INTRODUCTION:

Burn is still regarded as one of the emergency medicine affecting both genders and all ages groups in both developed and developing countries leading to physical and psychological disabilities with an increasing trend in mortality and morbidity¹. Burns are classified most commonly based on the depth of injury into first degree, second degree, third degree and forth degree. In the second-degree burn, injury extends to the superficial/deep dermis. Many of the burn injuries are due to disturbance in the normal protective and physiological functions of skin Partial thickness burns damage the epidermis and part of the dermis. Epidemiologically, it is the most commonly found type of burns compared to superficial and full thickness burns.¹Burns are treated with various of wound dressings. The dressings could be ranging from a cheap to expensive dressings. Wound care should be done in order to enhance wound healing process.

Evaporation is one of the problems, so a closed wound care is more preferable, especially a moist dressing.^2,3 A decent wound dressing could decrease the complications, including hyperpigmentation or formation of hypertrophic scar, while increase the healing process. From this statement, a proper selection of the wound dressing is important.⁴

Platelet-rich plasma (PRP) has been used in several surgeries and a positive result was found, especially regarding the healing process. However, the application of PRP in burns are not commonly performed. PRP is thought to be helpful in wound healing because PRP enhance the angiogenesis and fibroblast proliferation.⁵This study aims to understand the role of PRP on second degree of burn injury in a Wistar rats.

MATERIAL AND METHODS:

This experimental study was performed at the Pharmacy Laboratory in Faculty of Pharmacy and an ethical clearance has been obtained from the Experimental Research Ethical Committee of Universitas Sumatera Utara (0560/KEPH-FMIPA/2019). We performed a sample size estimation using Federer’s formula:

(n-1) (t-1) ≥ 15

n = sample from each groups

t=numbersof experimental groups

(n-1)(t-1) ≥15

(n-1)(2-t) ≥15

n-1 ≥15

n ≥16

Before the study began, animal test adjusted for one week with the condition of the room temperature (22-25 °C), under the cycle of 12hours light/ dark, given the food and the drinking water ad libitum. Male albino Wistar rats weighing 250 to 300grams was the inclusion criteria in this study. 40 rats were given the same water, food, temperature, humidity, and lighting conditions. After that, the rats were randomly divided into two groups, the PRP-applied groups (20 rats) and the control groups (20 rats). Another groups of Wistar rats were utilized as the donor for PRP preparation.

The burns induction was performed under anaesthesia the rats using ketamine hydrochloride 50milligrams per kilograms. The dorsum of the rats was shaved. Aseptic procedure was done, and 2x2cm iron plate was kept in boiling water, after that the plate was stroked to the area for 35 seconds with the weight of the iron plate. A histopathological analysis was done to confirm the partial-thickness burn injury. In the first group, we applied the PRP into the burn area and covered it with a transparent dressings, while the second group we applied a normal saline before the transparent dressings.

On the fourteenth day after the burn induction, the skin was harvested, fixed in 10% formaldehyde solution, then embedded in paraffin. For the analyses of collagen density, staining with Masson’s trichrome staining were performed. After that, a microscopic examination for collagen density was assessed by light microscope in 100 magnification areas.

Platelet-rich plasma was obtained following 20 step configurations of blood taken from the rats in donor group. First, a soft spin was performed (1500rpm, 10 minutes) in order to separate the plasma and the red blood cells. Second, a hard, long spin was done (1000 rpm, 15 minutes) to further separate the plasma into the platelet-rich plasma and platelet-poor plasma. Lastly, the PRP was mixed with 10% calcium chloride to activate platelets.

RESULT:

Animals used in research is a rat (Rattus norvegicus) Wistar male 150 – 200g. Before the study began, animal test In this study, 40 samples were analyzed on the 14th-day afterburn induction divided in to two groups: 20 samples of the control group and 20 samples of the treatment group. Research data for the entire observation period were in the form of fibroblast values, PMN, number of the blood vessel lumen and collagen deposition. Before doing the statistical analysis, a normality test was carried out using the Kolmogorov-Smirnov test.

Table 1: Characteristics of Research Samples

Characteristics	Control Group	PRP Group	p-value
Weight	276,2 (± 14,00)	278,7 (± 11,86)	0,609^a

^aIndependent T-test

The weight of the study sample in the control group had a mean weight of 276.2 (±14.00) grams compared to 278.7 (±11.86) grams in the PRP group. There were no significant differences in the two groups with a p-value of 0.609. Whereas in the normal dermis, obtained in the control group was 258.0 (±16.51) compared to 260.1 (±16.50), there is no statistically significance with the normal dermis (p value 0.704). Whereas when the burns were made, the wound condition was 204.8 (±16.40) in the control group while in the PRP intervention group was 242.0 (±16.63). A statistically significant difference was obtained with a p value of 0.001.

