1. Introduction
The second most common congenital craniofacial deformity is cleft lip and palate (CLP), which is caused by abnormal soft and hard organogenesis during embryonic development. The incidence of cleft lip with versus without cleft palate (CL/P) is 1:1000 [
1,
2]. The goal of early postnatal surgical procedures is to promote adequate palatal shelf growth and fusion. New opportunities for encouraging tissue growth at the site of the surgical repair for palatal clefts have emerged due to the advances in scaffold-based delivery technologies for precision tissue engineering [
3]. Asymmetries often shift as a child grows and develops, making the nasal deformity a daunting challenge. The repair of absent or asymmetric cartilage and the replacement of bone components are crucial for a successful treatment of the cleft lip and palate patients [
4,
5]. Avascular cartilage is mostly made up of extracellular matrix (ECM), which is maintained by a tiny number of local chondrocytes. The ECM is sustained by an array of growth factors and cytokines in healthy tissue [
6]. Despite the fact that multiple tissue factors are thought to have a role in the morphopathogenesis of CLP, research on human cleft nasal septum cartilage is limited due to ethical issues and a lack of available material.
Up to date, there are relatively few studies and data on remodeling factors, resorption factors, growth factors, and cytokines of cartilage due to the difficulty of acquiring hyaline cartilage of the nasal septum because of the ethical considerations.
Matrix metalloproteinases (MMPs) expression and their natural inhibitors (TIMPs) in craniofacial development is tissue specific. MMPs and TIMPs are believed to be necessary for the growth of the mammalian palate [
7].
MMPs degrade the ECM, causing cartilage damage and changing its biomechanical characteristics. MMPs are a type of protease involved in bone formation, angiogenesis, and connective tissue remodeling. In osteoarthritic cartilage, MMP-2, MMP-9, and MMP-13 have been found to be considerably overexpressed [
8]. MMP-2 is a proteinase that degrades undamaged type IV collagen and type I collagen that has been denatured in the extracellular compartment. MMP-2 mediated tissue remodeling plays a role in several physiological mechanisms, such as angiogenesis, neovascularization, and wound healing. Underactivity of MMP-2, either deficiency or insufficiency, has been linked to inflammation, metabolic dysregulation, and skeletal diseases [
9,
10].
MMP-8 can cleave type I–III collagens. MMP-8 has been found to participate in the breakdown of ECM and the degeneration of bone tissue. MMP-8 has the ability to generate and remodulate tissue via ECM breakdown, implying that MMP-8 could be an indication of active cartilage and might have a protective function [
8]. MMP-8 and MMP-9 overexpression increased cartilage damage and promoted degenerative alterations in knee structure and morphology in particular. In diabetic osteoarthritis (OA) rats, the overexpression of MMP8 and MMP9 enhanced the number of apoptotic chondrocytes [
11].
MMP-9 at increased levels, adds to cartilage degradation. Pro-inflammatory indicators including interleukin-1 (IL-1), interleukin-6 (IL-6), and C-reactive protein increase its expression [
12].
TIMPs have the capacity to stop MMPs from functioning. Furthermore, irrespective of their MMP neutralizing actions, TIMPs are thought to have implications of pluripotency on cellular functions, such as cell proliferation, movement, endurance, and differentiation [
13]. Tissue inhibitor of metalloproteinases 2 (TIMP-2) expression is lower in studies with pregnant pig mothers receiving hydroxy—methobutyrate supplementation for the skeletal development of their piglets, which could imply that these supporting tissues of animals are undergoing greater remodeling [
14]. The effect of a combination of anabolic growth factors and a protease inhibitor on an in vitro culture of articular chondrocytes revealed that TIMP-2 was beneficial in enhancing ECM synthesis [
15].
IL-1 is one of the most important pro-inflammatory cytokines implicated in cartilage degradation. It draws monocytes and neutrophils to the site of tissue damage, inducing MMPs, and disrupting homeostasis. IL-1 stimulates the production of other cytokines, such as IL-1, IL-6, and TNF- during the inflammatory process [
16]. This cytokine can trigger a slew of catabolic mediators in chondrocytes, the majority of which are involved in cartilage destruction, decreasing matrix formation, and chondrocyte apoptosis after a traumatic damage [
17,
18].
Chondrocytes can synthesize interleukin-10 (IL-10) and have the IL-10 receptor expressed on their cellular surface. In mechanically wounded cartilage, IL-10 therapy reduces posttraumatic cell death, matrix degradation, and chondrocyte dedifferentiation. In experimental models of OA, IL-10 inhibited matrix degrading enzymes and IL-1b expression produced by proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α). In addition, IL-10 enhanced proteoglycan production in an inflammatory setting increased the previously decreased biosynthetic activity of articular chondrocytes, displaying anti-apoptotic properties as well [
19].
