A single-cell mass cytometry platform to map the effects of preclinical drugs on cartilage homeostasis.

No disease-modifying drug exists for osteoarthritis (OA). Despite success in animal models, candidate drugs continue to fail in clinical trials owing to the unmapped interpatient heterogeneity and disease complexity. We used a single-cell platform based on cytometry by time-of-flight (cyTOF) to precisely outline the effects of candidate drugs on human OA chondrocytes. OA chondrocytes harvested from patients undergoing total knee arthroplasty were treated with 2 drugs, an NF-κB pathway inhibitor, BMS-345541, and a chondroinductive small molecule, kartogenin, that showed preclinical success in animal models for OA. cyTOF conducted with 30 metal isotope-labeled antibodies parsed the effects of the drugs on inflammatory, senescent, and chondroprogenitor cell populations. The NF-κB pathway inhibition decreased the expression of p-NF-κB, HIF2A, and inducible NOS in multiple chondrocyte clusters and significantly depleted 4 p16ink4a-expressing senescent populations, including NOTCH1+STRO1+ chondroprogenitor cells. While kartogenin also affected select p16ink4a-expressing senescent clusters, there was a less discernible effect on chondroprogenitor cell populations. Overall, BMS-345541 elicited a uniform drug response in all patients, while only a few responded to kartogenin. These studies demonstrate that a single-cell cyTOF-based drug screening platform can provide insights into patient response assessment and patient stratification.

Ononin ameliorates inflammation and cartilage degradation in rat chondrocytes with IL-1ß-induced osteoarthritis by downregulating the MAPK and NF-κB pathways.

BACKGROUND: Osteoarthritis (OA) treatment aims to improve inflammation and delay cartilage degeneration. However, there is no effective strategy presently available. Ononin, a representative isoflavone glycoside component extracted from natural Chinese herbs, exerts anti-inflammatory and proliferative effects. However, the therapeutic effect of ononin on chondrocyte inflammation remains unclear. METHODS: In this study, we explored the therapeutic effect and potential mechanism of ononin in OA by establishing an interleukin-1 beta (IL-1ß)-induced chondrocyte inflammation model. RESULTS: Our results verified that ononin alleviated the IL-1ß-induced decrease in chondrocyte viability, attenuated the overexpression of the inflammatory factors tumour necrosis factor α (TNF-α) and interleukin 6 (IL-6), and simultaneously inhibited the expression of cartilage extracellular matrix (ECM)-degrading enzymes such as matrix metalloproteinase-13 (MMP-13). Furthermore, the decomposition of Collagen II protein could be alleviated in the OA model by ononin. Finally, ononin improved chondrocyte inflammation by downregulating the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) signalling pathways. CONCLUSION: Our findings suggested that ononin could inhibit the IL-1ß-induced proinflammatory response and ECM degradation in chondrocytes by interfering with the abnormal activation of the MAPK and NF-κB pathways, indicating its protective effect against OA.

Engineering osteoarthritic cartilage model through differentiating senescent human mesenchymal stem cells for testing disease-modifying drugs.

Significant cellular senescence has been observed in cartilage harvested from patients with osteoarthritis (OA). In this study, we aim to develop a senescence-relevant OA-like cartilage model for developing disease-modifying OA drugs (DMOADs). Specifically, human bone marrow-derived mesenchymal stromal cells (MSCs) were expanded in vitro up to passage 10 (P10-MSCs). Following their senescent phenotype formation, P10-MSCs were subjected to pellet culture in chondrogenic medium. Results from qRT-PCR, histology, and immunostaining indicated that cartilage generated from P10-MSCs displayed both senescent and OA-like phenotypes without using other OA-inducing agents, when compared to that from normal passage 4 (P4)-MSCs. Interestingly, the same gene expression differences observed between P4-MSCs and P10-MSC-derived cartilage tissues were also observed between the preserved and damaged OA cartilage regions taken from human samples, as demonstrated by RNA Sequencing data and other analysis methods. Lastly, the utility of this senescence-initiated OA-like cartilage model in drug development was assessed by testing several potential DMOADs and senolytics. The results suggest that pre-existing cellular senescence can induce the generation of OA-like changes in cartilage. The P4- and P10-MSCs derived cartilage models also represent a novel platform for predicting the efficacy and toxicity of potential DMOADs on both preserved and damaged cartilage in humans.

