Categorization of collagen type I and II blend hydrogel using multipolarization SHG imaging with ResNet regression.

Previously, the discrimination of collagen types I and II was successfully achieved using peptide pitch angle and anisotropic parameter methods. However, these methods require fitting polarization second harmonic generation (SHG) pixel-wise information into generic mathematical models, revealing inconsistencies in categorizing collagen type I and II blend hydrogels. In this study, a ResNet approach based on multipolarization SHG imaging is proposed for the categorization and regression of collagen type I and II blend hydrogels at 0%, 25%, 50%, 75%, and 100% type II, without the need for prior time-consuming model fitting. A ResNet model, pretrained on 18 progressive polarization SHG images at 10° intervals for each percentage, categorizes the five blended collagen hydrogels with a mean absolute error (MAE) of 0.021, while the model pretrained on nonpolarization images exhibited 0.083 MAE. Moreover, the pretrained models can also generally regress the blend hydrogels at 20%, 40%, 60%, and 80% type II. In conclusion, the multipolarization SHG image-based ResNet analysis demonstrates the potential for an automated approach using deep learning to extract valuable information from the collagen matrix.

Exosomes Derived from Hypoxia-Cultured Human Adipose Stem Cells Alleviate Articular Chondrocyte Inflammaging and Post-Traumatic Osteoarthritis Progression.

Osteoarthritis (OA) is the most common age-related degenerative joint disease. Inflammaging, linking inflammation and aging, is found in senescent cells with the secretions of matrix-degrading proteins and proinflammatory cytokines. The senescence-associated secretory phenotype (SASP) plays a very important role in OA progression. However, there remains no effective way to suppress OA progression, especially by suppressing inflammaging and/or the chondrocyte SASP. Recent studies have shown that exosomes derived from hypoxia-cultured BMSCs can regenerate cartilage in OA animal models. Some reports have further indicated that exosomes secreted from MSCs contribute to the efficacy of MSC therapy in OA. However, whether hypoxia-cultured ADSC-secreted exosomes (hypoxia-ADSC-Exos) can alleviate the chondrocyte SASP or OA progression remains unclear. Accordingly, we hypothesized that hypoxia-ADSC-Exos have a beneficial effect on the normal functions of human articular chondrocytes (HACs), can attenuate the SASP of OA-like HACs in vitro, and further suppress OA progression in rats. Hypoxia-ADSC-Exos were derived from ADSCs cultured in 1% O2 and 10% de-Exo-FBS for 48 h. The molecular and cell biological effects of hypoxia-ADSC-Exos were tested on IL1-ß-induced HACs as OA-like HACs in vitro, and the efficacy of OA treatment was tested in ACLT-induced OA rats. The results showed that hypoxia-ADSC-Exos had the best effect on GAG formation in normal HACs rather than those cultured in normoxia or hypoxia plus 2% de-Exo-FBS. We further found that hypoxia-ADSC-Exos alleviated the harmful effect in OA-like HACs by decreasing markers of normal cartilage (GAG and type II collagen) and increasing markers of fibrous or degenerative cartilage (type I or X collagen), matrix degradation enzymes (MMP13 and ADAMT5), and inflammatory cytokines (TNFα and IL-6). More importantly, intra-articular treatment with hypoxia-ADSC-Exos suppressed OA progression, as evidenced by the weight-bearing function test and cartilage GAG quantification in ACLT rats. Moreover, through NGS and bioinformatic analysis, seven potential miRNAs were found in hypoxia-ADSC-Exos, which may contribute to regulating cellular oxidative stress and attenuating cell senescence. In summary, we demonstrated that hypoxia-ADSC-Exos, carrying potent miRNAs, not only improve normal HAC function but also alleviate HAC inflammaging and OA progression. The results suggest that hypoxia-ADSC-Exo treatment may offer another strategy for future OA therapy.

Polyvinyl Butyral Polymeric Host Material-Based Fluorescent Thin Films to Achieve Highly Efficient Red and Green Colour Conversion for Advanced Next-Generation Displays.

