Platelet-rich plasma and other blood derivatives are widely used in various fields of medicine and tissue engineering. They are natural cocktails of autologous growth factors growth factors cocktail, which may provide an alternative to recombinant protein formulations. Such derivatives blood have consistently demonstrated potentiation proliferation, migration and differentiation of stem cells. Here we review the spectrum of platelet-rich platelet-rich blood derivatives and discuss their current applications in tissue engineering and regenerative medicine, consider their effects on stem cells and highlight the current challenges applications.
Platelet-rich plasma is seen as a very promising regenerative medicine. Tissue engineering has traditionally stimulates cells using a single bioactive agent with key regenerative functions. However, natural tissue regeneration is based on a cocktail of signaling molecules and growth factors. During natural wound healing, activated platelets concentrate in the wound area and secrete a multitude of factors that play an important role in coordinating wound healing.
Using a single growth factor to control tissue regeneration represents too simplistic and ineffective an intervention. This is usually is usually solved by providing supraphysiological amounts of growth factor. As a consequence, a rapidly growing number of studies have investigated the efficacy of stem cell-based tissue engineering approaches with a natural growth factor cocktail, such as platelet concentrate. This has paved the way for improved stem cell function, including stem cell growth, viability, proliferation, differentiation and overall regenerative potential. Thus, platelet concentrates are widely used in medicine. Moreover, their use is supported by their availability, cost-effectiveness, wide range of applications and autologous nature. Indeed, several clinical applications have been reported platelet concentrates in the fields of dermatology, orthopaedics, dentistry and ophthalmology.
Platelet Rich Plasma
Platelet activation causes degranulation and subsequent release of trophic factors affecting wound healing, tissue repair, angiogenesis and stem cell behaviour. Two types of granules are present within platelets: alpha granules and dense granules. The alpha granules influence wound healing with several types of growth factors including: platelet-derived growth factor (PDGF), epithelial growth factor (EGF), vascular endothelial growth factor (VEGF), endothelial cell growth factor (ECGF), fibroblast growth factor (FGF), transforming growth factor-beta (TGF-β) and insulin-like growth factor (IGF). In general, these factors chemotactically attract and activate stem cells and induce their mitogenesis and differentiation. In contrast, dense granules promote tissue regeneration by secreting mediators such as serotonin and histamine, which increase vascular permeability and tissue perfusion. Several platelet-rich blood derivatives have been studied over the years. Above all, they have received considerable attention in tissue regeneration studies aimed at the healing of damaged soft and hard tissues.
Platelet-rich plasma (PRP) was introduced several decades ago. RPE is obtained by a two-step preparation of a blood sample for centrifugation, which is solidified with an anticoagulant. In the first centrifugation step, three layers are separated: plasma on top, red blood cells on the bottom, and the middle layer, which contains platelets and white blood cells. After the erythrocytes have been discarded, the residue is centrifuged a second time to ensure proper separation of the plasma. The OTPC is further obtained by discarding the plasma.
This process concentrates platelets by a factor of approximately 4-5 compared to untreated whole blood. By varying the separation protocol, different mixtures can be obtained. For example, different centrifugation settings (speed, time) can change the concentration of platelets. In one such approach, Amabel et al. investigated how centrifugation and temperature parameters affect the final product. The elimination of erythrocytes and leucocytes with minimal loss of platelets was considered an excellent result. The best results were obtained by centrifuging 300g for 5 min at 12 °C or 240g for 8 min at 16 °C for the first spin and 700g for 17 min at 12 °C for the second spin.
In general, longer centrifugal periods slightly increased platelet yield and decreased upper layer leucocyte concentrations. Thus, centrifugation parameters for PRP-Tubes can be used to control the number of leucocytes in the RBCs. Temperature has been shown to be essential in controlling platelet activation; Low temperatures slow platelet activation and prolong platelet viability. Despite its advantages, the preparation of OTPCs is based on several artisanal processing steps. This results in relatively high variability from batch to batch. It can be speculated that the lack of standardisation may explain some inconsistent clinical results.
Platelet Rich Plasma Fibrin
Several attempts have been made to develop new, easy-to-use products derived from platelets. This has resulted in platelet-rich fibrin (BTF), which is a one-step centrifuged product that does not require the addition of various chemicals. In particular, the blood is centrifuged after collection to prevent coagulation. Subsequently, the middle layer is separated from the other two layers. Centrifugation is usually carried out at 700g for 12 min to obtain standard BTF (C-BTF) or at 200g for 14 min to obtain activated BTF (A-BTF).
