Another look
Swiss medical devices for
autologous regenerative medicine.
REGEN LAB" PRODUCTS AT THE SERVICE
REGENERATIVE MEDICINE IN HUMANS
A/ PRP Classic or Standard
Summary
Regenerative medicine, based on the use of autologous tissues and embryonic, stem or differentiated cells, is attracting increasing interest.However, their preparation, in compliance with good practices and sanitary regulations, is a technical challenge. The objective of this manuscript is to present the design of reliable, CE-marked medical devices for the preparation of standardized platelet-rich plasma (PRP) and other autologous biologics for therapeutic use.
There are many processes for isolating PRP. Depending on the methodology used, the composition of PRP varies greatly in terms of platelet concentration, platelet quality and level of red and white blood cell contamination. This variability in PRP composition may affect clinical outcomes.
The devices presented here are based on a specific technology, patented worldwide, which allows the precise and reproducible separation of blood components according to their density using thixotropic separating gels in closed systems.
This allows the automated preparation of leukocyte-poor PRP with a standardized composition.
The production of PRP, in different forms, is a clinical asset to meet various therapeutic needs. That's why we offer solutions for preparing PRP in liquid or gel form, and PRP combined with hyaluronic acid. These biologics have been successfully used in many different therapeutic areas, resulting in over 150 published clinical studies.
Key words : platelet-rich plasma; PRP; tissue regeneration; cell therapy; regenerative medicine; autologous biologics; ortho-biologics; hyaluronic acid; HA; good manufacturing practices; GMP; medical devices.
Introduction
Major developments in regenerative medicine based on autologous tissues and cells (embryonic, stem or differentiated) have taken place since the beginning of the 20th century.
Platelet-rich plasma (PRP) therapy has gained popularity since the first reports of its clinical use in the 1980s and 1990s1. Reconstructive and orthopedic surgery techniques use the patient's own tissue, such as skin, fat, tendon or bone, to treat trauma or inherited defects. More recently, the idea of using cells or growth factors to stimulate tissue regeneration at the site of injury has emerged.
David R. Knighton described the first use of "locally acting growth factors obtained from human platelets and applied topically," highlighting the value of isolating growth factor-secreting platelets from whole blood to induce tissue regeneration, particularly in chronic wound healing. At the time, Knighton used laboratory techniques to prepare PRP (2,3).
PRP is easy to obtain, as it requires only a venipuncture. The blood components are then separated by centrifugation to obtain the fraction containing plasma and platelets. The relatively low cost and ease of use have facilitated the rapid expansion of PRP into medical practice (4,5).
The preparation of PRP has been greatly simplified in recent years with the development of commercial PRP preparation devices (6). These devices also allow for the preparation of PRP in compliance with health regulation and good practice requirements. As there are many publications in which PRP has been prepared using laboratory accessories, or tubes intended only for diagnostic use, most healthcare professionals are not aware that PRP for therapeutic use should only be prepared with certified medical devices intended for PRP preparation.
See, for example, Section 201(h) of the Federal Food, Drug, and Cosmetic Act, or Article 5 of the Medical Device Regulation 2017/745, for the United States or the European Union, respectively (7,8).
Standard process for the isolation of platelet-rich plasma (PRP).
There are many PRP preparation protocols, which differ in preparation devices, centrifugation conditions and operator dexterity, so that PRP is used to qualify biological products that vary widely in platelet concentration, quality and growth factor content, and level of contamination with pro-inflammatory red and white blood cells (9).
This wide variability in PRP preparations, and different treatment protocols, creates a challenge when trying to draw conclusions from the literature on the clinical benefits of PRP. For this reason, many PRP classification systems have been developed to facilitate the reporting of clinical study results (1,10).
However, the different technologies available produce mainly two types of PRP: plasma PRP and buffy coat PRP (10) (see Table 1).
Plasma PRP, is usually produced using low centrifugal force and/or very short centrifugation of a small volume of blood (usually less than 20 ml). Under these centrifugation conditions, the platelets still float in the plasma at the end of the centrifugation. They form a gradient, with the highest concentration of platelets near the white blood cell layer. With this technique, the PRP is generally a leukocyte-poor PRP (LP-PRP) with almost no red blood cell contamination and a platelet concentration factor of less than 3 times (3×) the baseline value in blood (10). For some devices, a second centrifugation is performed to increase the final platelet concentration beyond 3×, removing some of the platelet poor plasma (PPP). Therefore, these devices must process a larger volume of blood (60 ml) to obtain 6-7 ml of highly concentrated LP-PRP (11).
However, the composition of these LP-PRPs is highly operator dependent. Since there is no physical barrier between the plasma and the buffy coat, the PRP can easily be contaminated with blood cells if the operator gets too close to the buffy coat. On the other hand, if the operator stops collecting plasma too far from the buffy coat, the platelets with the highest density will not be recovered. The densest platelets are those richest in growth factors (12). Thus, this type of LP-PRP may contain less growth factors than other PRPs with the same platelet concentration, prepared using a technique that allows for more efficient recovery of platelets with the highest densities.