Rare-earth element-free fluorescent materials are eco-friendlier than other traditional fluorescent precursors, such as quantum dots and phosphors. In this study, we explore a simple and facile solution-based technique to prepare fluorescent films, which are highly stable under ordinary room conditions and show hydrophobic behaviour. The proposed hybrid material was designed with hybrid composites that use polyvinyl butyral (PVB) as a host doped with organic dyes. The red and green fluorescent films exhibited quantum yields of 89% and 80%, respectively, and both are very uniform in thickness and water resistant. Additionally, PVB was further compared with another polymeric host, such as polyvinylpyrrolidone (PVP), to evaluate their binding ability and encapsulation behaviour. Next, the effect of PVB on the optical and chemical properties of the fluorescent materials was studied using UV spectroscopy and Fourier transform infrared spectroscopy. The analysis revealed that no new bond was formed between the host material and fluorescent precursor during the process, with intermolecular forces being present between different molecules. Moreover, the thickness of the fluorescent film and quantum yield relation were evaluated. Finally, the hydrophobic nature, strong binding ability, and optical enhancement by PVB provide a powerful tool for fabricating a highly efficient fluorescent film with enhanced stability in an external environment based on its promising encapsulation properties. These efficient fluorescent films have a bright potential in colour conversion for next-generation display applications.

Characterization of collagen response to bone fracture healing using polarization-SHG.

In this study, we extend on the three parameter analysis approach of utilizing a noninvasive dual-liquid-crystal-based polarization-resolved second harmonic generation (SHG) microscopy to facilitate the quantitative characterization of collagen types I and II in fracture healing tissues. The SHG images under various linear and circular polarization states are analyzed and quantified in terms of the peptide pitch angle (PA), SHG-circular dichroism (CD), and anisotropy parameter (AP). The results show that the collagen PA has a value of 49.26° after 2 weeks of fracture healing (collagen type II domination) and 49.05° after 4 weeks (collagen type I domination). Moreover, the SHG-CD and AP values of the different collagen types differ by 0.05. The change tendencies of the extracted PA, SHG-CD, and AP parameters over the healing time are consistent with the collagen properties of healthy nonfractured bone. Thus, the feasibility of the proposed dual-liquid-crystal-based polarization-SHG method for differentiating between collagen types I and II in bone fracture healing tissue is confirmed.

Ablation of Discoidin Domain Receptor 1 Provokes an Osteopenic Phenotype by Regulating Osteoblast/Osteocyte Autophagy and Apoptosis.

Discoidin domain receptor 1 (DDR1) is a collagen receptor that belongs to the receptor tyrosine kinase family. We have previously shown that DDR1 plays a crucial role during bone development, resulting in dwarfism and a short stature in osteoblast-specific knockout mice (OKO mice). However, the detailed pathophysiological effects of DDR1 on bone development throughout adulthood have remained unclear. This study aims to identify how DDR1 regulates osteoblast and osteocyte functions in vivo and in vitro during bone development in adulthood. The metabolic changes in bone tissues were analyzed using Micro-CT and immunohistochemistry staining (IHC) in vivo; the role of DDR1 in regulating osteoblasts was examined in MC3T3-E1 cells in vitro. The Micro-CT analysis results demonstrated that OKO mice showed a 10% reduction in bone-related parameters from 10 to 14 weeks old and a significant reduction in cortical thickness and diameter compared with flox/flox control mice (FF) mice. These results indicated that DDR1 knockout in OKO mice exhibiting significant bone loss provokes an osteopenic phenotype. The IHC staining revealed a significant decrease in osteogenesis-related genes, including RUNX2, osteocalcin, and osterix. We noted that DDR1 knockout significantly induced osteoblast/osteocyte apoptosis and markedly decreased autophagy activity in vivo. Additionally, the results of the gain- and loss-of-function of the DDR1 assay in MC3T3-E1 cells indicated that DDR1 can regulate the osteoblast differentiation through activating autophagy by regulating the phosphorylation of the mechanistic target of rapamycin (p-mTOR), light chain 3 (LC3), and beclin-1. In conclusion, our study highlights that the ablation of DDR1 results in cancellous bone loss by regulating osteoblast/osteocyte autophagy. These results suggest that DDR1 can act as a potential therapeutic target for managing cancellous bone loss.

