blaha et al. [48]	1	1	1	1.0
Derelli Tufekci et al. [49]	1	0	1	0.66
De aquino et al. [50]	1	1	0	0.66
Leslie et al. [51]	1	1	1	1.0
Letra et al. [52]	1	0	1	0.66
Li et al. [53]	1	1	1	1.0
Souza et al. [54]	1	1	1	1.0
Smane et al. [55]	1	1	1	1.0
Smane et al. [56]	0	1	1	0.66
Smane and Pilmane [57]	1	1	1	1.0
Rahimov et al. [58]	1	1	1	1.0
Souza et al. [59]	0	1	1	0.66
Wang et al. [60]	1	1	0	0.66
Ge at al. [61]	0	1	1	0.66
Alghonemy and Ashmay, [62]	1	1	1	1.0

Growth factors

Fibroblast growth factor (FGF)

In developing primordia of the face, FGFR1 and FGFR2, the receptors for most FGF ligands, are broadly expressed. The numerous FGFR family members of signalling molecules have been linked to birth abnormalities that impact the craniofacial structures [51, 58]. FGF receptor genes 1, 2, and 3 are often linked to craniosynostosis syndrome. According to a study by Rahimov [58] nearly 76% of the instances in Apert syndrome and mutations in FGF receptor 2 (FGFR2) genes are linked to craniosynostosis and almost all the cases exhibited Byzantine arch-shaped palate.

In a study by Wang et al. [60] 297 case parent trios were gathered from four populations i.e. from Maryland (United States), Taiwan, Singapore and Korea. The authors analyzed 111 markers in 10 FGF/FGFR (FGFBP1, FGFR1, FGFR2, FGF2, FGF3, FGF4, FGF9, FGF10, FGF18, and FGF19) genes using 297 case-parent trios from an international survey. Single nucleotide polymorphisms (SNPs) in 7 genes showed linkage and correlation with unidentified causative variations for OFCs. Genes in the FGF/FGFR pathway are promising targets for OFCs due to their impact on craniofacial development. Three SNP haplotypes in FGFR1 (rs6987534, Type="Italic"> rs6474354, and rs10958700) were found to be significant (p = 0.04) among Asian trios.

A study in the Chinese Han community comprised 602 patients with non-syndromic OFCs and 605 controls as NC children. Only three of ten SNPs selected from the FGF and FGFR genes (FGF2/rs1048201, FGF5/rs3733336, and FGF9/rs546782) were linked to non-syndromic OFC susceptibility. The number of protective alleles (rs1048201-T – OR = 0.83, rs3733336-G – OR = 0.72, and rs546782-T – OR = 0.57) were dropped with the reported odds ratios in combination analysis; however, this was not statistically significant following the initial comparison. It has been suggested that, FGFR2 was detected as the most potential FGF which is mostly associated with non-syndromic OFCs among other FGFs studied in Han Chinese [61]. Krivicka et al. [18] demonstrated that, FGFR1 expression was variable in cleft affected tissue, including epithelium, connective tissue, and blood vessel walls. It was more apparent in the epithelium. In addition, FGFR1-containing epithelial cells were statistically significant in patients with cleft palate (p = 0.004) and the expression of basic fibroblast growth factor (bFGF) in cleft palate tissue was similar. However, no significant association with bFGF was observed by the authors (p = 0.0210). The five tissue samples from cleft lip patients revealed bFGF expression in a range of sebaceous gland cells. In rare cases, mild expression was also observed in the connective tissue and vascular smooth muscle cells.

Transforming growth factor (TGF)

TGFα, a significant growth factor, may lead to OFCs in humans. The allele frequencies of TGFα and genotype were estimated in non-syndromic OFCs Turkish patients and NC group. The ORs and CIs were calculated between TGFα polymorphisms and NC group. The frequency of the A allele was noted in 50% of OFC cases and 2% of NC children (OR = 9.87, 95% CI: 3.4–36.11.4.11, p = 0.002). There was a substantial difference in the frequency of TGFα polymorphisms between non-syndromic OFC cases and NC children. TGFα gene polymorphism may increase susceptibility to non-syndromic OFCs among Turkish patients [49]. It has been revealed that TGFα plays an essential role in endochondral ossification and predominantly regulates the proliferation, differentiation, and carcinogenesis of epithelial cells. TGFα is a well-documented genetic modulator of non-syndromic OFCs humans [47, 63, 64].