Ghanaati et al. reported that speed and time do not affect the concentration of monocytes and stem cells but do affect those of platelets and neutrophils. As a result, A-BTF contains more platelets, most of which are found in the distal layer of BTF, while C-BTF contains more neutrophils. This type of white blood has the potential to enhance angiogenesis by expression of the enzyme matrix metalloproteinase.
Thus, the incorporation of neutrophils into BTF can be considered if angiogenesis is of interest. BTF can release a large number of growth factors, including TFR-β1, FRT and FRES. The main difference between BTF and OTPK lies in their fibrin architectures. In BTF, this network gradually accumulates during centrifugation and in the absence of anticoagulants. This leads to a dense fibrin structure and in BTF it acts as a network in which platelets and
leucocytes are captured during centrifugation. This collector property of the fibrin network enhances the gradual release of growth factors and other mediators, leading to long-term maintenance and stimulation of stem cells by BTF. Indeed, the signatures of the release of growth factors, such as TFRβ and FRT, are different in OTPK and BTF. In OTPK, the release of TFRβ and FRT was clearly reduced after the first day, whereas BTF showed release of significant amounts of TFRβ and FRT for up to 2 weeks.
Ehrenfest et al. confirmed this difference in the release profiles of leucocyte FRES in BTF compared to OTPC. Together, these studies showed that BTF membranes were able to release more growth factors over a longer period of time. In addition to standard BTF preparations, an injection form (I-BTF) can also be obtained by compressing BTF membranes between metal sheets. Advantageously, this injectable material can coagulate immediately after injection to form a biomaterial and can also be combined with any biomaterial of choice for non-covalent incorporation.
The role of leukocytes
In addition to platelets, white blood cells contribute to the chemical composition of OTPCs and BTFs with secretory molecules such as interleukin 1β (IL1β), IL4, IL6 and tumour necrosis factor α (TNFα), which influence wound inflammation, vascularisation and regeneration. As a result, OTPCs and BTFs have been divided into two main groups based on whether or not they contain leukocytes. Although leucocytes are present in traditional OTPCs and BTFs (called L-OTPCs and L-BTFs), plasmapheresis of these OTPCs leads to „pure“ leucocyte-free OTPCs and BTFs (P-OTPCs and P-BTFs). Since inflammation is one of the main stages of wound healing, leukocytes can be seen as an interesting source of cells to initiate and regulate the tissue regeneration cascade. In addition, they can control excessive inflammation by the timely release of anti-inflammatory cytokines such as IL-4, IL-10 and IL-13. Indeed, white blood cells provide an immune regulatory role and release large quantities of FRES and other cytokines. Regardless, the most commonly used platelet-rich blood derivatives rely on limited leucocyte preparations (L-OTPK and L-BTF).
The activity of growth factors
In addition to the composition of OTPC and BtF, their method of delivery plays a key role in their ability to affect stem cells as well as their clinical outcome. A hydrogel system using alginate carriers to release OTPC growth factor showed that the pellets or capsules had different release profiles of FFR, TFR-β1 and IGF-1. Alginate beads showed a higher release of TFR-β1, whereas capsules favoured the release of FGF; Alginate capsules showed higher levels of SaOS-2 cell proliferation compared to pellets and control group. Using the same cell line, Celotti and colleagues used a TFRβ neutralising antibody that reduces cell proliferation. In addition, long-term controlled release of OTPK and BTF can be developed. The fibrin architecture is thicker and more robust in BTF than OTPC. Lyophilised OTPC scaffolds achieved up to 35 days of protein release in culture and stimulation of cell proliferation.
In addition, it was possible to supplement OTPC with heparin-conjugated fibrin biomaterial providing delayed release of FRF-2, FRT-BB and FRES. In a wound closure model in mice, this combination provided faster wound closure and regeneration, highlighting the potential role of OTPC in the treatment of chronic skin wounds. The ability to control the composite release of OTPC and BTF, as well as its time window, could thus improve the cellular approach in tissue engineering.
Platelet-rich plasma in tissue engineering
Platelet-rich plasma and fibrin-rich plasma have shown promising results in various applications of stem cell tissue engineering and regenerative medicine, but results have been inconsistent. More than a dozen large clinical trials are currently underway to investigate the regenerative potential of OTPC, which will help to identify the potential benefits and pitfalls of this blood derivative. This section of the review aims to discuss advances in our understanding of the effects of OTPC on cell proliferation, differentiation, growth factor release, inflammation and chemotaxis.
