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Medicine, Research & Experimental
Conducting experimental research is crucial in advancing medical knowledge and improving patient outcomes. This includes preclinical assessments such as in vitro and in vivo studies. Platelet-rich fibrin (PRF), a blood by-product, has garnered attention in the medical and dental fields due to its potential for tissue regeneration and wound healing. Animal models, such as rabbits and rats, have been utilized to create PRF and investigate its properties and applications. PRF has shown promise in reducing inflammation, promoting tissue repair, and expediting wound healing in the dental and medical fields. To provide guidelines for PRF animal research, this narrative review compares existing evidence and highlights the importance of standardizing animal models, following ethical considerations and ensuring transparency and accountability. The authors emphasize the necessity to use the correct relative centrifugal force (RCF), standardize centrifugal calibration, and report detailed information about blood collection and centrifuge parameters.
- platelet-rich fibrin
- animal models
- experimental research
- rcf
- g-force
- L-PRF
- PRF
1. Introduction
Translational research is a critical process that involves the translation of basic scientific discoveries into practical applications that benefit human health [1]. It consists of bridging the gap between basic research and clinical practice by using experimental studies to develop new techniques and procedures in the medical field. Experimental studies play a crucial role in the development of new medical techniques and procedures, as they provide the necessary evidence to support the effectiveness and safety of these innovations. For example, clinical trials are essential to testing the efficacy of new treatments and drugs before they are approved for use in patients. Advancing medical knowledge and developing new treatments that improve patient outcomes would be impossible without experimental studies, including important results derived from in vitro and in vivo preclinical assessments. The presence of experimental research in translational research is a vital step in ensuring that scientific discoveries are translated into practical applications that improve human health [2,3].
In the past two decades, platelet-rich fibrin (PRF) has become increasingly popular in medicine and dentistry for its potential use in wound healing and tissue regeneration [4,5,6,7,8]. However, PRF’s development deviated from the typical research process as it was first used in humans before being studied in vitro and with animal models [9]. This unconventional approach was taken to enhance the research and gain a better understanding of the blood by-product. This sequence of application was likely due to the urgent need for effective treatments in human patients, leading to the rapid adoption of PRF in clinical practice. As more knowledge and experience were gained, researchers began to study PRF in vitro and in animal models to better understand its mechanisms of action and optimize its use [10,11]. Such studies have yielded valuable insights into the properties and potential applications of PRF, paving the way for further translational research to develop new techniques and procedures [12,13,14,15].
In order to improve the techniques and discover new clinical applications, animal models, such as rabbits [16,17,18,19,20] and rats [21,22,23], have been used to produce PRF and investigate its properties and potential applications. The high concentrations of platelets, growth factors, and other bioactive molecules make PRF useful in promoting tissue regeneration and enhancing bone growth [11]. In the dental field, the blood by-product has been used in various procedures to reduce inflammation, accelerate wound healing, and improve outcomes. In the medical field, PRF has been used to treat chronic wounds, burns, and musculoskeletal injuries, with promising results in enhancing tissue repair and reducing inflammation [24,25].
Fibrin research has shown that fibrin clots in mammals such as mice, rats, and rabbits can exhibit different fiber diameters and clot densities compared to human clots. These differences have been considered when using animal models to study blood clotting and related disorders in humans, as they can inform therapeutic development and enhance our understanding of blood clotting mechanisms in general [26,27].
Compared to platelet-rich plasma (PRP), PRF does not necessitate the addition of anticoagulants or other external factors during its preparation. This simplifies the process and potentially mitigates the risk of adverse effects related to additives [27]. Nevertheless, the anticoagulants in PRP can aid researchers in acquiring the appropriate amount of this blood derivative for use in animal models, giving them more time during the blood draw.
The major concern about PRF’s production using animal models is the necessity to have fast blood collection, which is essential in producing high-quality PRF as it minimizes the risk of clotting and ensures that the concentration of platelets and other bioactive molecules remains high. However, in some animal species, such as rabbits and rats, blood coagulates quickly, making it necessary to collect blood rapidly to avoid clotting. In addition, tubes with clot activators, such as silica, can also aid in blood coagulation and make PRF’s production difficult. In addition, access to appropriate vessels (veins or arteries) from animals can be challenging, and the amount of blood that can be obtained may be limited. In this context, PRF has been investigated through very diverse animal models with a great heterogeneity of methodologies, with little help available for the choice of the best protocol for the production of this blood byproduct.
2. Animal Research
Animal care is a crucial aspect of any animal study, as researchers are responsible for ensuring that their subjects are treated humanely and with the utmost respect. This consists of providing appropriate housing, nutrition, and care to minimize any potential harm that may be inflicted on them during the study. In addition, by establishing an ethical committee for animal studies, researchers can ensure that all aspects of the study are reviewed by an independent body to ensure compliance with ethical standards and regulations [28].