Design, Manufacturing, and Control of a Pneumatic-Driven Passive Robotic Gait Training System for Muscle-Weakness in a Lower Limb.

We designed and manufactured a pneumatic-driven robotic passive gait training system (PRPGTS), providing the functions of body-weight support, postural support, and gait orthosis for patients who suffer from weakened lower limbs. The PRPGTS was designed as a soft-joint gait training rehabilitation system. The soft joints provide passive safety for patients. The PRPGTS features three subsystems: a pneumatic body weight support system, a pneumatic postural support system, and a pneumatic gait orthosis system. The dynamic behavior of these three subsystems are all involved in the PRPGTS, causing an extremely complicated dynamic behavior; therefore, this paper applies five individual interval type-2 fuzzy sliding controllers (IT2FSC) to compensate for the system uncertainties and disturbances in the PRGTS. The IT2FSCs can provide accurate and correct positional trajectories under passive safety protection. The feasibility of weight reduction and gait training with the PRPGTS using the IT2FSCs is demonstrated with a healthy person, and the experimental results show that the PRPGTS is stable and provides a high-trajectory tracking performance.

Intra-articular low-dose parathyroid hormone (1-34) improves mobility and articular cartilage quality in a preclinical age-related knee osteoarthritis model.

AIMS: Osteoarthritis (OA) is prevalent among the elderly and incurable. Intra-articular parathyroid hormone (PTH) ameliorated OA in papain-induced and anterior cruciate ligament transection-induced OA models; therefore, we hypothesized that PTH improved OA in a preclinical age-related OA model. METHODS: Guinea pigs aged between six and seven months of age were randomized into control or treatment groups. Three- or four-month-old guinea pigs served as the young control group. The knees were administered 40 µl intra-articular injections of 10 nM PTH or vehicle once a week for three months. Their endurance as determined from time on the treadmill was evaluated before kill. Their tibial plateaus were analyzed using microcalculated tomography (µCT) and histological studies. RESULTS: PTH increased the endurance on the treadmill test, preserved glycosaminoglycans, and reduced Osteoarthritis Research Society International score and chondrocyte apoptosis rate. No difference was observed in the subchondral plate bone density or metaphyseal trabecular bone volume and bone morphogenetic 2 protein staining. CONCLUSION: Subchondral bone is crucial in the initiation and progression of OA. Although previous studies have shown that subcutaneous PTH alleviates knee OA by improving subchondral and metaphyseal bone mass, we demonstrated that intra-articular PTH injections improved spontaneous OA by directly affecting the cartilage rather than the subchondral or metaphyseal bone in a preclinical age-related OA model. Cite this article: Bone Joint Res 2021;10(8):514-525.

Dual-LC PSHG microscopy for imaging collagen type I and type II gels with pixel-resolution analysis.

Collagen of type I (Col I) and type II (Col II) are critical for cartilage and connective tissues in the human body, and several diseases may alter their properties. Assessing the identification and quantification of fibrillar collagen without biomarkers is a challenge. Advancements in non-invasive polarization-resolved second-harmonic generation (PSHG) microscopy have provided a method for the non-destructive investigation of collagen molecular level properties. Here we explored an alternative polarization modulated approach, dual-LC PSHG, that is based on two liquid crystal devices (Liquid crystal polarization rotators, LPRs) operating simultaneously with a laser scanning SHG microscope. We demonstrated that this more accessible technology allows the quick and accurate generation of any desired linear and circular polarization state without any mechanical parts. This study demonstrates that this method can aid in improving the ability to quantify the characteristics of both types of collagen, including pitch angle, anisotropy, and circular dichroism analysis. Using this approach, we estimated the effective pitch angle for Col I and Col II to be 49.7° and 51.6°, respectively. The effective peptide pitch angle for Col II gel was first estimated and is similar to the value obtained for Col I gel in the previous studies. Additionally, the difference of the anisotropy parameter of both collagen type gels was assessed to be 0.293, which reflects the different type molecular fibril assembly. Further, our work suggests a potential method for monitoring and differentiating different collagen types in biological tissues, especially cartilage or connective tissue.