TGFβ1 is a secreted protein that binds to TGFβR2 and promotes cell proliferation, differentiation, and growth in several tissues such as bone, epithelium, and connective tissue. A cross-sectional study on Brazilians was conducted that included children without clefts serving as controls. The association between tooth agenesis and non-syndromic OFCs was investigated. A total of 216 patients with tooth agenesis and 243 children with non-syndromic OFCs were included. A genotypic analysis was conducted using genetic polymorphisms in TGFβ1 (rs1800470 and rs4803455), TGFβR2 (rs3087465 and rs764522), and other receptor genes. There was no link between tooth agenesis and the investigated genetic variants. When tooth agenesis and non-syndromic OFCs were combined, a marginal correlation was detected with rs1800470 in TGFβ1 (p = 0.06) [53]. Smane & Pilmane [57] studied the Latvian population and found TGFβ3 positive osteocytes in all unilateral OFC in humans. The unilateral OFCs had significantly more TGFβ3-positive cells than the bilateral OFCs and NC groups (p < 0.001). This could be owing to insufficient proliferation, differentiation, and cell death in the most severe type of cleft, i.e. bilateral OFC.

Nestin and cluster of differentiation 34 (CD34)

Nestin and CD34 are crucial for tissue regeneration. These signalling molecules were employed to identify oral mucosa stem cells and angiogenesis markers in facial clefts from damaged tissues. There were 29 OFC patients enrolled in the investigation. After primary and repeated plastic surgery repair, tissue samples were collected from 10 complete unilateral and 19 complete bilateral cleft palate children ranging in age from 3 months to 9 years. Immunohistochemistry was used to determine the levels of nestin and CD34. The oral mucosa displayed moderate to abundant nestin immunoreactive structures. CD34 antibodies were observed in all microvessels of the lamina propria of tissues affected by OFCs [56]. Balode and Pilmane [46] discovered that in the blood vessels of OFC tissue, connective tissue infiltrates the hyaline cartilage of the intranasal septum with CD34-positive endothelial cells in numbers ranging from zero to numerous.

Bone morphogenic protein (BMP)

BMPs are secreted signalling molecules from the TGFβ family of growth factors. They regulate essential developmental processes such cell proliferation, differentiation, and cell death. Inactivating the type 1 BMP receptor gene (BMPR1a) in the orofacial primordia can result in bilateral OFCs and tooth agenesis, while deleting its ligand BMP4 results in isolated cleft lip [58]. In a study by Smane & Pilmane [57] on Latvian individuals found BMP2/4-positive bone cells in all hard tissue samples, including osteoblasts and osteocytes. BMP2/4-positive bone cells were present in all unilateral OFC patients ranged from moderate to numerous. In another study, the authors have identified moderately immunoreactive cells that frequently expressed BMP 2/4 in hair follicles and sebaceous glands. Moreover, a significant variation was observed in the number of positive cells in an OFC impacted epithelium (p = 0.031) [18].

Matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP)

The matrix metalloproteinases (MMPs) are responsible for tissue remodelling which induce epithelial differentiation and morphogenetic variations during palatal development [65]. The activity of MMP is regulated by an inhibitory factor i.e. TIMP 2. Balode and Pilmane [46] examined 17 surgical records from 15 Latvian kids aged 6 to 17 years old (11 boys and 4 girls), 10 with unilateral OFCs and 5 with bilateral OFCs. During OFC rhinoplasties, tissue was extracted from the apex of the nose.

TIMP2, MMP2, and MMP9 were detected in hyaline cartilage chondrocytes and connective tissue cells, particularly those near blood vessels and nerve bundles. The number of cells observed varied from few to plentiful. These structures demonstrate how neoangiogenesis and the tissue remodelling pattern balance have intensified in nasal tissue impacted by OFCs [57]. Another study has demonstrated that there were varying numbers of MMP−2 positive oral epithelial cells in all specimens from the NC group and complete bilateral OFC group, particularly in the basal cell layer, connective tissue cells, and salivary gland cells. However, no significant differences were observed in means between the complete bilateral OFCs and the NC group (p = 0.09 & p = 0.77), respectively [45].

Vascular endothelial growth factor (VEGF)

During craniofacial development, it’s crucial to assess blood vessel density and the potential for angiogenesis. CD34 and VEGF are the most prominent markers used for this purpose [66]. Balode and Pilmane [46] found that VEGF was present in blood vessels, particularly in neoangiogenesis sites. The VEGF was also observed in both blood vessel endothelium and chondrocytes of hyaline cartilage. Positive structures were noticed in connective tissue which included TGFβ1, MMP2, and TIMP2. The presence of VEGF positive cells varied from moderate to considerable. However, the researchers identified a moderately positive association in the OFC group between VEGF and TIMP2 (r_s =0.535; p = 0.27).

