Complex tissue engineering concepts have to be evaluated in terms of their clinical applicability. Thus, the overall goal should be to establish a method that could ideally be completed within a short time span before or during intended regenerative surgical procedures. Over the last few years, Choukroun's PRF has proven to be a method that comes close to the ideal concept of guided (“smart”) tissue engineering. As previously shown, this form of PRF contains growth factors such as TGF-β1 and PDGF-AB²³  and can contribute to tissue regeneration in terms of osteoblast, prekeratinocyte, and gingival fibroblast differentiation.¹⁸ 

Even though the experiments mentioned above show the promising capacities of this concept in terms of contribution to successful tissue regeneration, up until now, there have been no studies showing the extent to which the cells are distributed into the fibrin scaffold in relation to the two essential parameters centrifugation speed and centrifugation time.

In the present work, we have shown that T-lymphocytes, B-lymphocytes, stem cells, and monocytes are found in both groups within the first 25–30% proximal part of the clot. There are no statistically significant differences between both groups in terms of distribution of those cells.

The tissue regeneration or repair process requires harmonious reaction of various types of cells, including immune response cells (neutrophils, macrophages, lymphocytes), epithelial cells, fibroblasts, and stem cells, as well as other cells relevant for the tissue in question. The PRF scaffold concept seems to be an ideal source of components for the healing process. An autologous blood-derived scaffold can be a unique source of hematopoietic stem cells (HSCs), which are of major importance in regenerative medicine. In the past decade, many studies highlighted the vast differentiation potential of HSCs.^28,29  A recent review by Ogawa et al presented strong evidence for the pluripotent capacity of HSCs and summarized work that has been done in this field.³⁰  Apart from their ability to replenish the majority of cell types in the body, stem cells also play a role as immune modulators³¹ : They can target B-lymphocytes and stimulate antibody secretion, inhibit or even lead to apoptosis of T-lymphocytes, and induce immune tolerance.

Other cells that can be observed in these advanced fibrin clots are B- and T-lymphocytes. Lymphocytes are responsible for specific and nonspecific intervention in tissue response for injury, although they are not prominent in the first phase of tissue repair. A study by Boyce et al revealed that CD8+ T-lymphocytes decreased wound healing, whereas B-lymphocytes were associated with an increased healing.^32,33 

In contrast to the cells mentioned above, platelets are distributed more evenly throughout the entire clot. It appears that a decrease in centrifugation speed and an increase in centrifugation time results in higher platelet concentrations in the distal part of clot, although this observation was not definitively proven. Platelets, providing the name for these fibrin-rich scaffolds (ie, platelet-rich fibrin) are not only present in the coagulation pathway or primary wound closure but also have a vast regenerative potential by releasing a broad spectrum of cytokines, chemokines, growth factors, and other mediators. Platelets are able to release, amongst others, molecules such as von Willebrand factor, P-selectin, fibronectin, VEGF, platelet-derived endothelial growth factor (PDEGF), vitronectin, and fibrinogen. With these different growth factors, adhesion molecules, and other mediators, platelets have the ability to initiate and modulate host immune responsiveness through influencing neutrophils, monocytes, and endothelial cells, as well as lymphocytes. Upon stimulation, platelets actively participate in pathogen detection, capturing, and sequestration. They can even induce the death of infected cellular targets. Thanks to this wide range of activities, platelets are visible at each step of regeneration.³⁴ 

Monocytes are also essential for tissue healing. They migrate into the inflamed area after the influx of neutrophils, where they then become macrophages.³⁵  Macrophages are multifunctional cells that represent distinct phenotypes. They have substantial roles in foreign body response, osteogenesis, and angiogenesis as they respond to inserted biomaterials.^36,37  Through expression of VEGF, PDGF, FGF, TGFα and –β, and other biologically active molecules (eg, BMP-2), macrophages support cell proliferation and tissue restoration following injury.^36,38  They are seen throughout all the processes of tissue repair from early inflammation through tissue-remodeling and scar formation.³⁸ 