Simvastatin Enhances the Chondrogenesis But Not the Osteogenesis of Adipose-Derived Stem Cells in a Hyaluronan Microenvironment.

Directing adipose-derived stem cells (ADSCs) toward chondrogenesis is critical for ADSC-based articular cartilage regeneration. Simvastatin (SIM) was reported to promote both chondrogenic and osteogenic differentiation of ADSCs by upregulating bone morphogenetic protein-2 (BMP-2). We previously found that ADSC chondrogenesis is initiated and promoted in a hyaluronan (HA) microenvironment (HAM). Here, we further hypothesized that SIM augments HAM-induced chondrogenesis but not osteogenesis of ADSCs. ADSCs were treated with SIM in a HAM (SIM plus HAM) by HA-coated wells or HA-enriched fibrin (HA/Fibrin) hydrogel, and chondrogenic differentiation of ADSCs was evaluated. SIM plus HAM increased chondrogenesis more than HAM or SIM alone, including cell aggregation, chondrogenic gene expression (collagen type II and aggrecan) and cartilaginous tissue formation (collagen type II and sulfated glycosaminoglycan). In contrast, SIM-induced osteogenesis in ADSCs was reduced in SIM plus HAM, including mRNA expression of osteogenic genes, osteocalcin and alkaline phosphatase (ALP), ALP activity and mineralization. SIM plus HAM also showed the most effective increases in the mRNA expression of BMP-2 and transcription factors of SOX-9 and RUNX-2 in ADSCs, while these effects were reversed by CD44 blockade. HAM suppressed the levels of JNK, p-JNK, P38 and p-P38 in ADSCs, and SIM plus HAM also decreased SIM-induced phosphorylated JNK and p38 levels. In addition, SIM enhanced articular cartilage regeneration, as demonstrated by implantation of an ADSCs/HA/Fibrin construct in an ex vivo porcine articular chondral defect model. The results from this study indicate that SIM may be an enhancer of HAM-initiated MSC-based chondrogenesis and avoid osteogenesis.

An Intermediate Concentration of Calcium with Antioxidant Supplement in Culture Medium Enhances Proliferation and Decreases the Aging of Bone Marrow Mesenchymal Stem Cells.

Human bone marrow stem cells (HBMSCs) are isolated from the bone marrow. Stem cells can self-renew and differentiate into various types of cells. They are able to regenerate kinds of tissue that are potentially used for tissue engineering. To maintain and expand these cells under culture conditions is difficult-they are easily triggered for differentiation or death. In this study, we describe a new culture formula to culture isolated HBMSCs. This new formula was modified from NCDB 153, a medium with low calcium, supplied with 5% FBS, extra growth factor added to it, and supplemented with N-acetyl-L-cysteine and L-ascorbic acid-2-phosphate to maintain the cells in a steady stage. The cells retain these characteristics as primarily isolated HBMSCs. Moreover, our new formula keeps HBMSCs with high proliferation rate and multiple linage differentiation ability, such as osteoblastogenesis, chondrogenesis, and adipogenesis. It also retains HBMSCs with stable chromosome, DNA, telomere length, and telomerase activity, even after long-term culture. Senescence can be minimized under this new formulation and carcinogenesis of stem cells can also be prevented. These modifications greatly enhance the survival rate, growth rate, and basal characteristics of isolated HBMSCs, which will be very helpful in stem cell research.

Intra-Articular Injection of (-)-Epigallocatechin 3-Gallate (EGCG) Ameliorates Cartilage Degeneration in Guinea Pigs with Spontaneous Osteoarthritis.

