Intra-Articular Hyaluronic Acid in Osteoarthritis and Tendinopathies: Molecular and Clinical Approaches.

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.

Platelet-Rich Plasma Gel Matrix (PRP-GM): Description of a New Technique.

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.

How centrifugation influences the recovery and soy peptone incorporation in hyaluronic acid coils from fermentation.

Hyaluronic acid from fermentation (bio-HA) is a high-added-value biopolymer with a wide range of applications in aesthetic and medical areas. Bio-HA is a capsular exopolysaccharide highly hydrophilic with a coiled structure and high negative charge density at physiological pH. Centrifugation, currently used to separate microbial cells after fermentation, should significantly affect the bio-HA interactions with the medium components. Although the literature shows various centrifugation conditions for bio-HA, there is no concern about its recovery and characterization. We examined how centrifugation influences its recovery and characterized interactions with soy peptone (SP), used as a nitrogen source. We collected experimental data according to a statistical central composite design (CCD) planning with replicates at the central point with time (8-22 min) and spin (582xg to 12,402xg) as independent variables. The obtained results show bio-HA recoveries ranged from 45 ± 0.5-100% as dosed in the supernatant. The incorporation of amino acids and peptides from SP increased HA coils' density, causing losses due to sedimentation at higher spins and times. Furthermore, SP incorporation also changed the coils' size regarding HA standards of molar mass 106,105 and 104 Da. We conclude centrifugation should significantly impact downstream processing and the total cost of bio-HA production.

Spinal Reflex Recovery after Dorsal Rhizotomy and Repair with Platelet-Rich Plasma (PRP) Gel Combined with Bioengineered Human Embryonic Stem Cells (hESCs).

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.

Hyaluronic acid and fibrin from L-PRP form semi-IPNs with tunable properties suitable for use in regenerative medicine.

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.

Leukocyte-rich PRP for knee osteoarthritis: Current concepts.

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.

Production of humic acids by solid-state fermentation of Trichoderma reesei in raw oil palm empty fruit bunch fibers.

Humic acids (HA) are organic macromolecules of high structural complexity and are primarily obtained from non-renewable carbon sources such as peat and coal. HA is widely used in agriculture but is known to have therapeutic properties, which are still poorly explored. Previous studies have shown the potential of biotechnological processes in the production of HA in submerged fermentation (SF) and solid-state fermentation (SSF) using pre-treated fibers of oil palm of empty fruit bunch (EFB) for the cultivation of Trichoderma strains. EFB is an agro-industrial residue that is readily available at a low cost. The present study aimed to study the production of HA by Trichoderma reesei in the SSF of raw fibers of EFBs from two different palm oil producers. Profiles of HA production, cellular protein, pH, glucose, moisture, and oxygen transfer were obtained during SSF by EFB with and without lipids, as well as, images by electron microscopy of fibers. Results showed efficient HA production in the raw fibers of the EFBs. HA production followed the cellular protein (6 g HA per 100 g of fibers) evolution of the fermentations in the absence of lipids, while the composition of lipids greatly affected its production. The best HA production (350 mg HA per 100 g of fibers) was obtained from EFB that was richer in lignocellulosics and the residual lipids were similar to the fractions of palm and kernel oils, while EFB with lower lignocellulosic presented a production 110 mg HA per 100 g of fibers.

Nanostructured lipid carriers loaded with free phytosterols for food applications.

The objective of this study was to develop nanostructured lipid carriers (NLCs) with free phytosterols (FP) using conventional fats and oils. Lipid matrices (LMs) and NLCs were produced with high oleic sunflower oil, fully hydrogenated canola (CA) and crambe (CR) oils by high-pressure homogenization (HPH). The NLCs were evaluated for hydrodynamic diameter (Z-ave), polydispersity index (PDI), and zeta potential (ZP). The melting behavior and polymorphism were investigated for both, the LMs and NLCs. The NLCs presented particle sizes ranging from 148.23 to 342.10 nm, PDI from 0.275 to 0.481, and ZP between -22.27 and -29.70 mV. The NLCs presented higher thermal resistance than that of the LMs. The use of CA and CR separately in the NLC formulations favored the incorporation of FP. The LMs and NLCs presented crystals in ß-form and in mixtures of ß' and ß forms. The developed NLCs can be used for food enrichment, such as spreads, margarine, and beverages.

