BACKGROUND: Cells, growth factors (GFs), and scaffold are three essential factors for tissue engineering. Our previous studies suggested that multiple applications of human amnion growth factors (AGF) into osseous defects could “mimic in-utero” growth. However, micro-gaps still exist between the scaffold and recipient tissue. We hypothesized that hyaluronic acid (HA) could act an accessory scaffold and gradually release active components of AGF and improve bone healing. MATERIALS AND METHODS: Calvaria from 50 7–9-day old CD1 neonatal mice were harvested, and a 2 mm defect punch made in each one. A type I collagen membrane with AGF alone or with HA at different concentrations applied over the defect. The culture medium was changed every 2-3 days and collected for alkaline phosphatase (ALP) and protein analysis. RESULTS: A single dose of AGF combined with 0.125% HA increased cellular infiltration into the defect area more than AGF with no HA or a lower concentration of HA (0.0625%). A single dose of AGF with HA can improve bone healing. CONCLUSION: A single dose of AGF with HA as an extra scaffold and a carrier can achieve bone formation like multiple dosages of AGF and reduce the number of clinical applications needed.

Within the last decade, neurodegenerative diseases such as Alzheimer’s and Parkinson’s have emerged as one of the top 5 leading causes of death globally, and there is currently no cure. All neurodegenerative diseases lead to loss of the functional cells in the nervous system, the neurons. One therapeutic approach is to replace the damaged and lost neurons with new, healthy neurons. Unfortunately, this is a difficult endeavor since mature neurons are not capable of cell division. Instead, researchers are turning to neural stem cells, which are able to self-renew and be rapidly expanded before being differentiated into functional cell phenotypes, such as neurons, allowing for large numbers of cells to be generated in vitro. Controlled differentiation of human neural stem cells into new neurons has been of interest due to the immense potential for improving clinical outcomes. Adult neural stem cell behavior, however, is not well understood and the transplanted stem cells are at risk for tumorigenesis. The focus of this dissertation is the development of engineered biomaterials as tools to study human neural stem cell behavior and neurogenesis (differentiation). A novel cell penetrating peptide was developed to enhance intracellular delivery of retinoic acid, a bioactive lipid known to induce differentiation. A hydrogel platform fabricated from hyaluronic acid, a naturally-occurring polysaccharide found in brain extracellular space, was designed to serve as a biomimetic soft substrate with similar mechanical properties to the brain. The biological behavior of the stem cells was characterized in response to chemical and physical cues. / Bioengineering

<p dir="ltr">Tissue engineering utilizes polymers, cells, and other bioactive factors to promote regeneration within damaged tissue. The main works in this thesis employ naturally derived polymers for use in tissue engineering and explores ways to recapitulate native environments <i>in vitro</i>.</p><p dir="ltr">Collagen (col) is the most prevalent protein in the body. Col type I, II, and III are all fibril-forming collagens that provide structure to tissues. All three types polymerize <i>in vitro</i> to form hydrogels, and these hydrogels have often been studied for use in tissue engineering. Other applications include <i>in vitro </i>tissue models for studies on drug diffusion and drug delivery. Blending collagen types is of particular interest as col I is easier to source and is therefore cheaper than other collagen types. However, to confer biological signals to tissues where col II or III are more abundant (e.g., cartilage or cardiac tissue, respectively), col II or III can be added to col I to form col I/II or col I/III gels, respectively. Additionally, adding multiple types of col to hydrogel models better recapitulates the native environment and can better capture effects on drug diffusion. In this work, compared to col I alone, col I/II hydrogels polymerize more slowly, form more fibril bundles, result in softer hydrogels, and impede transport of larger macromolecules. On the other hand, col I/III gels polymerize at a similar rate to col I, create heterogenous fibril structures, are oftentimes stiffer than col I, and also impede transport of larger macromolecules. Additionally, this work explored the effect of polymerization temperature on blended gel polymerization and properties.</p><p dir="ltr">The second work evaluates col I/II hydrogels for a specific application: cartilage tissue engineering for osteoarthritic applications. Col II is the primary protein found in cartilage. Other components include: glycosaminoglycans, such as hyaluronic acid (HA) and chondroitin sulfate, chondrocytes (cartilage cells), and other small signaling molecules. Building on prior work in the group, high molecular weight hyaluronic acid (HA) was added to col I/II hydrogels, and cartilage differentiation of mesenchymal stem cells (MSCs) was assessed under ideal laboratory conditions and under pro-inflammatory, osteoarthritic conditions (i.e., cytokine-supplemented media of oncostatin M (OSM) at 10 ng/mL and tumor necrosis factor-α (TNF-α) at 20 ng/mL). The addition of HA did not dramatically impact cartilage differentiation of MSCs, however, HA did mitigate the effect of inflammation via downregulation of a degradative enzyme. HA had little impact on inflammatory cytokine production of interleukin (IL)-6 or IL-8, both of which are upregulated during osteoarthritis. However, a linear model suggests that HA and IL-8 are strongly correlated. Thus, this system should be explored further with different HA concentrations or presentations (e.g., chemically modified).