Platelet The Effect of Hyaluronic Acid and Platelet-Rich Plasma on Soft Tissue Wound Healing : An Experimental Studyon Rabbits

Aims: This study aimed to evaluate the benefits of using platelet– rich plasma and hyaluronic acid when used alone or in combination with each other on wound healing. Materials and methods: The study performed on 15 adult male rabbits divided into three group according to healing periods (3,7,14 days), each group containing 5 animals. Four longitudinal incisions were made on the back of each rabbit, 2 in the right and 2 in the left. A distance of about 1.5 cm was left between one incision and another. The defects were filled with platelet–rich plasma, hyaluronic acid, or mixture of both, and the last incision served as control. Animals were sacrificed at 3rd, 7th, and 14th day after intervention, and biopsy were taken. Histopathological analyses were performed to evaluate the effect of these materials on acceleration of wound healing of skin. Statistical analyses was performed with Analysis of Variance and Duncan's Multiple Range Analysis Test, using a significant level of p ≤ 0.01. Results: Histopathological analysis revealed significant improvement of wound healing when treated with PRP, and with mixed application of PRP and HA compared with control and HA groups in 3rd and 7th day postoperatively, while there was no significant differences in wound healing between all groups at the end of the second week. Conclusions: The use of PRP alone or in combination with HA will accelerate wound healing , while the use of low concentration of HA alone presents no improvement of wound healing in comparison with normal one.


INTRODUCTION
Wound healing is a specific biological process related to general phenomenon of growth and tissue regeneration.It is characterized by the formation of granulation tissue consisting of inflammatory cells, newly formed blood vessels and fibroblasts embedded in a loose collagenous extracellular matrix.Reepithelialization, angiogenesis and matrix deposition are critical events controlling this process. (1)he science of wound healing is advancing rapidly particularly as a result of new therapeutic approaches such as laser, skin substitutes, gene and stem cell therapy. (2)latelet rich plasma is a new application of tissue engineering and a developing area for clinicians and researchers. (3)Platelet rich plasma is a platelet concentrate easily obtained by centrifugation of autologous blood and enriched with growth factors.These growth factors were described to be released from alpha granules when platelet activation is induced by Ca ions from extracellular media.Platelet growth factors play an important role in angiogenesis and tissue regeneration by controlling cell migration, differentiation and proliferation. (4)latelet rich plasma also provides a sterile field as part of its antimicrobial activity. (5)ound healing involves secretion of glycosaminoglycans (GAGs) by fibroblasts which form a hydrophilic matrix suitable remodeling during healing. (6)Hyaluranan, also called sodium hyaluronate (Hy) or Hyaluronic acid, (7) is a high molecular weight glycosaminoglycan (10 4 to 10 7) composed of repeated non-sulfated disaccharide units of N-acetylglucosamine and D-glucoronic acid. (8)It is one of extracellular matrix and plays an important role in the morphogenesis and tissue healing (9) .It is thought to stimulate migration, adhesion, proliferation and cell differentiation leading to acceleration in the wound healing process.
According to our knowledge the present study is the first designed to evaluate the effect of mixing platelet rich plasma and Hyaluronic acid together on wound healing of skin in rabbits as an accelerating factor of wound healing.

