Use of platelet‐rich plasma to facilitate wound healing

Abstract

Platelet‐rich plasma (PRP) is widely used nowadays in different fields of medicine, affecting physiological processes including tissue regeneration. The use of PRP in maxillofacial surgical interventions and its efficiency in the improvement of postoperative wound healing were analysed. Patients undergoing plastic and reconstructive surgeries in the maxillofacial region were recruited: 50 patients were enrolled into a control group (received no PPRP injection) and 50 patients were enrolled into a treatment group, where PRP was applied during the surgical procedure. Evaluation of treatment outcomes was carried out by determination of IL‐1β, TNFα, and IL‐6 cytokines levels in the wound‐drain fluid. The stages of wound healing were assessed by cytological analyses and ultrasound within a month period. The use of the PRP has substantially positive effects, contributing to the improvement of the healing process. In the treatment group, fibroblasts, macrophages, and collagen fibres appeared and their quantities increased earlier than when compared with control group patients. The concentration of IL‐1β and TNFα in wound fluid on day 1 and day 5 after operation was higher for the treatment group as opposed to the control group, which was linked to the influence of PRP on inflammatory and granulation phases of the healing process. An ultrasound examination showed less oedema and infiltration in the tissues around the wound of the treatment group.

1. INTRODUCTION

Platelet‐rich plasma (PRP) is an autologous platelet concentrate rich in biologically active substances, inducing immune cell reactions, which suppresses inflammation and accelerates tissue reparation and wound healing.1 PRP is extracted from patients' own blood for treatment purposes. It contains platelet releases enriched with various cytokines, growth factors, chemokines, and fibrin.2, 3 Platelet growth factors play a key role in the healing process and tissue formation.4

One of the crucial issues in surgery is the development of effective methods of improving healing of postoperative wounds, especially of patients with high risk of complications in the early postoperative period and long‐running processes of regeneration. Existing meta‐analyses show that PRP has effectively proven itself in the treatment of wounds of various aetiologies (acute, chronic‐vascular and neurotrophic, and postburn).3, 4, 5 The PRP is a convenient and safe method, accelerating regenerative processes that activate all parts of the natural regeneration processes.6, 7

Disregarding the fact that there are plenty of positive clinical studies that reaffirm the positive effect of PRP on healing processes, there are a number of studies where the authors have concluded that there are no substantial differences between two groups under analysis and indicated limited efficiency of plasma8 or no differences in the speed of wound healing in compared groups,9, 10 which points to the fact that more in‐depth research is required in this area. According to various studies under review, there are a number of inconclusive results of the use of PRP in maxillofacial surgical interventions, especially in bone surgeries.11, 12 Nevertheless, the effectiveness of the use of PRP in the case of surgical interventions (plastic and reconstructive surgeries of the facial and neck soft tissues) of patients with delayed regeneration was not observed.

For many patients, a wound that heals slowly is a serious issue and concern. For patients with comorbidities, having non‐healing or poorly healing wounds, cell proliferation is reduced. In these cases, keratinocytes do not proliferate and migrate properly, which destabilise wound healing by inhibiting epidermalisation.13 There are a number of studies showing that comorbidities (endocrine diseases, diseases of the cardiovascular system), repeatedly performed operations, and damaging lifestyle habits impair wound healing, and could lead to complications in the early operative period, such as suppuration of the wound, divergence of sutures, and development of infection.14, 15 Development of wound infection reduces fibroblast migration and proliferation, which leads to a downregulation of collagen synthesis and a delayed healing process. The results of treatment after the development of complications are not satisfactory. The aesthetic appearance of scars is noticeably different from surrounding soft tissues and causes dissatisfaction of patients.

In the current study, patients with predicted high risk of complications and delayed healing were enrolled. We have conducted studies to verify the influence of various risk factors on the development of complications in the early postoperative period at the City Hospital N5. 211 cases of patients who had undergone plastic and reconstructive operations were analysed to deduce the logistic regression equation that could be used to predict the probability of complications and delayed wound healing in the early postoperative period.