</p><p dir="ltr">The last primary chapter of this thesis provides depth to the pro-inflammatory, osteoarthritic model used in the previous chapter. Different pro-inflammatory environments are studied using cytokines found in OA. MSC pellets (used in literature as controls to confirm chondrogenic potential of MSCs) were used to evaluate these inflammatory environments since MSCs are commonly used in tissue engineering. Six treatments were studied: negative control (without the chondrogenic growth factor TGF-β3), positive control (with the chondrogenic growth factor TGF-β3), and four cytokine treatments all with TGF-β3. First, IL-1β at 10 ng/mL was utilized as a comparison to literature. The other three cytokine groups used TNF-α at 20 ng/mL and OSM at 10 ng/mL individually or combined to form the main experimental group, OSM+TNF-α. All cytokine treatment groups limited cartilage production, but OSM decreased production to a statistically lesser extent than other cytokine groups. This trend was similar to observations made via immunostaining of cartilage matrix and gene expression analysis of aggrecan. Furthermore, OSM+TNF-α statistically lowered aggrecan gene expression. In terms of degradation, when compared to all other groups, OSM dramatically increased the protein expression of the degradative enzyme matrix metalloproteinase-13 (MMP-13). Evaluation of inflammatory markers (IL-6 and IL-8) revealed no signal for OSM-treated pellets. TNF-α yielded some signal after 1 week in culture but no signal after two weeks. IL-1β and OSM+TNF-α both resulted in sustained IL-6 and IL-8 expression, however, IL-1β exhibited large variance. Thus, each cytokine contributes to various pathways that are present in OA. Since the combination of OSM and TNF-α appeared to lower cartilage gene expression and resulted in sustained and reproducible IL-6 and IL-8 production, it may serve as a better model of OA than a single cytokine such as IL-1β.</p>

Targeted delivery aids in minimizing most of the drug-originated systemic toxic effects as well as improving the pharmacokinetic properties of anticancer therapeutics. Tumor targeting using hyaluronic acid (HA) as the targeting ligand has attracted a great deal of interest among a host of strategies developed to target the overexpressed tumor specific receptors. HA is an endogenous molecule that possesses a lot of biological functions in the human body. The role of HA synthases, HA degrading enzymes and the interaction of HA with its primary receptor CD44 in tumor metastasis and angiogenesis is really complex and controversial to date. However, overexpression of CD44receptors on tumor surface has been well studied, which have been utilized to direct tumor targeted drugs. Most of the HA based targeting systems were HA drug conjugates and surface modified colloidal carriers which required covalent modification. The lack of accurate structural characterization of these systems resulted in modification of HA binding sites that could affect the efficient cellular uptake. LbL technique is a simple and facile method to incorporate several materials into polyelectrolyte assemblies for drug delivery applications. HA being a negatively charged polysaccharide can be easily incorporated into such systems without any covalent modification. Although HA based polyelectrolyte multilayer films and microcapsules have been reported in combination with polycations like PAH, PLL and chitosan, their application as targeted drug delivery systems have not yet been explored. Herein, two LbL architectures with HA as the terminal layer have been investigated as targeted drug carriers, which can recognize overexpressed CD44 receptors in metastatic breast cancer cells. In the first part of the thesis, a novel polyelectrolyte nanocapsule system composed of biopolymers HA and protamine sulphate (PR) as the wall components was prepared and characterized. These pH and enzyme responsive nanocapsules were then utilized for efficient loading and release of anticancer drug doxorubicin (dox). Higher drug release was observed in simulated intracellular conditions like acidic pH and presence of hyaluronidase enzyme as compared to physiological pH. In the second part of the thesis, dox incorporated bovine serum albumin (BSA) nanoparticles modified with HA-Poly(l-Lysine) multilayers were developed and characterized. The drug release pattern of the dox loaded BSA nanoparticles was found to depend on the presence of a protease enzyme trypsin than pH variations. Both of these drug delivery systems were then evaluated for their cell targeting efficiency and cytotoxicity in CD44+ positive metastatic breast cancer cell line MDA MB 231. The final layer HA facilitated targeted delivery of these drug carriers via CD44 receptor mediated endocytosis. The enhanced cellular uptake followed by sustained delivery of dox by virtue of slow intracellular enzymatic degradation of the drug carriers resulted in their improved cytotoxicity as compared to free dox. Further in vitro biodistribution and tumor suppression efficiency of both the systems were studied in breast cancer xenograft models using BALB/c nude mice. Enhance accumulation of dox in the tumor tissue and significant tumor reduction were observed when treated with encapsulated dox using the HA based nanocarriers as opposed to free dox.

