PRP, first developed in the 1970s, is prepared by centrifuging whole blood to achieve platelet concentrations exceeding 1000 × 10⁹/L, compared to 150–450 × 10⁹/L in whole blood (Zhang et al., 2025; Gruber, 2000). This concentrate rapidly releases growth factors such as PDGF and VEGF, but its therapeutic potential is constrained by transient bioavailability (<7 days) and the absence of a fibrin scaffold. The later introduction of PRGF in the 1990s addressed PRP’s thrombin dependency by utilizing endogenous coagulation pathways for platelet activation (Anitua et al., 2023; Okada et al., 2016). Although PRGF demonstrates improved biocompatibility, inconsistent growth factor release profiles and lack of structural support still limit its utility in tissue regeneration. Both formulations continue to serve as mainstream therapies in orthopedic and sports medicine applications for acute injuries.

PRF, developed by Choukroun in the early 2000s, constitutes a structural improvement upon PRP (Fursel et al., 2021). Its preparation through low-speed centrifugation yields a dense fibrin gel that facilitates sustained growth factor release over 14–21 days, though its low mechanical strength restricts application to superficial tissues. Subsequent modifications such as Advanced PRF (A-PRF), centrifuged at 1300–1500 rpm, improve platelet distribution for superior regenerative outcomes (Chmielewski et al., 2024), whereas injectable PRF (i-PRF), introduced in 2013, maintains fluidity for deep tissue administration (Farshidfar et al., 2000). Titanium-enriched PRF (T-PRF), developed in 2014, enhances implant osseointegration through titanium interactions (Shirbhate and Bajaj, 2022), and horizontal centrifugation in Horizontal Platelet-Rich Fibrin (H-PRF) prolongs growth factor retention (Qiu et al., 2023).

Later innovations like lyophilized PRP (Ly-PRP) and lyophilized PRF (Ly-PRF) employ freeze-drying for extended preservation (Silva et al., 2016; Ngah et al., 2021; Anitua et al., 2025), while albumin-bound PRF (Alb-PRF, 2015–2020) incorporates albumin to bolster scaffold stability and growth factor protection (Fujioka-Kobayashi et al., 2021). These developments collectively overcome PRF’s mechanical and storage constraints while broadening its clinical applications.

CGF, introduced in 2006, utilizes variable-speed centrifugation (2400–2700 rpm) to produce a fibrin matrix with greater CD34⁺ stem cell and leukocyte content than PRF (Xiao et al., 2024). The resulting scaffold exhibits elevated growth factor concentrations and a porous fibrin architecture, albeit with reduced mechanical stability for large bone defects. CGF combines the bioactivity of PRP with the sustained release profile of PRF, demonstrating particular efficacy in soft tissue regeneration and periodontal therapies.

Research on PL began comparatively late, with the first studies appearing in the late 1990s. PL consists of platelet components released through physical disruption (e.g., freeze-thaw cycles or sonication) or chemical lysis (e.g., hypotonic treatment), which ruptures cell membranes while preserving cytoplasmic contents, organelle fragments, and platelet-derived extracellular vesicles (p-EVs). Unlike standard PCs, PL contains higher growth factor concentrations and exerts biological effects without requiring intact platelets. This cell-free preparation functions as a bioactive supplement with broad applications in tissue engineering and cell culture (Lunardon et al., 2024; Bordin et al., 2022).

Extracellular vesicles (EVs), including exosomes and microparticles, are membrane-bound structures released by cells. Although exosomes and microparticles differ in biogenesis, physical properties, and biological functions, both mediate intercellular communication by transporting molecular cargo. This conserved signaling capability enables EVs to modulate various physiological and pathological processes. Research on platelet-derived microparticles and exosomes has gained significant attention in recent years. Although EVs were first identified in the 1980s, detailed investigations of p-EVs only commenced in the early 2000s. Advances in isolation techniques, such as ultracentrifugation and nanoparticle tracking analysis, have progressively enhanced both fundamental understanding and practical applications of these vesicles (Guo et al., 2024; Yadav et al., 2023; Wei et al., 2022).