Inhibition of SYK and cSrc kinases can protect bone and cartilage in preclinical models of osteoarthritis and rheumatoid arthritis.

The pathophysiology of osteoarthritis (OA) includes the destruction of subchondral bone tissue and inflammation of the synovium. Thus, an effective disease-modifying treatment should act on both of these pathogenetic components. It is known that cSrc kinase is involved in bone and cartilage remodeling, and SYK kinase is associated with the inflammatory component. Thus the aim of this study was to characterize the mechanism of action and efficacy of a small molecule multikinase inhibitor MT-SYK-03 targeting SYK and cSrc kinases among others in different in vitro and in vivo arthritis models. The selectivity of MT-SYK-03 kinase inhibition was assayed on a panel of 341 kinases. The compound was evaluated in a set of in vitro models of OA and in vivo OA and RA models: surgically-induced arthritis (SIA), monosodium iodoacetate-induced arthritis (MIA), collagen-induced arthritis (CIA), adjuvant-induced arthritis (AIA). MT-SYK-03 inhibited cSrc and SYK with IC50 of 14.2 and 23 nM respectively. Only five kinases were inhibited > 90% at 500 nM of MT-SYK-03. In in vitro OA models MT-SYK-03 reduced hypertrophic changes of chondrocytes, bone resorption, and inhibited SYK-mediated inflammatory signaling. MT-SYK-03 showed preferential distribution to joint and bone tissue (in rats) and revealed disease-modifying activity in vivo by halving the depth of cartilage erosion in rat SIA model, and increasing the pain threshold in rat MIA model. Chondroprotective and antiresorptive effects were shown in a monotherapy regime and in combination with methotrexate (MTX) in murine and rat CIA models; an immune-mediated inflammation in rat AIA model was decreased. The obtained preclinical data support inhibition of cSrc and SYK as a viable strategy for disease-modifying treatment of OA. A Phase 2 clinical study of MT-SYK-03 is to be started.

Targeted treatment for osteoarthritis: drugs and delivery system.

The management of osteoarthritis (OA) is a clinical challenge due to the particular avascular, dense, and occluded tissue structure. Despite numerous clinical reports and animal studies, the pathogenesis and progression of OA are still not fully understood. On the basis of traditional drugs, a large number of new drugs have been continuously developed. Intra-articular (IA) administration for OA hastens the development of targeted drug delivery systems (DDS). OA drugs modification and the synthesis of bioadaptive carriers contribute to a qualitative leap in the efficacy of IA treatment. Nanoparticles (NPs) are demonstrated credible improvement of drug penetration and retention in OA. Targeted nanomaterial delivery systems show the prominent biocompatibility and drug loading-release ability. This article reviews different drugs and nanomaterial delivery systems for IA treatment of OA, in an attempt to resolve the inconsonance between in vitro and in vivo release, and explore more interactions between drugs and nanocarriers, so as to open up new horizons for the treatment of OA.

Kaempferia parviflora Extract Alleviated Rat Arthritis, Exerted Chondroprotective Properties In Vitro, and Reduced Expression of Genes Associated with Inflammatory Arthritis.