Osteoarthritis (OA) is the most prevalent joint disease that causes an enormous burden of disease worldwide. (-)-Epigallocatechin 3-gallate (EGCG) has been reported to reduce post-traumatic OA progression through its anti-inflammatory property. Aging is the most crucial risk factor of OA, and the majority of OA incidences are related to age and not trauma. In this study, we assess whether EGCG can ameliorate cartilage degradation in primary OA. In an in-vitro study, real-time PCR was performed to assess the expression of genes associated with human articular chondrocyte homeostasis. A spontaneously occurring OA model in guinea pigs was used to investigate the effect of EGCG in vivo. OA severity was evaluated using Safranin O staining and Osteoarthritis Research Society International (OARSI) scores, as well as by immunohistochemical (IHC) analysis to determine the protein level of type II collagen (Col II), matrix metalloproteinase 13 (MMP-13), and p16 ink4a in articular cartilage. In the in-vitro study, EGCG increased the gene expression of aggrecan and Col II and decreased the expression of interleukin-1, cyclooxygenase 2, MMP-13, alkaline phosphatase, Col X, and p16 Ink4a; EGCG treatment also attenuated the degraded cartilage with a lower OARSI score. Meanwhile, IHC results showed that EGCG exerted an anti-OA effect by reducing ECM degradation, cartilage inflammation, and cell senescence with a less-immunostained Col II, MMP-13, and p16 Ink4a. In conclusion, these findings suggest that EGCG may be a potential disease-modifying OA drug for the treatment of primary OA.

The relationship between basic group resonance and quantum yield of high efficiency red light fluorescent solutions.

Compared with rare earth elements and heavy metal elements, rare-earth-element-free fluorescent films can greatly reduce environmental hazards. In this study, we use a solution method to produce the fluorescent films. The film thickness is 10 µm, which can maintain fluorescent light intensity in an environment with an average humidity of 55.1 (RH%) after encapsulation. We also find that the type of solvent affects the resonance position of the C[triple bond, length as m-dash]N functional group in DCJTB at a wavenumber of 2196 (cm-1), measured with Fourier transform infrared spectroscopy. The functional group is affected by the polar effect with its displacement decreasing with the quantum yield. Finally, we successfully made a fluorescent solution with a resonance displacement of only 12.8 (cm-1) for the C[triple bond, length as m-dash]N functional group with the quantum yield being as high as 81.3% and a fluorescent film with a quantum yield as high as 84.8%.

Intra-Articular Injection of (-)-Epigallocatechin 3-Gallate to Attenuate Articular Cartilage Degeneration by Enhancing Autophagy in a Post-Traumatic Osteoarthritis Rat Model.

(-)-Epigallocatechin 3-gallate (EGCG) is the main active green tea catechin and has a wide variety of benefits for health. Post-traumatic osteoarthritis (PTOA) occurs as a consequence of joint injuries that commonly happen in the young population. In this study, we investigated the effects of EGCG on PTOA prevention by using the anterior cruciate ligament transection (ACLT)-OA model and further investigated the roles of autophagy in OA treatment. Our results showed that intra-articular injection of EGCG significantly improved the functional performances and decreased cartilage degradation. EGCG treatment attenuated the inflammation on synovial tissue and cartilage through less immunostained cyclooxygenase-2 and matrix metalloproteinase-13. We further noted EGCG may modulate the chondrocyte apoptosis by activation of the cytoprotective autophagy through reducing the expression of the mTOR and enhancing the expression of microtubule-associated protein light chain 3, beclin-1, and p62. In conclusion, intra-articular injection of EGCG after ACL injury inhibited the joint inflammation and cartilage degradation, thereby increasing joint function. EGCG treatment also reduced the chondrocyte apoptosis, possibly by activating autophagy. These findings suggested that EGCG may be a potential disease-modifying drug for preventing OA progression.

Discoidin Domain Receptors 1 Inhibition Alleviates Osteoarthritis via Enhancing Autophagy.