Buffy coat PRP is produced by high force centrifugation which concentrates the platelets in the buffy coat with the white blood cells. Depending on the device, the entire buffy coat or part of it is collected in varying volumes of plasma. The resulting PRP is a leukocyte-rich PRP (LR-PRP) with a platelet concentration factor greater than 4×. It contains concentrated pro-inflammatory white blood cells and a variable level of red blood cells and, therefore, is often reddish (10). A high volume of blood (25-60 ml or more) is required to produce 1-6 ml of PRP-LR, as the PPP must be removed to achieve a platelet concentration factor greater than 3×. Hourglass-shaped buffy coat collection devices are designed to collect all or most of the buffy coat. The operator, using the device's screw system, must move the buffy coat area into the narrowest part of the device, before collecting it. The composition of the resulting PRP is therefore operator-dependent and is contaminated with a high concentration of pro-inflammatory white blood cells (13). Devices that contain a floating tray or buoy of a specific density allow physical separation of blood components during centrifugation. Platelets and plasma, which have a lower density than the physical separator, are collected in a separate compartment of the device, from which the PRP is collected.
Although the physical separator retains most of the red and white blood cells, the resulting PRP is nevertheless a LR-PRP with a variable level of contamination by red blood cells and, consequently a pro-inflammatory PRP (14).
Computer-assisted automated systems are smaller versions of the apheresis machines used in blood donation centers to prepare platelet concentrates for transfusion. After separation by centrifugation with a variable g-force in a specific machine, the blood components pass through light sensors. The change in color and turbidity of the sample triggers a switch that sends the blood components into different bags or into a syringe. With this type of device, the composition of the PRP obtained depends on the setting. The operator programs the device according to the type of final product desired. However, it is not possible to set the cell contamination level below a certain threshold. For example, the minimum hematocrit setting is 2 % in one type of device (14). Therefore, the PRP produced with these devices is usually a leukocyte-rich PRP. It should be noted that the use of parameters that minimize red and white blood cell contamination induces a significant decrease in platelet recovery (<50%) and probably also the loss of the densest platelets (14)
A. Collection
Manufacture of innovative devices for the standardized preparation of PRP
To meet the need for standardized PRP preparations, Regen Lab, a Swiss pharmaceutical and medical device company, has developed complex polymer-gel separation systems that efficiently recover platelets and plasma and remove red and white blood cells in an automated closed-loop system. This innovative technology combines the advantages, without the disadvantages, of the buffy coat and plasma PRP preparation methods.
These devices address the various challenges of effective PRP preparation in accordance with international medical device regulations, which means that the devices are effective for PRP isolation and safe for patients and operators. It also implies that the manufacturer must comply with all standards and requirements related to the manufacture of medical devices. In addition, these devices must meet the needs of clinicians in various therapeutic areas.
1. Design and manufacture of medical devices for safe and effective preparation of PRP.
1.1 Essential requirements for the manufacturer
In addition to international medical device regulations, a medical device manufacturer must follow numerous standards and guidelines. As far as ISO standards are concerned, the list is not intended to be exhaustive. The manufacturer must have a quality management system certified to ISO 13485 and a risk management system (ISO 1471). It must perform ongoing clinical evaluation (ISO 14155) and post-market surveillance (ISO/TR 20416) to verify the safety and performance, including clinical benefit, of the device when used as intended by the manufacturer. Manufacturing processes must be validated.
The manufacturer must ensure that its devices are manufactured in a controlled environment, e.g., in clean rooms (ISO 14644, ISO 14698) with approved, pharmaceutical grade or biocompatibility tested materials (ISO 10993).
In addition, PRP preparation devices must be manufactured to be sterile (ISO 11737, 17665, 11137), packaged to maintain sterility throughout the life of the devices (ISO 11607), and labeled appropriately (ISO 15223). Second, in order to be marketed, a medical device must be approved by the health authorities in each country where it is marketed. Regulations differ from country to country, but the main requirements for approval are always safety and effectiveness of the devices.
In the European Union, there is a single regulation (the MDR 2017/745, which replaces the MDD 93/42 EEC) applicable for all members and for other countries outside the Union, such as Switzerland, that have decided to follow this regulation.
Under this regulation, medical devices for the preparation of PRP are classified as IIa or IIb and must be certified by a notified body.
Health authorities must also conduct a regulatory audit of manufacturers. To simplify this process, the Medical Device Single Audit Program (MDSAP) has been put in place. It allows for a single regulatory audit of the device manufacturer by a recognized auditing organization that meets the relevant requirements of the regulatory authorities participating in the program. The members of the MDSAP program are Australia, Brazil, Canada, Japan and the United States. Other countries, such as the United Kingdom and the European Union, are, for now, only observers. RegenLab's quality management system is certified under this program, as MDSAP certification is mandatory for the marketing of medical devices in Canada as of 2019.
