"Experimental comparative study between autologous activated platelet-rich plasma (a-prp) and non-activated platelet-rich plasma (aa-prp) in wound healing of skin on rabbit model"
Creators: name: Dr. HARDIK RASIKLAL PATEL
name: Dr. ADITYA G. PUNDKAR
name: Dr. Sandeep Shrivastava
name: Dr. Suyash Yashwant Ambatkar
name: Dr. Rohan Ajay Chandanwale
Publisher: name: Zenodo
Year: 2023

description: ABSTRACT
Wound healing is multifaceted biological process crucial for tissue repair and regeneration. Platelet-rich plasma has gained considerable attention as potential therapeutic tool to expedite this intricate process. Our study delves into the comparative effects of autologous activated platelet-rich plasma (A-PRP) and non-activated platelet-rich plasma (AA-PRP) in wound healing, utilizing a rabbit model to mimic human skin responses.
In this experiment, New Zealand White rabbits were used as the chosen animal model, representing an essential step towards understanding clinical applications of PRP. Skin wounds were deliberately created on the rabbits' dorsal areas, after which they were segregated into two groups: one treated with A-PRP, the other with AA-PRP. Both types of PRP were derived from the rabbits' own blood through a centrifugation process according to STARS therapy protocol, with A-PRP being subsequently activated using calcium chloride. Wound healing progress was tracked over time, employing diverse evaluation methods, including wound size measurement, rate of regeneration, Quality of skin regeneration, histological analysis.
Our findings demonstrated significant disparities in wound healing between A-PRP and AA-PRP treatment groups. Wounds treated with A-PRP exhibited an accelerated rate of closure, a remarkable increase in collagen deposition, and notable improvements in histological characteristics, notably enhanced angiogenesis and reduced inflammation, compared to those treated with AA-PRP.
In conclusion, our experimental comparative study highlights substantial potential of autologous activated PRP as wound healing accelerator in a rabbit skin model, surpassing the outcomes observed with non-activated PRP. Results underscore clinical promise of A-PRP as an efficacious therapeutic approach for wound healing. However, further research is essential to confirm these outcomes and to explore the underlying mechanisms responsible for these differences, shedding light on the broader applications of PRP in wound management. This study contributes to our understanding of PRP's therapeutic potential, paving the way for future investigations in the field.
CTRI Registration number – REF/2023/08/072364 (Date- 25/08/2023)
Protocol version v1 dated 17/10/2023
Keywords -Autologous Activated PRP, wound healing, Rabbits,
 