The overall aim of this thesis was to establish strategies to minimize secondary damage to the injured spinal cord. Secondary damage that follows spinal cord injury (SCI) involves inflammatory and excitotoxic pathways. Regulation of these pathways using immunomodulatory and neuroprotective substances potentially protects the injured spinal cord from further damage. We also developed and studied resorbable biomaterials to be used as carriers for potential neuroprotectants to the injured spinal cord. We used transversal spinal cord slice cultures (SCSCs) derived from postnatal mice as a model. SCSCs were maintained on different biomaterials and were studied after treatment with immunomodulatory and/or neurotrophic factors. They were further excitotoxically injured and subsequently treated with interleukin-1 receptor antagonist (IL1RA) or by neural crest stem cell (NCSC)-transplantation. The results show that biocompatible and resorbable hydrogels based on hyaluronic acid (HA) preserved neurons in SCSCs to a much higher extent than a conventional collagen-based biomaterial or standard polyethylene terephthalate (PET) membrane inserts. Glial activation was limited in the cultures maintained on HA-based hydrogel. The anti-inflammatory factor IL1RA protected SCSCs from degenerative mechanisms that occur during in vitro incubation, and IL1RA also protected SCSCs from excitotoxic injury induced by N-Methyl-d-Aspartate (NMDA). IL1RA specifically protected neurons that resided in the ventral horn, while other neuronal populations such as dorsal horn neurons and Renshaw cells did not respond to treatment. Finally, transplantation of NCSCs onto excitotoxically injured SCSCs protected from neuronal loss, apoptosis and glial activation, while NCSCs remained undifferentiated. The results presented in this thesis indicate that carriers based on HA seem to be more suitable than conventional collagen-based biomaterials since they enhance neuronal survival per se. The observed neuroprotection is likely due to biomechanical properties of HA. IL1RA protects SCSCs from spontaneous degeneration and from NMDA-induced injury, suggesting that excitotoxic mechanisms can be modulated through anti-inflammatory pathways. Different neuronal populations are affected by IL1RA to various degrees, suggesting that a combination of different neuroprotectants should be used in treatment strategies after SCI. Finally, NCSCs seem to protect SCSCs from excitotoxic injury through paracrine actions, since they remain undifferentiated and do not migrate into the tissue during in vitro incubation. It seems that combinations of neuroprotectants and carrier substances should be considered rather than one single strategy when designing future treatments for SCI. Incorporation of neuroprotectants such as IL1RA combined with stem cells in injectable biocompatible carriers based on HA is the final goal of our group in the treatment of SCI.