2. MATERIALS AND METHODS

Patients (n = 100) from the department of maxillofacial surgery of the City Hospital N5 (Almaty, Kazakhstan) aged 18 to 60 years undergoing plastic and reconstructive surgeries on soft tissues in the maxillofacial area were enrolled. Study design—a randomised controlled clinical trial. Patients were randomised into two groups intraoperatively based on the use of PRP during surgical procedures. The inclusion criteria were for patients undergoing plastic and reconstructive operation in the maxillofacial area between June 1, 2017 and June 1, 2018. Exclusion criteria were used for patients with platelet dysfunction syndrome, haemodynamic instability, local infection at the site of the procedure, systemic use of coriticosteroids within 2 weeks, recent fever, and cancer. 50 patients, 26 males and 24 females aged 43 ± 6 (21‐60), were included in the treatment group. For these patients in addition to the traditional set of therapeutic measures, an injection of autologous PRP during the surgical procedure was used to optimise the wound‐healing process. Other 50 patients, 27 females and 23 males aged 41 ± 5 (19‐60) years, formed the control group. Patients in the control group underwent a traditional set of treatment methods, which consisted of surgical measures and subsequent conservative treatment.

All patients had been predicted to have a high risk of complications in the early postoperative period and delayed healing based on the logistic regression equation (p = $\frac{1}{1+e^{-d}}$) that was obtained earlier in the retrospective study. In the logistic regression equation, d is the value of the discriminant function, which is equal to the sum of variables. The variables in the equation are given in Table 1, where B—coefficient for the constant, SE—standard error, Wald—Wald chi‐square test, DF—degrees of freedom for the Wald chi‐square test, Exp (B)—exponentiation of the B coefficient (odds ratio), and CI—confidence interval.

Table 1.

The variables in equation

	B	SE	Wald	DF	Value	Exp (B)	95% CI exp (B)
							Lower	Upper
D—diabetes/cardiac ischaemia/arterial hypertension	1.8	0.6	9.9	1.0	0.0	6.3	2.0	19.7
S—size of incision	0.4	0.1	20.8	1.0	0.0	1.5	1.3	1.8
N—number of previous operations	0.0	0.0	32.1	1.0	0.0	1.0	1.0	1.0
C—constant	0.4	1.0	0.2	1.0	0.7	1.5	0	0

Evaluation of treatment outcomes was carried out by showing the concentration of proinflammatory cytokines (IL‐1β, TNFα, and IL‐6) in the wound‐drain fluid within 2 hours and then on the 1st, 3rd, and 5th days after the operation with sandwich enzyme‐linked immunosorbent assay (ELISA); conducting cytological analyses on the 1st, 3rd, 5th, 7th, 10th, 21st, 24th, and 30th days and ultrasound examination, including color Doppler ultrasound, within 1 month on Aloka A6 (Japan, Tokyo). Swabs from the contents of postoperative wounds of the soft tissues in the maxillofacial region were taken with a sterile metal spatula, transferred to adhesive slides. The prepared swabs were dried, fixed in alcohol‐acetone (1:1) for 5 minutes, and stained with methylene blue by May‐Grunwald (15 minutes) and azur‐eosin by Romanovsky‐Giemsa (30 minutes). The morphometric system of Leica company (a DM 1000 microscope and a DFC‐320 digital camera) was used for photographing the results. Images of cells were obtained with the use of the aforementioned complex. For evaluation of cytokines, sterile disks with a diameter of 5.0 mm, made of chromatographic paper, were placed on the postoperative wound surface (Figure 1). Having been imbued with liquid contents, they were air dried at room temperatures, placed in plastic sterile test tubes, diluted in 1 cc of normal saline, and stored at −18°C. The multiplicity of dilution of the eluted material was taken into account in the calculation of the concentration of proinflammatory cytokines.

Figure 1.

The method of sampling of wound‐drain fluid with chromatographic paper

The results of cytological analyses and measurement of cytokines were supported by the ultrasound. Examination was performed with the use of Aloka A6 (Japan) equipped with a 5 to 8 MHz‐wide linear sensor. Ultrasound scans were made on the 5th, 10th, 14th, 21st, and 30th days in both groups. Doppler color ultrasound was also used to measure the flow diameter and velocities—peak systolic velocity (PSV), end diastolic velocity (EDV) of the facial artery and its branches (labial, angular), temporal and occipital arteries, and resistive index (RI) in postoperative wounds on day 10.