Basic fibroblast growth factor (bFGF) can enhance the proliferation of chondrocytes and mesenchymal cells, as well as chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in the culture of chondrocytes and BMSCs for cartilage tissue engineering. bFGF increases chondrogenesis while inhibiting osteogenesis and protects cartilage from injury [
20]. It has been widely used in tissue engineering to increase chondrocyte proliferation, angiogenesis and healing of wounds via influencing epithelial cells, smooth muscle cells, fibroblasts and endothelial cells as bFGF can stimulate cell mitosis, and thus cell proliferation [
21].
Transforming growth factor β (TGF-β) promotes collagen, fibronectin, and proteoglycan synthesis while inhibiting collagen breakdown by lowering MMP activity and boosting TIMP activity [
22]. When chondrocytes were stimulated mechanically, they produced more pro-osteoclastic factors, such as transforming growth factor 1 (TGF-1), which increased condylar subchondral bone resorption by boosting osteoclastogenesis. These findings back up the theory that cartilage alterations occur before subchondral bone modificas, and so play a key role in mechanical loading [
23].
The objective of our current research is triggered by the lack of research in the cartilage tissue of CLP patients and aims to estimate the relative number and presence of tissue factors (MMP-2, MMP-8, MMP-9, TIMP-2, IL-1α, IL-10, bFGF, and TGFβ1) in the CLP patients’ cartilage throughout the first and second plastic rhinoplasty and alveolar osteoplasty.
3. Results
3.1. MMP-2
MMP-2 positive cells were detected in all cartilage tissue samples from the first-time surgery CLP group, the second-time surgery CLP group and the control group. The first-time surgery CLP group had a range of MMP-2 positive chondrocyte numbers, from + to +++, in the second-time surgery CLP group it varied from ++/+++ to +++/++++, and from +/++ to +++ in the control group (see
Table 3) (see
Figure 1A,A1).
The medial value of the positive cells for MMP-2 in the first-time surgery and the second-time surgery CLP group was +++ (SD = 0.89; SD = 0.41); however, in the control group it was lower—++ (SD = 0.46) number of positive structures (see
Table 3).
A statistically significant greater number of the positive chondrocytes for MMP-2 was found in the first-time surgery CLP group relative to the control group (U = 92.0; p = 0.011) and in the second-time surgery CLP group in comparison to the control group (U = 7.5; p = 0.003). The first-time surgery CLP group and the second-time surgery CLP group did not statistically significantly differ from one another (U = 117.0; p = 0.959).
3.2. MMP-8
MMP-8 positive cells were detected in all cartilage tissue samples from the first-time surgery CLP group, the second-time surgery CLP group, and the control group. The number of MMP-8 positive chondrocytes in the first-time surgery CLP group differed from ++ to ++++, in the second-time surgery CLP group—from ++/+++ to ++++, and in the control group—from ++ to +++/++++ (see
Table 3) (see
Figure 1B,B1).
The medial value of the MMP-8 positive cells in the first-time surgery CLP group and the control group was +++/++++ (SD = 0.68; SD = 0.45), but in the second-time surgery CLP group it was lower—+++ (SD = 0.53) number of positive structures (see
Table 3).
No statistically significant difference between all of the research groups was obtained. Between the first-time surgery and the second-time surgery CLP groups (U = 81.5; p = 0.198), between the first-time surgery CLP group and the control group (U = 168.5; p = 0.630), and between the second-time surgery CLP group and the control group (U = 19.5; p = 0.085).
3.3. MMP-9
All specimens had cells that were MMP-9 positive. The number of MMP-9 positive chondrocytes in the first-time surgery CLP group were +++/++++, in the second-time surgery CLP group it was ++/+++ to +++/++++, and it ranged from + to +++ in the control group (see
Table 3) (see
Figure 1C,C1).
The medial value of MMP-9 positive chondrocytes was the same in the first-time surgery and the second-time surgery CLP groups—+++ (SD = 0.58; SD = 0.38), but in the control group it was lower—+/++ (SD = 0.52) (see
Table 3).
The first-time surgery CLP group and the second-time surgery CLP group did not have any statistically significant difference between the numbers of MMP-9 positive chondrocytes (U = 117.0; p = 0.959). Both the first-time surgery CLP group and the second-time surgery CLP group had significantly more MMP-9 positive chondrocytes than the control group, (U = 31.5; p = 0.000) and (U = 4.5; p = 0.001), respectively.
3.4. TIMP-2
Each sample of cartilage tissue revealed cells that were TIMP-2 positive. Chondrocytes that were TIMP-2 positive in the first-time surgery CLP group differed from +/++ to ++++, in the second-time surgery CLP group—from ++/+++ to +++/++++, while in the control group it differed from +/++ to +++/++++ (see
Table 3) (see
Figure 1D,D1).
The medial value of the first-time surgery CLP group was lower than in the other groups—a ++ (SD = 0.65) number of TIMP-2 positive chondrocytes compared to +++ (SD = 0.29) in the second-time surgery CLP group, and ++/+++ (SD = 0.55) in the control group (see
Table 3).