Hyaluronan (HA) is a negatively charged extracellular matrix (ECM) component with water-attracting properties. It is the dominating ECM component in the renal medullary interstitium, where the amount changes in relation to hydration status: it increases during hydration and decreases during dehydration. It has, therefore, been suggested that HA participates in the regulation of renal fluid handling by changing the permeability properties of the interstitial space. This thesis investigates potential mechanisms for such a role in renal fluid regulation. The results demonstrate that the high renal HA content of late nephrogenesis decreases during the completion of kidney development in the rat, which takes place in the neonatal period. The heterogenous distribution of HA is mainly established during the first three weeks after birth. On day 21, the HA content is similar to that in the adult rat. The process is dependent on normal Ang II function. It primarily involves a reduction of HA synthase 2 expression and an increase of medullary hyaluronidase 1.  The cortical accumulation of HA that results from neonatal ACE inhibition can partly explain the pathological condition of the adult kidney, which causes reduced urinary concentration ability and tubulointerstitial inflammation. It is possible to reduce renomedullary HA with the HA synthesis inhibitor 4-MU, and the kidney’s ability to respond to a hydration challenge will then be suppressed, without affecting GFR.  The investigation of renomedullary interstitial cells (RMIC) in culture, shows that media osmolality and hormones of central importance for body fluid homeostasis, such as angiotensin II, ADH and endothelin, affect HA turnover through their effect on the RMICs, in a manner comparable to that found in vivo during changes in hydration status.  In established streptozotocin-induced diabetes, HA is regionally accumulated in the kidney, proteinuria and polyuria, reduced urine osmolality, and reduced response to ADH V2 activation will occur. As opposed to the proteinuria, the HA accumulation is not sensitive to mTOR inhibition, suggesting an alternate pathway compared to other ECM components  Taken together, the data suggest that during normal physiological conditions, renomedullary interstitial HA participates in renal fluid handling by affecting the interstitial prerequisites for fluid flux across the interstitial space. This is possible due to the water-attracting and physicochemical properties of this glycosaminoglycan. During pathological conditions, such as diabetes, the elevated interstitial HA can contribute to the defective kidney function, due to the proinflammatory and water-attracting properties of HA.

Hyaluronic acid is commonly used in dermatological fillers in the form of gels. It is established how these gels' firmness is affected by the amount of cross linker and hyaluronic acid respectively. However, the effect of hydroxide ions in solution is rather unknown. This thesis examines how the alkalinity of the solvent affects the intrinsic viscosity of 3 MDa hyaluronic acid by using the method of Ubbelohde capillary viscometry. Sodium hydroxide solutions between 2 and 10 wt% were prepared to study the variation in intrinsic viscosity at concentrations relevant for cross linking (1&lt;wt%). From these respective solutions, four solutions of different mass concentrations of hyaluronic acid were made. The flow time of respective samples were measured between two points in the capillary viscometer in a controlled temperature of 25 °C with an SI Viscoclock to ensure a high accuracy.From the resulting flow times, the intrinsic viscosity was calculated. The intrinsic viscosity varied between 0,55 and 0,70. The relation between intrinsic viscosity and hydroxide ion concentration had a correlation coefficient r &lt; 0,001. No trend could be ensured as the confidence interval for the intrinsic viscosity at the different concentrations was too large.

O uso concomitante da enzima hialuronidase (H) ao anestésico local (AL) é muito utilizado para melhorar a eficácia anestésica em oftalmologia; em odontologia, no entanto, não mostrou vantagens. Um novo protocolo foi testado com o objetivo de prolongar a anestesia local sem a realização de complementação anestésica. Esta possibilidade seria especialmente interessante para pacientes que apresentam restrição de dose máxima recomendada de AL ou vasoconstritor (ex. cardiopatas). Utilizou-se cloridrato de lidocaína 2% com epinefrina para bloqueio sensitivo, motor e proprioceptivo no nervo ciático em ratos (Truant,1958). Hialuronidase 75 UTR (unidade de turbidade reduzida) foi injetada no mesmo local 30 minutos após o início da analgesia (antes do término do efeito anestésico), utilizando-se a pata contralateral como controle (injeção de solução anestésica e veículo da hialuronidase solvente). A duração do bloqueio