2.1. Protocol for obtaining PRP

Vacuum tubes with sodium heparin gel were used for venous blood sampling (9‐27 mL). On average, 1 tube of 9 mL was required for a wound not exceeding 10 cm, 2 tubes for 10 to 20 cm wounds, and 3 tubes for large wounds (>20 cm). Tubes filled with venous blood were centrifuged for 5 minutes at a speed of 3000 rotations per minute. Thereafter, two fractions of blood samples were visible in the tubes: erythrocyte‐leukocyte clot and plasma enhanced with platelets in the lower third, corresponding to 600 000, in the middle—200 000, and at the top—50 000 per 1 μL. A syringe was used to take the lower third of the resulting plasma above the separation layer, which was subsequently injected intradermally after suturing the wound, leaving 0.5 cm from the edge of the wound. Injections of 0.1 to 0.2 mL of the autologous plasma were performed with a 30G needle syringe. Distance between injections was 1.5 to 2 cm. The remaining plasma was applied to the sterile gauze and put over the postoperative wound.

2.2. Statistical analysis

Statistical analysis was made using the SPSS software package (IBM Corp., Released 2012, IBM SPSS Statistics for Windows, Version 21.0, Armonk, NY). Distribution of all parameters was tested by the Kolmogorov–Smirnov method. Variables and outcomes between two groups were compared by the Mann–Whitney U test as the resultant distribution of parameters in two groups was not normal. Statistical data were presented as Median (Minimum ‐ Maximum) and frequencies with percentages. The difference of parameters with a P value < 0.05 was set as statistically significant.

2.3. Ethical considerations

All participating patients signed informed consent forms to be eligible for research. Ethics approval was obtained from Local Ethics Commission at S.D. Asfendiyarov Kazakh National Medical University (protocol of approval N5 [56], May 31, 2017, Almaty, Kazakhstan). The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki.

3. RESULTS

3.1. Part I

3.1.1. Cytological analyses and determination of proinflammatory cytokines IL‐1β, TNFα, and IL‐6

Epithelial cells of different stages of variations were detected on the swabs, including those that were dystrophically altered, with neutrophil invasion and contaminated with microorganisms. In addition, mononuclear cells with cytoplasm, holonuclear mononuclear cells, segmented neutrophils and lymphocytes, erythrocytes, and collagen fibres, as well as coccal microflora were detected. Six stages of epithelial cell differentiation for the convenience of counting were identified.

For both groups, a large amount of neutrophils was found on the 3rd day. The difference in the amount of cells was not significant, although quantity of neutrophils was higher for the treatment group (Figure 2). In this period of time after operation, the cytograms were dominated by segmented neutrophils, erythrocytes. Lymphocytes and mononuclears were also detected.

Figure 2.

Swab of the wound‐drain fluid on day 3. Coloring on May‐Grunwald and Romanovsky‐Giemsa. Magnification ×630. (A) Control group, (B) treatment group

The significant difference was found on day 5. Fibrin fibres, microflora, represented mainly by cocci were found in the control group, such as macrophages, destroyed mononuclear cells, and neutrophils phagocyting microorganisms. Separate fibroblasts appeared in the wound‐drain fluid of treatment group patients in addition to emergence of fibroblast swabs presented by macrophages and mostly destroyed neutrophils (Figure 3).

Figure 3.

Swabs of the wound‐drain fluid on day 5. Coloring on May‐Grunwald and Romanovsky‐Giemsa. Magnification ×630. (A) Control group, (B) treatment group

A large number of segmented neutrophils, mostly destroyed in the state of karyorexis, karyopicosis and cytolysis, and macrophages and lymphocytes were detected on the 7th day in swabs from wounds of the control group. There was a coccal microflora as well. The number of macrophages and fibroblasts increased in the treatment group in comparison with the 5‐day period. Macrophages, neutrophils, and phagocyting microorganisms were identified. Segmented neutrophils and erythrocytes also were detected (Figure 4). The increase of fibroblasts in the cytogram of the treatment group indicates the onset of the next granulation phase of wound healing. Occasionally, separate collagen fibres were found.