1.2 Design of user-friendly devices for the preparation of standardized PRP
The manufacturer must determine the intended use and functional requirement specifications of its devices. In this case, the devices are intended for the preparation of standardized autologous PRP, and the primary functional requirements are that the devices be sterile, single-use, designed for bedside use by physicians to obtain a small amount of venous blood.
Sterility:
Medical devices are packaged in individual blisters and are sterilized by exposure to a minimum gamma irradiation dose of 25 kGy or, for devices containing hyaluronic acid, by moist heat. To maintain the sterility of the biological sample, a device intended for the preparation of PRP at the patient's bed must operate in a closed circuit (Figure 1).
This also has the advantage of minimizing the risk of blood exposure for the operator. Once the PRP is prepared, it is collected using a syringe connected to a transfer device. The use of these accessories ensures that the biological sample is not exposed to air and reduces the risk of microbial contamination. The tube holder and transfer device have an internal needle that does not cut into the tube cap. Thus, when the tube is removed from the accessories, it is still airtight. These tubes are made of pharmaceutical grade borosilicate glass type I (Ph. Eur &USP) with cerium and closed with bromobutyl rubber stoppers, tested for biocompatibility according to ISO 10993.
Anticoagulant :
To maintain the PRP in liquid form until it is used, a PRP device must contain a reversible anticoagulant. The use of a reversible anticoagulant is more convenient, provided that the anticoagulant does not have a side effect on the patient. Sodium citrate was chosen because citrate-based anticoagulants are fully reversible. Compared to ACD-A (acid citrate dextrose solution A), which is an acidic anticoagulant (pH 4.5-5) often used in the preparation of PRP, sodium citrate has the advantage of having a neutral pH (pH 7) and not containing any sugar (15). Therefore, sodium citrate is more physiological and has no side effects on the patient. It is pre-dosed in the tubes, thus avoiding the risks related to the handling of the anticoagulant by the operator.
Figure 1. Standardized PRP production process. Regen PRP tubes are designed for use in closed systems to maintain sterility of biological samples.
(A) The venipuncture is performed, and the desired number of Regen PRP tubes are filled with whole blood. The vacuum inside the tubes allows for automatic collection of the required volume of blood (approximately 10 ml).
(B) The tubes are carefully inverted three times to mix the blood with the anticoagulant.
(C) The tube is centrifuged with a relative centrifugal force of 1500 g for 5 to 9 minutes.
(D) After centrifugation, the blood is fractionated; red and white blood cells are trapped under the gel, and platelets settle on the surface of the gel.
(E) The tube is gently shaken to resuspend the platelets.
(F) The resulting PRP is collected using a syringe connected to a transfer device.
2. Standardized preparation of PRP with separating gels:2.
The main challenge when designing a device for PRP preparation is to find an efficient technology to isolate PRP. In order to be operator independent, physical separation is required to mechanically isolate platelets and plasma from other blood components.
As mentioned above, other devices on the market have been designed with floating tablets or buoys, or use computer-aided systems with a light sensor and valves, to physically separate blood components.
Compared to these technologies, the use of polymer separator gels has many advantages.
It can be used in small blood collection tubes, reducing the volume of blood needed, gel separation requires only a short centrifugation (5 or 9 min, depending on the type of gel) and it allows precise separation of blood components at the cellular level (16).
These separating gels are designed with a specific density; they are lighter than unwanted red and white blood cells and heavier than platelets and plasma.
These gels also have thixotropic properties that allow them to become fluid when subjected to a centrifugal force of 1500× g, and return to their original consistency when centrifugation is complete.
The separating gels used in RegenLab devices are biocompatible according to ISO 10993 and chemically inert, thus safe for the patient. During centrifugation, the blood components are separated according to their specific gravity and form distinct layers, which are the plasma, the buffy coat and the red cell pellet.
The buffy coat is a whitish area that contains platelets and white blood cells. The separating gel, thanks to its thixotropy, becomes fluid, migrates to the top of the device and intercalates precisely within the buffy coat at its own specific density.
At the end of the centrifugation, the separating gel returns to its solid consistency and forms a solid barrier that mechanically separates the blood components. It isolates platelets and plasma in the upper part of the tube, while unwanted red and white blood cells are trapped under the separating gel in the lower part.
Due to the centrifugal force, the platelets form a thin sediment on the top surface of the gel. To obtain PRP, the tube must be gently shaken to resuspend the platelets in the plasma (Figure 2). At this point, the preparation is ready to be used by the physician.
Because it is the polymer gel that separates the blood components based on their density, PRP isolation in these devices is specific and operator-independent, and is therefore reliable and reproducible.