Kaempferia parviflora Wall. ex Baker (KP) has been reported to attenuate cartilage destruction in rat model of osteoarthritis. Previously, we demonstrated that KP rhizome extract and its active components effectively suppressed mechanisms associated with RA in SW982 cells. Here, we further evaluated the anti-arthritis potential of KP extract by using multi-level models, including a complete Freund's adjuvant-induced arthritis and a cartilage explant culture model, and to investigate the effects of KP extract and its major components on related gene expressions and underlying mechanisms within cells. In arthritis rats, the KP extract reduced arthritis indexes, with no significant changes in biological parameters. In the cartilage explant model, the KP extract exerted chondroprotective potential by suppressing sulfated glycosaminoglycans release while preserving high accumulation of proteoglycans. In human chondrocyte cell line, a mixture of the major components equal to their amounts in KP extract showed strong suppression the expression of genes-associated inflammatory joint disease similar to that of the extract. Additionally, KP extract significantly suppressed NF-κB and MAPK signaling pathways. The suppressing expression of necroptosis genes and promoted anti-apoptosis were also found. Collectively, these results provided supportive evidence of the anti-arthritis properties of KP extract, which are associated with its three major components.

Evaluation of Virola oleifera activity in musculoskeletal pathologies: Inhibition of human multiple myeloma cells proliferation and combination therapy with dexamethasone or bortezomib.

ETHNOPHARMACOLOGICAL RELEVANCE: Virola oleifera (Schott) A.C. Smith, Myristicaceae, has been widely used in traditional medicine in Brazil to treat rheumatic pain, joint tumours, skin diseases, halitosis, bronchial asthma, haemorrhoids, and intestinal worms. Recently, research data showed the antioxidant properties in several oxidative stress-related models. However, there is no experimental evidence supporting its potential use in managing rheumatic diseases and bone malignancies. AIMS OF THE STUDY: To evaluate the therapeutic potential of the resin from Virola oleifera in joint and bone diseases, namely arthritis, osteosarcoma, chondrosarcoma, and multiple myeloma. MATERIALS AND METHODS: To determine Virola oleifera resin (VO) effects on arthritis-associated inflammation and cartilage degradation, the LPS-induced NO production, and mRNA and protein expression of ADAMTS5, MMP13, COL2, and ACAN, were evaluated in chondrocytes (ATDC5 and TC28 cell lines). The cytotoxic effects of VO (0.05-50 µg/ml) on multiple myeloma (ARH-77), osteosarcoma (SAOS-2), and chondrosarcoma (SW-1353) cell lines were analysed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The VO effects, combined with dexamethasone or bortezomib, were evaluated in a multiple myeloma cell line. The mechanisms of VO, alone or in combination with bortezomib, were determined by cell cycle analysis through flow cytometry, while expression levels of p-Akt/Akt, p-ERK/ERK, p-p38/p38 MAPK, Bax, Bcl-2, and cleaved-caspase-3/caspase-3 proteins by Western blot. RESULTS: VO had no significant effect on LPS-induced NO production in chondrocytes at non-cytotoxic concentrations. VO treatment diminished the mRNA levels of metalloproteinases and ECM components; however, any significant effect was observed on the protein expression levels. The cell viability of a multiple myeloma cell line was strongly reduced by VO treatment in a dose- and time-dependent manner, while osteosarcoma and chondrosarcoma cell lines viability was significantly affected only by the highest dose assessed. In multiple myeloma cells, VO leads to G2/M cell cycle arrest. Furthermore, it synergizes with dexamethasone by increasing cell toxicity. Finally, VO reverts bortezomib activity by counteracting ERK1/2, Bax, and caspase-3 activation. CONCLUSIONS: The current work supports the ethnopharmacological use of Virola oleifera (Schott) A.C. Smith in bone and joint diseases, but there is no evidence for the amelioration of arthritis-associated inflammatory or catabolic processes. Our data also supports the potential use of Virola oleifera as adjuvant therapy to optimize the pharmacologic effects of current chemotherapeutic drugs. However, possible herb-drug interactions should be considered before clinical application.

Rheumatoid Arthritis: Applicability of Ready-to-Use Human Cartilaginous Cells for Screening of Compounds with TNF-Alpha Inhibitory Activity.