sensitivo foi avaliada através da ausência do reflexo de retirada da pata, utilizando-se um analgesímetro. O bloqueio motor foi avaliado pela duração da claudicação e da ausência do reflexo de estiramento da pata, enquanto o bloqueio proprioceptivo foi avaliado pela perda dos reflexos de salto e do reposicionamento da pata (Thalhammer et al., 1995). Foi também estudada a alteração tecidual induzida pela hialuronidase nos períodos de 1 h, 24 h, 48 h e 72 h pós-injeção. Foram avaliados os grupos: (1) falso operado (Sham); (2) AL +H; (3) AL+ solvente (Solv) e (4) Solv+Solv. Concluiu-se que a hialuronidase prolonga a duração de ação anestésica local quando injetada isoladamente antes da regressão do bloqueio de condução do nervo ciático de rato. Em todos os grupos analisados, exceto o grupo falso-operado, observou-se reação inflamatória após as injeções. Esta inflamação foi mais acentuada no grupo hialuronidase, que mostrava sinais de regressão após 72 horas. É possível que o mecanismo de ação da hialuronidase envolva a desorganização do tecido conjuntivo na região da injeção, facilitando a difusão da solução anestésica residual até o nervo. / The concomitant use of the enzyme hyaluronidase (H) and local anesthetics (LA) is widely employed in ophthalmology in order to improve the effectiveness of anesthesia; in dentistry, however, this association did not seem advantageous. A new protocol was tested with the aim of drawing out local anesthesia without supplementary anesthesia. This possibility is especially interesting for patients with restriction of maximum recommended dose of LA or vasoconstrictor due to pathological conditions (e.g. heart disease). We used 2% lidocaine hydrochloride with epinephrine for sensitive, motor and proprioceptive blockade of the sciatic nerve in rats (Truant, 1958). Hyaluronidase 75 UTR was injected 30 minutes after the beginning of the anesthesia (before the recovery of the sensory function), using the contralateral limb as control (injection of LA plus the H vehicle solvent). The duration of the sensitive blockade was evaluated through the absence of the paw withdrawal reflex, using an analgesymeter. The motor blockade was evaluated by the duration of claudication (complete absence of extensor postural thrust) and by the absence of the paw stretching reflex, while the proprioceptive blockade was evaluated by the absence of hopping and tactile placing response (Thalhammer et al., 1995). Histological changes induced by H were analyzed 1h, 24h, 48h, and 72 h after the injection in the following groups: (1) Sham; (2) LA + H; (3) LA + H solvent (solv) and (4) Solv + Solv. We concluded that H draws out local anesthesia when injected before the recovery of the sciatic nerve blockade in the rat. In all groups studied, with the exception of the Sham, there was an inflammatory reaction after the injections. Inflammation was more intense after H injection, showing signs of regression after 72 hours. It is possible that the mechanism of action of H involves disorganization of the connective tissue, thus facilitating the diffusion of the residual anesthetic solution to the nerve.

Enfermidade muito observada nas articulações dos equinos, o processo degenerativo articular crônico na articulação tibio-társica pode ser de origem primária, ou secundária à osteocondrose dissecante. A confirmação diagnóstica é feita por avaliação radiográfica. Importantes aliados diagnósticos são a avaliação do líquido sinovial, a avaliação ultrassonografia e a cirurgia de video-artroscopia, sendo esta última o tratamento para a enfermidade. Por sua evolução crônica e às vezes assintomática, a osteocondrose dissecante causa alterações no ambiente articular, como a perda de matriz cartilagínea, o que limita o potencial atlético do animal, acarretando em queda na performace e perdas econômicas. É comum o uso terapêutico intra-articular no periodo pós operatório recente de substâncias autólogas como o plasma rico em plaquetas (PRP), bem como o uso ácido hialurônico (HA), como adjuvante para o tratamento cirúrgico. O presente trabalho objetiva comparar e avaliar estes tratamentos. Foram utilizados 18 equinos, com idade variando entre 2 a 6 anos de idade, da raça Brasileiro de Hipismo, com peso corpóreo entre 400 e 650 kg, oriundos do mesmo criatório, com osteocondrose dissecante, na crista intermédia do osso tíbia. Estes animais no décimo dia pós operatório são dividos em três grupos experimentais: grupo AH, grupo PRP e grupo RCL como controle. Dados clínicos, ultrassonográficos e laboratoriais foram coletados em tempos definidos. Os resultados demonstraram que os animais do grupo controle - grupo RCL, que receberam solução de ringer com lactato intraarticular, apresentaram respostas clínicas e laboratoriais que indicaram menor indução do processo inflamatório ao ambiente articular quando comparado aos grupos PRP e AH. Os