The resulting PRP is a standardized PRP plasma with high platelet recovery (>80%) without specific loss of the densest platelets, low leukocyte levels, with specific depletion of pro-inflammatory white blood cells and virtually no red blood cells.
Red blood cells are undesirable in PRP because their degradation releases free radicals that induce oxidative stress and components, such as heme from hemoglobin, that are deleterious to cells [17,18].
Leukocytes can be divided into three main populations in the blood: granulocytes (65%), lymphocytes (30%) and monocytes (5%).
3. Different types of separator gels that differ in specific density have been developed.
This allows either the recovery of platelets only (RegenBCT/A-CP) or platelets and mononuclear white blood cells (lymphocytes and monocytes) (RegenTHT) with high efficiency. Even in the latter case, the resulting PRP is still an LP-PRP because the final concentration of total white blood cells remains below the baseline level in the blood. Depending on the type of gel separator, the recovery of mononuclear white blood cells ranges from 20 to 80 % (device performance data on file, available upon request). We believe that lymphocytes and monocytes present in the preparation may improve wound healing through their effects on modulation of inflammation, tissue remodeling and repair, and phenotypic presentation of macrophages (19). It has been shown previously that the difference in leukocyte concentration in PRP can affect the polarization of macrophages. Studies have shown that Rich PRP (LR-PRP) mainly increases the expression of M1 macrophages, while LP-PRP significantly induces the activity of M2 macrophages (20). It was concluded that the LP-PRP not only promotes cell proliferation through growth factors, but recruits repair cells throughout the tissue and bloodstream to promote tissue repair.
Other studies have demonstrated the beneficial effects of mononuclear cells (21-23). Monocytes are associated with increased cell metabolism and collagen production in fibroblasts, as well as decreased release of the antiangiogenic cytokines interferon-g and IL-12 (21,22). Previous studies have shown that platelets activate lymphocytes to help stimulate collagen production via increased IL-6 expression (21,22). Today, in clinics, there is still debate about whether leukocytes should be included in PRP. Some argue that leukocyte concentration should be reduced for intra-articular applications (24,25) and increased for tendon repair, for example (26). On the other hand, granulocytes, the main population of leukocytes, are pro-inflammatory cells. They are filled with granules that contain powerful destructive enzymes, such as peroxidases, proteases and lysosomal enzymes. The release of these molecules is crucial in fighting bacterial infection in open wounds; however, it has a deleterious effect in aseptic wounds. It has been shown in neutrophil-depleted mice that the absence of neutrophils, the most abundant type of granulocytes, does not affect skin healing and may even accelerate it (27). Thus, specific depletion of granulocytes in PRP could prevent adverse inflammatory reactions. Since the total volume of plasma is recovered on the separator gel, the platelet concentration factor in PRP prepared with RegenLab devices is approximately 1.5 to 1.7 times the baseline value in whole blood.
Due to the high quality of the recovered platelets, this low-concentration PRP has proven to be effective in all therapeutic areas in which it has been tested (see below). However, if deemed necessary, some of the platelet-poor plasma can be removed prior to the platelet resuspension step. The platelet concentration factor can thus be increased up to 3 to 4 times the baseline value in the blood.
PRP containing high quality platelets at a concentration slightly above the physiological value is relevant for therapeutic use, as it does not affect tissue homeostasis, and the low level of cellular contamination (mainly lymphocytes and monocytes) reduces the risk of adverse inflammatory reactions.It is important to note that too high a platelet concentration (more than 5 times the baseline value) has been shown to produce suboptimal results or cytoxic effects in vitro (28,29) and in animal models (30).
Comparative clinical studies also favor a less concentrated PRP (31,32). The benefits of using highly concentrated PRP versus PRP with a lower platelet concentration factor have not been demonstrated. The so-called therapeutic platelet concentration of 1 billion per ml (4 to 5 times higher than baseline values) (33) has never been demonstrated in comparative clinical studies and may be limited to leukocyte-rich PRP, as a higher platelet concentration may be required to offset the negative effects of pro-inflammatory white blood cells.
4. The know-how developed for the isolation of PRP with the gel separator can be used for the preparation of other biological products.
The gel that allows you to recover blood mononuclear cells (MNC) in addition to platelets can be used to process bone marrow aspirate for the preparation of bone marrow cell concentrates.
In this tube, bone marrow stem cells are recovered in the mononuclear cell fraction on the separator gel.
Recently, we have developed a new gel separator to isolate cell-free plasma for the preparation of convalescent plasma for patients who have recovered from an infection.
5. Production of PRP in different forms to meet therapeutic needs
Anticoagulation with sodium citrate is reversible. Citrate prevents clotting only by binding to plasma calcium ions in the blood sample used for PRP preparation. Calcium ions are essential cofactors in the coagulation cascade. Thus, when liquid PRP is injected into the tissues, it coagulates thanks to the supply of calcium ions by the interstitial fluid.