In the context of modern drug discovery, there is an obvious advantage to designing phenotypic bioassays based on human disease-relevant cells that express disease-relevant markers. The specific aim of the study was to develop a convenient and reliable method for screening compounds with Tumor Necrosis Factor-alpha (TNF-α) inhibitory activity. This assay was developed using cryopreserved ready-to-use cartilage-derived cells isolated from juvenile donors diagnosed with polydactyly. It has been demonstrated that all donor (10 donors) cells were able to respond to TNF-α treatment by increased secretion of pro-inflammatory cytokine IL-6 into subcultural medium. Inhibition of TNF-α using commercially available TNF-α inhibitor etanercept resulted in a dose-dependent decrease in IL-6 production which was measured by Enzyme-Linked Immunosorbent Assay (ELISA). TNF-α dependent IL-6 production was detected in the cells after both their prolonged cultivation in vitro (≥20 passages) and cryopreservation. This phenotypic bioassay based on ready-to-use primary human cells was developed for detection of novel TNF-α inhibitory compounds and profiling of biosimilar drugs.

Curcumin prevents tension-induced endplate cartilage degeneration by enhancing autophagy.

AIMS: Intermittent cyclic tension stimulation(ICMT) was shown to promote degeneration of endplate chondrocytes and induce autophagy. However, enhancing autophagy can alleviate degeneration partly. Studies have shown that curcumin can induce autophagy and protect chondrocytes, we speculated that regulation of autophagy by curcumin might be an effective method to improve the stress resistance of endplate cartilage. In this study, human cervical endplate cartilage specimens were collected, and expression of autophagy markers was detected and compared. MAIN METHODS: Human cervical endplate chondrocytes were cultured to establish a tension-induced degeneration model, for which changes of functional metabolism and autophagy levels were detected under different tension loading conditions. Changes in functional metabolism of endplate chondrocytes were observed under high-intensity tension loading in the presence of inhibitors, inducers, and curcumin to regulate the autophagy level of cells. In addition, a rat model of lumbar instability was established to observe the degeneration of lumbar disc after curcumin administration. KEY FINDINGS: Through a series of experiments, we found that low-intensity tension stimulation can maintain a stable phenotype of endplate chondrocytes, but high-intensity tension stimulation has a negative effect. Moreover, with increasing tension intensity, the degree of degeneration of endplate chondrocytes was gradually aggravated and the level of autophagy increased. Besides, curcumin upregulated autophagy, inhibited apoptosis, and reduced phenotype loss of endplate chondrocytes induced by high-intensity tension loading, thereby relieving intervertebral disc degeneration induced by mechanical imbalance. SIGNIFICANCE: Curcumin mediated autophagy and enhanced the adaptability of endplate chondrocytes to high-intensity tension load, thereby relieving intervertebral disc degeneration.

Herbal Remedies as Potential in Cartilage Tissue Engineering: An Overview of New Therapeutic Approaches and Strategies.

It is estimated that by 2023, approximately 20% of the population of Western Europe and North America will suffer from a degenerative joint disease commonly known as osteoarthritis (OA). During the development of OA, pro-inflammatory cytokines are one of the major causes that drive the production of inflammatory mediators and thus of matrix-degrading enzymes. OA is a challenging disease for doctors due to the limitation of the joint cartilage's capacity to repair itself. Though new treatment approaches, in particular with mesenchymal stem cells (MSCs) that integrate the tissue engineering (TE) of cartilage tissue, are promising, they are not only expensive but more often do not lead to the regeneration of joint cartilage. Therefore, there is an increasing need for novel, safe, and more effective alternatives to promote cartilage joint regeneration and TE. Indeed, naturally occurring phytochemical compounds (herbal remedies) have a great anti-inflammatory, anti-oxidant, and anabolic potential, and they have received much attention for the development of new therapeutic strategies for the treatment of inflammatory diseases, including the prevention of age-related OA and cartilage TE. This paper summarizes recent research on herbal remedies and their chondroinductive and chondroprotective effects on cartilage and progenitor cells, and it also emphasizes the possibilities that exist in this research area, especially with regard to the nutritional support of cartilage regeneration and TE, which may not benefit from non-steroidal anti-inflammatory drugs (NSAIDs).