Figure 4.

Swabs of the wound surface on day 7. Coloring on May‐Grunwald and Romanovsky‐Giemsa. Magnification ×400. (A) Control group, (B) treatment group

On days 14 to 21, segmental neutrophils and macrophages were detected in small amounts in the swabs taken from the wounds of the control group. Fibroblasts predominated among the cellular elements. The amount of collagen fibres was increasing. Separate epithelial cells started to appear. In the treatment group, the number of fibroblasts and collagen fibres was much greater than for the patients of the control group. The number of epithelial cells has also increased (Figure 5).

Figure 5.

Swabs of the wound surface on day 21. Coloring on May‐Grunwald and Romanovsky‐Giemsa. Magnification ×200. (A) Control group, (B) treatment group

On the 30th day, collagen fibre clumps, fibroblasts, and individual epithelial cells located in clots were found in the control group. In addition, rare segments of neutrophils and macrophages were found. Epithelial cells were detected in large numbers in swabs of the treatment group. The pictures of swabs were similar to a normal skin, epithelial cells with a pycnotic nucleus and non‐nuclear cells predominated in the cytograms (Figure 6).

Figure 6.

Swabs of the wound surface on day 30. Coloring on May‐Grunwald and Romanovsky‐Giemsa. Magnification ×200. (A) Control group, (B) treatment group

According to the cytology in the treatment group, the epithelialisation occurred in 11 (22%) cases within 7 to 10 days, in 27 (54%) cases within 10 to 14 days, in 9 (18%) cases within 14 to 16 days, and in 3 (6%) cases within 16 to 21 days. On the other side of the analysis was the control group where in 2 (4%) cases wound epithelialisation ended on 7 to 10 days, in 7 (14%) cases on 10 to 14 days, in 32 cases (64%) within 14 to 16 days, in 5 (10%) cases on days 16 to 21, and in 3 (6%) cases on 21 to 30 days.

Wound exudates have been shown to contain proinflammatory cytokines, such as interleukin‐1β (IL‐β), interleukin‐6 (IL‐6), and tumor necrosis factor alpha (TNF‐α) (Table 2). The concentration of IL‐1β, TNFα, and IL‐6 in wound fluid in the treatment group after 2 hours and on the 1st day after the operation was higher than in the control group, which confirms the stimulation of the inflammatory phase of wound healing by PRP. On the 3rd day, there was no significant difference between concentrations of IL‐1β, TNFα cytokines in the treatment and control groups, respectively, but the level of IL‐6 was three times higher in the control group. The level of cytokines became lower in dynamics for both groups. On the 5th day, concentration of IL‐1β and TNFα in the treatment group increased again, while the level of the same cytokines was lower in the control group. It was objectively accompanied by visible healing of wounds and correlated with the appearance of granulation tissues in the treatment group according to the results of the cytology. This fact indicates that the activation and proliferation of fibroblasts has begun, not the lengthening of the inflammatory phase of healing. An increase of concentration of IL‐6 in the control group was observed on day 5, which correlated with the presence of large amounts of neutrophils and represented the lengthening of the inflammatory phase.

Table 2.

The concentration of cytokines in wound‐drain fluid (in pg/mL)

	Group	Days
		In 2 h	1st day	3rd day	5th day
IL‐1β	Treatment	66.5 (32.1‐234.7)	35.7 (35.1‐105.2)	8.1* (3.5‐12.1)	34.9 (0.3‐36.2)
	Control	50.1 (17.5‐201.5)	22.6 (18.9‐89.3)	6.8* (4.4‐19.1)	22.3 (6.7‐27.6)
TNF‐α	Treatment	89.3 (24.5‐129.1)	102.3 (1.2‐201.1)	27.4* (2.78‐34.5)	38.3 (17.4‐101.2)
	Control	76.2 (17.4‐100.7)	99.8 (3.4‐116)	29.2* (2.7‐99.8)	30.1 (2.7‐99.8)
IL‐6	Treatment	81 (10.3‐379.7)	72.4 (20.2‐110)	26.8 (3.5‐156.7)	14 (1.1‐197)
	Control	98.1 (11.2‐451.5)	101.7 (26.3‐189.7)	88.3 (11.2‐189.1)	92 (2.57‐475.6)

