Fast Green Staining Research Articles

Abstract 17122: Cardioprotection With Proangiogenic Nanomaterials Formulated With CHIR99021 and FGF1

Introduction: We aimed at to investigate whether activation of both FGF1 and Wnt1 synergistically enhances angiogenesis and render the cardioprotection in hearts with post infarction LV remodeling. Materials and methods: CHIR99021 (a Wnt1 activator) and FGF1 were encapsulated into poly lactic-co-glycolic acid nanoparticles (CHIR99021/FGF1-NPs). Ischemic heart models were generated in C57BL/6 mice or Yorkshire swine by ligation of the left anterior descending coronary. The mice were randomly divided into 6 groups: Myocardial infarction (MI) Only (n=11), MI with nanoparticles (CHIR99021 or/and FGF1 loaded) injection (n=12, each), MI with non-loaded nanoparticles injection (n=11) and Sham group (n=10). The swine were divided into 3 groups: ischemic reperfusion injury (IRI) (n=4), IRI with CHIR99021/FGF-NPs injection (n=4) and Sham group (n=4). Left ventricle function 4 week after surgery were evaluated by Echocardiography. Cell proliferation was assessed via immunostaining in ischemic hearts and in cultivated human umbilical vein endothelial cells (HUVECs) and Human vascular smooth muscle cells (HVSMCs) using the following markers: Ki67, Brdu, PH3, and Aurora B. Apoptosis was determined with TUNEL staining. Cardiac fibrosis was evaluated via Sirius red and Fast green staining (mice) and magnetic resonance imaging (swine). Results: CHIR99021/FGF1-NPs treatment attenuated fibrosis and preserved heart contractile function in mice and swine models of postinfarction LV remodeling. The proportion of cells that expressed cell cycle markers (Ki67; BrdU; Aurora B and PH3) was significantly greater in CHIR99021+FGF1 treatment group than CHIR99021, FGF1 and PBS treatment group. The proportion of apoptotic cells was significantly smaller in CHIR99021+FGF1 treatment group than in groups of control, VEGF, CHIR99021, or FGF1 treatment alone. Conclusions: CHIR99021 and FGF synergistically activate cell cycle and reduce apoptosis, which accompanied by a significant improvement of cardiac function.

Salvianolate reduces atrial fibrillation through suppressing atrial interstitial fibrosis by inhibiting TGF-β1/Smad2/3 and TXNIP/NLRP3 inflammasome signaling pathways in post-MI rats

BackgroundSalvianolate is the main water-soluble bioactive compound of Salvia Miltiorrhiza Bunge and is now clinically used in the treatment of cardiovascular diseases in China. However, its applications in the prevention of atrial interstitial fibrosis (AIF) and atrial fibrillation (AF) are not fully revealed. PurposesTo investigate the preventive effect of salvianolate on the pathogenesis of AF in post-myocardial infarction (MI) rats and to elucidate the potential mechanisms. Materials and methodsRats underwent left anterior descending coronary artery ligation were randomized into four groups and administered intraperitoneally with vehicle (MI group, n = 13), or 10, 20 and 40 mg/kg salvianolate (Sal-L, Sal-M and Sal-H group, n = 13, 14 and 13 respectively) for totally five weeks. Rats underwent sham operation was used as control group (Sham, n = 10). Then, echocardiography and AF inducibility test were detected. Tissues and serum were collected for Sirius red and fast green counter stain or hematoxylin-eosin to assess atrial interstitial fibrosis and hypertrophy, or for western blot and ELISA analysis. ResultsSalvianolate injection significantly improved cardiac function, reduced left atrial enlargement and P-wave duration, and decreased not only the vulnerability to AF but also AF duration. Histologic analysis showed that salvianolate mitigated AIF and atrial hypertrophy. Western blot analysis found that salvianolate inhibited the TGFβ1/Smad2/3 mediated-collagen deposition and inhibited the TXNIP/NLRP3 inflammasome /IL-1β and IL-18 signal pathway. ELISA analysis showed that salvianolate significantly reduced the serum concentrations of BNP, IL-6, CRP and TGFβ1. ConclusionsSalvianolate may constitute a novel upstream therapy for AF by suppressing AIF. The underlying mechanism may be attributable to its inhibitory effects on TGF-β1/Smad2/3 and TXNIP/NLRP3 inflammasome signaling pathway.