Platelet-rich plasma has been shown to increase proliferation in all cell types studied, including differentiated cells such as osteoblast-like cells and chondrocytes, periodontal ligament cells, tendon cells, preadipocytes and endothelial cells as well as multipotent cells such as mesenchymal stem cells and stem cells derived from fat cells.
OTPC mediates this increase in proliferation in a dose-dependent manner. Osteoblasts have been shown to increase proliferation with OPPC, although one study showed that there is a greater increase in cell proliferation in platelet-poor plasma (50%). In alveolar bone cells, higher concentrations of OTPC suppressed proliferation, whereas low concentrations (1-5%) stimulated proliferation. In any case, OTPC has a stimulating effect on the proliferation of stem cells such as mesenchymal stem cells (MSCs) (Figure 2a), also showing a dose-dependent effect. Murphy et al. showed that RCTs obtained from human umbilical cord blood had higher proliferation rates in MSCs compared to normal blood. Small concentrations (0.1%) of umbilical cord OPPC had maximal increase in proliferation compared to different combinations of recombinant growth factors.
Although stimulation of stem cell proliferation may be a multifactorial process due to the numerous different proliferation-stimulating molecules, Stat3/p27Kip1 cell cycle progression has been identified as a potential mechanism. Moreover, OTPC can also indirectly stimulate proliferation by enhancing stem-cell adhesion, highlighting the use of OTPC in enhancing stem-cell communication in biomaterials. In addition to proliferation, OTPK can increase the rate of cell growth by reducing cell death. In particular, OTPK supplementation decreased the expression and apoptosis of Bcl-2.
Various experimental animal models have confirmed the potential and limitations of OPPK for cell proliferation. In a diabetic rat femur fracture model (non-critical), the addition of OTPK to the fracture site enhanced cell proliferation. In another study, OTPC released from gelatin gels implanted into the bone defect promoted bone regeneration (Figure 2b). However, in the long bone defect model, OPPC combined with a collagen framework had no significant effect on bone volume, mineral density or mechanical stiffness. These potentially contradictory reports may indicate the severe effects of different growth factor formulations among individual patients, highlighting the irregularity of the composition of OTPC and its clinical implications. Taken together, these reports indicated that OTPC has a stimulatory effect on cell proliferation in various tissues. Nevertheless, further efforts are needed to understand how different OPPC preparations, individual variations and the specific composition of OPPC contribute to these effects. Moreover, little is known about the proliferative effects and mechanisms of BTFs acting on stem cells.
Platelet-rich plasma can have a strong effect on the differentiation of various stem cells. The isolation of OTPC has shown a particular propensity to stimulate stem cell differentiation into skeletal cell types such as cartilage, bone, blood vessel and tendon.
Periodontal ligament cells as well as the osteoblast-like cell lines SaOS-2 and HOS showed increased alkaline phosphatase activity and increased differentiation into mineralized tissue-forming cells when exposed to OTPC. Moreover, these cell lines rapidly increased the expression of osteogenic differentiation markers such as osteopontin, osteoprotegerin and transcription factor smallness #2. In adipose-derived stem cells at later stages (21 days), OTPK was found to stimulate osteogenic differentiation by increasing alkaline phosphatase, osteopontin, osteocalcin and the transcription factor of smallness in a dose-dependent manner. Interestingly, exposure to OTPC not only increased the osteogenic potential of stem cells but also decreased their probability of lipogenic differentiation (Figure 2c). It is suggested that OTPC manifests its osteogenic stimulation through synergistic effects with bone morphogenetic protein #2, #4, #6 and #7 (MBK).
Indeed, MBK has a stronger effect on osteoblast differentiation than OTPK, but shows increased effects when OTPK is added. This makes OTPC a suitable synergistic agent to control stem cell osteogenesis for bone tissue applications. In addition to osteogenesis, the effects of OTPC on stem cell chondrogenesis have been well characterised. The addition of OTPC to growing chondrogenic progenitor cells allows long-term maintenance of chondrogenic potential. Moreover, the addition of OTPC to chondrogenic media increased the chondrogenic potential of MSCs. The incorporation of OTPC into a polyglycolic acid-hyaluronic structure increased the expression of the chondrogenic markers collagen II and IX, aggrecan and cartilage oligomeric matrix protein. In addition, the addition of platelet lysate affected MSCs encapsulated in dextran-thiramine by their chemotactic recruitment and by improving their subsequent adhesion and efficiency of chondrogenic differentiation. The effects of BTF on chondrogenesis and osteogenesis remain largely unexplored. Moreover, the contribution of OTPC and BTF to the differentiation of stem cells into various tissues has not been systematically investigated and therefore remained largely unknown.