Discussion

In this systematic review, we thoroughly analyzed and found a variety of growth factors and receptors that play essential roles in the molecular and cellular mechanisms underlying the pathogenesis of orofacial clefts. Majority of the studies were conducted in Europe that include Latvia, Czech Republic, Switzerland and Turkey. Few studies were from United States, Brazil, and Asian countries such as Taiwan, Singapore, South Korea, Philippines and China.

OFC-impacted tissue revealed statistically significant spike in FGFR1 expression in the epithelium, both in general and individually. FGFR1 is the major FGF/FGFR receptor, having the lowest affinity for βFGF and a vast distribution throughout the human body. The βFGF interaction generates a wide range of cellular activities [18, 67, 68]. In assessing transmission distortion of individual SNPs among these 10 FGF/FGFR genes, two separate markers in FGF19 exhibited nominally significant evidence of linkage and association (P < 0.05) with OFCs across Asian trios, while another SNP was significant exclusively among Maryland trios. A 3-SNP haplotype in FGFR1 (rs6987534, rs6474354, and rs10958700) was significant (p = 0.04) among Asian trios. The authors have also found maternal genotypic influences on FGF2 and FGF10 markers in Asian trios [60]. In a previous study on Han Chinese population, 10 mRNA-binding site SNPs in seven FGF/FGFR genes were identified and successfully genotyped in a case-control investigation. One SNP (rs2241286) was excluded and of the remaining 9 SNPs only 3 SNPs (FGF2/rs1048201, FGF5/rs3733336, and FGF9/rs546782) are associated with non-syndromic orofacial clefts [52]. Similarly in another case-control study using RNA-Seq, the authors were able to identify 32 mRNAs associated with FGF/FGFR members. After conducting further association analysis, it was concluded that FGFR2 has greater potential than other FGF/FGFR genes found within the Chinese non-syndromic OFCs [59]. Two of the studies did not find any genetic association of FGF12 polymorphisms and OFCs between Brazilians and in different populations assessed [9, 50].

The TGF growth factor family and its receptors play a vital role in craniofacial development. Previous research on animal models and in vivo investigations has considerably contributed to our current understanding of the TGF signalling pathway’s role in OFCs. TGFα was one of the first genes shown to be associated with non-syndromic OFCs. It is significantly expressed during palatal fusion and facilitates ECM biosynthesis [69–72]. TGFβ and VEGF and can cause endotheliocyte proliferation, budding, and neoangiogenesis, as evidenced by the presence of glomus bodies in several samples collected. TGFβ1-positive cells were found to be similar in both NC and OFC affected tissue [46].

A study explored the relationship between tooth agenesis and non-syndromic OFCs. In addition, the correlation between both conditions and genetic variants affecting growth factors was investigated. Only rs1800470 in TGFβ1 showed a marginal connection with tooth agenesis in children with non-syndromic OFCs. TGFβ1 regulates odontoblast differentiation and root formation during odontogenesis [54]. Correspondingly a study demonstrates that the TGFα Hinfl gene polymorphism may be related with non-syndromic OFC susceptibility, thereby contributing to the occurrence of non-syndromic OFCs in Turkish patients. Another investigation on Latvian population has detected 10 cases with TGFβ3-positive structures around blood vessels and five cases with few TGFβ3-positive macrophages in the visual field. The TGFβ superfamily of cytokines includes over 30 growth factors that regulate various cellular and physiological processes, including embryonic development, angiogenesis, and wound healing [14, 56]. Likewise, TGFβR3 was initially identified as a TGFBR2 coreceptor. TGFBR3 binds to all three TGFβ isoforms and modulates their interaction with other TGFβ superfamily signalling receptors, although lacking a functional kinase domain. Investigators revealed that elevated TGFβR3 expression in cleft affected individuals indicates aberrant tissue growth and altered TGFβ signalling pathways in postnatal pathogenesis, which might result in poor mucosa after surgical closure of malformations [16]. Furthermore, a comparison was made between complete unilateral and bilateral OFC patients from the tissue samples obtained for osteoplasty has shown a moderate decrease in TGFβ3 osteoblasts and osteocytes in patients with complete bilateral compared to OFC patients and NC group. This could be attributed to insufficient cell proliferation, differentiation, and apoptosis in the most severe type of cleft i.e. complete bilateral OFC [57].

