RESUMO

Musculoskeletal diseases continue to rise on a global scale, causing significant socioeconomic impact and decreased quality of life. The most common disorders affecting musculoskeletal structures are osteoarthritis and tendinopathies, complicated orthopedic conditions responsible for major pain and debilitation. Intra-articular hyaluronic acid (HA) has been a safe, effective, and minimally invasive therapeutic tool for treating these diseases. Several studies from bedside to clinical practice reveal the multiple benefits of HA such as lubrication, anti-inflammation, and stimulation of cellular activity associated with proliferation, differentiation, migration, and secretion of additional molecules. Collectively, these effects have demonstrated positive outcomes that assist in the regeneration of chondral and tendinous tissues which are otherwise destroyed by the predominant catabolic and inflammatory conditions seen in tissue injury. The literature describes the physicochemical, mechanical, and biological properties of HA, their commercial product types, and clinical applications individually, while their interfaces are seldom reported. Our review addresses the frontiers of basic sciences, products, and clinical approaches. It provides physicians with a better understanding of the boundaries between the processes that lead to diseases, the molecular mechanisms that contribute to tissue repair, and the benefits of the HA types for a conscientious choice. In addition, it points out the current needs for the treatments.

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Several musculoskeletal conditions are triggered by inflammatory processes that occur along with imbalances between anabolic and catabolic events. Platelet-rich plasma (PRP) is an autologous product derived from peripheral blood with inherent immunomodulatory and anabolic properties. The clinical efficacy of PRP has been evaluated in several musculoskeletal conditions, including osteoarthritis, tendinopathy, and osteonecrosis. When used in combination with hyaluronic acid (HA), a common treatment alternative, the regenerative properties of PRP are significantly enhanced and may provide additional benefits in terms of clinical outcomes. Recently, a new PRP-derived product has been reported in the literature and is being referred to as "plasma gel". Plasma gels are obtained by polymerizing plasmatic proteins, which form solid thermal aggregates cross-linked with fibrin networks. Plasma gels are considered to be a rich source of growth factors and provide chemotactic, migratory, and proliferative properties. Additionally, clot formation and the associated fibrinolytic reactions play an additional role in tissue repair. There are only a few scientific articles focusing on plasma gels. Historically, they have been utilized in the fields of aesthetics and dentistry. Given that the combination of three products (PRP, HA, and plasma gel) could enhance tissue repair and wound healing, in this technical note, we propose a novel regenerative approach, named "PRP-HA cellular gel matrix" (PRP-GM), in which leukocyte-rich PRP (LR-PRP) is mixed with a plasma gel (obtained by heating the plasma up) and HA in one syringe using a three-way stopcock. The final product contains a fibrin-albumin network entangled with HA's polymers, in which the cells and biomolecules derived from PRP are attached and released gradually as fibrinolytic reactions and hyaluronic acid degradation occur. The presence of leukocytes, especially monocytes and macrophages, promotes tissue regeneration, as type 2 macrophages (M2) possess an anti-inflammatory feature. In addition, HA promotes the viscosuplementation of the joint and induces an anti-inflammatory response, resulting in pain relief. This unique combination of biological molecules may contribute to the optimization of regenerative protocols suitable for the treatment of degenerative musculoskeletal diseases.

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Dorsal root rhizotomy (DRZ) is currently considered an untreatable injury, resulting in the loss of sensitive function and usually leading to neuropathic pain. In this context, we recently proposed a new surgical approach to treat DRZ that uses platelet-rich plasma (PRP) gel to restore the spinal reflex. Success was correlated with the reentry of primary afferents into the spinal cord. Here, aiming to enhance previous results, cell therapy with bioengineered human embryonic stem cells (hESCs) to overexpress fibroblast growth factor 2 (FGF2) was combined with PRP. For these experiments, adult female rats were submitted to a unilateral rhizotomy of the lumbar spinal dorsal roots, which was followed by root repair with PRP gel with or without bioengineered hESCs. One week after DRZ, the spinal cords were processed to evaluate changes in the glial response (GFAP and Iba-1) and excitatory synaptic circuits (VGLUT1) by immunofluorescence. Eight weeks postsurgery, the lumbar intumescences were processed for analysis of the repaired microenvironment by transmission electron microscopy. Spinal reflex recovery was evaluated by the electronic Von Frey method for eight weeks. The transcript levels for human FGF2 were over 37-fold higher in the induced hESCs than in the noninduced and the wildtype counterparts. Altogether, the results indicate that the combination of hESCs with PRP gel promoted substantial and prominent axonal regeneration processes after DRZ. Thus, the repair of dorsal roots, if done appropriately, may be considered an approach to regain sensory-motor function after dorsal root axotomy.