Nevertheless, for certain therapeutic applications, such as wound care, health care providers must obtain PRP gels or clots.
Coagulation of citrated PRP can be induced by activators, such as thrombin, calcium solution or a combination of both. There is often confusion between platelet activation and coagulation activation. There is a misconception that PRP must be activated to trigger the release of growth factors. This stems from early in vitro experiments, where the addition of activators, such as calcified thrombin, was necessary to extract growth factors from platelets (34).
Platelets are not simple vesicles filled with growth factors but functional entities that release growth factors in a controlled manner in response to local signals. Thus, in vitro, in the absence of activation, platelets do not release their growth factors. High doses of calcified thrombin are therefore required to induce complete platelet degranulation and uncontrolled growth factor release.
Endogenous platelet activation occurs when PRP is injected into the patient's tissue (10). Platelets are physiologically activated by contact with extracellular matrix proteins (e.g., collagen) at the injection site. For each stage of the healing process, platelets secrete different cocktails of growth factors, in response to local signals, to stimulate organized tissue repair. Exogenous activation is only necessary to obtain a gel PRP that coagulates rapidly at the injection site, or to obtain a fibrin clot, platelet-enriched autologous fibrin sealant or suturable fibrin membranes (35,36). These types of products are used, for example, to treat hard-to-heal wounds (37). This prevents the PRP from spreading and ensures a localized action.
We recommend the use of autologous serum that contains autologous thrombin at the physiological level to activate the PRP, alone or in combination with a pharmaceutical grade calcium solution.
This serum is prepared from the patient's blood using a specific device that also uses gel separator technology, but in a tube without anticoagulant.
The use of autologous thrombin allows the physiological formation of a fibrin clot in which platelets secrete growth factors in a controlled and sequential manner throughout the process of replacing the clot with new tissue.
6. Therapeutic areas in which clinical studies have been published.
Biological products prepared with Regen Lab devices are used in many therapeutic areas such as: sports medicine, orthopedic surgery, skin care and wound care, among others. More than 150 clinical studies with positive results have been published, see Table 2 (full list available on request).
7. Innovative biological combinations with hyaluronic acid for synergistic tissue regeneration
Endogenous hyaluronic acid (HA), one of the main components of the extracellular matrix, is a polysaccharide that belongs to the glycosaminoglycan family and is composed of a basic unit of two sugars, glucuronic acid and N-acetylglucosamine.
HA serves to maintain a highly hydrated environment, regulate osmotic balance, absorb shock, fill space, and serve as a lubricant. It plays a role in cell migration and physiological angiogenesis. Among its unique characteristics, its biocompatibility and biodegradability are very important for its clinical use. HA generally exists in high molecular weight form in the synovial fluid that surrounds joints, cartilage, and tissues of the eye and skin (38). It is considered a key player in the tissue regeneration process (39,40). It has been shown to modulate inflammation, cell migration and angiogenesis, which are the main phases of wound healing (41). The biological properties of HA are related to its molecular size: high molecular weight HA exhibits anti-inflammatory, immunosuppressive and anti-angiogenic properties, while low molecular weight HA exhibits potent pro-inflammatory and pro-angiogenic molecules.
Exogenous HA can be processed and functionalized by physical and chemical modifications and cross-linking to generate versatile HA-based hydrogels with various clinical applications (42). HA has many qualities, such as moisturizing and anti-aging effects, which recommend it over other substances used in skin regeneration (38,43).
The molecular weight of HA influences its penetration into the skin and its biological activity (43). It can be injected intradermally or used topically. HA is commonly used in other clinical applications, including intra-articular injections, ophthalmic surgery and tissue engineering (vascular, skin, cartilage, bone) (44).
Cellular Matrix® :
Many physicians are interested in combining PRP with HA. Because PRP and HA target different pathways and have different functions, when used together, they can have a synergistic effect as a therapeutic approach for healing, inflammation or analgesic purposes.
Cellular Matrix is the first and, to date, the only CE-certified medical device that allows the combination of HA and PRP in compliance with medical device and health regulations. HA creates a cell-friendly matrix in which platelets are suspended. This biologically enriched network facilitates the migration and proliferation of cells to the treated site.
Indeed, HA is an implantable medical device and PRP is a biological drug. Therefore, healthcare professionals are not expected to prepare their own PRP-HA mixture by combining any HA with any PRP, as modifications of medical devices or biological drugs are not permitted. This device has been designed to allow the rapid and safe preparation of PRP in the presence of high-quality, non-cross-linked HA produced by bacterial fermentation, using technology similar to that used for PRP isolation, but with HA preloaded into the tube (Figure 3).
Conclusion
Regenerative medicine encompasses a wide range of techniques aimed at repairing or even replacing damaged or aged tissue.
Of these, autologous platelet-rich plasma is one of the simplest and most effective. This approach is based on the intrinsic capacity of the human body to self-repair and the role of platelets in this process.
