Metabolic and cytoprotective effects of in vivo peri-patellar hyaluronic acid injections in cultured tenocytes

Abstract

The purpose of this study was to investigate tenocyte mechanobiology after sudden-detraining and to examine the hypothesis that repeated peri-patellar injections of hyaluronic acid (HA) on detrained patellar tendon (PT) may reduce and limit detrained-associated damage in tenoctyes.

Twenty-four male Sprague–Dawley rats were divided into three groups: Untrained, Trained and Detrained. In the Detrained rats, the left tendon was untreated while the right tendon received repeated peri-patellar injections of either HA or saline (NaCl). Tenocyte morphology, metabolism and synthesis of C-terminal-propeptide of type I collagen, collagen-III, fibronectin, aggrecan, tenascin-c, interleukin-1β, matrix-metalloproteinase-1 and-3 were evaluated after 1, 3, 7 and 10 days of culture. Transmission-electronic-microscopy showed a significant increase in mitochondria and rough endoplasmic reticulum in cultured tenocytes from Detrained-HA with respect to those from Detrained-NaCl. Additionally, Detrained-HA cultures showed a significantly higher proliferation rate and viability, and increased synthesis of C-terminal-Propeptide of type I collagen, fibronectin, aggrecan, tenascin-c and matrix-metalloproteinase-3 with respect to Detrained-NaCl ones, whereas synthesis of matrix-metalloproteinase-1 and interleukin-1β was decreased. Our study demonstrates that discontinuing training activity in the short-term alters tenocyte synthetic and metabolic activity and that repeated peri-patellar infiltrations of HA during detraining allow the maintenance of tenocyte anabolic activity.     

关节腔内注射透明质酸钠与富血小板血浆治疗膝关节骨性关节炎的比较

文题释义： 富血小板血浆：1993年HOOD等首先提出富血小板血浆的概念,人全血经过离心后获得富含血小板的血浆,在其中加入凝血酶后可变为胶状物,因此也被称为富血小板凝胶。富血小板血浆中含有大量的生长因子如血小板源性生长因子、转化生长因子β、胰岛素样生长因子1等,可促进骨再生。富血小板血浆的制备方法尚未形成统一的标准,主要有血浆分离置换法和密度梯度离心法2种方法。 透明质酸钠：是一种葡聚糖醛酸,广泛存在于胎盘、羊水、晶状体、关节软骨、皮肤真皮层等组织。它是关节腔滑液的主要成分,对关节起润滑作用,可以减少组织间的磨擦,关节腔内注入透明质酸钠的作用：明显改善滑液组织的炎症反应,增强关节液的黏稠性和润滑功能,保护关节软骨,促进关节软骨的愈合与再生,缓解疼痛,增加关节的活动度。  背景：富血小板血浆因其富含生长因子,在组织修复及再生中起促进作用,其已逐渐应用于临床治疗骨性关节炎。透明质酸钠可改善滑液组织的炎症反应,保护关节软骨,促进关节软骨的愈合与再生,缓解疼痛。 目的：对比观察富血小板血浆、透明质酸钠治疗膝关节骨性关节炎的疼痛和功能改善程度。 方法：符合标准的膝关节骨性关节炎患者被随机分配到富血小板血浆组和透明质酸钠组。富血小板血浆组患者在21 d内接受3次关节穿刺注射富血小板血浆治疗,透明质酸钠组患者在35 d内接受5次关节穿刺注射透明质酸钠治疗。注射前以及注射后2,4,6个月随访评估疼痛及功能的改善程度,所使用的评估量表包括可视量化分级量表、膝关节和骨性关节炎症状系统分级表、HSS评分量表。 结果与结论：富血小板血浆组最终有35例患者,透明质酸钠组有36例患者进入结果分析。在6个月的随访期末,两组患者的疼痛、功能症状都有所减轻。膝关节损伤和骨性关节炎症状系统分级评分表明,相对于透明质酸钠组,富血小板血浆治疗关节炎分级Ⅱ级患者更有效。最后1次治疗后,富血小板血浆治疗组患者可视量化分级量表分值相对于初始值减少50%的患者数明显多于透明质酸钠组。两组患者治疗后2,4,6个月HSS评分差异无显著性意义(P > 0.05)。结果表明,富血小板血浆治疗方法适用于骨性关节炎分级较低的患者。 ORCID: 0000-0003-4453-6221(孙仁义) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Evaluation of intra-articular delivery of hyaluronic acid functionalized biopolymeric nanoparticles in healthy rat knees

