The Effect of Primary Suture In Cleft Lip On Healing of the Surgical Wound And the Role of Matrix Metalloproteinases

INTRODUCTION

Non-syndromic orofacial clefts are among the most common congenital disorders. Annual incidence in newborns with facial clefts in the Czech Republic is relatively stable and fluctuates around a long-term average of 1.8 per 1000 labours (1, 3). With the development of surgical procedures, partial successes in the results of this surgical procedure and the development of anaesthesia, the timing of surgical intervention is also gradually changing. According to information from various cleft centres, there are still discussions whether to perform surgery of the lip early after birth (i.e. within 8 days) or according to the usual protocol – i.e. when the child is around 3 months of age. Healing of surgical wound in early lip suture after birth is faster and is characterised by less significant scars than those from operations performed when the child is older (2, 3).

Normal wound healing, after plastic surgery for example, is the process which results in the production of a scar. Its size depends not only on the age of the patient but also on his or her individual predisposition. In extreme cases a hypertrophic or even keloid scar can develop. The whole process of wound healing in bigger children and adults is relatively well known and described (4, 5).

Wound healing in the foetal period takes place in a different way; healed foetal tissue has a normal dermal architecture. The delayed inflammatory cell response is significantly suppressed in the healing process. Foetal tissue of the skin is different from adults in the composition of extracellular matrix (ECM) (9, 10, 11).

The matrix metalloproteinases (MMP) play an important role in wound healing. They are contained in extracellular matrix and have a function in all phases of healing and remodelation of extracellular matrix (5). MMP is a group of zinc-dependant enzymes which are structurally similar. MMP split various components of ECM and participate in various physiological processes, where degradation and synthesis of ECM takes place (5). Their presence and expression changes in the course of gestation (9). MMP also play a role in various inflammatory processes, during tumour growth and other pathological processes. MMPs are regulated in the organism by various mechanisms. Tissue inhibitors of matrix metalloproteinases (TIMP) are among the important regulators. Scarless wounds have an increased ratio of MMP/TIMP – there is more favourable remodelation and lower accumulation of collagen taking place. Increased concentrations of MMP and TIMP have also been observed in association with rupture of cell membranes (6).

MMP could also play a role in the aetiology of clefts (7). Palatogenesis in mammals is a complex process that includes regulated interactions between epithelial and mesenchymal cells of the palate, which result in remodelation of extracellular matrix and subsequent fusion of palate arches (7).

Brown et al. report an experiment in mice where increased level of MMP-3 during palatogenesis resulted in successful joining of palate arches compared with mice with lower levels of MMP-3 (7).

There have not yet been sufficient studies to determine the extent to which some mechanisms of scarless healing also persist in the postnatal period and the extent to which it would be possible to activate these as a part of a certain therapeutic process.

We decided to analyze the components of ECM in the tissue samples harvested from the edges of the cleft, which are removed during the primary reconstruction of the lip as redundant. The goal was to determine concentration of MMP-1, MMP-2 and MMP-3.

HYPOTHESIS

The aim of this study was to verify the hypothesis that the level of MMP in patients with facial clefts who undergo earlier operations is higher than that in patients who are operated upon at the age of about 3 months, and therefore the healing of the wound is favourably affected (13).

MATERIAL AND METHODS

In the group of 220 early operated patients who underwent surgery by the 8^th day after birth, samples of tissues have been collected from 17 patients, who have been studied (10 boys, 7 girls). The control group contained 13 patients (6 boys and 7 girls) who underwent surgery between the 2^nd and 4^th month of age, i.e. with a greater time delay since birth (Table 1, the spectrum of cleft disorders in studied patients).

The surgical procedure was uniform; one surgeon performed all operations. Primary lip suture was performed using the modified method according to Tennison (2).

Harvested tissue was stored by freezing at -70 ľC. After thawing the tissue sample was weighed in the first step of the analysis and inserted for extraction to cacodylate buffer, where it was disintegrated with scissors. There was four times the weight of moist tissue of cacodylate buffer used for extraction. Extraction took place over 24 hours at a temperature of 2–8°C. After extraction supernatant was separated with centrifugation (30 minutes, 13000 G) and used for further analyses.

Concentration of MMP-1 and MMP-3 were determined with the ELISA method, using the Amersham Biotrak Activity Assay kits (GE Healthcare UK). Due to the absence of published data about concentration of MMP in the tissues of child clefts in the early gestation period, the concentration of total MMP-1 was determined with the ELISA method (range from 0.1–50 ng/ml with sensitivity 0.1 ng/ml, measured at 405 nm). Concentrations of MMP-3 were determined using the ELISA method (range 0.25–32 ng/ml with sensitivity 0.1 ng/ml, measured at 405 nm) and concentrations of MMP-2 using the ELISA method (range 0.19–12 ng/ml, sensitivity 0.19 ng/ml, measured at 405 nm). Measured concentrations of MMP-1 and MMP-3 were subsequently related to the concentration of total protein in the sample. To determine total protein in the tissues the Lowry method was chosen (extent 0.01–1 mg/l, 750 nm). The method was validated on concentration levels of 0.40 mg/l (CV₁ = 13.43%) and 0.85 mg/l (CV₂ = 18.25%).