Regenerative effects of hyperbaric oxygen therapy and platelet-rich plasma on the osteochondral defects of rats.

OBJECTIVES: This study aims to investigate the effects of hyperbaric oxygen (HBO) therapy and platelet-rich plasma (PRP) on the regeneration of osteochondral defects of the rats, and the synergistic effect of this combined treatment. MATERIALS AND METHODS: This randomized, controlled, and interventional animal study was conducted between May 2014 and August 2014 Osteochondral regeneration was evaluated in four treatment groups (control, PRP, HBO, and HBO+PRP groups) at the 30th day after iatrogenic injury. Thirty-two female Wistar albino rats (weighing 248-305 g) underwent arthrotomy and osteochondral surgery on left knees. The regenerations of defects were then examined histologically by the modified version of O'Driscoll score. RESULTS: Groups that were treated with either HBO or PRP alone regenerated significantly better than the control group (p=0.01), while no significant difference was found between the HBO- and PRP-treated groups (p>0.05). The defects in group 4 (treated with both HBO and PRP) regenerated significantly better than the control group, the HBO-treated group alone, and the PRP-treated group alone (p=0.01). CONCLUSION: The results of this study showed a synergistic effect of HBO and PRP on knee cartilage regeneration. However, the possible underlying mechanisms should be the subject of future researches. The aggregation and activation of growth factors released from platelets whose activation is increased in the hyperbaric environment may explain this effect. This may result in a better regeneration than the effect of PRP or HBO alone.

COMBSecretomics: A pragmatic methodological framework for higher-order drug combination analysis using secretomics.

Multi drug treatments are increasingly used in the clinic to combat complex and co-occurring diseases. However, most drug combination discovery efforts today are mainly focused on anticancer therapy and rarely examine the potential of using more than two drugs simultaneously. Moreover, there is currently no reported methodology for performing second- and higher-order drug combination analysis of secretomic patterns, meaning protein concentration profiles released by the cells. Here, we introduce COMBSecretomics (https://github.com/EffieChantzi/COMBSecretomics.git), the first pragmatic methodological framework designed to search exhaustively for second- and higher-order mixtures of candidate treatments that can modify, or even reverse malfunctioning secretomic patterns of human cells. This framework comes with two novel model-free combination analysis methods; a tailor-made generalization of the highest single agent principle and a data mining approach based on top-down hierarchical clustering. Quality control procedures to eliminate outliers and non-parametric statistics to quantify uncertainty in the results obtained are also included. COMBSecretomics is based on a standardized reproducible format and could be employed with any experimental platform that provides the required protein release data. Its practical use and functionality are demonstrated by means of a proof-of-principle pharmacological study related to cartilage degradation. COMBSecretomics is the first methodological framework reported to enable secretome-related second- and higher-order drug combination analysis. It could be used in drug discovery and development projects, clinical practice, as well as basic biological understanding of the largely unexplored changes in cell-cell communication that occurs due to disease and/or associated pharmacological treatment conditions.

Glycitin Suppresses Cartilage Destruction of Osteoarthritis in Mice.