3.2. Part II

3.2.1. Ultrasound examination

Wounds that were scanned on days 5, 10, 14, 21, and 30 were compared with the results of cytological measurements. A comparison demonstrates the correlation between two techniques (r = 0.9, P < 0.05). Emergence of granulation tissue and collagen in wounds, as well as the epithelisation were identified by both techniques. Ultrasound scans of wounds in the first few days after operations has shown echo‐poor picture that represented early granulation tissue. According to the cytological results, the formation of granulation tissue occurs within 3 to 5 days and consists of macrophages and fibroblasts. As the wound fills with granulation tissues, further deposition of fibroblasts that are responsible for synthesis of collagen in the wound occurs. Thus, changes occurring after day 5 in the wound could be clearly seen by ultrasound when fibroblasts have synthesised a fibrous extracellular matrix. The difference between ultrasound scans performed in two groups is shown in Table 3. Oedema and infiltration were still pronounced in 44% of cases in the control group as opposed to 18% in the treatment group up until the 10th day. It should also be noted that on the 10th and 14th days, lymphostasis was more frequent in the control group—16% cases versus 6% in the treatment group respectively. On day 21, 14% of the control group retained oedema, while in the treatment group only 4% of patients had oedema. Figure 7 presents the ultrasound picture of a patient of the treatment group on day 21 where oedema and infiltration were less pronounced. The echonegative structure is 7 mm in length and 3 mm in depth on the scan as the corresponding infiltration is determined. To compare, Figure 8 represents cases of patients of the control group on day 21 with pronounced oedema, infiltration of 11 mm in length and 6 mm in depth, and the presence of the lymphostasis.

Table 3.

Ultrasound scans at different stages of wound healing

	Control group	Treatment group
Granulation phase	Collagen accumulation starts to be visible only on the 10th to 14th day	Ultrasound scans of wounds in the initial days after operations show echo‐poor picture that identifies early granulation tissue on the 5th day. Visible collagen accumulation starts from the 10th day. Increase in echogenicity appears on 14th, 21st days, respectively.
Epithelisation	A line of echogenic tissue was identified in regularity on the 21 to 24th days at the surface of the tissue that confirms reepithelisation.	A line of echogenic tissue was identified on the 10th to 14th day at the surface of the tissue that confirms reepithelisation. Starting from the 21st and regularly on the 30th day, there was almost no significant difference.
Oedema	Pronounced	Not pronounced
Infiltration	Pronounced	Not pronounced
Lympostasis	Pronounced	Not pronounced

Figure 7.

Ultrasound scan of soft tissues of maxillofacial area on day 21 of patient of the treatment group

Figure 8.

Ultrasound scan of soft tissues of maxillofacial area on day 21 of patient of the control group

Depending on the type and place of operational interventions of 56 patients (28 of control, 28 of treatment group), indicators of superficial temporal artery, posterior auricular artery, and occipital artery were measured, in 82 (41 of control, 41 of treatment group) of facial artery, 24 (12 of control, 12 of treatment group) of labial artery. The areas of the maxillofacial region served by different vessels are shown in Figure 9, where A—facial artery and vein, B—labial arteries and veins, C—superficial temporal artery and vein, D—posterior auricular artery and vein, and E—occipital artery and vein. Figure 10 represents ultrasound pictures with indicators of blood flow such as PSV, EDV, and RI of the facial artery on day 10 in the treatment group (A) and control group (B). In the treatment group, these indicators were equal to the following values PSV—15.0 cm/s, EDV—6.5 cm/s, RI—0.5 versus PSV—25.9 cm/s, EDV—6.7 cm/s, and RI—0.7 in the control group. Increase of the PSV verifies that the vessel diameter does not correspond to the normal blood flow, which can be explained by persistent oedema and tissue infiltration in the control group. Figure 11 represents ultrasound pictures with the same indicators of facial vein. In the treatment group, indicators were as follows: PSV—10.1 cm/s, EDV—6.2 cm/s, and RI—0.385 and in the control group PSV—17.1 cm/s, EDV—8.6 cm/s, and RI—0,5. The difference is noticeable in terms of PSV and EDV parameters, which corresponds to the change of normal blood flow. The Doppler ultrasound in the assessment of blood flow in different vessels supplying regions of the maxillofacial area of the control group shows that the PSV has increased and RI slightly decreased as opposed to the results of the treatment group (see Table 4). It was observed that the neovascularisation appeared earlier for the treatment group in contrast to the control group. It was clearly visible on day 10 in direct contrast to day 14 for the control group.