MATERIALS AND METHODS
The present study was implemented in the Department of Oral and Maxillofacial Surgery, Dentistry College, Mosul University.Fifteen healthy local adult male rabbits weighing (1.5-2.5kg) were included in the research, they were divided randomly into three groups according to the healing period (3,7, and 14 days), each containing five animals: Group I: Consisted of 5 animals, these animals sacrificed 3 days after operation.Group II: Consisted of 5 animals, these animals sacrificed 7 days after operation.Group III: Consisted of 5 animals which were sacrificed 14 days after operation.
All of the animals were housed under standard conditions , fed the same diet and kept in the same environment during the study period.The materials tested in the study were autologous platelet rich plasma (PRP) and Hyaluronic acid (HA) (Crespine gel/, Biopolymer GmbH8 and Co.KG D-56410 Montabaur, Germany).Prior to surgery, the PRP was obtained by aspiration of two milliliters of autologous blood drawn from the heart of each rabbit (cardiac puncture was done with 23 gauge needle) and combined with 0.5ml of an anticoagulant citrate dextrose phosphate (ACD-A) in sterile tube.The blood was centrifuged at 2000 rpm for 10min.to separate plasma containing platelet from R.B.C., the plasma was drawn off the top and centrifuged for an additional 10min at 3200 rpm to separate the platelets and the plasma containing platelets was drawn from bottom for another cycle of centrifuge at 4000 rpm for 5min.At the time of application, the PRP about (50µl) was combined with equal volume of sterile solution of 10% CaCl2 (a citrate inhibitor that allow the plasma to coagulate and cause activation of platelet to release the growth factors). (10)General anesthesia was obtained by an intramuscular injection of ketamine hydrochloride general anesthetic solution (50 mg/kg of body weight) and xylazin at dose of 0.5, 0.3ml/kg of body weight (11,12) .Complete anesthesia had been obtained within 5min, the surgical field was shaved , cleaned and disinfected with povidone iodine solution and covered with sterile towel.After that the rabbit was laid on its abdomen and given local anesthesia in the area (about 0.25ml of lidocaine 2% with epinephrine 1:80,000,New Stetic com.Colombia), four small incisions (about 1.5 cm length, 0.5 cm depth) were made in the skin of the rabbit back using blade no.15.(a distance of about 2cm was left between one incision and another), the dermis and epidermis were reflected gently by blunt dissector.For the control site, the defect was filled with gelatin sponge alone, whereas in the remaining defects the gelatin sponge was used as a vehicle for the application of autologous PRP(25μl), Hyaluronic Acid (0.01ml), or a mixture of autologous PRP and hyaluronic acid.Following application of materials, the wounds were irrigated with normal saline and closed with one stitch black silk suture (Nantong Jianan Medical Products Co.,LTD.China) and disinfected by povidone iodine ,then the animal was given 0.5 ml oxytetracycline (Iran).I.M. postoperatively (and continuously for three days).A number was inserted in the rabbit`s ear for labeling the date of operation were done for each animal and the animal was isolated for the remaining time of the operation day.The suture removal was done after 7 days.
In group I,II, and III the animals slaughtered at day 3,7,and 14 subsequently following surgery.Following slaughter, biopsy specimens were taken and subjected to histopathological examination.The histopathological examination was performed by two specialists of oral pathology using double blind checking manner.Wound healing was measured by assessment of reepithelialization and inflammatory response to surgery.
To measure the degree of reepithelialization, the criteria established by Sinha and Gallagher were used: (13) -Grade 0 : Reepithelialization at the edge of the wound.
-Grade 1: Reepithelialization covering less than half of the wound.
-Grade 2: Reepithelialization covering more than half of the wound.
The degree of inflammation was measured with the resolution phases of inflammatory processes described by Cotran et al: (14) -Grade 1: Acute inflammation (pyogen membrane is formed).
-Grade 4, Resolution and cicatrization (reduction or disappearance of chronic inflammation, although occasional round cells may persist).
The data were analyzed using SPSS under window program (version 12.0) .A descriptive statistic was made for each variable.The following statistical tests were used: One way analysis of variance test (ANO-VA).Duncan's multiple range analysis.Statistical significant difference was accepted for P ≤ 0.01.

RESULTS
Among the 15 rabbit involved in the study, no animal died throughout the period of the research and all of them remained apparently healthy.All of the wounds healed normally with no post operative complications Reepithelialization:at the 3rd post operative day, the reepithelialization was faster (Reepithelialization covering more than half of wound as shown in (Figure 1) in incisions treated with PRP (mean= 3.0) and mixed application (Hyaluronic acid and PRP) (mean= 3.0) than in those treated by hyaluronic acid alone (mean = 0.6) and control (mean = 0.6), where thin epidermis at the edges of the wound in half of cases was noticed, and in other half the wound showed reepithelialization covering less than half of wound (Figure 2).At the 7th post operative day, there was increased epithelial thickness in wound defects treated by PRP and with mixed application (mean=3.60), the reepi-thelialization was grade 3 (i:e) the reepithelialization covering the entire wound although of irregular thickness (Figure 4).Analysis of variance (ANOVA) and Duncan's multiple range analysis showed significant differences (P≤0.01) between study groups Figure (3), Table (1).
By the end of 2nd week most of all treated cases showed grade 4 of reepithelialization (i:e) reepithelialization covering the entire wound, normal thickness Figure (6).and only few cases (2 of control, 2 of hyaluronic acid, and 1 of mixed treated wound defects) showed grade 3 of thelialization.Analysis of variance (ANOVA) and Duncan's multiple range analysis reveled no significant differences among study groups at P ≤ 0.01 Figure (3), Table (1).