Osteoarthritis (OA), a chronic joint disease, is characterized by cartilage surface erosion, subchondral bone rebuilding, and formation of osteophytes. To date, the nosogenesis and underlying mechanisms of OA have not yet been elucidated. However, it is widely accepted that TNF-α is a crucial cytokine in the development of OA. Glycitin, a natural isoflavone extracted from legumes, affects physiological reactions and pathological responses. Recently, the anti-inflammatory effect of glycitin has been reported. However, the function of glycitin in cartilage degeneration in OA remains to be investigated. In the current study, primary murine chondrocytes were isolated and stimulated by TNF-α to evaluate the anti-inflammatory effects and protective function of glycitin in chondrocytes. In vivo, the ACLT mouse model, a frequently-used OA model, was used to further examine the therapeutic role of glycitin in cartilage degeneration and inflammation in OA. Consequently, glycitin functions were examined both in vivo and in vitro. Moreover, the underlying mechanism of action of glycitin was investigated and was found to involve the NF-κB signaling pathway. Collectively, this study suggests that glycitin can be potentially used for the treatment of joint degenerative diseases, including OA.

A biological extract of turmeric (Curcuma longa) modulates response of cartilage explants to lipopolysaccharide.

BACKGROUND: Turmeric is commonly used as a dietary treatment for inflammation, but few studies have evaluated the direct effect of turmeric on cartilage. The purpose of this study was to characterize cartilage explants' inflammatory responses to lipopolysaccharide in the presence of a simulated biological extract of turmeric. METHODS: Turmeric was incubated in simulated gastric and intestinal fluid, followed by inclusion of liver microsomes and NADPH. The resulting extract (TURsim) was used to condition cartilage explants in the presence or absence of lipopolysaccharide. Explants were cultured for 96 h (h); the first 24 h in basal tissue culture media and the remaining 72 h in basal tissue culture media containing TURsim (0, 3, 9 or 15 µg/mL). Lipopolysaccharide (0 or 5 µg/mL) was added for the final 48 H. media samples were collected immediately prior to lipopolysaccharide exposure (0 h) and then at 24 and 48 h after, and analyzed for prostaglandin E2 (PGE2), glycosaminoglycan (GAG), and nitric oxide (NO). Explants were stained with calcein-AM for an estimate of live cells. Data were analyzed using a 2-way repeated measures (GAG, PGE2, NO) or 1-way ANOVA without repeated measures (viability). Significance accepted at p < 0.05. RESULTS: TURsim significantly reduced PGE2, NO and GAG, and calcein fluorescence was reduced. CONCLUSIONS: These data contribute to the growing body of evidence for the utility of turmeric as an intervention for cartilage inflammation.

Does cartilage ERα overexpression correlate with osteoarthritic chondrosenescence? Indications from Labisia pumila OA mitigation.

Chondrosenescence (chondrocyte senescence) and subchondral bone deterioration in osteoarthritic rats were analyzed after treatment with the estrogenic herb Labisia pumila (LP) or diclofenac. Osteoarthritis (OA) was induced in bilaterally ovariectomized (OVX) rats by injecting mono-iodoacetate into the right knee joints. Rats were grouped (n = 8) into nontreated OVX+OA control, OVX+OA + diclofenac (5 mg/kg) (positive control), OVX+OA + LP leaf extract (150 and 300 mg/kg) and healthy sham control. After 8 weeks' treatment, their conditions were evaluated via serum biomarkers, knee joint histology, bone histomorphometry, protein and mRNA expressions. The LP significantly reduced cartilage erosion, femur bone surface alteration, bone loss and porosity and increased trabecular bone thickness better than diclofenac and the non-treated OA. The cartilage catabolic markers' (matrix metalloproteinase (MMP)-13, RUNX2, COL10a, ERa, CASP3 and HIF-2 alpha) mRNA expressions were down-regulated and serum bone formation marker, PINP, was increased by LP in a dose-dependent manner. The LP (containing myricetin and gallic acid) showed protection against chondrosenescence, chondrocyte death, hypoxia-induced cartilage catabolism and subchondral bone deterioration. The bone and cartilage protective effects were by suppressing proteases (collagen break-down), bone resorption and upregulating subchondral bone restoration. The cartilage ER alpha over-expression showed a strong positive correlation with MMP-13, COL10 alpha1, histological, micro-computed tomography evidence for cartilage degradation and chondrosenescence.

Chondroprotective effect of Nigella sativa oil in the early stages of osteoarthritis: an experimental study in rabbits.