Figure 9.

Areas of face served by different vessels (Frank H. Netter, Interactive Atlas of Human Anatomy, 2003 Icon Learning Systems LLC)

Figure 10.

Color Doppler ultrasound of blood flow in facial artery (A) Control group, (B) treatment group

Figure 11.

Color Doppler ultrasound of blood flow in facial vein (A) Control group, (B) treatment group

Table 4.

Color Doppler ultrasound results at the day 10 after operation

Group	Flow diameter (mm)	PSV (cm/s)	EDV	RI
Superficial temporal artery
Treatment group	1.8 (1.7‐1.9)	17.7 (16.5‐24.6)	13.7 (12.8‐14.3)	0.72 (0.69‐0.77)
Control group	2.0 (1.8‐2.2)	18.9 (17.8‐25.1)	11.3 (10.9‐12.8)	0.68 (0.64‐0.73)
Facial artery
Treatment group	2.1 (1.6‐2.7)	18.3 (17.9‐20.1)	16.2 (15.4‐16.9)	0.8 (0.7‐0.89)
Control group	1.9 (1.8‐2.0)	21.84 (19.3‐22.4)	12.8 (12.6‐13.2)	0.7 (0.67‐0.81)
Labial artery
Treatment group	1.2 (0.8‐1.6)	12.8 (8.7‐17.2)	6.8 (6.1‐7.2)	0.75 (0.72‐0.78)
Control group	1.6 (0.9‐1.9)	13.1 (8.9‐17.4)	5.6 (5.4‐6.2)	0.74 (0.71‐0.77)
Posterior auricular artery
Treatment group	1.1 (0.9‐1.4)	19.8 (19.1‐19.9)	13.1 (12.8‐13.9)	0.76 (0.73‐0.77)
Control group	1.0 (0.8‐1.6)	24.3 (19.9‐25.1)	10.3 (10.1‐12.5)	0.72 (0.7‐0.76)
Occipital artery
Treatment group	1.4 (1.1‐1.6)	28.3 (27.8‐29.1)	24.2 (23.8‐24.9)	0.7 (0.6‐0.72)
Control group	1.3 (1.1‐1.5)	32.4 (30.1‐33.2)	20.1 (19.9‐22.1)	0.6 (0.5‐0.69)
Vascularisation in the wound
Treatment group	Small vessels are visible in 45 cases
Control group	Small vessels are visible in 33 cases

EDV, end diastolic velocity; PSV, peak systolic velocity; RI, resistive index.

4. DISCUSSION

In the current study, the injection of PRP resulted in a significant improvement of wound healing after plastic and reconstructive interventions in the maxillofacial area. It has been shown that the use of autologous PRP has a considerably positive effect on postoperative wound healing of patients with concomitant diseases and in cases with delayed healing that was predicted before surgical interventions. None of the studies in the current literature evaluated the cytological analyses, ultrasound examination, and determination of cytokine concentration simultaneously within one study.