animais que receberam PRP apresentaram maior efusão sinovial e resposta positiva ao teste de flexão quando comparados aos animais que receberam AH; ao passo que a administração intra-articular de AH induziu maiores alteração citológica do líquido sinovial quando comparadas aos animais que receberam PRP. / One of the most commom diseases in the equine joint, the degenerative joint disease in the tibio tarsal joint could be a primary disease, or secondary to osteochondrosis dissecans. The diagnosis is made by radiographic study. Allies in diagnosing this disease includes sinovial fluid evaluation, ultrasonographic evaluation and video arthroscopy surgery, which is the way to treat this disease. The osteochondrosis dissecans has a chronic evolution, and several times asymptomatic, which causes an alteration on articular environment that contributes to a loss in the cartilage matrix. Resulting in the limited development of the athletic potential of the animal, poor performance and economic losses. Is common to use therapeutical autholog substances intra articular as the platelet rich plasma, and hialuronic acid, in the early post surgical treatment as an adjuvant therapy to the surgical treatment. This study reaches to compare and evaluate those treatments. Were used 18 horses, with ages between 2 to 6 years old, Brasileiro de Hipismo breed, weighting between 400 and 650 kg, from the same breeding center, with osteochondrosis dissecans fragments from the intermediate tibial crest. Those patients, 10 days after the video arthroscopy surgery, were divided in three experimental groups: AH group, PRP group, and RCL group as control group. Laboratorial data, clinical signs and ultrassonographic exam were performed. The results proved that RCL group had show less induction of inflamatory process on articular enviroment, when we compare with PRP group and AH group. The PRP group had shown more synovial effusion, and more sensibility to the flexion test when we compare to AH group; but the AH group had shown more laboratorial alterations when we compare to the PRP group.

Schwere Infektionen und Abstoßungen sind die häufigsten Komplikationen nach Lebertransplantation. Hyaluronsäure (HA) als eine Komponente der extrazellulären Matrix wird aus dem Blutkreislauf praktisch nur durch die Endothelzellen der Lebersinusoide (SEC) entfernt. Somit ist die Konzentration von HA im Plasma abhängig vom HA-Einstrom in den Blutkreislauf (z.B. bei vermehrter HA-Produktion der Fibroblasten nach Zytokinstimulation) und der metabolischen Funktion der SEC. Es wird angenommen, daß die SEC zu den ersten Angriffspunkten einer Abstoßungsreaktion in der Leber gehören. Bei 81 Patienten mit 85 Lebertransplantationen wurde zusätzlich zu den Routineparametern die Plasma-HA bestimmt. 28 der 81 Patienten (34,6%) entwickelten eine akute Abstoßung. 14 Patienten konnten erfolgreich mit Methylprednisolon (steroid-sensible akute Abstoßung, AR) behandelt werden, während 14 Patienten eine zusätzliche Therapie mit FK506 oder OKT3 (steroid-resistente akute Abstoßung, SR) benötigten. 4 Patienten entwickelten innerhalb des ersten postoperativen Jahres eine frühe chronische Abstoßung (CR). Bei 10 Patienten wurde eine schwere postoperative Infektion beobachtet, 11 Patienten entwickelten eine milde oder asymptomatische Cholangitis, während 37 Patienten einen unauffälligen postoperativen Verlauf hatten. Mittlere HA Spiegel waren bei den Patienten mit AR gegenüber den Patienten mit unauffälligem Verlauf erhöht. Ein weiterer Anstieg von HA konnte bei den Patienten mit SR (p / Severe infections and rejections are the most frequent complications following liver transplantation. Hyaluronic acid (HA) as a component of the extracellular matrix is cleared from the circulation only by sinusoidal liver cells (SEC). So the concentration of plasma HA depends upon the flow of HA in the circulation (e.g. increased HA-production in fibroblasts after cytokine stimulation and release) and the metabolic function of the SEC. SECs are suspected to be the first targets in allograft liver rejection. 81 patients with 85 liver transplantations were monitored for routine parameters and plasma HA on a daily basis. Of 81 patients, 28 patients (34,6%) developed acute rejection. 14 patients were successfully treated with methylprednisolone (steroid-sensitive acute rejection, AR), while 14 patients required additional treatment with FK506 or OKT3 (steroid-resistant acute rejection, SR). 4 patients developed an early chronic rejection (CR) within the first postoperative year. 10 patients developed a severe postoperative infection, 11 patients had a mild or asymptomatic cholangitis, while 37 patients had an uneventful postoperative course. Mean HA levels were elevated in patients with AR compared with patients with an uneventful course. A further increase in HA was noticed in patients with SR (p