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Oral hyaluronic acid (HA) is a ubiquitous biopolymer that has gained attention as a treatment for local or systemic diseases. Here, we prepared and characterized structures of free HA (f-HA) with a high (>105 Da), intermediate (≤105 Da), and low (≤104 Da) average molar mass (MM); nanoparticles crosslinked with adipic dihydrazide (n-HA); and mixed formulations (mixed-HA) containing f-HA and n-HA. MM distribution determined the structure, hydrodynamic diameter, and zeta potential of the f-HAs. Crosslinking changed the physicochemical properties in n-HA. In vitro tack adhesion assays, using mucin tablets or a viable rat intestinal mucosa, showed better mucoadhesion with f-HA (intermediate MM) and mixed-HA (25% n-HA), especially in the jejunum segment. High MM f-HA presented negligible mucoadhesion. n-HA showed the deepest diffusion into the porous of the membranes. In vivo results showed that, except for high MM f-HA, there is an inverse relationship between rheological changes in the intestinal membrane macerates resulting from mucoadhesion and the effective intestinal permeability that led to blood clearance of the structures. We conclude that the n-HA formulations are promising for targeting other tissues, while formulations of f-HA (intermediate MM) and mixed-HA are better for treating dysbiosis.

Assuntos

RESUMO

Autologous leukocyte- and platelet-rich plasma (L-PRP) combined with hyaluronic acid (HA) has been widely used in local applications for cartilage and bone regeneration. The association between L-PRP and HA confers structural and rheological changes that differ among individual biomaterials but has not been investigated. Therefore, the standardization and characterization of L-PRP-HA are important to consider when comparing performance results to improve future clinical applications. To this end, we prepared semi-interpenetrating polymer networks (semi-IPNs) of L-PRP and HA and characterized their polymerization kinetics, morphology, swelling ratio, stability and rheological behavior, which we found to be tunable according to the HA molar mass (MM). Mesenchymal stem cells derived from human adipose tissue (h-AdMSCs) seeded in the semi-IPNs had superior viability and chondrogenesis and osteogenesis capabilities compared to the viability and capabilities of fibrin. We have demonstrated that the preparation of the semi-IPNs under controlled mixing ensured the formation of cell-friendly hydrogels rich in soluble factors and with tunable properties according to the HA MM, rendering them suitable for clinical applications in regenerative medicine.

Assuntos

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Knee osteoarthritis is a major painful and debilitating orthopaedic disease affecting a large number of adult individuals on a global scale. Over the years, this severe condition has been widely studied and while many alternatives have been utilized, platelet-rich plasma (PRP) remains one of the most popular solutions among researchers and clinicians alike. While there are different formulations and techniques involved in the preparation of PRP, produced either manually or via the use of commercial kits, the presence of leukocytes in a PRP mixture is a factor that raises concern due to their well-known pro-inflammatory activity. Although it is reasonable to worry about this, it should be taken into consideration that in order for the healing process to occur, the inflammatory phase is necessary. Leukocytes present in the inflammatory phase release both pro and anti-inflammatory molecules and, when combined with activated platelets, their potential increases. Additionally, due to the macrophage's plasticity to switch from the subtype 1 to subtype 2, it is suggested that the inclusion of the components from the buffy coat layer in a PRP mixture, classifying it as leukocyte-rich platelet-rich plasma or L-PRP, may provide benefits instead of detriments, from a standpoint of the regenerative potential of PRP.

RESUMO

Platelet-rich plasma (PRP) associated with high molecular weight hyaluronic acid (HA) has been clinically used for tissue regeneration in orthopedics. Despite the recognized beneficial clinical outcomes (e.g., early pain control, improvement of patients' functional limitation and longer-term effectiveness compared to PRP and HA alone in mild and moderate osteoarthritis treatments), its use is still challenging and controversial due to lack of standardization of association practical protocols. Moreover, most studies neglect the matrix structure, that generates the ultimate properties of the association among platelets, fibrin network and the microparticles. In the present work, we aimed to analyze the influence of the PRP/HA association with a controlled matrix structure on the stability, rheological behavior, release of growth factors and in vitro proliferation of human adipose-derived mesenchymal cells (h-AdMSCs). The attenuation of the negative charge of HA was also evaluated. Pure PRP (P-PRP) (i.e., plasma enriched with platelets and poor in leukocytes) was prepared by centrifugation and activated with serum and calcium chloride (AP-PRP). Autocrosslinked hyaluronic acid (AHA) was prepared by organocatalyzed auto-esterification and structured in microparticles (MPAHA) by shearing. The attenuation of the negative charge of MPAHA was performed with chitosan (CHT) by polyelectrolyte complexation yielding MPAHA-CHT. The results showed that microparticles (MPs) have viscoelastic properties, extrusion force and swelling ratio appropriate for injectable applications. The association of AP-PRP with the controlled structure of MPAHA and MPAHA-CHT formed a matrix composed of platelets and of a fibrin network with fibers around 160 nm located preferably on the surface of the MPs with an average diameter of 250 µm. Moreover, AP-PRP/MPAHA and AP-PRP/MPAHA-CHT associations were non-toxic and supported controlled growth factor (PDGF-AB and TGF-ß1) release and in vitro proliferation of h-AdMSC with a similar pattern to that of AP-PRP alone. The best h-AdMSC proliferation was obtained with the AP-PRP/MPAHA-CHT75:25 indicating that the charge attenuation improved the cell proliferation. Thus, the association of AP-PRP with the controlled structure of HA can be a valuable approach for orthopedic applications.