There is growing interest in the use of standardized PRP, alone or in combination, in regenerative medicine as it represents a safe and natural treatment and has, to date, shown promising results in a wide range of therapeutic indications.
Many medical devices for the preparation of PRP are on the market. They vary greatly in terms of technology and final PRP composition. We discussed their specifics and limitations in relation to our technology.
PRP is now a key player in the medical world with millions of patients treated each year.
However, limitations related to the lack of a standardized procedure for its preparation make it difficult to compare the available clinical data. The technology described in this publication provides a solution to the challenge of standardizing PRP preparations for therapeutic use.
References
- Wu, P.I.; Diaz, R.; Borg-Stein, J. Platelet-Rich Plasma. Physical medicine and rehabilitation clinics of North America 2016, 27, 825-853, doi:10.1016/j.pmr.2016.06.002.
- Knighton, D.R.; Hunt, T.K.; Thakral, K.K.; Goodson, W.H., 3rd. Role of platelets and fibrin in the healing sequence: an in vivo study of angiogenesis and collagen synthesis. Ann Surg 1982, 196, 379-388, doi:10.1097/00000658-198210000-00001.
- Knighton, D.R.; Ciresi, K.F.; Fiegel, V.D.; Austin, L.L.; Butler, E.L. Classification and treatment of chronic nonhealing wounds. Successful treatment with autologous platelet-derived wound healing factors (PDWHF). Ann Surg 1986, 204, 322-330, doi:10.1097/00000658-198609000-00011.
- Labusca, L.S.; Cionca, D. Clinical review about the role of platelet rich plasma for the treatment of traumatic and degenerative musculoskeletal disorders. Ortho & Rheum Open Access J 2016, 2OROAJ.MS.ID.55589.
- Cohn, C.S.; Lockhart, E. Autologous platelet-rich plasma: evidence for clinical use. Current opinion in hematology 2015, 22, 527-532, doi:10.1097/MOH.0000000000000183.
- Engebretsen, L.; Steffen, K.; Alsousou, J.; Anitua, E.; Bachl, N.; Devilee, R.; Everts, P.; Hamilton, B.; Huard, J.; Jenoure, P.; et al. IOC consensus paper on the use of platelet-rich plasma in sports medicine. Br J Sports Med 2010, 44, 1072-1081, doi:10.1136/bjsm.2010.079822.
- Federal Food, Drug, and Cosmetic Act (FD&C Act). As Amended Through P.L. 117-103, Enacted March 15, 2022 U.S. Department of Health and Human Services.
- REGULATION (EU) 2017/745 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 5 April 2017 on medical devices, amending Directive 2001/83/EC, Regulation (EC) No 178/2002 and Regulation (EC) No 1223/2009 and repealing Council Directives 90/385/EEC and 93/42/EEC.
- Harmon, K.; Hanson, R.; Bowen, J.; Greenberg, S.; Magaziner, E.; Vandenbosch, J.; Harshfield, D.; Shiple, B.; Audley, D. Guidelines for the Use of Platelet Rich Plasma. The International Cellular Medical Society 2011.
- DeLong, J.M.; Russell, R.P.; Mazzocca, A.D. Platelet-rich plasma: the PAW classification system. Arthroscopy : the journal of arthroscopic & related surgery : official publication of the Arthroscopy Association of North America and the International Arthroscopy Association 2012, 28, 998-1009, doi:10.1016/j.arthro.2012.04.148.
- Maisel-Campbell, A.L.; Ismail, A.; Reynolds, K.A.; Poon, E.; Serrano, L.; Grushchak, S.; Farid, C.; West, D.P.; Alam, M. A systematic review of the safety and effectiveness of platelet-rich plasma (PRP) for skin aging. Arch Dermatol Res 2020, 312, 301-315, doi:10.1007/s00403-019-01999-6.
- Corash, L.; Tan, H.; Gralnick, H.R. Heterogeneity of human whole blood platelet subpopulations. I. Relationship between buoyant density, cell volume, and ultrastructure. Blood 1977, 49, 71-87.
- Oh, J.H.; Kim, W.; Park, K.U.; Roh, Y.H. Comparison of the Cellular Composition and Cytokine-Release Kinetics of Various Platelet-Rich Plasma Preparations. The American journal of sports medicine 2015, 43, 3062-3070, doi:10.1177/0363546515608481.
- Degen, R.M.; Bernard, J.A.; Oliver, K.S.; Dines, J.S. Commercial Separation Systems Designed for Preparation of Platelet-Rich Plasma Yield Differences in Cellular Composition. HSS journal : the musculoskeletal journal of Hospital for Special Surgery 2017, 13, 75-80, doi:10.1007/s11420-016-9519-3.