Main body
Introduction
Our complex skin is made up of the epidermis and dermis, which also contain the subcutaneous fat or dermal adipocyte layer. The skin is vulnerable to environmental elements such water evaporation, the penetration of harmful UV radiation, and bacterial invasion. Additionally, the skin protects the underlying organs, which is another crucial job for the organism's existence. It acts as a barrier between the body and the outer environment, leaving it constantly open to harm.(1)
Inflammation, blood clotting, cellular proliferation, and extracellular matrix (ECM) remodelling are some of the overlapping mechanisms that contribute to the healing of wounds, which is a conserved evolutionary process that occurs across species. However, the outcome of skin damage healing varies depending on the species.(2)
Squamous epithelium that is stratified makes up mammalian epidermis. This depends on the basal layer's continued expansion and differentiation. Upward protrusions are made by differentiated basal epidermal cells. They develop into granular layer and suprabasal cells before differentiating into enucleated corneocytes, which make up the stratum corneum. Being the skin's top layer, the epidermis is regularly vulnerable to various types of harm. If the damaged skin is not re-epithelialized, the loss of the organ's barrier function, dehydration, infection, or even death could occur. The migration and multiplication of epithelial cells are necessary for the quick healing of the wound site and the restoration of the barrier function, both of which are essential for the organism's survival. Numerous investigations have shown that resident epithelial stem cells support the re-epithelialization of adult skin. (1,3)
The use of growth factors accelerates skin regeneration. Although mechanism of action is still not understood, local administration of growth factors has significant therapeutic potential in skin regeneration. Growth factor administration has been shown in numerous investigations to quicken skin regeneration. Important facets of soft tissue and hard tissue repair are regulated by growth factors and their receptors. Platelets are a plentiful and practical supply of the complicated growth factors needed for wounds to heal on their own. Despite their function in clot formation, platelets can act as a source of cytokines and growth factors. The typical platelet, which has 58 to 198 granules, can release a lot of growth factors when stimulated by an agonist. (4)
These substances are released from the platelets as a result of activation followed by degranulation. In addition to pharmacological substances like calcium ionophorous, calcium chloride, physiological agents like thrombin, collagen, serotonin, adenosine diphosphate, platelet factor activator, thromboxane, and epinephrine also trigger platelet activation. The effects of each of those substances are assumed to be unchained by interacting with receptors present in the plasmatic membrane of platelets. Platelets change morphologically after activation and form pseudopods, which promote platelet aggregation and later platelet granule degranulation(5)
Growth factors are endogenous signalling molecules that regulate cellular actions like migration, proliferation, and differentiation that are important for wound healing. Exogenous growth factor injection is inefficient in clinically due to its short in vivo half life, limited skin absorption, clearance by exudation before reaching the wound area, and other unfavourable side effects.(6)
One method of delivering the growth factors found in platelets is by concentrating them. This concentrated form of platelets that is produced after blood platelets are processed is known as platelet rich plasma (PRP). Platelets and plasma, two of the four fundamental parts of whole blood, make up PRP.(7)
Platelet-rich plasma (PRP), first identified in 2007, is a mixture of platelets contained in a small volume of plasma with a large number of growth factors (GFs) required for bone growth and regeneration. The PRP contains about 15 growth factors. The three most important among them are platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), and transforming growth factor, along with their isoforms (TGF). The platelet's alpha granules produce these growth factors (50–80 per platelet). Recent studies have shown demonstrated that platelet stimulation, in addition to releasing growth factors, also causes the release of cytokines, enzymes, proteins, fibrinolytic and anti-fibrinolytic proteins via a mechanical or chemical process. Collagen, thromboxane, calcium, magnesium, serotonin, and other chemicals that cause platelets to aggregate help to activate them. These growth elements are instantly released as a result, and they exhaust themselves after 24 hours. Accessibility, ease of isolation, superior handling and storage properties, and usage in tissue engineering are all advantages of using PRP in tissue regeneration. PRP also eliminates the chance of immunological rejection and the spread of illness because it is autologous.(7,8)
                   Despite the fact that Autologous Activated PRP has been utilised to heal wounds in people, particularly those that have been ulcerating for a long time, there are very few references discussing its usage in experimental animals. Numerous experimental studies conducted abroad have focused on the application of autologous activated PRP in both soft and hard tissue healing in goats, horses, and pigs, all of which have had positive results. Surprisingly few references exist for clinical studies conducted in India using autologous activated PRP to treat animal wounds. Because of this, the current clinical trial's objective is to generate more compelling data in favour of employing autologous activated platelet rich plasma in rabbit skin regeneration.
 
This protocol is reported in line with the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines.
 
AIM AND OBJECTIVES
AIM: 
"To study the role of autologous activated platelet-rich plasma (a-prp) and non-activated platelet-rich plasma (aa-prp) for tissue engineering and regeneration of the skin in rabbit model"
 
OBJECTIVES:
The objectives of the study will be to evaluate the role of Activated PRP in terms of
1. Rate of regeneration
2. Quality of skin regeneration
3. Development of PRP protocols for Activated Platelet-Rich Plasma
4.Analysis of Activated Platelet-Rich Plasma infiltrated wounds in Rabbit model
 
Protocol*
Study setting
Study was conducted in Central Preclinical (Animal) Research Facility, Datta Meghe Institute of Higher Education and Research, Sawangi (Meghe) after receiving approval from the Institutional Animal Ethics Committee of Datta Meghe Institute of Higher Education and Research. It will take place over 2 years (August 2022 - July 2024). 
This study will Experimental Prospective study and include a sample size of 8 New Zealand Rabbits who meet the inclusion criteria.
MATERIALS AND METHODS
Study subjects: New Zealand Rabbits with surgically crafted fresh 3 cm diameter full-thickness subcutaneous deep wounds will be included in this study.
Sample size: The sample size for this study will 8 (Per rabbit 2 wounds were be made)
Duration of data collections and follow up: August 2022 to July 2024
Data analysis: September 2022 to August 2024
Method: 8 rabbits will prospectively enrolled who underwent 2 wound creation each. Fresh Autologous Non-Activated PRP will be done by Sandeep's technique for assisted regeneration of skin (STARS)technique protocol. Then activated with 10% calcium chloride. The wounds will enrolled into Two groups
Group A : Fresh Autologous Activated PRP infiltration every 4th day according to STARS technique protocol
Group B : Fresh Autologous Non-Activated PRP infiltration every 4th day according to STARS technique protocol
Endpoint: till wound completely heals.
 