Platelet microparticles are small vesicles shed from the cell membrane during platelet activation or apoptosis, typically with diameters ranging from 100 to 1000 nm. They are formed by the encapsulation of cytoplasmic components within the platelet membrane and retain partial platelet membrane proteins like CD41 and CD62P. on their surface. These microparticles contain various bioactive molecules, including growth factors, cytokines, lipids, and nucleic acids. They can act as carriers for intercellular communication, transmitting platelet information to other cells and thereby regulating cellular functions and behaviors (Guo et al., 2024).

Platelet exosomes represent a smaller EV population (30–150 nm) released via exocytosis, distinguished by their endosomal origin and surface markers including CD63, CD81.These vesicles carry abundant nucleic acids (mRNA, miRNA), proteins, and lipids that contribute to intercellular signaling, immune modulation, and tissue regeneration processes (Wei et al., 2022).

The current classification system for PCs primarily focuses on preparation techniques and compositional profiles but fails to adequately capture their functional heterogeneity. While PRP, PRF, A-PRF, and CGF differ in preparation methods and structural properties, existing taxonomies poorly characterize their therapeutic differences, particularly regarding physical states (liquid versus solid matrices) and growth factor release kinetics (immediate versus sustained).

Significant variation in cellular composition, especially leukocyte and erythrocyte concentrations, complicates standardization efforts. Leukocytes—mainly neutrophils and monocytes—exhibit context-dependent therapeutic effects, offering benefits in some clinical scenarios while potentially causing harm in others. In infected or inflammatory environments, they contribute to antimicrobial defense and immunomodulation through cytokine release, whereas under sterile conditions, leukocyte-derived reactive oxygen species and pro-inflammatory mediators may hinder tissue regeneration by amplifying oxidative stress. This biological duality highlights the need for strategic leukocyte management across PC formulations. PRP often reduces leukocyte content to minimize inflammation, whereas PRF and CGF retain leukocytes to prolong cytokine activity. PLEXOs represent a further refinement, leveraging platelet-derived exosomes (30–150 nm) to deliver targeted molecular payloads while avoiding leukocyte-related complications. Thus, optimal leukocyte incorporation must be carefully matched to the specific pathophysiology, emphasizing the importance of precision medicine in PC selection.

The failure to recognize the value of PL, platelet microparticles, and platelet exosomes overlooks a critical avenue for innovation in platelet-based therapeutics. These platelet-derived components contain diverse bioactive molecules—growth factors, signaling molecules, nucleic acids, and proteins—that regulate cell proliferation, immune modulation, tissue repair, targeted drug delivery, and diagnostic applications. Current research limitations and technological barriers have prevented their full characterization and utilization, obscuring their translational potential. This gap hinders advances in platelet product development for regenerative medicine, precision medicine, and multimodal disease management.

Inconsistent preparation protocols further complicate the field. While traditional classification systems outline platelet product manufacturing methods, procedural variations and the absence of standardized protocols yield substantial batch-to-batch and intermanufacturer heterogeneity in product quality and biological activity. Such variability compromises clinical performance while introducing unnecessary therapeutic uncertainty.

Current generation classification systems offer limited practical guidance for clinical decision-making. These frameworks fail to address critical questions regarding optimal product selection, administration protocols, or expected therapeutic outcomes for specific diseases or anatomical sites. Without such evidence-based recommendations, clinicians struggle to maximize the therapeutic potential of platelet-derived therapies.

The heterogeneity in PCs terminology and generational classification mirrors advancements in production methods—including anticoagulant-free processing and low-speed centrifugation—and diverse functional objectives like sustained release or structural support. Yet existing classification systems exhibit three key shortcomings: overlapping functional profiles across generations, insufficient exploitation of platelet lysates and derivatives, and variable efficacy due to inconsistent manufacturing standards. Furthermore, they provide no systematic guidance for clinical implementation. Establishing standardized nomenclature, production protocols, and functional validation would significantly enhance their utility in regenerative medicine.

Classic PCs (PRP/PRGF), presented as liquid bioactive solutions. Centered on platelet aggregation effects, these concentrates contain high concentrations of platelets, red blood cells, white blood cells, and plasma proteins. Their mechanism relies primarily on platelet aggregation, which induces the transient release of growth factors such as PDGF and TGF-β from α-granules upon activation with calcium ions or collagen. This process stimulates osteoblast and fibroblast activity, though the therapeutic effects remain short-lived.