Inflammation
At 3rd post operative day after operation, a significant increase in number of new vascularization, inflammatory cell, collagen arrangement, and number of fibroblast was noticed in wound defects treated with PRP and mixed application (mean=2.60)(Figure 7), when compared with both control and wounds treated by hyaluronic acid only (mean=1.40), which showed marked acute inflammation (Figure 8).At day 7th following operation, statistically significant differences were noticed (P ≤ 0.01) (Table 2) between PRP and mixed application (these samples showed the most resolved phases of acute inflammatory process and there was predominance of chronic inflammation i.e. proliferation of fibroblast) (mean=3.80) Figure (11) when compared to control sample (mean=2.80)and hyaluronic acid alone (mean=2.60) which showed predominance of granulation tissue and beginning of resolution of acute inflammatory process Figure (10).By the end of 2nd week, the wounds in all defects showed complete resolution of inflammatory process, however only a few cases (2 in control, 2 in hyaluronic acid treated wound, and 1 case in PRP and with mixed sample) showed prevalence of chronic inflammation ( where fibroblast proliferation had begun to take place) Figure ( 12) .(ANOVA) and Duncan's multiple range test showed no significant differences (P≤0.01) Figure ( 9), Table (2).

DISCUSSION
Recent in vivo and in vitro studies have suggested that various autocrine and/or paracrine growth factors are present in the serum play important roles in the wound healing of various tissue, including soft and hard tissue. (15)The action of these growth factors vary with regard to environment in which the growth factors are placed, it may be complimentary or even inhibitory.A platelet rich plasma (PRP) which is known to contain multiple growth factors which are especially important in wound healing and may contribute to tissue formation and epithelialization.Autologus platelet concentrate or PRP is a concentration of at least five autologus growth factors and has been shown to accelerate wound healing.PRP is appealing in that it is an easily derived in the initiation of healing response. (16)nother important factor is hyaluronic acid (HA) which is naturally biocompatible, biodegrdable and lacks immunogencity, (17) and its one major member of GAG present in the skin.During the inflammatory phase intact HA in the blood clot of wound helps in the physical stabilization of the matrix.It also stimulates cell infiltration and migration, and controls the degradation of fibrin.The degradation products of HA-fibrin matrix act as regulator molecules of wound healing process. (18)valuation Of Reepithelialization It has been demonstrated that the wounds treated by PRP with or without subsequent application of HA at the 3rd and 7th postoperative day healed faster than in control wound and wound treated with HA alone and there was a significant increase in epithelial thickness, while there was no significant difference at 14th postoperative day among all treated wounds, this may be attributed to the diverse biological effects associated with PRP through a variety of growth factors present in αgranules of platelet and most of these factors may have action on multiple cell and tissue type.Among these factors epithelial growth factor (EGF), which stimulate angiogenesis, endothelial chemotaxis, epithelial and mesenchymal cell mitogenesis, and collagen synthesis. (19)EGF act upon the basal cell at wound edge to promote an epithelial proliferation which migrate into nutrient granulation tissue on the surface of the cell adhesion molecules in the clot.So it stimulate the basal cell of skin to lay down the specific component of basement membrane. (3)valuation of inflammation The wounds treated with PRP alone and with mixed application of both PRP and HA showed the most resolved phase within the acute inflammatory phase during the first week than those obtained with HA treated wounds and with those left to heal on their own.While all the wounds were completely healed at the end of second week postoperatively.The faster resolution of acute inflammation in PRP treated wound is attributed to the secretary proteins contained in the αgranules of platelet including platelet derived growth factor (PDGF), (20) vascular endothelial growth factor, (21) and transforming growth factor-β (TGF-β). (19,20) hese growth factors aid healing by attracting un-differentiated cells in the newly formed matrix and triggering cell division. (20)RP may suppress cytokine release and limit inflammation, interacting with macrophages to improve tissue healing and regeneration, (22) and promote new capillary growth. (23,24) garding the effect of HA on tissue regeneration it is related to the interaction of HA with CD44 which drive the