In addition to ethical oversight, guidelines are essential in any animal study. The ARRIVE guidelines were developed to provide a checklist of information that should be included in reports of in vivo experiments [29]. These guidelines help to ensure transparency and consistency in reporting by providing a standardized format that contains information about animal welfare, housing, and health status. These guidelines also ensure that researchers provide sufficient detail on their experimental design, methods, and results, allowing others to reproduce the study if necessary. They are of special interest in platelet concentrate research as these products depend on the content of several growth factors and inflammatory mediators whose production and release may be affected by different biological factors, health status, and stress.
Similarly, the PREPARE guidelines were developed to provide a framework for the planning phase of animal studies [30]. By considering all the factors that may influence the study’s outcome during the planning phase, researchers can ensure their study is well-designed and scientifically rigorous. This includes considering factors such as the choice of animal model, sample size, experimental design, and statistical analysis.
The importance of these guidelines cannot be overstated, as they provide clear and concise instructions for the ethical use of animals in research. They promote transparency, consistency, and accountability, ultimately enhancing the research findings’ credibility.
Sample size calculation is another critical aspect of studies involving animals [31,32]. By determining the minimum number of animals required to achieve statistical power, researchers can ensure that their research is adequately powered to detect any effects of the intervention or treatment being tested. Furthermore, sample size calculations consider factors such as the expected effect size, variability, and significance level to ensure the study is well-designed and scientifically rigorous. Adequate sample sizes also help to minimize the number of animals used in the study, reducing any potential harm that may be inflicted on them.
3. Considerations on Blood Collection
When collecting blood from animals for research, it is essential to prioritize the safety and well-being of the animals, which requires adhering to similar guidelines as those followed in human blood donation. One fundamental recommendation is the “10% rule”, which states that the amount of blood drawn should not exceed 10% of the animal’s total blood volume [33,34]. For instance, a 2 kg rabbit has an average blood volume of approximately 55–70 mL per kilogram of body weight, translating to a total blood volume of around 110–140 mL. Hence, based on the 10% rule, a maximum of 11–14 mL of blood can be safely collected from this rabbit to avoid any adverse effects. By following these guidelines, researchers can collect blood from animals safely and humanely in a manner that does not jeopardize their health. In addition, ensuring safe and adequate blood collection involves adhering to proper techniques and equipment, and selecting the appropriate vessel and blood flow for the study type. The choice of vessel and blood flow depends on the desired blood by-product and its specific requirements
4. Venipuncture of Rats
Blood collection in rats is a common procedure in many research studies that require blood samples for analysis (e.g., cytokines and growth factors) or for blood concentrate production [40,41,42,43,44,45,46].
Blood collection from the tail vein is relatively easy and minimally invasive, making it a preferred option for many researchers. The rat’s tail is first warmed with a heat lamp to dilate the veins, and sometimes a small incision is made to collect the blood. This method is used for collecting small amounts of blood and is suitable for tests that require minimal manipulation of the blood sample.
Once the blood sample is collected for PRF production, it is essential to understand that the coagulation time for rat blood is around 2–5 min, which is faster than the human coagulation time of 5–10 min [65,66,67,68,69,70]. Considering this information, the researcher needs a fast blood collection from a vessel with satisfactory blood flow. After that, the tube must go quickly to the centrifugation process.
In terms of the number of cells in the blood, rats have a higher number of red blood cells and a lower number of white blood cells compared to humans. It is important to note that the platelet count in rats can vary widely depending on various factors such as age, sex, strain or breed, and health status. The platelet count in rats can range between 600,000 and 1,500,000. Therefore, it is recommended to consider these factors when interpreting the platelet count in rats for PRF production.
5. Venipuncture of Rabbits
Rabbits are another animal model used to study different types of blood concentrates [74,75,76,77,78,79]. Considering the animal’s anatomy, there are several areas where blood can be collected from rabbits.
The ear vein is a less-invasive method for collecting small volumes of blood from rabbits. This method is relatively easy, making it a preferred option for many researchers. The rabbit’s ear usually is warmed to dilate the veins to collect the blood. The ear vein is the most common area used by researchers to produce PRF. However, this area does not offer a large amount of blood in a favorable flow to produce the PRF membrane. Therefore, it should not be considered the first area of election to produce more than one or a larger PRF membrane.
Intracardiac function is a more invasive method of blood collection that involves accessing the heart directly. It requires surgical skills and experience and should be performed under anesthesia. As was described for the rat model, this method allows for the collection of larger volumes of blood and is more suitable for extensive manipulation of the blood sample.