The transition from first-generation PCs (PRP/PRGF) to second-generation fibrin-based scaffolds (PRF/CGF) reflects a paradigm shift in regenerative therapeutics. Unlike first-generation products, which relied on anticoagulant protocols to enrich platelets at the cellular level, second-generation approaches optimized structural properties by developing endogenous fibrin matrices. PRF preserves fibrin networks at low temperatures to facilitate prolonged growth factor release, while CGF utilizes variable-speed centrifugation to concentrate platelet microparticles and modulate growth factor profiles. This shift from liquid suspensions to three-dimensional bioactive scaffolds redefined therapeutic strategies, replacing transient growth factor delivery with microenvironment-mimicking tissue engineering.

Although second-generation technologies improved functionality, their dependence on cellular integrity led to advanced derivatives such as A-PRF, i-PRF, and T-PRF. These variants modify fibrin architecture—exemplified by H-PRF’s horizontal centrifugation—or enhance stability through methods like albumin incorporation in Alb-PRF, while maintaining the focus on structural optimization. This progression highlights a key trend in regenerative medicine: advancing from cellular concentration toward biomaterial-driven, spatiotemporally controlled tissue repair, a principle further refined by third-generation cell-free exosome therapies.

EVs and exosomes provide unique benefits as cell-free systems that avoid complications from platelet aggregation. p-EVs therapeutics, particularly PLEXOs, represent a transformative development in regenerative medicine compared to conventional approaches (Wan et al., 2024; Gardin et al., 2022; Wei et al., 2024; Gupta et al., 2025; Li et al., 2024). These platforms, ranging from fibrin-based scaffolds like PRF and CGF to engineered exosomes, enable the targeted delivery of physiologically balanced growth factors, miRNAs, and signaling molecules with precise spatiotemporal control. Their autologous origin guarantees immune compatibility, while exosomes exploit intrinsic targeting mechanisms to efficiently cross biological barriers, including the blood-brain barrier. Although regulatory classification and cost-effectiveness remain unresolved, platelet-derived systems mark a paradigm shift from empirical cell transplantation to controlled, patient-specific tissue regeneration. By integrating natural healing processes with bioengineering precision, these therapeutics are reshaping regenerative therapy strategies.

This evolution expands the scope of regenerative medicine, yet each generation of platelet-derived therapeutics faces distinct standardization hurdles. The first generation contends with concentration variability due to centrifuge parameters, which closed systems can minimize. The second generation overcomes fibrin structure inconsistencies caused by manual preparation through automated standardization. The third generation resolves low exosome purity by implementing advanced extraction and purification methods, setting critical benchmarks for clinical-grade exosome therapy.

While first-generation PCs (PRP/PRGF) remain clinically valuable for their rapid growth factor release, their transient therapeutic effects limit long-term efficacy. To address this, emerging nano-engineering strategies focus on sustained delivery systems.

Although PRF and CGF achieve the sustained release of growth factors through structural optimization, their dependence on the integrity of the cytoskeleton limits their application scenarios. Current research focuses on structural optimization using cross-linking agents to enhance scaffold stability against enzymatic degradation (Karimi et al., 2022). Particularly promising is the integration with gelatin methacryloyl (GelMA), a photo-crosslinkable hydrogel that enables 3D printing of porous scaffolds (80%–90% porosity) (Liang et al., 2023). This combination approach has demonstrated particular potential for complex wound healing applications, such as diabetic foot ulcers, by providing both structural support and controlled bioactive release.

PLEXOs show promise as cell-free therapeutic carriers, but their clinical translation requires addressing several key challenges. Dual-targeted exosomes (CD47 + RGD) overcome critical limitations in targeted delivery and immune evasion by simultaneously preventing macrophage phagocytosis through CD47’s “do not eat me” signal and enhancing cellular uptake via RGD-mediated integrin binding (Xie et al., 2023). This dual-targeting strategy increases exosome accumulation at disease sites, facilitating tissue regeneration. CRISPR-associated protein 9 (CRISPR - Cas9) technology further expands therapeutic potential by enabling precise exosomal cargo modifications, such as miR-34a knockout to suppress tumor metastasis (Aslan et al., 2024). The SpyTag/SpyCatcher system permits covalent attachment of functional proteins to exosome surfaces, creating versatile platforms for vaccines, targeted therapies, and diagnostic applications (Zheng et al., 2024). These engineered exosomes can respond to environmental stimuli or enzymatic triggers, advancing precision medicine approaches.