Comparing the second-time surgery CLP group to the control group, it was found that there were considerably more TIMP-2 positive chondrocytes in the second-time surgery CLP group (U = 16.0; p = 0.044). When comparing the first-time surgery CLP group to the second-time surgery CLP group—a substantial increase in the number of TIMP-2 positive chondrocytes was seen (U = 53.5; p = 0.021). Between the first-time surgery CLP group and the control group, there was no statistically significant difference (U = 162.5; p = 0.523).
3.5. IL-1α
IL-1α positive chondrocytes were found in every cartilage tissue sample. The number of IL-1α positive chondrocytes in all the groups varied from ++ to ++++ (see
Table 3).
The medial value of the IL-1α positive cells differed in all groups—+++/++++ (SD = 0.68) in the first-time surgery CLP group, +++ (SD = 0.63) in the second-time surgery CLP group and ++/+++ (SD = 0.19) in the control group (see
Table 3) (see
Figure 2A,A1).
No differences were found between the first-time surgery CLP group and the second-time surgery CLP group (U = 97.5; p = 0.407), the first-time surgery CLP group and the control group (U = 165.5; p = 0.476), and between the second-time surgery CLP group and the control group (U = 23.5; p = 0.179).
3.6. IL-10
Cells that were IL-10 positive were found in all cartilage samples, and the number of IL-10 positive chondrocytes in the first-time surgery CLP group was ++ to ++++, in the second-time surgery CLP group it was ++/+++ to +++/++++, and in the control group—from +/++ to +++ (see
Table 3) (see
Figure 2B,B1).
The medial value of IL-10 positive cells was ++/+++ (SD = 0.62) in the first-time surgery CLP group and +++ (SD = 0.35) in the second-time surgery CLP group, in comparison to ++ (SD = 0.16) in the control group (see
Table 3).
Between the first-time surgery CLP group and the control group, a statistically significant difference was discovered (U = 82.0; p = 0.004), as well as between the second-time surgery CLP group and the control group (U = 7.0; p = 0.003), where the count of IL-10 positive cells was higher in the CLP groups in comparison to the control group. However, no statistically significant difference between the first-time surgery and the second-time surgery CLP groups was found (U = 87.5; p = 0.243).
3.7. bFGF
All cartilage tissue samples from the CLP groups and the control group had bFGF positive cells. The number of bFGF positive chondrocytes in the first-time surgery CLP group was the most variable—from + to ++++, in the second-time surgery CLP group it varied from +++ to +++/++++, while in the control group it ranged from +/++ to +++ (see
Table 3) (see
Figure 2C,C1).
The medial value of the first-time surgery CLP group’s positive chondrocytes was +++ (SD = 0.87), in the second-time surgery CLP group—+++/++++ (SD = 0.27), compared to ++ (SD = 0.60) in the control group (see
Table 3).
The first-time surgery CLP group (U = 80.5; p = 0.005) and the second-time surgery CLP group (U = 4.500; p = 0.001) had a considerably larger number of chondrocytes that were bFGF positive when compared to the control group. The number of chondrocytes that were positive for bFGF in the first-time surgery CLP group and the second-time surgery CLP group did not significantly differ statistically (U =1 01.5; p = 0.626).
3.8. TGFβ1
All cartilage tissue samples from the CLP groups and the control group contained TGFβ1 positive cells. In the CLP groups, the quantity of TGFβ-1 positive chondrocytes ranged from 0/+ to +++, whereas in the control group, it ranged from +/++ to +++/++++ (see
Table 3) (see
Figure 2D,D1).
The medial value of TGFβ1 positive chondrocytes in the first-time surgery CLP group was +++ (SD = 0.88), while in the second-time surgery CLP group it was +++/++++ (SD = 0.64), and in the control group there was a ++/+++ (SD = 0.66) number of TGFβ1 positive chondrocytes (see
Table 3).
A higher amount of TGFβ1 positive chondrocytes was observed in the second-time surgery CLP group compared to the control group (U = 14.0; p = 0.027). There was no difference observed between the first-time surgery CLP group and the control group (U = 163.5; p = 0.461) and between the first-time surgery CLP group and the second-time surgery CLP group (U = 69.0; p = 0.073).
3.9. Statistical Data
Statistically significant (
p < 0.05) strong (
rs = 0.6–0.79) correlations were found between IL-1α and IL-10 (
rs = 0.698;
p = 0.000); IL-1α and MMP-8 (
rs = 0.604;
p = 0.000) in the first-time surgery CLP group (see
Table 4).
Statistically significant (
p < 0.05) strong (
rs = 0.6–0.79) correlation was found between IL-1α and IL-10 (
rs = 0.877;
p = 0.010) in the second-time surgery CLP group (see
Table 5).
Statistically significant (
p < 0.05) strong (
rs = 0.6–0.79) correlations were found between MMP-2 and TIMP-2 (
rs = 0.867;
p = 0.001); MMP-2 and MMP-8 (
rs = 0.743;
p = 0.009); MMP-9 and IL-1α (
rs = 0.850;
p = 0.001) in the control group (see
Table 6).