- Gorgu, M.; Gokkaya, A.; Dogan, A. Comparison of Two Anticoagulants for Pain Associated with Platelet-Rich Plasma Injections. Aesthetic Plast Surg 2020, 44, 955-961, doi:10.1007/s00266-019-01541-z.
- Bowen, R.A.; Remaley, A.T. Interferences from blood collection tube components on clinical chemistry assays. Biochemia medica 2014, 24, 31-44, doi:10.11613/BM.2014.006.
- Larsen, R.; Gouveia, Z.; Soares, M.P.; Gozzelino, R. Heme cytotoxicity and the pathogenesis of immune-mediated inflammatory diseases. Front Pharmacol 2012, 3, 77, doi:10.3389/fphar.2012.00077.
- Boswell, S.G.; Cole, B.J.; Sundman, E.A.; Karas, V.; Fortier, L.A. Platelet-rich plasma: a milieu of bioactive factors. Arthroscopy 2012, 28, 429-439, doi:10.1016/j.arthro.2011.10.018.
- Brancato, S.K.; Albina, J.E. Wound macrophages as key regulators of repair: origin, phenotype, and function. Am J Pathol 2011, 178, 19-25, doi:10.1016/j.ajpath.2010.08.003.
- Nishio, H.; Saita, Y.; Kobayashi, Y.; Takaku, T.; Fukusato, S.; Uchino, S.; Wakayama, T.; Ikeda, H.; Kaneko, K. Platelet-rich plasma promotes recruitment of macrophages in the process of tendon healing. Regenerative therapy 2020, 14, 262-270, doi:10.1016/j.reth.2020.03.009.
- Yoshida, R.; Murray, M.M. Peripheral blood mononuclear cells enhance the anabolic effects of platelet-rich plasma on anterior cruciate ligament fibroblasts. Journal of orthopaedic research : official publication of the Orthopaedic Research Society 2013, 31, 29-34, doi:10.1002/jor.22183.
- Naldini, A.; Morena, E.; Fimiani, M.; Campoccia, G.; Fossombroni, V.; Carraro, F. The effects of autologous platelet gel on inflammatory cytokine response in human peripheral blood mononuclear cells. Trays 2008, 19, 268-274, doi:10.1080/09537100801947426.
- Zhou, Y.; Zhang, J.; Wu, H.; Hogan, M.V.; Wang, J.H. The differential effects of leukocyte-containing and pure platelet-rich plasma (PRP) on tendon stem/progenitor cells - implications of PRP application for the clinical treatment of tendon injuries. Stem cell research & therapy 2015, 6, 173, doi:10.1186/s13287-015-0172-4.
- Kim, J.H.; Park, Y.B.; Ha, C.W.; Roh, Y.J.; Park, J.G. Adverse Reactions and Clinical Outcomes for Leukocyte-Poor Versus Leukocyte-Rich Platelet-Rich Plasma in Knee Osteoarthritis: A Systematic Review and Meta-analysis. Orthopaedic journal of sports medicine 2021, 9, 23259671211011948, doi:10.1177/23259671211011948.
- Cavallo, C.; Filardo, G.; Mariani, E.; Kon, E.; Marcacci, M.; Pereira Ruiz, M.T.; Facchini, A.; Grigolo, B. Comparison of platelet-rich plasma formulations for cartilage healing: an in vitro study. The Journal of bone and joint surgery. American volume 2014, 96, 423-429, doi:10.2106/JBJS.M.00726.
- Fitzpatrick, J.; Bulsara, M.; Zheng, M.H. The Effectiveness of Platelet-Rich Plasma in the Treatment of Tendinopathy: A Meta-analysis of Randomized Controlled Clinical Trials. The American journal of sports medicine 2017, 45, 226-233, doi:10.1177/0363546516643716.
- Dovi, J.V.; Szpaderska, A.M.; DiPietro, L.A. Neutrophil function in the healing wound: adding insult to injury? Thromb Haemost 2004, 92, 275-280, doi:10.1267/THRO04080275.
- Graziani, F.; Ivanovski, S.; Cei, S.; Ducci, F.; Tonetti, M.; Gabriele, M. The in vitro effect of different PRP concentrations on osteoblasts and fibroblasts. Clinical oral implants research 2006, 17, 212-219, doi:10.1111/j.1600-0501.2005.01203.x.
- Yoshida, R.; Cheng, M.; Murray, M.M. Increasing platelet concentration in platelet-rich plasma inhibits anterior cruciate ligament cell function in three-dimensional culture. Journal of orthopaedic research : official publication of the Orthopaedic Research Society 2014, 32, 291-295, doi:10.1002/jor.22493.
- Fleming, B.C.; Proffen, B.L.; Vavken, P.; Shalvoy, M.R.; Machan, J.T.; Murray, M.M. Increased platelet concentration does not improve functional graft healing in bio-enhanced ACL reconstruction. Knee surgery, sports traumatology, arthroscopy: official journal of the ESSKA 2015, 23, 1161-1170, doi:10.1007/s00167-014-2932-6.