Masson Trichrome’s staining in control group

Masson Trichrome’s staining in PRP group

Table 2: Collagen Density of Research Samples

Characteristic	Control group	PRP group	P value
Collagen density	79,5 (± 7,21)	93,1 (± 4,58)	0,001^a

^aIndependent T-test

The density of collagen in the control group with an average of 79.5% (±7.21%) was obtained compared to the PRP group of 93.1% (±4.58%). The result showed a statistically significant difference where p value 0.001 is obtained.

DISCUSSION:

Our study proves the effect of topical PRP administration on healing process of mid-dermal degree burn wound in Wistar rats with an increase of collagen density on 14th days with histological staining of Masson’s Trichrome (MT). Henderson mentioned that the use of autologous PRP topical gel accelerates wound healing by stimulating the inflammatory response that causes an increase in extracellular matrix and tissue granulation, vascularity and fibroblast proliferation, and collagen production⁶. In a study conducted by Ozcelik in 2016, there were no significant differences, although the value of fibroblasts was higher in the treatment group⁷. Several in vitro studies of PRP and wound healing suggest that PRP stimulates fibroblast proliferation^8,9,10. PRP can stimulate fibroblasts depending on its concentration, at a concentration of 5% will promote the proliferation of dermal fibroblasts significantly but not at a concentration of 20% or higher^11,12,13.

In the initial phase of wound healing, there are an increase of neutrophils and macrophages, cells that stimulate debris and bacterial phagocytosis while producing and releasing growth factors¹⁴. There are several studies that support that the application of PRP to the location of the graft accelerates the wound healing process, promotes epithelialization and angiogenesis in the skin graft and donor site^15,16. Ozcelik stated that the collagen deposition in the control group was lower than the treatment group, but not statistically significant⁷. It is consistent with this study, where an increase in collagen density was obtained in the treatment group given PRP compared to the control group. However, in our study significant results were obtained. Ozcelik in 2016 was found that vascular proliferation was more numerous in the study group but not statistically significant⁷. PRP enhances epithelial cell differentiation and collagen organization. PRP also increases the integration of biological mesh networks^16,17. These results support Hao et al who found that using PRP with xenogeneic asenular dermal matrix for the treatment of second-degree burns can reduce infection rates and accelerate wound healing¹⁸. This is caused by the reduced inflammatory phase and the absence of prolonged inflammation in wounds treated with PRP^7,16. Not all research states that PRP will be able to accelerate wound healing, in studies conducted with full thickness thickness excision in rabbits and then treated with PRP did not show any epithelialisation differences in the control group or treatment group¹⁹.

The advantages expectation from a dressing are the ability to prevents fluid loss and as microorganism barrier, supports cell proliferation in wound healing process, allows proliferation of blood vessels, keratinocyte adhesion and differentiation, but it is not yet available at this time. PRP has been proven to accelerates the process of wound healing in acute trauma wounds, chronic wounds that did not heal, and incision wounds, and effective in soft or hard tissue reconstruction^5,16,20,21. Wounds treated using PRP showed a short inflammatory phase with out prolonged time of inflammation²¹. Hao et al. showed that using PRP with xenogenic acenular dermal matrix for the treatment of second-degree burns could reduce infection rates and accelerate the wound healing process²¹.

CONCLUSION:

In our study, we found there were statistically significant differences in the increase in collagen density between the PRP intervention group versus the control group. PRP can accelerate the healing process of burn wound. The use of PRP in patients with extensive burn wound can be the treatment option for this cases. Further research is needed to know the administration and effect of PRP in human burn wound. We still need further research about topical PRP application on deeper-degree burn wound, such as deep dermal and full thickness degrees in experimental animals and humans and effectivity of repeated application of PRP.

ACKNOWLEDGEMENT:

The authors are grateful to the authorities of Universitas Prima Indonesia for the facilities.

CONFLICT OF INTEREST:

This study was supported by TALENTA Universitas Sumatera Utara.

REFERENCES:

1. Sabarahi, S. 2010. Principles and Practice of Burn Care. New Delhi: Jaypee Ltd.

2. Moenadjat, Y. 2003. Burn Injury: Clinal Practice. Faculty of Medicine, Universitas Indonesia. Jakarta. p: 4, 23-28. Herndorn, D.N. 2002. Total Burn Care. 2nd ed. Saunders. London. p: 101-169.

3. Civelek A, Ak K, Kurtkaya O, Tekeli A, et al. Effect of a low molecular weight heparin molecule, dalteparin, on cellular apoptosis and inflammatory process in an incisional wound-healing model. Surg Today. 2007;37: 406-11.

4. Kazakos K, Lyras DN, Tilkeridis VK, Tryfonidis M. The use of autologous PRP gel as an aid in the management of acute trauma wounds. Injury. 2009; 40: 801-5.

5. Henderson JL, Cupp CL, Ross EV, Shick PC, Keefe MA, Wester DC. The effects of autologous platelet gel on wound healing. Ear Nose Throat J. 2003 Aug;82(8): 598-602.