Platelet-rich plasma is also known to stimulate angiogenesis, which is integral to tissue regeneration and stem cell recruitment. It increases migration and vessel formation in human umbilical vein endothelial cells. Platelet-rich plasma showed in co-culture of endothelial progenitor cells (EPCs) and dental pulp stem cells increased secretion of FRES and FRT, thereby stimulating vasculogenesis and stimulating EPCs to form vascular-like structures. The fact is that platelet-rich plasma contains high levels of angiopoietin 1, which mediates angiogenesis. Inhibition of Ang1-Tie2 signalling inhibited the pro-angiogenic effect of OTPCs. Interestingly, platelet-rich plasma prevented endotoxin-induced pulmonary oedema through the same pathway, which may be due to the potential of OTPC to stabilise vascular integrity and permeability. OTPC reduced the destruction of cellular integrity induced by inflammatory cytokines. Indeed, the addition of OTPC has been consistently associated with improved angiogenesis in various models and applications. Consequently, blood derivatives such as OTPC can be considered as an autologous competitor to the traditionally used recombinant FRES protein for the induction of blood vessel formation in implanted bioengineered constructs.
Chemotaxis and inflammation
In addition to their direct effects on proliferation, differentiation and angiogenesis, OTPC and BTF also affect wound healing indirectly through chemotactic cell recruitment and local control of the inflammatory environment. Indeed, OTPC has been reported to chemotactically recruit human MSCs. Another promising chemotactic effect was also observed in a rat tendon healing model, where OTPC was able to recruit circulating blood cells and aid in the initial stages of tendon healing. In addition, it has been shown that OTPC can attract peripheral blood monocytes in a dose-dependent manner, which also leads to changes in the pro-inflammatory cytokine release profile of monocytes. Several research groups have reported the ability of OTPC to mitigate inflammation. Activated OTPC showed high levels of hepatocyte growth factor (HGF), IL-4 and TNFα. In chondrocytes, high levels of FRG and TNF-α induced by OTPC decreased the transactivating activity of NF-κB, acting as an anti-inflammatory trigger. Furthermore, in IL1B-induced osteoarthritic chondrocytes, OTPC reduced NF-κB activation levels and had multiple anti-inflammatory effects. In another osteoarthritis model using osteoarthritic cartilage and synovium from patients, OTPC, with or without leukocytes, had similar anti-inflammatory effects. Moreover, in tendon cells treated with IL-1B, OTPC induced expression of FRES, cytokine A5 and FRG and reduced the pro-inflammatory cytokines IL-6, IL-8 and MCP-1.
Taken together, the aforementioned reports highlight the role of OTPC in growth factor release and its importance in chemotaxis and inflammation. Not surprisingly, platelet-rich plasma is being considered for the treatment of inflammation and pain in certain diseases such as osteoarthritis.
Neuroprotection and other effects
In organ cultures of the cerebral and spinal cortex, OTPC has been found to stimulate axonal growth mediated by IGF-1 and ERF, but not by FRT-AB. In addition, inhibition of TRF-β1 leads to faster axonal growth, indicating that TRF-β1 inhibits or impairs axonal growth. In addition to axonal growth, OTPK has neuroprotective properties. In a rat model of ischaemic cerebral infarction, OTPC systemically or locally provided a neuroprotective effect. However, the infarct volume reduction effect was higher with local intra-arterial infusion of OTPC lysate compared to systemic administration. In addition, OTPC minimised neurological deficits in the same model.
OPPC also showed antimicrobial activity by inhibiting bacterial growth. Agar plates with OTPC inhibited the initial bacterial growth of Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus faecalis. This inhibition is mediated by the release of antibacterial chemokine ligands 1, 3 and 5. Interestingly, OTPC has been shown to enhance pluripotency markers and reduce the expression of specific markers in human MSCs, human muscle progenitor cells and adipose stem cells. Moreover, OTPC has been shown to increase cell viability during cryopreservation. Thus, OTPC enhances stem cell expansion by minimising the loss of stem cell viability.