We recently reported that the chondrocyte-specific knockout of discoidin domain receptors 1 (Ddr1) delayed endochondral ossification (EO) in the growth plate by reducing the chondrocyte hypertrophic terminal differentiation, and apoptosis. The biologic and phenotypic changes in chondrocytes in the articular cartilage with osteoarthritis (OA) are similar to the phenomena observed in the process of EO. Additionally, autophagy can promote chondrocyte survival and prevent articular cartilage from degradation in OA. On this basis, we explored the effect of Ddr1 inhibition on OA prevention and further investigated the roles of autophagy in treating OA with a Ddr1 inhibitor (7 rh). The anterior cruciate ligament transection (ACLT)-OA model was used to investigate the role of 7 rh in vivo. Forty 8-week-old mice were randomly assigned to four groups, including the sham group, ACLT group, and two treated groups (ACLT with 7 rh 6.9 nM or 13.8 nM). According to the study design, normal saline or 7 rh were intra-articular (IA) injected into studied knees 3 times per week for 2 weeks and then once per week for 4 weeks. The results showed that 7 rh treatment significantly improved the functional performances (the weight-bearing ability and the running endurance), decreased cartilage degradation, and also reduced the terminal differentiation markers (collagen type X, Indian hedgehog, and matrix metalloproteinase 13). Moreover, 7 rh decreased chondrocyte apoptosis by regulating chondrocyte autophagy through reducing the expression of the mammalian target of rapamycin and enhancing the light chain 3 and beclin-1 expression. These results demonstrated that the IA injection of 7 rh could reduce the chondrocyte apoptosis and promote chondrocyte autophagy, leading to the attenuation of cartilage degradation. Our observations suggested that the IA injection of 7 rh could represent a potential disease-modifying therapy to prevention OA progression.

Green Tea Catechin (-)-Epigallocatechin-3-Gallate (EGCG) Facilitates Fracture Healing.

Green tea drinking can ameliorate postmenopausal osteoporosis by increasing the bone mineral density. (-)-Epigallocatechin-3-gallate (EGCG), the abundant and active compound of tea catechin, was proven to be able to reduce bone loss and ameliorate microarchitecture in female ovariectomized rats. EGCG can also enhance the osteogenic differentiation of murine bone marrow mesenchymal stem cells and inhibit the osteoclastogenesis in RAW264.7 cells by modulation of the receptor activator of nuclear factor-kB (RANK)/RANK ligand (RANKL)/osteoprotegrin (OPG) (RANK/RANKL/OPG) pathway. Our previous study also found that EGCG can promote bone defect healing in the distal femur partially via bone morphogenetic protein-2 (BMP-2). Considering the osteoinduction property of BMP-2, we hypothesized that EGCG could accelerate the bone healing process with an increased expression of BMP-2. In this manuscript, we studied whether the local use of EGCG can facilitate tibial fracture healing. Fifty-six 4-month-old rats were randomly assigned to two groups after being weight-matched: a control group with vehicle treatment (Ctrl) and a study group with 10 µmol/L, 40 µL, EGCG treatment (EGCG). Two days after the operation, the rats were treated daily with EGCG or vehicle by percutaneous local injection for 2 weeks. The application of EGCG enhanced callus formation by increasing the bone volume and subsequently improved the mechanical properties of the tibial bone, including the maximal load, break load, stiffness, and Young's modulus. The results of the histology and BMP-2 immunohistochemistry staining showed that EGCG treatment accelerated the bone matrix formation and produced a stronger expression of BMP-2. Taken together, this study for the first time demonstrated that local treatment of EGCG can accelerate the fracture healing process at least partly via BMP-2.

Interactive Learning for Identifying Relevant Tweets to Support Real-time Situational Awareness.