There are a number of studies showing that the use of autologous PRP can improve the results of wound‐healing processes, as well as in cases of poorly healing wounds. Several controlled studies have given statistically significant evidence that the use of PRP leads to the acceleration of wound healing.16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 There are three controlled studies on humans that reported of improvement of wound healing with regard to healing time, the quality of life, and reduction of pain.17, 18, 25 According to a prospective controlled study of Hom et al, epithelialisation and granulation formation appeared 3 days earlier in the experimental group where PRP was used,16 while it was established in our study that granulation formation also started 3 days earlier and epithelialisation 4 to 6 days earlier in the treatment group. Spyridakis et al in a trial of 52 patients asserts that complete healing occurs within 24 days as opposed to more than 30 days in the control group,17 which was close to our results. Time period characterising wound healing according to cytology in our study was shorter than in the study of Kazakos et al, which showed significant disparities in the wound healing time in a controlled study of 59 patients: 21.26 days in the experimental group with PRP in contrast to 40.6 days in the control group.18

In the following experimental studies on animals, the acceleration of wound healing after use of PRP with the help of histological techniques of measurement was shown.25, 26, 27, 28 These experimental works demonstrated the considerable impact of PRP on epithelialisation of wounds. In accordance with previous articles, positive results of the PRP application on the overall improvement of wound healing on humans were found. Dionyssiou et al supported the findings in accelerating the healing by PRP with the results of the histological study; according to which, the use of PRP as in our study contributed to the early appearance of granulation tissues, acceleration of epithelialisation, and stimulation of angiogenesis processes in wounds.29 PRP has proven itself in Kim et al and Carter et al studies showing the acceleration, proliferation, and migration of keratinocytes, and a significant reduction in the surface and depth of the wounds.13, 24

Wound cytokine concentrations have been previously assessed in a number of studies. Increase of proinflammatory cytokines, especially IL‐1β and TNF‐α in acute wounds, was identified in some of them.27, 28 The levels of IL‐1β, TNF‐α, and IL‐6 increase after operation and can be detected in wound fluid.30, 31, 32, 33 IL‐1β is not detected on an intact skin, but a significant expression of this cytokine occurs within 30 to 90 minutes on damaged tissues.34 Confirmation of our findings regarding the increase in cytokines levels after injury was found in the review of Pastar et al where the fact of release of IL‐1 and TNFα by keratinocytes immediately after injury is emphasised.35 In a study of Di Vita et al, it was also mentioned that IL‐1 increases in patients on postoperative days 1 and 2.28 The synthesis of IL‐1 is necessary for the development of local inflammation and for the acute phase response. The main cells producing IL‐1 after the skin damage are neutrophils, monocytes, and macrophages. According to the literature, this fact explains the increase of IL‐1 and TNFα in the current study for treatment and control groups. An increase of IL‐1β and TNF‐α on the 1st day correlated with an increase of neutrophils in cytological swabs, which at a first glance can be regarded as strengthening of the inflammatory reaction in the current research. Trengove et al reported that the proinflammatory cytokines IL‐1, IL‐6, and TNF‐α were found in higher concentrations in wound fluid from non‐healing wounds compared with healing wounds.36 According to Kuffler, leukocytes in PRP contribute to the increased production of inflammatory cytokines, such as IL‐1β and TNF‐α, which increases inflammation.37 This proposition has weight as the use of PRP increases the concentration of growth factors locally. This leads to the migration of cells to the lesion focus. Results of current research correspond to and match with the results of other studies and literature concerning the role of proinflammatory cytokine in the process of wound healing—especially in inflammatory and proliferation phases.38, 39, 40, 41 Everts et al, conclude that during the first 2 days of wound healing, an inflammatory process is initiated by migration of neutrophils. Activated macrophages release cytokines.42 However, in our study, an increase of IL‐1β and TNF‐α especially on day five correlated with an increase of fibroblasts and a decrease in the number of neutrophils. According to cytological analyses, it was an indication of the beginning of the proliferation phase in the treatment group. The confirmation of the role of cytokines in fibroblast proliferation was found in a study of Inoue et al43 where authors have shown that the presence and level of cytokines within burn blister fluids play a role in fibroblast proliferation. Angiogenesis and emergence of fibroblasts start on the 3rd day, followed by collagen synthesis ongoing from day 3 to day 5.41 Thus, through current findings, we can see that a recurrence of IL‐1β and TNF‐α in wound fluid in the treatment group on day 5 is higher than in the control group, which is a presumed signal of the intensity of the wound‐healing process, followed by epithelialisation.

