A technical note on contamination from PRF tubes containing silica and silicone

Abstract

Background: Platelet-rich fibrin (PRF) has been widely utilized in modern medicine and dentistry owing to its ability to rapidly stimulate neoangiogenesis, leading to faster tissue regeneration. While improvements over traditional platelet rich plasma therapies (which use chemical additives such as bovine thrombin and calcium chloride) have been observed, most clinicians are unaware that many tubes utilized for the production of 'natural' and '100% autologous' PRF may in fact contain chemical additives without appropriate or transparent knowledge provided to the treating clinician. The aim of this overview article is therefore to provide a technical note on recent discoveries related to PRF tubes and describe recent trends related to research on the topic from the authors laboratories.

Methods: Recommendations are provided to clinicians with the aim of further optimizing PRF clots/membranes by appropriate understanding of PRF tubes. The most common additives to PRF tubes reported in the literature are silica and/or silicone. A variety of studies have been performed on their topic described in this narrative review article.

Results: Typically, PRF production is best achieved with plain, chemical-free glass tubes. Unfortunately, a variety of other centrifugation tubes commonly used for lab testing/diagnostics and not necessarily manufactured for human use have been utilized in clinical practice for the production of PRF with unpredictable clinical outcomes. Many clinicians have noted an increased variability in PRF clot sizes, a decreased rate of clot formation (PRF remains liquid even after an adequate protocol is followed), or even an increased rate in the clinical signs of inflammation following the use of PRF.

Conclusion: This technical note addresses these issues in detail and provides scientific background of recent research articles on the topic. Furthermore, the need to adequately select appropriate centrifugation tubes for the production of PRF is highlighted with quantitative data provided from in vitro and animal investigations emphasizing the negative impact of the addition of silica/silicone on clot formation, cell behavior and in vivo inflammation.

Keywords: A-PRF; I-PRF; L-PRF; Platelet rich fibrin; Platelets.

Fig. 1

Final sizes of PRF clots produced utilizing 3 different centrifugation tubes in each of 3 different centrifugation devices (a) total of 9 tested groups). (A) Final PRF clot weight sizes depicted when different groups of tubes are used. (B) Final PRF clot weight sizes depicture when different centrifugation systems were utilized. Notice that in general, the IntraSpin centrifugation device produced slightly large clots (roughly 15%), whereas the glass tubes (Process for PRF, Salvin) produced the largest PRF clots (roughly 200–250% larger than those produced with plastic silica-coated IntraSpin tubes). Reprinted with permission from Miron et al. 2019 [11]

Fig. 2

In this experiment, PRF clots were produced in 3 different commercially available tubes containing silica. Following centrifugation, clots were removed, the PRF clots were enzymatically digested, and ‘leftover’ remaining silica particles were visually assessed by scanning electron microscopy (SEM). SEM observations of silica microparticles contained in a Neotubes, b Vacuette tubes and c Venoject II tubes at low (top) and high magnification (bottom). Note the high incorporation of silica microparticles detached from PRF tube walls into PRF clots. Reprinted with permission from Tsujino et al. 2019 [10]

Fig. 3

Microstructural images of human periosteal cells treated with silica microparticles. The cells treated with silica microparticles derived from Neotubes (1:8 dilution) for 24 h were fixed and examined using SEM at a low magnification and b high magnification. Note that the cells rapidly incorporated silica with high affinity. Similar observations were obtained from four other independent experiments, including those involving Vacuette’s silica. Reprinted with permission from Masuki et al. 2020 [12]

Fig. 4

Fluorescence visualization of apoptosis in human periosteal cells treated with silica microparticles. The cells were treated with silica microparticles derived from Neotubes for 24 h. The fixed cells were probed with PE-conjugated annexin V for detection of phosphatidylserine on the cell surface, which is accepted as a marker of apoptosis, and are shown at a low magnification and b high magnification. The cells were counterstained with FITC-conjugated phalloidin to visualize cytoskeletal polymerized actin. Similar observations were obtained from four other independent experiments, including those involving Vacuette’s silica. Reprinted with permission from Masuki et al. 2020 [12]

Fig. 5

Comparative analysis of PRF tube clot weights and sizes from 6 individuals after centrifugation utilizing the same centrifugation speed and time and either plain glass tubes or silica-coated plastic tubes. a In general, the silica-coated plastic tubes on average decrease the final weight of PRF-based matrices nearly twofold. b Over a 10-day period, while both PRF clots are slowly and gradually degraded over time, significantly increased amounts of membrane remain at 0, 3 and 7 days. Both glass tubes and silica-coated plastic tubes were filled to 9 mL (Reprinted with permission from Miron et al. 2021 [13]

Fig. 6

Effect of the addition of silicone to glass tubes. (A) Comparative weight analysis of PRF clots produced in plain glass tubes versus those produced in A-PRF glass tubes coated with silicone. Clot membranes increase more than 200% in plain glass tubes. Silicone may be more detrimental to standard clot size than silica (Reprinted with permission from Miron et al. 2021 [13]

Fig. 7

Experimental setup describing the orientation of PRF membranes during histological assessment. The proximal surface describes the inner tube wall (generally receiving the smallest g-force), whereas the distal surface is the outer tube wall, where cells generally accumulate during centrifugation at high g-force. a Regions in compressed A‐PRF or CGF matrix. This image is the proximal surface. b Centrifugal force and distal and proximal surfaces of A‐PRF or CGF matrix. A‐PRF, advanced platelet-rich fibrin; CGF, concentrated growth factors. Reprinted with permission from Takahishi et al. 2019 [14]

Fig. 8

Distribution of PDGF‐BB in A‐PRF and CGF matrices. a, d Region 1, b, e region 2, and c, f region 3. a–c Low magnification, d–f high magnification. Note that the majority of cells and growth factor accumulate on the back distal surfaces of PRF tubes. A‐PRF, advanced platelet‐rich fibrin; CGF, concentrated growth factors; PDGF‐BB, platelet‐derived growth factor‐BB. Reprinted with permission from Takahishi et al. 2019 [14]

Fig. 9

Macroscopic observation of a compressed and fixed A-PRF membrane. a This PRF membrane was divided into seven pieces, designated regions 1 to 7, where region 1 represents the region closest to the red blood cell fraction. b Microscopic observation of A-PRF cross-sections obtained from individual regions. Cross-sections were stained with hematoxylin and eosin (HE). c To confirm morphological similarity, the size of sections was modified to adjust their lengths at similar levels. Arrows represent the direction of gravity force. Summary of platelet distribution under various conditions. Asterisks represent wide-open spaces, and platelets were distributed sparsely. Double asterisks represent wider spaces than single asterisks. Bar scales are 200um. In general, it was found that cells were more evenly distributed at low centrifugation speeds, whereas high-speed centrifugation typically led to more cells accumulated at the back distal surfaces and closer to the buffy coat. Reprinted with permission from Tsujino et al. 2019 [15]