One of the most important findings of the present study is that changing the centrifugation protocol in terms of centrifugation time and speed leads to a different distribution pattern for neutrophilic granulocytes. Neutrophilic granulocytes are most commonly considered to be early inflammatory cells due to their phagocytotic capacity, degranulation, and neutrophilic extracellular traps.^40,41  However, recent studies have shown that neutrophilic granulocytes have tissue regeneration properties as well. Neutrophils also facilitate trafficking of monocytes into the wound to phagocytose inflammatory remnants (necrotic and apoptotic cells).^40,42  Moreover, they also participate in the process of wound debridement by secreting several proteases, including matrix metalloproteinase 9 (MMP9), an extracellular matrix digesting enzyme. Furthermore, neutrophilic granulocytes expressing MMP9 play a part in the process of revascularization of the tissue defect by being recruited, for example, by VEGF-A.^17,40  Neutrophilic granulocytes and monocytes/macrophages are in mutual communication, and their interplay contributes to further differentiation towards a pro- or anti-inflammatory state of the macrophages. Additionally, recent studies of Tan et al revealed a potential contribution of neutrophils to inflammatory lymphogenesis by VEGF-A modulation and VEGF-D secretion in a murine model.⁴³  These cells modulate the innate as well as adaptive immune response in a direct and indirect manner by crosstalk with B- and T-lymphocytes.⁴⁴  Thus, the distribution of neutrophilic granulocytes within the A-PRF clot might be the basis for a better functionality of the transplanted (but also resident) monocytes/macrophages and lymphocytes and their deployment to support tissue regeneration.

Aside from the neutrophilic granulocyte's role in early inflammation and its potential regenerative properties, it has to be questioned why granulocytes are capable of migrating deeper into the matrix of the clot. One approach to addressing this question is the neutrophilic diameter. Thus, with an average diameter of 8.5–10 μm neutrophilic granulocytes are remarkably smaller than monocytes (diameter of 15–20 μm⁴⁵) and therefore could be more prone to penetrate deeper into the clot during the centrifugation process. If this, however, is true, it needs to be determined whether the weight of neutrophils correlates with their diameter. Basically, differential centrifugation is based on the differences in the sedimentation rate of the (biological) particles of difference size, shape, and density, as well as conditions of centrifugation. However, this present study failed to uncover why the neutrophilic granulocytes, in particular, “react” toward modifications in the centrifugation protocol. Therefore, further research has to be performed to validate these findings and give a scientific explanation for the different neutrophil behavior.

Among the ideas that are presently being established in the field of tissue regeneration—such as the use of co-cultures or monocultures, for example with osteoblasts or fibroblasts³—the PRF concept could be an additional component suitable for clinical applications. This concept of generating a fibrin-based cell-seeded matrix solely by drawing blood and centrifugation for 12–14 minutes is truly revolutionary in terms of clinical practicability, as it can be handled and modified easily in a short period of time and provides the defect not only with a matrix permitting cell migration into the defected area but also providing the wound with crucial biological cues, potentially accelerating the wound-healing process: These include platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), platelet factor 4 (PF4), IL-1, vascular endothelial growth factor (VEGF), epidermal growth factor, endothelial cell growth factor (ECGF), platelet-derived endothelial growth factor (PDEGF), insulin-like growth factor, osteocalcin, osteonectin, fibrinogen, vitronectin, fibronectin, and thrombospondin.⁶  Furthermore, the fact that it is a matrix that is generated by the patient's own (ie, autologous) blood, there is virtually no risk of a rejection reaction (foreign body response).

The future of clinically applicable tissue engineering concepts will most likely be spearheaded by cell-based tissue constructs that can be generated in a short time span and in close proximity to the patient. Nevertheless, it still has to be determined how these constructs, including Choukroun's variation of PRF, will interact in vitro and in vivo after combination with cultured osteoblasts, fibroblasts, or a mixture with osteoconductive granules in vivo. It still remains to be shown in well-designed clinical studies that this fibrin-based matrix can facilitate guided bone-tissue engineering with simultaneous avoidance of obstacles such as infection, rejection, and cumbersome isolation methods. It is hoped that such clinical studies will also reveal to what extent the neutrophilic granulocytes contribute to the regenerative properties of the PRF.

The present data show that specific cell types are distributed differentially depending on the (cumulative) centrifugal force. This concept enables the optimal scaffold or composites to be tailored for specific clinical applications. These powerful composites can contribute to wound healing and tissue repair as well as tissue regeneration. Additionally, A-PRF appears to be an ideal provider of autologous cells (especially neutrophils and macrophages), thus enabling mutual stimulation, thereby creating a synergistic relationship in the interest of tissue regeneration.