The effectiveness of allogeneic mesenchymal stem cells therapy for knee osteoarthritis in pigs.

Intraarticular injection of the mesenchymal stem cells (MSCs) has shown to be successful for treating osteoarthritis (OA). Nevertheless, many studies have been focusing on autologous MSCs. The following study investigates the safety and effectiveness of intraarticular injection of allogenic MSCs in a pig OA model. Superparamagnetic iron oxide (SPIO) nanoparticles were labelled with bone marrow-derived mesenchymal stem cells (BM-MSCs) to allow cells tracking using magnetic resonance imaging (MRI). A pig OA model was established by bilateral medial meniscectomy. Next, SPIO-BM-MSCs were injected into the right knee, while the left knee was left untreated. MRI and radiography were used to assess the degree of OA and to evaluate the effectiveness of allogenic MSCs. Hematoxylin and eosin (H&E), safranin-o fast green staining, toluidine blue, and immunohistochemical staining were used to evaluate the therapeutic effect of the injections. At concentration of ≤20 µg/mL, SPIO caused no toxicity to BM-MSCs. Four weeks after surgery, OA changes were observed on MRI scan. The SPIO labeled BM-MSCs were found moving towards the impaired part of the cartilage 8 to 24 h after injections. In addition, no significant differences between the right side (therapeutic side) and the left side (untreated side) were observed following histological and immunohistochemistry analysis. The suitable concentration of SPIO for labelling BMSCs was 20 µg/mL, while the allogenic MSCs could move towards and accumulate around the impaired cartilage. No significant difference was found between treatment and control group.

Exosomes derived from miR-92a-3p-overexpressing human mesenchymal stem cells enhance chondrogenesis and suppress cartilage degradation via targeting WNT5A

BackgroundWNT5A is known to be involved in the pathogenesis of osteoarthritis. This study investigated the molecular mechanism of exosomal miR-92a-3p and WNT5A in chondrogenesis and cartilage degeneration.MethodsExosomal miR-92a-3p expression was assessed in vitro in a human mesenchymal stem cell (MSC) model of chondrogenesis and in normal and OA primary human chondrocytes (PHCs). MSCs and PHCs were treated with exosomes derived from MSC-miR-92a-3p (MSC-miR-92a-3p-Exos) or its antisense inhibitor (MSC-anti-miR-92a-3p-Exos), respectively. Small interfering RNAs (siRNAs) and luciferase reporter assay were used to reveal the molecular role of exosomal miR-92a-3p and WNT5A in chondrogenesis. The protective effect of exosomes in vivo was measured using Safranin-O and Fast Green staining and immunohistochemical staining.ResultsExosomal miR-92a-3p expression was elevated in the MSC chondrogenic exosome, while it was significantly reduced in the OA chondrocyte-secreted exosome compared with normal cartilage. Treatment with MSC-miR-92a-3p-Exos promoted cartilage proliferation and matrix genes expression in MSCs and PHCs, respectively. In contrast, treatment with MSC-anti-miR-92a-3p-Exos repressed chondrogenic differentiation and reduced cartilage matrix synthesis by enhancing the expression of WNT5A. Luciferase reporter assay demonstrated that miR-92a-3p suppressed the activity of a reporter construct containing the 3’-UTR and inhibited WNT5A expression in both MSCs and PHCs. MSC-miR-92a-3p-Exos inhibit cartilage degradation in the OA mice model.ConclusionsOur results suggest that exosomal miR-92a-3p regulates cartilage development and homeostasis by directly targeting WNT5A. This indicates that exosomal miR-92a-3p may act as a Wnt inhibitor and exhibits potential as a disease-modifying osteoarthritis drug.

Facet joint degeneration in adolescent idiopathic scoliosis.