The aim of this study is to evaluate the toxicity of nanoparticles of poly(D,L-lactic acid) (PLA) or poly(D,L-lactic-co-glycolic acid) (PLGA) covered by chemically esterified amphiphilic hyaluronate (HA) which will be used for intra-articular injection as a drug carrier for the treatment of arthritis (RA) and/or osteoarthritis (OA). PLA and PLGA are FDA approved polymers that are already used for the preparation of nano or microparticles. HA is a natural polysaccharide already present in the articulations known to interact with the CD44 receptors of the cells (especially chondrocytes). Therefore, we can envisage that the HA covering can improve the interactions between the cells and the nanoparticles, leading to better targeting or biodistribution. The knee of healthy male rats was injected one to two times weekly, with various concentrations of nanoparticles encapsulating Dextran-FITC. The synovial membranes and the patellae were collected aseptically and histologically analyzed to assess the effects and localization of the nanocapsules in the knee joint. We did not observe significant modifications in the synovial membranes (weak hyperplasia) or patellae integrity after local administration of nanodevices into the rats. While we found some nanoparticles in the synovial membrane, none were detected in the patellae. Moreover, the histological observations for patellae were confirmed by radiosulfate intake, which depicted no decrease in proteoglycans biosynthesis in nanoparticles treated animals. Concerning the safety towards synovial membranes, we also had a look at the inflammatory response after injections of nanoparticles covered by amphiphilic HA or polyvinyl alcohol (PVA) by monitoring the mRNA expression levels of some specific early cytokines (IL-1β and TNF-α). Once again, no differences were observed between the control rats and the rats treated with nanoparticles. Considering these preliminary results obtained in healthy rats, we can establish that neither the amphiphilic HA-covered PLGA nanoparticles nor their degradation products induce major modifications of articular tissues functions, while injected into the knee of healthy rats. These results should be confirmed in OA or RA rat models, in order to confirm that nanoparticles do not worsen already altered (degenerative or inflamed) articular tissues. Once confirmed, such tuneable nanoparticles could be proposed as a safe drug delivery system for the treatment of articular disease, allowing a wide range of encapsulating molecules.     

Serum hyaluronic acid in patients with disseminated neoplasm.

Hyaluronic acid concentrations were measured by a laser nephelometric assay in serum samples from 50 patients with advanced disseminated neoplasm and 50 healthy controls matched for age and sex. The identity of hyaluronic acid was confirmed by a combination of electrophoretic and enzymatic techniques. The mean serum hyaluronic acid concentration for the control group was 1.09 mg/l, with a range of 0-4 mg/l. The mean concentration for patients with neoplastic disease was 10.38 mg/l, with a range of 0-100 mg/l. Sixty two per cent of the patients with disseminated neoplasm had serum hyaluronic acid concentrations above the control range. There was no correlation between the increased concentration of hyaluronic acid and tumour type, serum bilirubin, serum alkaline phosphatase, or serum urea concentrations. There was a higher incidence of hypercalcaemia in patients with increased hyaluronic acid concentrations, but the correlation between hyaluronic acid and calcium concentrations was not significant. In view of the possible role of hyaluronic acid in cellular differentiation and morphogenesis the finding of increased hyaluronic acid concentrations in patients with advanced neoplastic disease may be of fundamental importance in cancer biology.     

Degradation of hyaluronic acid by polymorphonuclear leukocytes

Degradation (depolymerization) of hyaluronic acid is readily accomplished by superoxide-ion-generating systems, especially those which beget secondary free radicals. It has been presumed, but not confirmed, that this is the mechanism by which neutrophils might alter synovial fluid viscosity. We have demonstrated, in a neutrophil (PMN) superoxide system, physical disruption of the hyaluronate macromolecule using column chromatography and by measurement of intrinsic viscosity. In addition, comparison of calibrated free radical fluxes between a cell-free superoxide system and a neutrophil system revealed very close parallels in iron requirement, inhibition by free radical scavengers, and magnitude of effect. It is concluded that oxygen-derived free radicals are probably the major, if not sole, mechanism by which neutrophils might degrade hyaluronate.     