Fgfr1 and Fgfr2 were found to be expressed in MEE cells during palatal shelf fusion. FGF7 and FGF10-FGFR2 have excellent bonding specificity and were only detected in the palatal mesenchyme during palatogenesis [73]. FGF7 and FGF10-FGFR2 have excellent bonding specificity and were only detected in the palatal mesenchyme during palatogenesis. Fgf7 was originally expressed in the nasal aspect of the mesenchyme prior to shelf elevation, and later intensified in the oral side as well as the adjacent region of MEE [74].

CD34 is a marker of angiogenesis and microvascular density in the oral mucosa, both in normal and pathological situations. The researchers have identified numerous CD34-positive microvessels in the lamina propria of OFC affected tissue. Correspondingly the CD34-positive endothelial cells were also observed in the blood vessels of cleft affected tissue thereby invading the hyaline cartilage of the intranasal septum. Cells ranged from relatively small to numerous across all study groups examined [46, 57].

Nestin, a type VI intermediate filament protein, was first identified as a marker of brain stem and progenitor cells. It appears in several human embryonic, fetal, and adult tissues. It is transiently produced in undifferentiated cells, including newly formed blood vessels [75]. An investigation on Latvian children has demonstrated nestin expression in all unilateral and bilateral OFC tissues. It has been suggested that nestin-expressing cells are crucial for proliferation, differentiation, and migration after re-activation [46].

Nestin expression in both unilateral and bilateral OFC affected tissue suggests a potential increase in tissue regeneration. CD34-positive oral mucosa cells imply enhanced angiogenesis, while sporadic TGFβ3 expression suggests no significant role in the growth and development of cleft-affected progenitor cells [56].

BMPs have a critical role in several biological processes during palatogenesis, including cell proliferation, apoptosis, ECM formation, cell migration, epithelial mesenchymal transition, and basement membrane disintegration [16–18]. The authors revealed that the conditional inactivation of the type 1 BMP receptor gene in the orofacial primordia leads to bilateral OFCs and tooth agenesis, whereas conditional deletion of its ligand BMP4 in the same tissue results in isolated OFCs alone [43, 58].

BMPs belong to the TGFβ superfamily. BMP ligands (e.g., BMP2, BMP4, BMP7) bind to BMP receptors, forming hetero-multimers with BMP type II receptors and BMP type I receptors (subsequently phosphorylate SMAD1/5/9 to transduce the signals [76]. BMP signaling regulates proliferation, cell death, differentiation, and cell fate specification during embryogenesis, including craniofacial development in addition to osteogenesis [77].

MMPs are proteases that include zinc (II) (Zn2 + ions) and three histidine residues in their catalytic regions. MMPs target the ECM. Over 20 MMPs have been found in humans [72]. Among 20 studies selected in this review, 6 articles were discussed about MMPs and TIMPs. In a large case-control dataset from Brazil, the researchers noticed 45 SNPs in 14 MMP and TIMP genes involved in craniofacial development as potentially suitable candidates for OFCs [6]. The other studies have also shown strong association between the MMP2 and TIMP2 in patients with all types of clefts [46, 48, 52, 55, 57].

A systematic review has reported that a variety of MMPs and TIMPs can have an impact on OFC-affected tissues and cells. The authors demonstrated the association between apoptosis in cleft-affected cells and three MMPs (MMP-2, MMP-3, and MMP-9). MMP-3 rs3025058 and MMP-9 rs17576 polymorphisms were linked to the risk of OFCs, according to the individual investigations. TIMPs inhibit MMPs, cell growth activity and angiogenesis. But also have the ability to increase the cell growth and regulate apoptosis. Apoptosis is a morphological process that has a significant role in embryonic development, morphogenesis, tissue regeneration, and normal tissue turnover. Anti-apoptotic alterations in genes involving apoptosis may be the cause for OFC susceptibility. TIMP-2 appears to have antiapoptotic characteristics in apoptosis regulation. Thus, the overexpression of growth factors, MMPs, TIMPs, and apoptosis may be linked to pathogenesis of OFCs [56, 78, 79].

The VEGF primarily affects vascular endothelial cells. However, it has a limited impact on other cells, such as stimulating monocyte/macrophage migration. In vitro, VEGF increases endothelial cell proliferation and migration. It also improves microvascular permeability [72]. The researchers anticipated the presence of VEGF in the OFC tissue since it influences bone growth, blood vessel design, and maturation during palatogenesis. OFC’s higher vascular density can be explained by the greater number of VEGF-positive cells compared to healthy controls, as the growth factor stimulates neoangiogenesis, increases blood vessel permeability, and causes hypertrophy. VEGFα, a growth factor associated in angiogenesis and ossification is essential for palate development. The VEGFα conditional knockout (CKO) palatal shelves showed significantly decreased vascularization, indicating a non-cell autonomous involvement of VEGFα signaling from cranial neural crest derived cells to the endothelium during vessel formation. Defective vascular development was linked to poor intramembranous ossification of maxillary and palatal mesenchyme [46, 80, 81].