PURPOSE: Nigella sativa oil possesses a well-known ability to protect certain organs from oxidative, neoplastic, and inflammatory damage. This study investigated the potential chondroprotective effects of intraarticular injections of Nigella sativa oil in a rabbit osteoarthritis model. METHODS: Osteoarthritis models were created by performing anterior cruciate ligament transections in 20 New Zealand rabbits. Rabbits were randomly divided into two groups of 10 and given intraarticular injections in their right knees weekly for 5 weeks, beginning in the third week post-operation. Injections given to the first group contained whole Nigella sativa oil, whereas the second group was injected with a saline solution. Knee joints were harvested 8 weeks after surgery. Knee joint surfaces were examined macroscopically, and medial femoral condyle sections were examined microscopically. RESULTS: There was a statistically significant difference in the macroscopic grading results of the groups, with the Nigella sativa group having better results (p=0.001). The Nigella sativa group also received significantly better total Osteoarthritis Research Society International (OARSI) scores (p=0.035). CONCLUSIONS: Intraarticular administration of Nigella sativa oil has the potential to protect cartilage from degeneration in the early stages of osteoarthritis.

Electrospun PCL nanofibers blended with Wattakaka volubilis active phytochemicals for bone and cartilage tissue engineering.

In the present study, the polycaprolactone (PCL) nanofibers were investigated as a carrier to deliver phytochemicals for bone and cartilage tissue engineering. The PCL nanofibers was blended with phytochemicals hexadecanoic acid, octadecanoic acid and N,N-diisopropyl (2,2,3,3,3-pentafluoropropyl) amine isolated from a medicinal plant, Wattakaka volubilis. The scaffolds were characterized using scanning electron microscope (SEM) and Fourier transform infrared (FTIR) spectroscopy. The average diameter of control and phytochemical loaded nanofiber was 208 ±â€¯9.6 nm and 316 ±â€¯7.0 nm respectively. Biodegradation rate of nanofibers, impact of nanofiber on meniscus and osteoblast cell growth was analyzed using 3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) assay, DNA content and extra cellular matrix secretion. Hoechst stain and SEM images were used to visualize and monitor the cell growth on PCL scaffold. The phytochemicals incorporated PCL nanofibers enhanced the growth and proliferation of primary human meniscus and osteoblast like cells and hence may be suitable scaffold for bone and cartilage tissue engineering applications.

Docosahexaenoic acid inhibits bone remodeling and vessel formation in the osteochondral unit in a rat model.

OBJECTIVES: We aimed to determine whether bone remodeling and vessel formation in the osteochondral unit are suppressed by supplementing with docosahexaenoic acid in anterior cruciate ligament transection (ACLT)-induced rats. METHODS: Twelve-week-old male Sprague Dawley rats were randomized to sham-operated, ACLT-operated and treated with vehicle, or ACLT-operated and treated with DHA groups. Micro-architecture and vasculature in the tibial osteochondral unit were examined by micro-CT, as well as by histomorphometry. To evaluate the effects of DHA in vitro, we conducted functional and expressional assays in RAW264.7 cells and HUVECs. Finally, we used OARSI-modified Mankin criteria and histological analyses to assess the status of the cartilage layer. RESULTS: Microstructural parameters in the osteochondral unit showed that bone mass loss and angiogenesis were less in DHA-treated rats than in vehicle-treated rats. Immunofluorescence-positive cells labeled with TRAP, RANKL, CD31, and endomucin agents in the osteochondral unit of ACLT-operated rats were reduced in the DHA-treated group compared with the vehicle-treated group. Furthermore, the number of TRAP-stained cells, areas of bone resorption pits, and mRNA expression of TRAP, CTSK, MITF, and NFATC1 were reduced in RAW264.7 cells treated with RANKL + DHA compared with those treated with only RANKL. Tube formation, proliferation and migration of HUVECs, and VEGF-C mRNA and VEGFR2 protein expression were inhibited by DHA. The decrease in OARSI score, and MMP-13 and collagen X expression suggested that DHA attenuated cartilage degeneration. CONCLUSIONS: DHA has the ability to restrain bone remodeling and vessel formation in the osteochondral unit, which may contribute to protection of cartilage.