The selective suppression of immunogenicity by hyaluronic acid

A hyaluronidase-sensitive component of human peritoneal fluid from a patient with Wilms' tumor when injected into rabbits has been shown to suppress the formation of humoral precipitating antibodies to certain major classes of proteins present in the fluid. Furthermore, it has been found that hyaluronic acid, when included with certain test antigens (serum albumin, fetuin) or antigen mixtures (tumor isolates or mixtures of albumin, immunoglobulin G and immunoglobulin M), produces a marked distortion or complete blockage of immunoelectrophoresis precipitin arcs, as well as altered gel chromatography elution profiles. These findings that hyaluronic acid can interfere profoundly with both the elicitation of a complete antibody response and the formation of "normal" patterns of antigen-antibody precipitates in laboratory tests supports the possibility that this polysaccharide may play an immuno-regulatory role by masking potential immunogens. Consideration of the mechanisms for these in vivo and in vitro effects suggests that there may be some common basis in an "excluded volume" property of the hyaluronate, but this does not appear sufficient to explain the complexity and selectivity of the observed phenomena.     

Enhanced humoral immune response in rats by hyaluronic acid

The effect of hyaluronic acid (HA) on antibody responses to egg albumin, dog albumin and birch pollen proteins, was studied in rats. The highest antibody responses to dog and egg albumin were obtained in rats immunized with a coprecipitate of the antigen with HA. However, pretreatment of rats with a subcutaneous injection of HA up to three (3) days prior to the priming injection of dog albumin also significantly enhanced the secondary antibody responses to that antigen. Possible explanations of this stimulatory effect on the humoral immune response are discussed.     

Stimulation of in vivo angiogenesis by cytokine-loaded hyaluronic acid hydrogel implants

Crosslinked hyaluronic acid (HA) hydrogels were evaluated for their ability to elicit new microvessel growth in vivo when preloaded with one of two cytokines, vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF). HA film samples were surgically implanted in the ear pinnas of mice, and the ears retrieved 7 or 14 days post implantation. Histologic analysis showed that all groups receiving an implant demonstrated significantly more microvessel density than control ears undergoing surgery but receiving no implant (p < 0.01). Moreover, aqueous administration of either growth factor produced substantially more vessel growth than an HA implant with no cytokine. However, the most striking result obtained was a dramatic synergistic interaction between HA and VEGF. Presentation of VEGF in crosslinked HA generated vessel density of NI = 6.7 at day 14, where NI is a neovascularization index defined below, more than twice the effect of the sum of HA alone (NI = 1.8) plus VEGF alone (NI=1.3). This was twice the vessel density generated by co-addition of HA and bFGF (NI=3.4, p<0.001). New therapeutic approaches for numerous pathologies could be notably enhanced by the localized, synergistic angiogenic response produced by release of VEGF from crosslinked HA films.     

Prolongation of sciatic nerve blockade by in situ cross-linked hyaluronic acid

Controlled release technology has been applied extensively in providing prolonged duration local anesthesia. Here we used modified hyaluronic acids (HAs; hydrazide and aldehyde) that cross-link upon mixing, as the vehicle for bupivacaine. We assessed the formulations' efficacy and biocompatibility in a rat model of sciatic nerve blockade. We found that 2% (w/v) cross-linked HA doubled the duration of block of 0.1%, 0.25%, and 0.5% (w/v) bupivacaine, without a statistically significant increase in myotoxicity. 1% (w/w) cross-linked HA also prolonged nerve block, but unmodified HA, and both modified HAs did not. HA itself was associated with a mild to moderate inflammatory response with macrophages and lymphocytes. Cross-linked HA is an effective and biocompatible vehicle for enhancing local anesthetic efficacy.     