RESUMO

Spinal dorsal roots can be affected by a wide range of lesions, leading to a significant loss of proprioceptive information transmission and greatly affecting motor behavior. In this context, the reimplantation of lesioned roots with platelet-rich plasma (PRP) may allow nerve regeneration. Therefore, the present study evaluated sensorimotor improvement following dorsal root rhizotomy and repair with PRP. For this purpose, female Lewis rats were subjected to unilateral rhizotomy (RZ) of the L4-L6 dorsal roots and divided into the following groups: (1) the unlesioned control group; (2) the group that underwent rhizotomy (RZ) without repair; and (3) the group that underwent RZ followed by root repair with PRP. PRP was obtained from human blood and characterized regarding platelet concentration, integrity, and viability. Reflex arc recovery was evaluated weekly for eight weeks by the electronic von Frey method. The spinal cords were processed 1 week postlesion to evaluate the in vivo gene expression of TNFα, TGF-ß, BDNF, GDNF, VEGF, NGF, IL-4, IL-6, IL-13 by qRT-PCR and eight weeks postlesion to evaluate changes in the glial response (GFAP and Iba-1) and excitatory synaptic circuits (VGLUT1) by immunofluorescence. The results indicated that PRP therapy partially restores the paw withdrawal reflex over time, indicating the reentry of primary afferents from the dorsal root ganglia into the spinal cord without exacerbating glial reactivity. Additionally, the analysis of mRNA levels showed that PRP therapy has immunomodulatory properties. Overall, the present data suggest that the repair of dorsal roots with PRP may be considered a promising approach to improve sensorimotor recovery following dorsal rhizotomy.

Assuntos

RESUMO

Leukocyte and platelet-rich plasma (L-PRP) is an autologous product that when activated forms fibrin nanofibers, which are useful in regenerative medicine. As an important part of the preparation of L-PRP, the centrifugation parameters may affect the release of soluble factors that modulate the behavior of the cells in the nanofibers. In this study, we evaluated the influences of four different centrifugation conditions on the concentration of platelets and leukocytes in L-PRP and on the anabolic/catabolic balance of the nanofiber microenvironment. Human adipose-derived mesenchymal stem cells (h-AdMSCs) were seeded in the nanofibers, and their viability and growth were evaluated. L-PRPs prepared at 100× g and 100 + 400× g released higher levels of transforming growth factor (TGF)-ß1 and platelet-derived growth factor (PDGF)-BB due to the increased platelet concentration, while inflammatory cytokines interleukin (IL)-8 and tumor necrosis factor (TNF)-α were more significantly released from L-PRPs prepared via two centrifugation steps (100 + 400× g and 800 + 400× g) due to the increased concentration of leukocytes. Our results showed that with the exception of nanofibers formed from L-PRP prepared at 800 + 400× g, all other microenvironments were favorable for h-AdMSC proliferation. Here, we present a reproducible protocol for the standardization of L-PRP and fibrin nanofibers useful in clinical practices with known platelet/leukocyte ratios and in vitro evaluations that may predict in vivo results.

Assuntos

RESUMO

Hyaluronic acid (HA) is a ubiquitous polysaccharide with diverse biological functions. Is known that in the intestinal epithelium, the exogenous HA of molar mass ≥105 Da orally administered antagonizes TLR4 overexpression resulting from dysbiosis and promotes immunomodulation in multifactorial crosstalk, thus helping to treat or to prevent injuries. As macromolecules mediate cell signaling, the three-dimensional structure of HA plays a vital role in those functions. Introducing HA in terms of its molecular structure, its spatial architecture as dependent on pH, concentration and molar mass, occurrence, biological functions and turnover in the tissues, this review addresses the HA in the gastrointestinal system, the molecular dynamics of intestinal uptake and signaling, immunomodulation at intestinal and systemic levels and HA fate to other tissues. Finally, at the light of these behaviors, a nanotechnological approach is presented as progress in the field of the oral HA administration and discussed with perspectives for future developments.