Centrifugation Conditions in the L-PRP Preparation Affect Soluble Factors Release and Mesenchymal Stem Cell Proliferation in Fibrin Nanofibers.

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.

Structural Modifications and Solution Behavior of Hyaluronic Acid Degraded with High pH and Temperature.

Hyaluronic acid (HA) is a macromolecule with valuable benefits over its range of molar masses (MM). Degradation studies are relevant to maintain the same purity level in biomedical studies when using HA of different MM. We degraded HA via high pH and temperature and evaluated its MM, solution behavior, and structure over time. After 24 h, low MM HA was predominant, and the MM decreased from 753 to 36.2 kDa. Dynamic light scattering (DLS) showed a decrease in the number of HA populations, and the solution tended to be less polydispersed. The zeta potential varied from - 10 to - 30 mV, close to the stable range. FTIR showed that the primary structure of HA was affected after only 48 h of reaction. These results are relevant for the production of low MM HA to be used or mixed with high MM HA, generating structured biomaterials for biomedical applications.

Liposomal-based lidocaine formulation for the improvement of infiltrative buccal anaesthesia.

This study describes the encapsulation of the local anaesthetic lidocaine (LDC) in large unilamellar liposomes (LUV) prepared in a scalable procedure, with hydrogenated soybean phosphatidylcholine, cholesterol and mannitol. Structural properties of the liposomes were assessed by dynamic light scattering, nanoparticle tracking analysis and transmission electron microscopy. A modified, two-compartment Franz-cell system was used to evaluate the release kinetics of LDC from the liposomes. The in vivo anaesthetic effect of liposomal LDC 2% (LUVLDC) was compared to LDC 2% solution without (LDCPLAIN) or with the vasoconstrictor epinephrine (1:100 000) (LDCVASO), in rat infraorbital nerve blockade model. The structural characterization revealed liposomes with spherical shape, average size distribution of 250 nm and low polydispersity even after LDC incorporation. Zeta potential laid around -30 mV and the number of suspended liposomal particles was in the range of 1012 vesicles/mL. Also the addition of cryoprotectant (mannitol) did not provoke structural changes in liposomes properties. In vitro release profile of LDC from LUV fits well with a biexponential model, in which the LDC encapsulated (EE% = 24%) was responsible for an increase of 67% in the release time in relation to LDCPLAIN (p < 0.05). Also, the liposomal formulation prolonged the sensorial nervous blockade duration (∼70 min), in comparison with LDCPLAIN (45 min), but less than LDCVASO (130 min). In this context, this study showed that the liposomal formulations prepared by scalable procedure were suitable to promote longer and safer buccal anaesthesia, avoiding side effects of the use of vasoconstrictors.

Humic acids: Structural properties and multiple functionalities for novel technological developments.

Humic acids (HAs) are macromolecules that comprise humic substances (HS), which are organic matter distributed in terrestrial soil, natural water, and sediment. HAs differ from the other HS fractions (fulvic acid and humins) in that they are soluble in alkaline media, partially soluble in water, and insoluble in acidic media. Due to their amphiphilic character, HAs form micelle-like structures in neutral to acidic conditions, which are useful in agriculture, pollution remediation, medicine and pharmaceuticals. HAs have undefined compositions that vary according to the origin, process of obtainment, and functional groups present in their structures, such as quinones, phenols, and carboxylic acids. Quinones are responsible for the formation of reactive oxygen species (ROS) in HAs, which are useful for wound healing and have fungicidal/bactericidal properties. Phenols and carboxylic acids deprotonate in neutral and alkaline media and are responsible for various other functions, such as the antioxidant and anti-inflammatory properties of HAs. In particular, the presence of phenolic groups in HAs provides antioxidant properties due to their free radical scavenging capacity. This paper describes the main multifunctionalities of HAs associated with their structures and properties, focusing on human health applications, and we note perspectives that may lead to novel technological developments. To the best of our knowledge, this is the first review to address this topic from this approach.

In vitro performance of injectable chitosan-tripolyphosphate scaffolds combined with platelet-rich plasma.