Intra-articular ozone slows down the process of degeneration of articular cartilage in the knees of rats with osteoarthritis

BackgroundOsteoarthritis (OA) is a joint disease of multifactorial etiology, affecting mainly the knees. We aimed to evaluate the effects of two different doses of gaseous ozone intra-articularly on the knee cartilage morphology of rats with osteoarthritis (OA).MethodsThe articular lesion was induced by sodium monoiodoacetate (MIA). 40 Wistar rats were divided into 4 groups: G1 control (without lesion and without treatment), G2 articular lesion (AL) (only lesion MIA-induced), G3 AL + treatment with 5 μg/mL of ozone intra-articular, and G4 AL + treatment with 10 μg/mL of ozone intra-articular. The experiment was carried out for 60 days.ResultsBoth doses of ozone intra-articular demonstrated less reduction in joint space (G3 and G4) compared to the G2, formation of osteophytes, but without subchondral sclerosis. Ozone decreased the volumetric density of the articular lesion (VV(AL)) of tibial. The treatments recovered VV(AL) of the femur similar to G1. Ozone lower dose (G3) showed lower tibia and femur macroscopic scores.ConclusionIntra-articular gaseous ozone can delay the degeneration of articular cartilage and can represents an integrative therapy in the OA treatment of knee after 60 days of treatment. For the first time the role of ozone in articular cartilage degeneration was evaluated helping to understand this therapy.     

透明质酸生物活性的研究进展

透明质酸多糖（hyaluronic acid,HA）是一种大分子酸性粘多糖,由N-乙酰氨基葡萄糖（GlcNAc）与葡萄糖醛酸（GlcA）双糖单位聚合而成,即[（1—3）．B—D—GlcNAc（1—4）-D—D—GlcA],平均相对分子质量为10^5～10^7,其在医药、化妆品、食品等领域有广泛而独特的应用价值。一般是通过HA酶（HAase）的酶解途径或活性氧自由基（ROS）的氧化途径,HA经过降解可以得到低相对分子质量的透明质酸（LMWHA）和透明质酸寡聚糖（o—HA）。     

Rheologic changes in the synovial fluid of patients with gonarthritis induced by intraarticular infiltration of hyaluronic acid.

The aim of the present study was to evaluate possible changes in synovial fluid viscosity in gonarthritic patients treated with intraarticular infiltration of hyaluronic acid. Thirty patients with radiologically proven (Stage III Kellgren) serious gonarthrosis, local pain and functional limitation were enrolled. All patients had reported at least 4-5 episodes of hydrarthrosis during the previous 12 months. Therefore, two different phases of their illness could be observed: a relatively silent phase of hydrarthrosis and a symptomatic phase. According to the protocol, one sample of synovial fluid was collected for evaluation of baseline viscosity (pretreatment); immediately afterwards, intraarticular administration of high molecular weight sodium hyaluronate (one 20 mg vial/week for 3 weeks) was initiated. During the entire treatment period and for 3 weeks following the end of treatment, intraarticular synovial fluid samples (one sample per week for 3 treatment weeks followed by a further 3 weeks as control) were collected to perform rheologic assessment and viscosimetric analysis. The results of this preliminary study show that exogenous administration of high molecular weight sodium hyaluronate induced normalization of hyaluronic acid viscosity values in patients with high and low baseline hyaluronic acid viscosity values.     

Surface functionalization of hyaluronic acid hydrogels by polyelectrolyte multilayer films

Hyaluronic acid (HA), an anionic polysaccharide, is one of the major components of the natural extracellular matrix (ECM). Although HA has been widely used for tissue engineering applications, it does not support cell attachment and spreading and needs chemical modification to support cellular adhesion. Here, we present a simple approach to functionalize photocrosslinked HA hydrogels by deposition of poly(l-lysine) (PLL) and HA multilayer films made by the layer-by-layer (LbL) technique. PLL/HA multilayer film formation was assessed by using fluorescence microscopy, contact angle measurements, cationic dye loading and confocal microscopy. The effect of polyelectrolyte multilayer film (PEM) formation on the physicochemical and mechanical properties of hydrogels revealed polyelectrolyte diffusion inside the hydrogel pores, increased hydrophobicity of the surface, reduced equilibrium swelling, and reduced compressive moduli of the modified hydrogels. Furthermore, NIH-3T3 fibroblasts seeded on the surface showed improved cell attachment and spreading on the multilayer functionalized hydrogels. Thus, modification of HA hydrogel surfaces with multilayer films affected their physicochemical properties and improved cell adhesion and spreading on these surfaces. This new hydrogel/PEM composite system may offer possibilities for various biomedical and tissue engineering applications, including growth factor delivery and co-culture systems.     