METHOD:
BLOOD WITHDRAWAL:
Rabbits are readily disturbed during blood collection or clinical examinations and may scratch the person handling them or leap off the examination table. Rabbits will therefore be confined in a cage. Up to 5ml of blood will be collected from the auricular marginal veins of each ear using a 22G to 25G needle, a 22G butterfly attached to a syringe, or any similar device. The ear will be shaved to get rid of the fur and skin will be cleaned with alcohol before the blood collection process begins. 
The skin around the ears will be anaesthetized locally with a lotion that contains lidocaine because to its high level of sensitivity. A plastic sheet and an adhesive bandage served as a protective barrier around the creamed area. The entire thickness of the skin becomes numb after 45 minutes. For the next 60 minutes, the anaesthetic effect will longer.
Vessel dilution was obtained by ear massage. The vein will occlude with thumb and index finger, the needle will carefully introduce and blood will withdraw. The needle will remove, and at the site of venipuncture, cotton gauze will firmly apply for at least 1 minute or until the bleeding stopped to avoid the development of hematomas. For the following few hours, the rabbit was monitored to make sure that haemostasis would finish. Through auricular marginal venipuncture, approximately 10ml of blood were taken from each ear and distributed among four test tubes, each holding 6ml of CPDA anticoagulant solution.
STARS therapy protocol(9)
Similar to a local anaesthetic infiltration, this autologous PRP is transferred to a 20 ml ACD tube and locally injected into the wound edges with an equivalent quantity using a 22 G needle.
 Every fourth day (with a three-day break), the process is repeated.
 On alternate days, local dressing is done using only wet saline.
Diluted Cetrimide and gluconate solution of chlorhexidine (Savlon) are both
applied to dirty wounds
No other major surgical operation is performed, such as debridement or
reconstructions with flaps / grafts
No antibiotics are prescribed unless they are life-threatening or to treat any
co-morbid infections. To monitor the infection status of the wound, infected
wound swabs are taken for culture and sensitivity every week.
Unless the pain visual analogue scale score is 7 or more, no analgesics are given,
Only Vitamin C tablets are given twice daily
STARS treatment has distinct benefits because it focuses on the simple way of
preparation, uses a small amount of venous blood (only 20 ml) and infiltrates the margins of the wound. It is used as a definitive tissue repair and regeneration treatment without the use of intensive local care, antibiotics and other operations."
 
PREPARATION OF PRP (according to STARS technique protocol)
Preparation of PRP will be done through DOUBLE SPIN METHOD. The ACD tubes containing the blood will keep in a centrifuge machine and will be counterbalanced. The initial centrifuge cycle will perform for 10 min at 1200 rpm (soft spin). The whole blood will separate into upper straw-coloured plasma and a lower portion containing red blood cells. The upper part of plasma is called Platelet poor plasma due to its relatively low level of platelets and the lower portion including the buffy coat contains a high concentration of platelets. A 2ml syringe will used to withdraw the plasma from the tubes by moving the needle above downwards. The draw stopped when an RBC layer will reach or in the first 1 to 2mm of that layer. The plasma which will collected and then transferred into another plain tube. The same procedure will perform with the remaining tubes. The tubes containing plasma underwent a second spin for 10 minutes at 2000 rpm. After the spin, the tube consists of a fibrinogen rich upper layer of clear serum with very low platelet concentration and the bottom layer consisting of highly concentrated platelets.
With the same 2ml syringe, the top two-thirds of this liquid will be drawn off, leaving the tube with the remaining one-third filled with concentrated serum for platelets. To create the pale yellow-hued platelet rich plasma concentrate, the remaining one-third of the liquid was correctly combined. 
 