Self-Assembled Injectable Nanocomposite Hydrogels Coordinated by in Situ Generated CaP Nanoparticles for Bone Regeneration.

Due to the great similarity to the natural extracellular matrix and minimally invasive surgeries, injectable hydrogels are appealing biomaterials in cartilage and bone tissue engineering. Nevertheless, undesirable mechanical properties and bioactivity greatly hamper their availability in clinic applications. Here, we developed an injectable nanocomposite hydrogel by in situ growth of CaP nanoparticles (ICPNs) during the free-radical polymerization of dimethylaminoethyl methacrylate (DMAEMA) and 2-hydroxyethyl methacrylate (HEMA) matrix (PDH) for bone regeneration. The ICPNs are self-assembled by incorporation of poly-l-glutamic acid (PGA) with abundant carboxyl functional groups during the formation of carboxyl-Ca2+ coordination and further CaP precipitation. Furthermore, the carboxyl groups of PGA could interact with the tertiary amines of DMAEMA fragments and thus improve the mechanical strength of hydrogels. Upon mixing solutions of DMAEMA and HEMA bearing PGA, Ca2+, and PO43-, this effective and dynamic coordination led to the rapid self-assembly of CaP NPs and PDH nanocomposite hydrogels (PDH/mICPN). The obtained optimal nanocomposite hydrogels exhibited suitable injectable time, an enhanced tensile strength of 321.1 kPa, and a fracture energy of 29.0 kJ/m2 and dramatically facilitated cell adhesion and upregulated osteodifferentiation compared to hydrogels prepared by blending ex situ prefabricated CaP NPs. In vivo experiments confirmed the promoted osteogenesis, which shows a striking contrast to pure PDH hydrogels. Additionally, the methacrylate groups on the monomers could easily be functionalized with aptamers and thereby facilitate recognition and capturing of bone marrow stromal cells both in vitro and in vivo and strengthen the bone regeneration. We believe that our conducted research about in situ self-assembled CaP nanoparticle-coordinated hydrogels will open a new avenue for bone regeneration in the future endeavors.

In vitro chondroprotective potential of Senna alata and Senna tora in porcine cartilage explants and their species differentiation by DNA barcoding-high resolution melting (Bar-HRM) analysis.

Senna species and anthraquinone derivatives generated by these organisms, rhein and aloe-emodin, exert anti-inflammatory effects. These species present a similar morphology but produce different ingredients when they are used as medicinal products. In this study, a DNA barcoding- (Bar-) high-resolution melting (HRM) technique was developed using internal transcribed sequence 2 (ITS2) to differentiate between Senna alata and Senna tora as a result of significant differences in their melting profiles. We used this approach for confirmation of S. alata and S. tora raw materials, and we examined the chondroprotective properties of the ethanolic extracts of S. alata and S. tora using a porcine model of cartilage degradation induced by a combination of interleukin-17A (IL-17A) and IL-1ß. We found that both Senna ethanolic extracts, at a concentration of 25 µg/mL, effectively prevented cartilage degradation. Rhein and aloe-emodin were present in the extract of S. alata but not in that of S. tora. We observed a reduction in the release of sulfated glycosaminoglycans (S-GAGs) and hyaluronic acid (HA) into media in both treatments of Senna extracts, which indicated proteoglycan preservation in explant tissues. These results suggest that neither rhein nor aloe-emodin are the main factors responsible for cartilage-protecting properties. Taken together, results show that both S. alata and S. tora are promising for further development as anti-osteoarthritic agents and that Bar-HRM using ITS2 could be applied for species confirmation with Senna products.