RESULTS

Healing of surgical wound in the early primary suture of the lip took place without complications and took 3–4 days depending on the severity of the defect. In the second post-operation day the children were breast-fed or fed using a dummy. We have not encountered any perioperative or postoperative complication. Discharge took place between the 3^rd to 5^th day after the operation. Six months after the operation the scars on the lip were virtually stable and only minimally noticeable. In children operated between the 2^nd to 4^th month of age healing was also accomplished without any complications and took 5–7 days depending on the severity of the defect. Even in this case we have not encountered any perioperative or postoperative complication. In this group too the children were fed orally from the second postoperative day. Between the 6^th and 8^th month the scars were stable and had a favourable appearance. All patients remain in the care of a multidisciplinary team of the department, and their progress will be followed and documented.

Concentrations of total MMP-1 in children who underwent surgery during the first week after birth were 0.017 ± 0.023 μg/g of proteins (mean ± SD); in children who underwent surgery between the 2^nd and 4^th month the figures were 0.028 ± 0.026 μg/g of protein. Concentrations of total MMP-3 in children who underwent surgery during the first week after birth were 0.200 ± 0.142 μg/g of proteins; in children that underwent surgery between the 2^nd and 4^th month we established 0.155 ± 0.093 μg/g of proteins. There was no statisticaly significant difference at the significant level p<0.05 demonstrated between the concentrations of the matrix metalloproteinases in both groups.

Concentrations of MMP-2 were higher in all measured samples than the upper limit of the working range of the diagnostic kit; i.e. more than 12 ng/ml.

DISCUSSION

During the study of this first small group there were no statistically significant differences observed in the quantity of total protein and concentration of MMP between both groups, considered according to the timing of the surgery. Nevertheless, we think the benefit of the study is based on determination of the concentrations of total MMPs, which are expected to have an effect on wound healing with a minimal scar. After the determination of total MMP concentrations it will be necessary to extend the file of samples and extend the spectrum of examined MMP and measure concentration of active MMP.

During the operation only very small tissue particles were excised and removed. Removed pieces of tissues were variable: they contained skin, subcutaneous tissue, fat and mucosa. Therefore it was necessary to perform separation prior to analysis. The highest inaccuracy was caused by the presence of fatty tissue. Presence of adrenaline solution with water increased the inaccuracy during weighing and calculation of the quantity of MMP per moist tissue. Therefore all MMPs were finally related to the total protein. In the additional file, which will be studied, the technique of collection will be modified, to minimize these negative effects.

Foetal extracellular matrix is different from that of adults in the composition of collagen, hyaluronic acid and quantity of proteoglycans. Collagen type I is a basic component of ECM in a foetus and in adults (9). The quantity of collagen type III in ECM gradually decreases during the development of the foetus, as does the quantity of hyaluronic acid, which facilitates the movement of cells. The proteoglycans in ECM modulate the synthesis of collagen, maturation and degradation. Decorin and TGF-beta factor influence fibrillogenesis. MMP and TIMP contribute to the degradation of collagen (11).

Experimental studies in mice with a cleft have shown that TGF-beta reduces scaring after operation of cleft lip and has an effect on the expression of MMP-9 and its activity, while also causing expression of MMP-13 in palate fibroblasts (7, 8). In mice with a deficit of TGF-beta, TIMP-2 is missing, and a significantly smaller quantity of MMP-13 has also been observed (7). If we administer TGF-beta in an experiment, significant reduction of scar production will follow. Increased level of TGF-beta-3 reduces the accumulation of collagen I and also restricts differentiation of myofibroblasts, which synthesize it and thereby restrict production of scars (12).

CONCLUSION

There is a marked difference between the average levels of MMP-1 and MMP-3 in both groups, but no statistically significant differences were demonstrated in concentrations of MMP between the two groups related to the early timing of surgery or the quantity of total protein. Research will continue, involving a larger number of patients in both groups, and concentrations and activities of other MMPs will be determined. It may also be presumed that the changed technique of tissue harvest could affect the concentration of MMPs.

The study was prepared with the support of IGA 10012-4/2008 grant of the Ministry of Health of the Czech Republic.

Address for correspondence:

Jiří Borský, M.D.

Department of Plastic Surgery

3^rd Faculty of Medicine, Charles University Prague

and University Hospital Královské Vinohrady

Šrobárova 50

100 34 Prague 10

Czech Republic

E-mail: borsky.jiri@gmail.com