Platelet-rich plasma and fibrin-rich plasma have many clinical applications in regenerative medicine. Numerous in vitro and animal studies have reported that these blood-derived products can stimulate stem cell differentiation along some cell lines. Currently, OPPCs overloaded with stem cells are under clinical investigation. In particular, they are being investigated in maxillofacial procedures, orthopaedic surgeries, and wound or burn therapy. Platelet-rich plasma has been reported to speed up the recovery of chronic skin ulcers, as well as improve the fat grafting process. For example, patients with lower limb ulcers were treated with OTPK mixed with a base of adipose tissue, or a control treatment consisting of collagen mixed with hyaluronic acid. After 7 and 10 weeks, the wounds treated with OTPC were completely re-epithelialized in 61.1 and 88.9%, respectively, compared to 40 and 60% in the control group. A similar study was conducted to understand the effect of platelet-rich plasma on the treatment of various maxillofacial defects. Patients received either treatment with OTPC, fat grafting or fat grafting alone. One year after surgery, graft survival and maintenance of three-dimensional tissue structure was 70% in the group that received OTPC compared to 31% in the control group.
In orthopaedics, platelet-rich plasma is routinely included as a bioactive component. For example, in a clinical trial for the treatment of deformities, 21 patients underwent tibial osteotomy and MSC therapy in combination with OPPS injection, while 23 patients in the control group had tibial osteotomy with OPPS injection alone. This demonstrated that the addition of MSCs significantly reduced both pain and fibrotic cartilage formation (Figure 4). Similarly, Koh et al. treated patients with osteoarthritic knees using a combination of MSCs isolated from the intrapatellar fat pad with OTPC, while control patients received an injection of OTPC without the addition of stem cells. The mixture was injected into the osteoarthritic knees once a week for up to 3 weeks. Short-term results showed that the addition of MSCs reduced pain and improved joint function in treated patients. Although these particular studies investigated the potential therapeutic effects of MSCs, the clinical effects of OTCPharm were still to be fully investigated. Despite this, the researchers were able to confirm that they were right about the safety of the clinical use of OTCPharm.
In the field of maxillofacial surgery/dentistry, platelet-rich plasma has been studied to treat «black triangle» which is manifested by the appearance of interproximal distance between teeth. Ten patients received one to five injections and were followed for up to 69 months. All patients reported high satisfaction with the cosmetic results without any major side effects. Patients demonstrated varying degrees of regeneration and defect filling, with two patients showing complete regeneration (Figure 5). However, the role of progenitor cells in this regeneration remained largely unexplored.
Early clinical results have thus shown that platelet derived blood cells, such as OTPK and BTF, are promising adjuncts to modern stem cell-based therapies. In particular, they demonstrate that they represent a safe and readily available source of growth factors.
Although several reports have detailed the benefits of blood derivatives in orthopaedics, dermatology and vascular surgery, there are still several challenges to overcome.
The composition of blood derivatives is characterised by relatively strong intra-organismal variation. Moreover, the variation in composition correlates with age, gender and comorbidities of the patient. In addition, it remains unclear how these changes affect the behaviour of stem cells in vivo. Therefore, a more detailed characterisation of OTPCs and BTFs is imperative in order to provide more reliable and predictable clinical results. In addition to the intra-organismal variant, previous reports have emphasised that the composition in each patient is also influenced by the subsequent preparation technique.
Unfortunately, there is currently no universally accepted standardised protocol. These inconsistencies reduce the comparability and reproducibility of different RCT studies. These problems can be solved by a systematic high throughput analysis. Alternatively, it could be argued that the establishment of multiple donor pools could alleviate this problem, but this approach would be at the expense of its autologous nature.
In addition to the composition of the blood derivative, its delivery can greatly affect its effect on stem cells and tissue regeneration. In particular, biomaterial characteristics such as stiffness, degradability, porosity and bioactivity can likely alter the effect of the blood derivative on stem cells and tissue regeneration. Although several studies have begun to explore the concept of combining OTPCs and BTFs with biomaterials, integrated or systematic approaches have not been performed. In addition, biomaterials can be used to overcome the problem of prolonging the lifespan of OTPCs and BTFs. Currently, their supply is dependent on poorly controlled mass production. As a consequence, long-term treatment requires multiple treatments, e.g. multiple injections. This leads to highly fluctuating growth factor concentrations, which impairs clinical predictability. Biomaterials could act as controlled-release devices, which would allow sustained or even rapid delivery of these growth factor cocktails. In addition, it can be assumed that biomaterials can covalently bind specific growth factors to locally maintain high levels of these molecules.