This study aimed to evaluate the in vitro biological effectiveness of chitosan microparticles crosslinked with sodium tripolyphosphate (TPP) in combination with activated pure platelet-rich plasma (aP-PRP) as an injectable composite scaffold for growth factors release, cell proliferation and osteogenic differentiation. Two main novelties were addressed in the field of scaffolds in regenerative medicine: the first is the approach including simultaneously the three vertices of the proliferation triangle formed by the capabilities genic progenitor cells, conductive scaffolds and inductive growth factors, which are provided by platelet rich plasma (PRP); secondly, the approach of an injectable composite scaffolds formed by the fibrin network from aP-PRP and the chitosan microparticles crosslinked with TPP. The microparticles were prepared by vortexing the chitosan and TPP solutions. The ionic crosslinking of chitosan with TPP was made at mass ratios of 2:1, 5:1, and 10:1 at pH 4.0. P-PRP was obtained via the controlled centrifugation of whole blood. The composite scaffolds were prepared by adding the microparticles to immediately activated P-PRP. The results showed that the microparticles had adequate physicochemical and mechanical properties for injection. Furthermore, the microparticles controlled the release of growth factors from P-PRP. The proliferation of human adipose-derived mesenchymal stem cells was lower than in aP-PRP alone but significant at a 2:1 chitosan-TPP mass ratio. Osteogenic differentiation was stimulated at all studied mass ratios, as indicated by the alkaline phosphatase activity. These results offer perspectives for optimizing the composite scaffold, and to prove its potential as an injectable scaffold in regenerative medicine.

Sterilization of auto-crosslinked hyaluronic acid scaffolds structured in microparticles and sponges.

This work evaluated the effects of UV irradiation, plasma radiation, steam and 70% ethanol treatments on the sterilization and integrity of auto-crosslinked hyaluronic acid (HA-ACP) scaffolds structured in microparticles and sponges aiming in vivo applications for regenerative medicine of bone tissue. The integrity of the microparticles was characterized by rheological behavior, while for the sponges, it was characterized by scanning electron microscopy, Fourier transform infrared spectroscopy and differential scanning calorimetry. The effectiveness of the sterilization treatment was verified by the number of microorganism colonies in the samples after the treatments. In conclusion, plasma radiation was the best treatment for the sponges, while steam sterilization in the autoclave at 126°C (1.5 kgf/cm2) for 5 min was the best treatment for the microparticles.

Performance of PRP associated with porous chitosan as a composite scaffold for regenerative medicine.

This study aimed to evaluate the in vitro performance of activated platelet-rich plasma associated with porous sponges of chitosan as a composite scaffold for proliferation and osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells. The sponges were prepared by controlled freezing (-20, -80, or -196°C) and lyophilization of chitosan solutions (1, 2, or 3% w/v). The platelet-rich plasma was obtained from controlled centrifugation of whole blood and activated with calcium and autologous serum. The composite scaffolds were prepared by embedding the sponges with the activated platelet-rich plasma. The results showed the performance of the scaffolds was superior to that of activated platelet-rich plasma alone, in terms of delaying the release of growth factors and increased proliferation of the stem cells. The best preparation conditions of chitosan composite scaffolds that coordinated the physicochemical and mechanical properties and cell proliferation were 3% (w/v) chitosan and a -20°C freezing temperature, while -196°C favored osteogenic differentiation. Although the composite scaffolds are promising for regenerative medicine, the structures require stabilization to prevent the collapse observed after five days.

The crosslinking degree controls the mechanical, rheological, and swelling properties of hyaluronic acid microparticles.

Viscosupplements, used for treating joint and cartilage diseases, restore the rheological properties of synovial fluid, regulate joint homeostasis and act as scaffolds for cell growth and tissue regeneration. Most viscosupplements are hydrogels composed of hyaluronic acid (HA) microparticles suspended in fluid HA. These microparticles are crosslinked with chemicals to assure their stability against enzyme degradation and to prolong the action of the viscosupplement. However, the crosslinking also modifies the mechanical, swelling and rheological properties of the HA microparticle hydrogels, with consequences on the effectiveness of the application. The aim of this study is to correlate the crosslinking degree (CD) with these properties to achieve modulation of HA/DVS microparticles through CD control. Because divinyl sulfone (DVS) is the usual crosslinker of HA in viscosupplements, we examined the effects of CD by preparing HA microparticles at 1:1, 2:1, 3:1, and 5:1 HA/DVS mass ratios. The CD was calculated from inductively coupled plasma spectrometry data. HA microparticles were previously sized to a mean diameter of 87.5 µm. Higher CD increased the viscoelasticity and the extrusion force and reduced the swelling of the HA microparticle hydrogels, which also showed Newtonian pseudoplastic behavior and were classified as covalent weak. The hydrogels were not cytotoxic to fibroblasts according to an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay.