6. Ozcelik U, Ekici Y, Bircan HY, Aydogan C, Turkoglu S, Ozen O. Effect of Topical Platelet-Rich Plasma on Burn Healing After Partial-Thickness Burn Injury. Med Sci Monit. 2016 Jun 5; 22: 1903-9.

7. Shivhare Y, Singh P, Patil UK. Healing potential of Trichosanthes dioica Roxb on burn wounds. Research Journal of Pharmacology and Pharmacodynamics. 2010;2(2): 168-71.

8. Kakudo N, Minakata T, Mitsui T, Kushida S, Notodihardjo FZ, Kusumoto K. Proliferation-promoting effect of platelet-rich plasma on human adipose-derived stem cells and human dermal fibroblasts. Plast Reconstr Surg. 2008 Nov;122(5):1352-60. doi: 10.1097/PRS.0b013e3181882046.

9. Bernuzzi G, Tardito S, Bussolati O, Adorni D, Cantarelli S, Fagnoni F. Platelet gel in the treatment of cutaneous ulcers: the experience of the Immunohaematology and Transfusion Centre of Parma. Blood Transfus. 2010 Oct;8(4): 237-47. doi: 10.2450/2009.0118-09. P Borzini, P Mazzucco. Platelet rich plasma (PRP) and platelet derivatives for topical therapy. What is true?. ISBT Science Series (2007) 2, 272–281

10. Rachmasari A, Kartini RA, Alviani G, Solihah I. Healing effect of Spray gel collagen extract from Channa striata bone on burn in rats. Research Journal of Pharmaceutical Dosage Forms and Technology. 2019;11(4): 275-9.

11. Samein, L. H. Preparation and evaluation of nystatin-loaded solid-lipid-nanoparticles for topical delivery. Asian Journal of Pharmaceutical Research. 2014; 4(1), 44-51.

12. Adler SC1, Kent KJ. Enhancing wound healing with growth factors. Facial Plast Surg Clin North Am. 2002 May;10(2): 129-46.

13. Venter NG, Marques RG, Santos JS, Monte-Alto-Costa A. Use of platelet-rich plasma in deep second- and third-degree burns. Burns. 2016 Jun;42(4):807-14. doi: 10.1016/j.burns.2016.01.002. Epub 2016 Jan 25.

14. Pallua N1, Wolter T, Markowicz M. Platelet-rich plasma in burns. Burns. 2010 Feb;36(1):4-8. doi: 10.1016/j.burns.2009.05.002. Epub 2009 Jun 21.

15. Pauzi, M. F., Juhari, S. N., Ahmad, M. N., & Ghazali, F. C. (2018). Effects of Glycosaminoglycans Extract from sea Cucumber Stichopus vastus on Inflammatory Mediators and Tissue Healing on Rodents with full Thickness Burn Injury. Research Journal of Pharmacy and Technology, 11(9), 3874-3880.

16. Pintucci G, Froum S, Pinnell J, Mignatti P, Rafii S, Green D. Trophic effects of platelets on cultured endothelial cells are mediated by platelet-associated fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF). Thromb Haemost. 2002 Nov;88(5): 834-42.

17. Sowmya, M. V., and Mangayarkarasi, M. (2020). Effect of Physiotherapy Intervention after Platelet Rich Plasma Procedure in Subjects with Grade 3 Osteoarthritis Knee. Research Journal of Pharmacy and Technology, 13(5), 2065-2068.

18. Kazakos K1, Lyras DN, Verettas D, Tilkeridis K, Tryfonidis M. The use of autologous PRP gel as an aid in the management of acute trauma wounds. Injury. 2009 Aug;40(8):801-5. doi: 10.1016/j.injury.2008.05.002. Epub 2008 Aug 13.

19. Iesari S, Lai Q, Rughetti A, Dell'Orso L, Clemente K, Famulari A, Pisani F, Favi E. Infected Nonhealing Wound in a Kidney Transplant Recipient: Successful Treatment with Topical Homologous Platelet-Rich Gel. Exp Clin Transplant. 2017 Apr;15(2):222-225. doi: 10.6002/ect.2014.0236. Epub 2015 Jun 15.

20. Carter CA1, Jolly DG, Worden CE Sr, Hendren DG, Kane CJ. Platelet-rich plasma gel promotes differentiation and regeneration during equine wound healing. Exp Mol Pathol. 2003 Jun;74(3): 244-55.

21. Hao T, Zhu J, Hu W, Zhang H, Gao Z, Wen X. Autogenous platelet-rich plasma gel with acellular xenogeneic dermal matrix for treatment of deep II degree burns. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2010 Jun;24(6): 647-9.

Received on 02.08.2020 Modified on 10.10.2020

Research J. Pharm. and Tech. 2021; 14(7):3533-3536.

DOI: 10.52711/0974-360X.2021.00612