Fabrication of zein/hyaluronic acid fibrous membranes by electrospinning

Electrospinning of biopolymers, such as proteins and polysaccharides, has recently attracted much attention for the fabrication of scaffolds for tissue engineering. In this report, zein/hyaluronic acid (HA) blend fibrous membranes were electrospun and characterized. To facilitate the compatibility of zein and HA, poly(vinyl pyrrolidone) (PVP) was introduced into aqueous ethanol solutions of the blend. A series of zein/HA/PVP blend fibrous membranes with different volume ratios were successfully electrospun. The effect of blend composition on the morphology of electrospun fibrous membranes was investigated by scanning electron microscopy. The average diameter of blend fibers increased with increasing the content of zein component. The electrospun zein/HA/PVP fibrous membranes were then cross-linked by methylene diphenyl diisocyanate (MDI). The morphology of the cross-linked zein/HA/PVP fibrous membranes changed slightly compared with un-cross-linked membranes. Tensile tests demonstrated that the mechanical properties of the zein/HA/PVP fibrous membranes were improved by cross-linking.     

Fabrication of zein/hyaluronic acid fibrous membranes by electrospinning

Electrospinning of biopolymers, such as proteins and polysaccharides, has recently attracted much attention for the fabrication of scaffolds for tissue engineering. In this report, zein/hyaluronic acid (HA) blend fibrous membranes were electrospun and characterized. To facilitate the compatibility of zein and HA, poly(vinyl pyrrolidone) (PVP) was introduced into aqueous ethanol solutions of the blend. A series of zein/HA/PVP blend fibrous membranes with different volume ratios were successfully electrospun. The effect of blend composition on the morphology of electrospun fibrous membranes was investigated by scanning electron microscopy. The average diameter of blend fibers increased with increasing the content of zein component. The electrospun zein/HA/PVP fibrous membranes were then cross-linked by methylene diphenyl diisocyanate (MDI). The morphology of the cross-linked zein/HA/PVP fibrous membranes changed slightly compared with un-cross-linked membranes. Tensile tests demonstrated that the mechanical properties of the zein/HA/PVP fibrous membranes were improved by cross-linking.     

Collagen-based wound dressing: effects of hyaluronic acid and fibronectin on wound healing

Our previous studies have shown that a collagen-based wound dressing induces the spatial deposition of wound tissue. This study was conducted to determine the effects of hyaluronic acid and fibronectin on wound healing. These macromolecules play an important role in wound healing, embryonic development and cellular migration in vitro. The effects of the addition of varying levels of fibronectin and hyaluronate to a collagen sponge were studied. Low levels of both hyaluronate and fibronectin modified the structure of the implant, and resulted in increased chemoattraction, replication and collagen deposition in an in vivo wound healing model.     

Non-invasive investigation of liver disease in haemophilic patients.

Liver biopsy specimens previously taken from 16 haemophilic patients with chronic non-A, non-B hepatitis were reviewed. The degree of fibrosis correlated with serum procollagen III peptide (sPIIIP) concentrations, measured both at the time of biopsy and 4.25 years later. Two patients with extremely high sPIIIP concentrations had collateral veins on computed tomography, suggesting portal hypertension. Twenty eight of 47 patients (60%) had splenomegaly on computed tomography, and of 28 patients in whom intravenous contrast medium was used, seven (25%) had collateral oesophageal veins. Serum procollagen III peptide estimations and computed tomography, both non-invasive investigations, indicated that hepatic fibrosis and portal hypertension had developed in a proportion of haemophilic patients with non-A, non-B hepatitis. Infection with the human immunodeficiency virus (HIV) may modify the course of this presumably cytopathic virus infection of the liver.