Various domain users are increasingly leveraging real-time social media data to gain rapid situational awareness. However, due to the high noise in the deluge of data, effectively determining semantically relevant information can be difficult, further complicated by the changing definition of relevancy by each end user for different events. The majority of existing methods for short text relevance classification fail to incorporate users' knowledge into the classification process. Existing methods that incorporate interactive user feedback focus on historical datasets. Therefore, classifiers cannot be interactively retrained for specific events or user-dependent needs in real-time. This limits real-time situational awareness, as streaming data that is incorrectly classified cannot be corrected immediately, permitting the possibility for important incoming data to be incorrectly classified as well. We present a novel interactive learning framework to improve the classification process in which the user iteratively corrects the relevancy of tweets in real-time to train the classification model on-the-fly for immediate predictive improvements. We computationally evaluate our classification model adapted to learn at interactive rates. Our results show that our approach outperforms state-of-the-art machine learning models. In addition, we integrate our framework with the extended Social Media Analytics and Reporting Toolkit (SMART) 2.0 system, allowing the use of our interactive learning framework within a visual analytics system tailored for real-time situational awareness. To demonstrate our framework's effectiveness, we provide domain expert feedback from first responders who used the extended SMART 2.0 system.

(-)-Epigallocatechin-3-Gallate Decreases Osteoclastogenesis via Modulation of RANKL and Osteoprotegrin.

Osteoporosis is the second most common epidemiologic disease in the aging population worldwide. Previous studies have found that frequent tea drinkers have higher bone mineral density and less hip fracture. We previously found that (-)-epigallocatechin gallate (EGCG) (20⁻100 µmol/L) significantly suppressed receptor activator of nuclear factor-kB ligand (RANKL)-induced osteoclastogenesis and pit formation via inhibiting NF-κB transcriptional activity and nuclear transport of NF-κB in RAW 264.7 cells and murine primary bone marrow macrophage cells. The most important regulation in osteoclastogenesis is the receptor activator of nuclear factor-kB/RANKL/osteoprotegrin (RANK/RANKL/OPG) pathway. In this study, we used the coculture of RAW 264.7 cells and the feeder cells, ST2, to evaluate how EGCG regulated the RANK/RANKL/OPG pathway in RAW 264.7 cells and ST2 cells. We found EGCG decreased the RANKL/OPG ratio in both mRNA expression and secretory protein levels and eventually decreased osteoclastogenesis by TRAP (+) stain osteoclasts and TRAP activity at low concentrations-1 and 10 µmol/L-via the RANK/RANKL/OPG pathway. The effective concentration can be easily achieved in daily tea consumption. Taken together, our results implicate that EGCG could be an important nutrient in modulating bone resorption.

(-)-Epigallocatechin-3-gallate (EGCG) enhances healing of femoral bone defect.

BACKGROUND: Previously, we found that (-)-epigallocatechin-3-gallate (EGCG) enhanced osteogenic differentiation of murine bone marrow mesenchymal stem cells by increasing the mRNA expression of osteogenesis-related genes, alkaline phosphatase activity and eventually mineralization. We further found EGCG supplementation preserved bone mass and microarchitecture in female rats during estrogen deficiency in the proximal tibia and lumbar spine at least in part by increasing bone morphogenetic protein-2 (BMP2). BMP2 can enhance de novo bone formation. PURPOSE: In this study, we evaluate the effect of local EGCG application in de novo bone formation in bone defect healing. METHODS: Twenty-four rats aged 4 months were weight-matched and randomly allocated to 2 groups: defect control with vehicle treatment (control) and defect with 10 µM EGCG treatment (EGCG). Daily vehicle and EGCG were applied locally by percutaneous local injection 2 days after defect creation for 2 weeks. Four weeks after treatment, animals were sacrificed for micro-computed tomography (µ-CT) and biomechanical analysis. RESULTS: Local EGCG at femoral defect can enhance de novo bone formation by increasing bone volume and subsequently improve mechanical properties including max load, break point, stiffness, area under the max load curve, area under the break point curve and ultimate stress. CONCLUSIONS: Local EGCG may enhance bone defect healing via at least partly by the de novo bone formation of BMP-2.

(-)-Epigallocatechin-3-Gallate (EGCG) Enhances Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells.