maintenance and establishment of collagen synthesis and normal skin function.Hyaluronic acid is present in the extracellular matrix of basal keratinocytes and the structural integrity of dermal collagen fragments and fibroblast penetration. (25)Decreased levels of HA correlate to impaired local inflammatory response and reduced tissue repair due to degeneration of the granular tissue matrix. (25,26) cording to the scope of the present research the effect of PRP in wound healing showed similar positive result of PRP in acceleration of epithelial migration, the angiogenic response and tissue fill.However, these results agree with those studies induced by Lee et al, have noted acceleration of epithelialization in treatment of lower equine wound with PRP and faster resolution in inflammatory response, (27) and with the finding reported by Vaterio et al, who showed acceleration in skin reepithelialization by using PRP during fat grafting in patient who underwent plastic surgery. (28)Alsabaawe, and Hom et al, who stated that there was an increased of epithelialization rate and regression of acute inflammatory process in PRP treated skin wounds in rabbits during week 1 postoperative period compared to week 2 where there was no statistical differences found in epithelial thickness and inflammation stage between PRP treated wound and control, where the epithelialization returned to normal texture and there was improvement of wound healing in term of reducing the inflammatory process. (29,30)hese results came in agreement with the present study in that the epithelialization and inflammatory response reached the complete stage in wound healing at the end of second week.While Margolis and his colleagues (31) carried out a retrospective study in patients with diabetic neuropathic ulcers who receive an adjuvant therapy such as PRP, PDGF, or graft skins were found to have wound less likely to heal.Concerning the results gained by application of hyaluronic acid alone showed no significant differences in epithelial thickening rather than inflammatory response when compared to normal so there was no acceleration of wound healing.The explanation of this may be related to molecular weight specific, purity, and dose specific of hyaluronic acid.In this line Bettinger et al suggest that the exogenous HA retard healing of partial thickness wound in adult, (32) moreover, in another study induced by Icono et al revealed that the application of exogenous HA dampens the fibrotic healing response in injured fetal mouse limb organ culture. (33)Conversely, several authors claimed that the administration of exogenous HA of high concentration of high molecular weight to adult tissue injuries promotes rapid healing and decreased fibrosis that normally occure in skin burns, (34) also Ruggiero et al reported the effect of HA in scarless fetal wound healing in rabbit wounds showed increased granulation tissue and enhanced collagen deposition with fibrosis. (35)evertheless, the results of the present study reveal acceleration of wound healing in mixed application group (PRP and HA) it may be attributed to influence of PRP on wound healing rather than HA which act as a mucoadhesive and retain the therapeutic agent at its site of action/absorption. (36)t is the first time to evaluate the effect of both PRP and HA on skin wound healing so there is no available study that support this result.

CONCLUSION
Within the limits of the present study, it can be concluded that the PRP will accelerate wound healing when applied alone or in combination with hyaluronic acid.In addition, it is emphasized that there was no beneficial effect of using low concentration of HA on healing skin wounds as it was solely applied.

Figure ( 1 )
Figure(1): Photomicrograph in PRP,and mixed applied of PRP and HA cutaneous wound in rabbits at 3rd p.o (10X).reepithelialization cover more than half of wound.

Figure ( 2 )
Figure (2): Photomicrograph of control and HA cutaneous wound at 3rd p.o day in a rabbit (20X).Notice the epithelial covering is less than half of wound.

Figure ( 5 )
Figure (5): Photomicrograph of cutaneous wound in 7th p.o day of control and HA treated group rabbit (4X).Incomplete epithelization covering more than half of wound .

Figure ( 7 )
Figure (7): Photomicrograph in PRP and mixed application group of cutaneous wound in rabbit at 3rd p.o day (40X).Numerous newly formed blood vessels with increased inflammatory infiltrated.

s
Means with different letters were statistically significant at P< 0.01.

Figure ( 12 )
Figure(12): Photomicrograph in experimental all groups in cutanenous wound at 14th p.o day (40X).Shows resolution of chronic inflammation and heavy proliferation of fibroblast.
* Significant differences existed at p < 0.01

Table ( 2
): Analysis of Variance for Inflammation score.