The ultrasound examination could be useful for visualising processes that occur during the wound healing. It is a non‐invasive technique of measurement of clots, tissue granulation, and epithelialisation in the acute wound.44 Barret et al, used ultrasound not only to measure the healing process but also to predict surgical wound complications that might occur.45 Our findings in the current study were similar to the results of other authors who used ultrasound for assessment of wound healing and visualisation of fibrous granulation tissue, collagen accumulation in wound tissues.46, 47, 48, 49 Clinically, wound closure is defined by skin re‐epithelialisation without drainage or need for a bandage.50 This definition relies on a visual assessment of the wound. However, a healing skin may not possess the barrier function and the epithelium covering the wound is functionally deficient allowing the entry of microorganisms, which may result in postclosure wound complications.51, 52

In comparison with the Rippon et al study, the ultrasound measurements of fibrous granulation indicated the accumulation of granulation tissues in wounds during the healing process, which has also correlated with histology (r = 0.96, P > 0.001). Granulation tissue was identified as echo‐poor structures on days 2 and 3, an increase in echogenicity corroborated the collagen accumulation of up to 21 days.44

To improve healing, especially after reconstructive operations, tissues must be revascularised.53, 54 Consequently, particular attention to the assessment of vessels by color Doppler ultrasound was given. Arteries of the face run superficially, which could be easily demonstrated by Doppler ultrasound.55, 56 Doppler ultrasound is capable of providing data on the blood flow dynamics and aids in the assessment of wound severity and calculation of the healing process.57 In the current study, flow indicators of branches and main vessels of the face were observed. Doppler ultrasound was applied to assess vascularisation in several studies.52, 53, 55, 56, 58 The closest to the current study was the research of Ariji where indicators of blood flow in arteries feeding masseter muscle were measured, in healthy volunteers the means of the flow diameter, maximum and minimum velocities, and RI and pulsatility index were 1.8 mm, 24.6 cm/s, 5.1 cm/s, 0.80, and 2.51. Authors reported that for most patients with inflammation and inframuscular haemangiomas, these values increased.56

5. CONCLUSION

Local wound levels of cytokines correlated with the stage of wound healing, as the results of ultrasound scans and cytology measurements have shown. Appearance of the fibroblasts starting on the 5th day in the treatment group, early deposition of collagen and fibrin, which were pronounced between 14 and 21 days, is clear evidence of the activation of proliferation and early wound‐healing processes. In the treatment group, fibroblasts, macrophages, and collagen fibres appeared and their quantity increased 3 days earlier, as well as the epithelialisation that appeared 4 to 6 days earlier as opposed to the control group. All of the above highlights early healing and amelioration especially for patients with delayed healing conditions.

Measurement of the proinflammatory cytokines showed an increase of IL‐1β and TNF‐α on the 1st and the 5th day in the treatment group. Quantitative data were higher for the treatment group as opposed to the control group, which was linked to the influence of PRP on inflammatory and granulation phases of healing. In the control group, on the 5th day, a slight decrease of IL‐1β, TNFα and an increase of IL‐6 concentration correlated with an increase in the quantity of neutrophils, which confirms the lengthening of the inflammation phase.

Ultrasound scans helped to identify earlier granulation of tissue formation, collagen deposition, and earlier epithelisation in the treatment group. Ultrasound scan of soft tissuses of the maxillofacial area showed less‐pronounced oedema, infiltration, lack of lyphostasis, which indicates that PRP leads to a decrease of influence of factors that could lead to complications and delayed healing. According to measurements of parameters of blood flow in vessels of maxillofacial area, changes in the normal blood flow in the control group corresponded to the worsening conditions of the blood supply after surgical interventions. It was duly observed that the neovascularisation appeared earlier for the treatment group in contrast to the control group.

Overall, the use of PRP in maxillofacial surgical interventions is an effective method in the improvement of postoperative wound healing of patients with concomitant diseases and predictable complications in the early postoperative period.

Menchisheva Y, Mirzakulova U, Yui R. Use of platelet‐rich plasma to facilitate wound healing. Int Wound J. 2019;16:343–353. 10.1111/iwj.13034