Although OFCs exhibit a variety of phenotypic manifestations and treating heterogeneous groups as a single entity weakens the ability to detect effects. Hence, accurate phenotyping is essential to understand the epidemiology and etiology of any congenital abnormality [3].

Our study focused only on growth factors and receptors involving certain genes that are involved in the etiopathogenesis of non- syndromic OFCs. Hence, the gene sequencing, genetic-environment interactions candidate gene association, and genome wide association (GWA) research investigations were not been thoroughly studied as the number of available studies on this topic is limited due to ethical concerns, tissue availability, and differences in study methods.

Furthermore, prenatal biomarkers and interventions like as growth factor modification were not investigated. Because our findings do not include an examination of the maternal serum proteome or C-reactive protein (CRP), which are potential serum biomarkers for the prenatal diagnosis of non-syndromic OFCs.

Although, these are some of the limitations of this review. It was possible that our keywords search terms must have included the specific terms like genetic sequencing, linkage, environment factors and GWA studies would have provided deeper insights into genetic and environmental correlation.

Conclusion

This systematic review emphasizes the important roles that growth factors (FGF, TGF, Nestin, CD34, BMP, MMPs, TIMPs, and VEGF) and their receptors play in the pathophysiology of non-syndromic OFC. FGFR1 and TGFβ3 were strongly linked to OFC susceptibility. CD34 and VEGF revealed increased angiogenesis. BMP2/4, FGFR1, and TGFβ3 were all expressed at higher levels in unilateral OFC tissues, especially in bone cells, connective tissue, and epithelium.

Further research on growth factors in OFC patients should focus on their role in improving hard and soft tissue regeneration. Research should also look into how they affect midfacial growth modulation, and orthodontic tooth movement. Integrating growth factors with stem cells for tissue engineering, as well as exploring their genetic and epigenetic regulation, could lead to individualized regenerative treatment options.

Thus, the results of the various studies pointed out the requirement of conducting longitudinal studies using samples drawn from a larger number of people in various populations from multiple ethnicities and to investigate standardized approaches to reduce variation and facilitate more robust comparisons across populations. The findings should include all genetic study standards in order to produce validated data that would enhance our knowledge in understanding the pathogenesis and molecular basis of the disease.

Abbreviations

BMP

Bone morphogenic protein

BMPR

Bone morphogenic protein receptor

CD

Cluster of differentiation

CKO

Conditional knockout

CI

Confidence interval

CRP

C-reactive protein

ECM

Extra cellular matrix

FGF

Fibroblast growth factor

FGFR

Fibroblast growth factor receptor

GWA

Genome wide association

MEE

Medial edge epithelia

MES

Midline epithelial seam

MeSH

Medical Subject Heading

MMPs

Matrix metalloproteinases

NC

Non-cleft

NOS

Newcastle Ottawa Scale

OFCs

Orofacial clefts

OR

Odds ratio

PRISMA

Preferred reporting items for systematic review and meta-analysis

QUIN

Quality assessment tool for in-vitro studies

ROBINS-I

Risk of bias in non-randomised Studies of interventions

ROBIS

Risk of Bias in systematic review

RNA

Ribonucleic acid

mRNA

Messenger Ribonucleic acid

SNPs

Single nucleotide polymorphisms

TGF

Transforming growth factor

TIMP

Tissue inhibitor of metalloproteinase

VEGF

Vascular endothelial growth factor

Zn2+

 Zinc II

Authors’ contributions

MZU: Conceptualization, Design of the work, drafted the review, Screening, Data extraction, HBY: Data acquisition, data analysis, SKV: Software utilization, data analysis, JYA: Validation, Revising the draft, Data extraction, SDS: review & editing, data acquisition, SAQ: Funding Resources, Investigation, Review of the Draft, AM: review & editing, Software utilization, SS: Data extraction, Data analysis, review & editing, Final validation.

Funding

The authors extend their appreciation to the Deanship of Research and Graduate Studies at King Khalid University for funding this work through Large Research Project under grant number RGP2/542/46.

Data availability

The data utilized to support the study findings are included within this systematic review.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.