- Filardo, G.; Kon, E.; Pereira Ruiz, M.T.; Vaccaro, F.; Guitaldi, R.; Di Martino, A.; Cenacchi, A.; Fornasari, P.M.; Marcacci, M. Platelet-rich plasma intra-articular injections for cartilage degeneration and osteoarthritis: single- versus double-spinning approach. Knee surgery, sports traumatology, arthroscopy: official journal of the ESSKA 2012, 20, 2082-2091, doi:10.1007/s00167-011-1837-x.
- Rappl, L.M. Effect of platelet rich plasma gel in a physiologically relevant platelet concentration on wounds in persons with spinal cord injury. Int Wound J 2011, 8, 187-195, doi:10.1111/j.1742-481X.2011.00770.x.
- Marx, R.E. Platelet-rich plasma (PRP): what is PRP and what is not PRP? Implant dentistry 2001, 10, 225-228.
- Ross, R.; Glomset, J.; Kariya, B.; Harker, L. A platelet-dependent serum factor that stimulates the proliferation of arterial smooth muscle cells in vitro. Proceedings of the National Academy of Sciences of the United States of America 1974, 71, 1207-1210.
- Hersant, B.; SidAhmed-Mezi, M.; La Padula, S.; Niddam, J.; Bouhassira, J.; Meningaud, J.P. Efficacy of Autologous Platelet-rich Plasma Glue in Weight Loss Sequelae Surgery and Breast Reduction: A Prospective Study. Plastic and reconstructive surgery. Global open 2016, 4, e871, doi:10.1097/GOX.0000000000000823.
- Gumina, S.; Campagna, V.; Ferrazza, G.; Giannicola, G.; Fratalocchi, F.; Milani, A.; Postacchini, F. Use of platelet-leukocyte membrane in arthroscopic repair of large rotator cuff tears: a prospective randomized study. J Bone Joint Surg Am 2012, 94, 1345-1352, doi:10.2106/JBJS.K.00394.
- Hu, Z.; Qu, S.; Zhang, J.; Cao, X.; Wang, P.; Huang, S.; Shi, F.; Dong, Y.; Wu, J.; Tang, B.; et al. Efficacy and Safety of Platelet-Rich Plasma for Patients with Diabetic Ulcers: A Systematic Review and Meta-analysis. Advances in wound care 2019, 8, 298-308, doi:10.1089/wound.2018.0842.
- Neuman, M.G.; Nanau, R.M.; Oruna-Sanchez, L.; Coto, G. Hyaluronic acid and wound healing. Journal of pharmacy & pharmaceutical sciences : a publication of the Canadian Society for Pharmaceutical Sciences, Societe canadienne des sciences pharmaceutiques 2015, 18, 53-60, doi:10.18433/j3k89d.
- Abatangelo, G.; Vindigni, V.; Avruscio, G.; Pandis, L.; Brun, P. Hyaluronic Acid: Redefining Its Role. Cells 2020, 9, doi:10.3390/cells9071743.
- Litwiniuk, M.; Krejner, A.; Speyrer, M.S.; Gauto, A.R.; Grzela, T. Hyaluronic Acid in Inflammation and Tissue Regeneration. Wounds: a compendium of clinical research and practice 2016, 28, 78-88.
- Lierova, A.; Kasparova, J.; Filipova, A.; Cizkova, J.; Pekarova, L.; Korecka, L.; Mannova, N.; Bilkova, Z.; Sinkorova, Z. Hyaluronic Acid: Known for Almost a Century, but Still in Vogue. Pharmaceutics 2022, 14, doi:10.3390/pharmaceutics14040838.
- Ding, Y.W.; Wang, Z.Y.; Ren, Z.W.; Zhang, X.W.; Wei, D.X. Advances in modified hyaluronic acid-based hydrogels for skin wound healing. Biomaterials science 2022, 10, 3393-3409, doi:10.1039/d2bm00397j.
- Juncan, A.M.; Moisa, D.G.; Santini, A.; Morgovan, C.; Rus, L.L.; Vonica-Tincu, A.L.; Loghin, F. Advantages of Hyaluronic Acid and Its Combination with Other Bioactive Ingredients in Cosmeceuticals. Molecules 2021, 26, doi:10.3390/molecules26154429.
- Dovedytis, M.L., JZ; Bartlett, S. Hyaluronic acid and its biomedical applications: A review. Engineered Regeneration 2020, 1, doi:10.1016/j.engreg.2020.10.001.
Author contributions: Conceptualization and writing - preparation of original version, F.G., S.V., and S.B.; writing - revision and editing, F.G., S.V., and S.B.; visualization, S.B.; supervision, A.T. and S.B.; project administration, A.T. and S.B.; acquisition of funding, A.T. All authors have read and approved the published version of the manuscript.