6. Venipuncture of Dogs
Blood collection from dogs for experimental research involving blood concentrates is a common practice. The most commonly used methods for blood collection in dogs include venipuncture of the cephalic, saphenous (lower limb veins), or jugular veins.
The coagulation time in dogs is similar to that of humans, usually taking around 2 to 10 min. The number of cells in the blood, cytokines, and growth factors in dogs is also similar to those found in humans [110,111]. However, it is important to note that there can be individual variations in these factors between dogs, as well as between different breeds and ages.
When compared to other animals used in experimental research, such as rats or rabbits, dogs have a longer coagulation time. This difference should be considered when designing studies that involve clotting factors or blood coagulation.
7. Venipuncture of Pigs and Mini-Pigs
Blood collection in pigs and mini-pigs for studies in blood concentrates is a commonly used technique in experimental research [112,113,114,115,116,117]. The choice of blood collection site in pigs and mini-pigs will depend on the size and age of the animal.
Overall, pigs and mini-pigs are valuable models for experimental research. The use of pigs and mini-pigs in biomedical research can provide important insights into human health and disease. Careful consideration should be given to the choice of blood collection site to ensure minimal stress to the animal and maximum accuracy of results.
8. Venipuncture of Goats
Blood collection from goats for experimental research involving blood concentrates is an important practice. The most common method for blood collection in goats is through the jugular vein.
The coagulation time in goats is longer than that of humans, usually taking around 3 to 7 min. The number of cells in the blood, cytokines, and growth factors in goats are also similar to those found in humans. However, similar to dogs, there can be individual variations in these factors between goats, as well as between different breeds and ages [67,110].
When comparing platelet counts, goats exhibit a range similar to that of humans. On average, goats have between 100,000 and 500,000 platelets per microliter of blood, while humans typically possess 150,000 to 450,000 platelets per microliter of blood. This difference should also be taken into account when designing experiments that involve platelets [67].
It is important to note that goats are particularly useful as models for human research in studying blood products, particularly fibrinogen, due to the similarity of goat and human fibrinogen structures.
9. Considerations on the Relative Centrifugal Force
Producing blood concentrates, such as PRF, requires precise adherence to protocols; one of these is accurately calculating the relative centrifugal force (RCF) or g force required for the centrifugation process. Errors in RCF calculations can significantly affect the quality and reproducibility of the final product, making it vital to ensure accurate RCF calculations in every study. Standardizing RCF values across different studies can also enhance comparability and facilitate the transfer of scientific knowledge from animal to human studies.
The RCF value is determined by using a formula that considers the rotor’s radius and the revolutions per minute (rpm). The formula is an essential component of the blood concentrate production process, and any errors in calculating the RCF value can significantly impact the final product’s quality. Therefore, it is crucial to ensure that the formula is accurately applied in every study to produce reliable and reproducible results. In addition, it is recommended to use standardized methods for determining the RCF value to enhance comparability between different studies. Overall, correctly applying the RCF formula is critical for producing blood concentrates necessary for various scientific and clinical applications [122,123,124,125,126].
Communication of scientific and clinical protocols is crucial to ensure the reproducibility of human studies in a standard format. The principles of translational research determine that all details of a method of study should be reported to facilitate the transfer of scientific knowledge of animal studies to humans. However, inaccuracies in reports may impair the translation of research results. The PRF protocol is an example of such inaccuracy. The protocol reports the 400× g centrifugation RCF but does not specify the location of the rotor in which the RCF was measured (mm). Therefore, to standardize the terminology for PRF production, it is necessary to establish an approach that focuses on the RCF obtained from the minimum radius (×1 mm), average radius (×2 mm), and maximum radius (×3 mm).
Obtaining standardization in centrifugal calibration is crucial for reliable and reproducible results in the production of blood concentrate. The maximum RCF (×3 mm) is the most commonly used parameter for centrifuge calibration, as it ensures the best standardization in different models and brands.
In order to have accurate and consistent results in PRF production, it is recommended to follow the same standardized standard for human studies and use the maximum radius (×3 mm) to determine the RCF value. This value can be found in the literature [126] or obtained from the centrifuge manufacturer.
In addition to the use of standard measurements, it is also crucial to report all the details of the centrifuge protocol in a standardized format to ensure reproducibility and comparability between studies. This includes reporting the rotor’s location, the centrifuge’s time and speed, and other relevant details. Following these guidelines, researchers can improve the accuracy and reliability of their results and promote the advancement of translational research in blood concentrate production.
It was important to identify the correct protocol for each centrifuge. The maximum RCF (×3 mm) is commonly used for centrifugal calibration to obtain a more reliable standardization. Consequently, in the standardization published for human studies, the maximum radius should be considered to determine the value of the RCF in the production of blood concentrate using several centrifuges available on the market.
This entry is adapted from the peer-reviewed paper 10.3390/bioengineering10040482
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