Osteoporosis is the second most-prevalent epidemiologic disease in the aging population worldwide. Cross-sectional and retrospective evidence indicates that tea consumption can mitigate bone loss and reduce risk of osteoporotic fractures. Tea polyphenols enhance osteoblastogenesis and suppress osteoclastogenesis in vitro. Previously, we showed that (-)-epigallocatechin-3-gallate (EGCG), one of the green tea polyphenols, increased osteogenic differentiation of murine bone marrow mesenchymal stem cells (BMSCs) by increasing the mRNA expression of osteogenesis-related genes, alkaline phosphatase activity and, eventually, mineralization. We also found that EGCG could mitigate bone loss and improve bone microarchitecture in ovariectomy-induced osteopenic rats, as well as enhancing bone defect healing partially via bone morphogenetic protein 2 (BMP2). The present study investigated the effects of EGCG in human BMSCs. We found that EGCG, at concentrations of both 1 and 10 µmol/L, can increase mRNA expression of BMP2, Runx2, alkaline phosphatase (ALP), osteonectin and osteocalcin 48 h after treatment. EGCG increased ALP activity both 7 and 14 days after treatment. Furthermore, EGCG can also enhance mineralization two weeks after treatment. EGCG without antioxidants also can enhance mineralization. In conclusion, EGCG can increase mRNA expression of BMP2 and subsequent osteogenic-related genes including Runx2, ALP, osteonectin and osteocalcin. EGCG further increased ALP activity and mineralization. Loss of antioxidant activity can still enhance mineralization of human BMSCs (hBMSCs).

Parathyroid hormone-(1-34) ameliorated knee osteoarthritis in rats via autophagy.

Anterior cruciate ligament (ACL) tear can lead to osteoarthritis (OA). However, parathyroid hormone (PTH)-(1-34) was found to alleviate OA progression in a papain-induced OA model. Autophagy is a protective mechanism in normal cartilage, and its aging-related loss is linked with chondrocyte death and OA. Thus we examined the roles of autophagy in PTH treatment in OA after ACL transection (ACLT). Thirty-six rats were randomized into three groups: control group, ACLT-induced OA (OA) group, and OA with intra-articular PTH-(1-34) treatment (OA+PTH) group. Weight-bearing and treadmill tests were evaluated. Cartilage matrix was determined by a histological evaluation of glycosaminoglycan (GAG), Osteoarthritis Research Society International (OARSI) score, chondrocyte apoptosis, and immunohistochemistry. Rats in the OA group had significantly decreased weight bearing and running endurance. The histological results indicated that GAG, collagen type II, and chondrocyte autophagy had decreased but that the OARSI score, terminal differentiation markers (collagen type X and Indian hedgehog), and chondrocyte apoptosis had increased in the OA group. Additionally, PTH-(1-34) treatment significantly improved weight bearing and treadmill endurance, preserved GAG and collagen type II, and reduced the OARSI score and terminal differentiation markers. Finally, PTH-(1-34) ameliorated chondrocyte apoptosis by regulating the expression of autophagy-related proteins, through reducing mechanistic target of rapamycin (mTOR) and p62 and enhancing microtubule-associated protein-1 light chain 3 (LC3) and beclin-1. Reconstructive surgery after ACL rupture cannot prevent OA occurrence. Intra-articular PTH-(1-34) treatment can alleviate OA progression after ACLT and histological molecular changes. Possible mechanisms are reducing chondrocyte terminal differentiation and apoptosis, with increasing autophagy. NEW & NOTEWORTHY Anterior cruciate ligament (ACL) tear can lead to osteoarthritis (OA). Intra-articular parathyroid hormone (PTH)-(1-34) significantly improved weight bearing and treadmill endurance, preserved glycosaminoglycan and collagen type II, and reduced Osteoarthritis Research Society International (OARSI) score and terminal differentiation. Finally, PTH-(1-34) ameliorated chondrocyte apoptosis by regulating the expression of autophagy-related proteins, through reducing mechanistic target of rapamycin (mTOR) and p62 and enhancing microtubule-associated protein-1 light chain 3 (LC3) and beclin-1. PTH-(1-34) can alleviate OA progression after ACL transection. Possible mechanisms are reducing chondrocyte terminal differentiation and apoptosis, with increasing autophagy.