ACTIVATION METHOD
Platelet degranulation and growth factor release are induced using an activation technique. PRP can be activated in a variety of ways, including by encouraging platelet release of bioactive compounds and fibrinogen cleavage. The most frequently employed substances for this are thrombin and/or calcium chloride. PRP eventually forms a hydrogel after activation, and it has been suggested that this hydrogel secretes growth factors (10).
Calcium chloride is an inhibitor of citrate which enables the coagulation of plasma. Samples activated with 10% calcium chloride released growth factors at a significantly higher rate than collagen activated samples. According to anecdotal evidence, medical professionals in the cosmetic field frequently combine injectable 10% CaCl2 solution with PRP prior to facial injections. Some clinicians have suggested that the addition of a small amount of CaCl2 can help reduce the painful burning / stinging sensation that many patients experience during PRP injections. This phenomenon may be explained by the effects that CaCl2 solution has on the pH of PRP.
Storage
While stability studies have shown that a storage term of 6 hours at 20 C can be consistent with the maintenance of platelet functionality and growth factor release potential, prolonged storage times lead to a decrease in the concentration of growth factors within platelet granules(11). It is believed that a pH drop brought on by an increase in lactic acid generation in hypoxic settings is one of the primary causes of platelet viability loss (12). 
Agitation of the platelet concentrate makes sure that there is enough oxygen in the storage container and that the platelets are continuously oxygenated, which allows excess carbon dioxide to be evacuated (13). For the preservation of platelet concentrates, a temperature range of 21oC and 24oC is advised in order to prevent platelets from activating(14).
A appropriate amount of O2 (21 percent) should be given for optimal storage conditions, a temperature between 21oC and 24oC and not exceeding 6 hours of rest.
 
Inclusion Criteria:
Healthy rabbits of weight 1kg to 2.5kg
 
Exclusion Criteria:
o Weight less than 1kg
o Rabbits with Low platelet count (< 1,50.000)
o Rabbits with skin disease
 
Observation and Evaluation:
1. Analysis of Activated and Non-Activated Platelet-Rich Plasma infiltrated wounds in Rabbit model
2. Size of the wound – sequential tracing on paper in terms of area calculation (per square cm)
3. Serial/sequential photographs of all wounds just before dressing
4. Biopsy of skin regeneration at the end of treatment or 6 weeks whichever is earliest
 
STATISTICAL ANALYSIS:
Appropriate statistical test would be applied to analyse data.
 
Dissemination*
Planning to execute the findings in National Conference Proceedings and publish in an index journal.
Study status*
The process of recruiting New Zealand Rabbits is yet to begin.
 
DISCUSSION 
8 rabbits that each received two wounds will eventually be enrolled. Two groups will be formed.Every fourth day, Group A will receive fresh autologous activated PRP infiltration, while Group B will receive fresh autologous non-activated PRP infiltration. Then follow up will be done till wound completely heals. Wounds in various groups will be observed from day 0 to day 30. In my opinion there will be earlier healing of wounds in Group A as compared to group B. The healing process depends on the biological nature of PRP (15). The intradermal injection in the Rabbit of PRP into skin wounds greatly improved the healing process (16). PRP therapy frequently showed to be more successful in the healing process of an experimental wound in animals. The group receiving fresh autologous activated P.R.P infiltration will heal more quickly(17).Fresh Autologous Activated PRP will speed up the healing process and significantly reduce ulcers in acute and chronic wounds that are difficult to cure(18,19). Injection of autologous PRP subcutaneously at the wound margins will substantial improvement of wound re-epithelization and also reduced scar formation(20). PRP improves and hastens the healing process, thereby signifying its therapeutic ability on cutaneous lesions and its usage in patients with weakened wound healing(21).PRP will effective in the remodelling and cicatrisation processes of the skin grafts in rabbits compared with non-treated animals(22). PRP is a very innovative, promising therapy that can be used to provide a variety of vital, physiologically active growth factors to the location of the damage. It uses the patient's own blood (autologous), is highly straightforward and convenient to use, may be cost-effective, and is regarded as a very safe therapy (23). the use of Activated PRP gel as an enhanced therapy for surgical or non-healing wounds in horses, as well as in immunocompromised, diabetic, or elderly people, may result in the quality healing of acute wounds.(24).Chronic ulcers can be treated safely and effectively with autologous activated PRP(25). Autologous Activated PRP plays a role in wound contraction and collagen synthesis in addition to strongly promoting re-epithelialization and inducing angiogenesis(26).
Ethical considerations*
Institutional Animal Ethical Committee Approval has been obtained on 10/08/2022
 IAEC No – DMIMS/CPCSEA-IAEC/22-23/11
The study documentation will be treated as confidential information and safely archived with only the principal investigator having access. In case of any adverse effect, reporting will be done to the investigator and the subject will be treated accordingly and will be considered as drop out from the study.
Data availability –
Underlying data
As this is a study protocol, there are no data linked with this article
Competing interests* 
NONE
Grant information* 
Self-Funded, no external grant received 
Acknowledgements 
To all the staff in the Department of Orthopaedics at JNMC, AVBRH, Sawangi, Wardha, Maharashtra, India and Central Preclinical (Animal) Research Facility, Datta Meghe Institute of Higher Education and Research, Sawangi (Meghe), I would like to convey my gratitude for their support during the research and analysis.