The solid-state cultivation of Streptococcus zooepidemicus in polyurethane foam as a strategy for the production of hyaluronic acid.

Hyaluronic acid (HA) is a biopolymer with important applications in the pharmaceutical, medical, and cosmetic fields. This work explores the potentialities of a cylindrical polyurethane foam dowel with central aeration as a novel packed bed bioreactor for the production of HA. The goals were to provide a large surface area for oxygen transfer through the patches of liquid film that form in the pores of the foam in which cell proliferation and HA production occur and to easily recover the HA produced. The resulting yields of HA/cell were higher than 1, and the produced HA was completely recovered by pressing the foam. The external conditions that inhibit catabolism, the deviation of energetic metabolism toward the production of HA, were modulated by aeration and the initial glucose concentration. The production of HA was reproducible in 12 successive fermentation cycles. These findings contribute to the development of efficient strategies for the controlled production and recovery of HA.

Effects of soy peptone on the inoculum preparation of Streptococcus zooepidemicus for production of hyaluronic acid.

Soy peptone (SP) was studied as nutrient source in replacement of the conventional media as Brain-Heart Infusion (BHI) and sheep blood in the first seed culture medium in Petri plates of Streptococcus zooepidemicus. This substitution, aimed at meeting the claim of the pharmaceutical and cosmetics industries, for the removal of animal sources of the culture media used in obtaining their products for safety reasons. The animal sources were used as a control. The effects of this substitution were studied in fermentations carried out at 37°C and 150rpm in 250mL Erlenmeyer flasks containing 100mL culture medium containing glucose and SP only. The replacement of animal nutrient sources by SP to about twice the BHI concentration did not alter the amount of the produced HA, or caused deviations in the metabolism of the microorganism in favor of HA to the detriment of cell growth.

Effects of organic solvents on hyaluronic acid nanoparticles obtained by precipitation and chemical crosslinking.

Hyaluronic acid is a hydrophilic mucopolysaccharide composed of alternating units of D-glucuronic acid and N-acetylglucosamine. It is used in many medical, pharmaceutical, and cosmetic applications, as sponges, films, or particle formulations. Hyaluronic acid nanoparticles can be synthesized free of oil and surfactants by nanoprecipitation in organic solvents, followed by chemical crosslinking. The organic solvent plays an important role in particles size and structure. Therefore, this study aimed to investigate the influence of acetone, ethanol, and isopropyl alcohol on the synthesis and physico-chemical properties of hyaluronic acid nanoparticles. Particles were crosslinked with adipic hydrazide and chloride carbodiimide under controlled conditions. The nanoparticles obtained with all three studied solvents were moderately electrostatically stable. Experiments with acetone produced the smallest particle size (120.44 nm) and polydispersity (0.27). The size and polydispersity of hyaluronic acid nanoparticles correlated with the surface tension between water and the organic solvents, not with the thermodynamic affinity of water for the organic solvents.

Hyaluronic acid depolymerization by ascorbate-redox effects on solid state cultivation of Streptococcus zooepidemicus in cashew apple fruit bagasse.

The cashew fruit (Anacardium occidentale L.) has been used as a promising agricultural resource for the production of low-molecular weight (M(W)) hyaluronic acid (HA) (10(4)-10(5) Da). The cashew juice is a rich source of vitamin C containing, 1.2-2.0 g L(-1). This work explores the effects of the initial concentration of the ascorbate on the solid fermentation of the juice-moisturized bagasse from the cashew apple fruit. The results show that the M(W) reduction of HA is proportional to the initial ascorbate concentration. The presence of ascorbate did not influence the Streptococcus zooepidemicus metabolism. However, the HA productivity was increased from 0.18 to 0.28 mg g(-1) h(-1) when the ascorbate concentration ranged from 1.7 to 10 mg mL(-1). These findings contribute to the controlled production of HA in a low M(W) range, which is important in cell signalization, angiogenesis and nanoparticles production.