Adolescent idiopathic scoliosis (AIS) is a poorly understood deformity of the thoracolumbar spine which affects the intervertebral discs (IVDs) and the articular facet joints. The knowledge concerning facet joints in this context is very limited, although facet joint degeneration is a known contributor of back pain. In this study, a comprehensive investigation was performed to characterize the facet joint chondrocytes and extracellular matrix within the scoliotic spine. Surgically removed articular facet joint tissues were collected from patients undergoing spinal corrective surgery for AIS deformities, while non‐scoliotic articular facet joint tissues were obtained from cadaveric organ donors. Alterations in cartilage tissue structure were evaluated histologically with safranin‐O fast green and a modified OARSI grading scale. Pro‐inflammatory cytokines, matrix‐degrading proteases, and fragmented matrix molecules associated with cartilage degradation were analyzed by immunohistochemistry and western blotting. Safranin‐O fast green staining revealed that young scoliotic facet joints show clear signs of degeneration with substantial proteoglycan loss, similar to osteoarthritis (OA). The proteoglycan levels were significantly lower than in healthy asymptomatic non‐scoliotic control individuals. In comparison to controls, scoliotic articular facets showed increased cell density, increased expression of the proliferation marker Ki‐67, and higher expression of MMP‐3, MMP‐13, and IL‐1β. Expression and fragmentation of the small leucine‐rich proteins (SLRPs) chondroadherin, decorin, biglycan, lumican, and fibromodulin were analyzed with western blot. Chondroadherin and decorin were fragmented in cartilage from patients with a curve greater than 70°, whereas biglycan and fibromodulin did not show curve‐related fragmentation. AIS facet joint cartilage shows hallmarks of OA including proteoglycan loss, overexpression of pro‐inflammatory mediators, increased synthesis of matrix‐degrading proteases and fragmentation of SLRPs. As with patients with age‐related OA, the premature joint degeneration seen in scoliotic patients is likely to contribute to the pain perceived in some individuals.

Effect of epidermal growth factor receptor signaling pathway inhibitor gefitinib on fracture healing in rats

Objective To observe the effect of epidermal growth factor receptor (EGFR) signal pathway inhibitor gefitinib on femoral fracture healing in rats. Methods Eighty SD rats were divided into experimental group (n=40) and control group (n=40) by random number table. An open femur fractured model was established by cutting the middle part of the femoral shaft and placing 1 mm Kirschner wire through medial and lateral femoral patella incision. Rats in the experimental group were given gefitinib (100 mg· kg-1· d-1), an inhibitor of epidermal growth factor receptor (EGFR) signaling pathway, and rats in the control group were given equal doses of methylcellulose. The X-ray examination was performed before femoral and serum specimens were collected at 1, 2, 3, 4, and 6 weeks. Bone fracture healing was evaluated by Lane and Sandhu X ray scoring and biomechanical test. Then the tissue morphology was observed by HE staining, safranin fast green staining, and microCT detection. The bone volume (BV), bone volume fracture (BV/TV), trabecular number (Tb. N), trabecular thickness (Tb. Th), and trabecular separation (Tb. Sp) were measured by MicroCT. The levels of serum BALP, PINP, TRACP5b, and CTX were measured by Elisa. Results The X-ray scores of the experimental group (1.26±0.54, 4.94±1.16, and 8.23±1.17) were significantly higher than those in the control group (0.91±0.52, 4.11±1.18, and 7.08±0.86) at 1, 2 and 3 weeks after the fracture (P<0.05). The failure load of experimental group at 3 and 4 weeks after fracture [(38.65±1.07)N , (63.63±7.74)N] were significantly larger than those of the control group [ (29.47+ 1.00)N, (45.42+ 3.26)N](P<0.05). The callus formation in the experimental group was obvious and the healing process of the fracture was faster than that of the control group according to HE and safranin fast green staining. The results of microCT showed that in the experimental group, BV was significantly higher at 2 and 3 weeks after fracture [(59.30±6.54)mm3 and (43.08±2.33)mm3] than those in control group [(42.39±7.82)mm3 and (33.43±5.94)mm3]; BV / TV at 2, 3, and 4 weeks after fracture[(0.61±0.06)%, (0.55±0.05)%, and (0.53±0.04)%] were significantly higher than those in the control group [(0.48±0.07)%, (0.44±0.07)%, and (0.43±0.03)%]; Tb. N at 2, 3, and 4 weeks after fracture [(2.05±0.11)/mm, (1.86±0.18)/mm, and (2.034 ±0.26)/mm]were significantly higher than those in control group [(1.63±0.21)/mm, (1.32±0.21)/mm, and (1.65±0.08)/mm)]; Tb. Th at 2, 3, and 4 weeks after fracture [(0.33±0.02)mm, (0.33±0.03)mm, and (0.27±0.02)mm] were significantly higher than those in the control group [(0.27 ±0.03)mm, (0.28±0.02)mm, and 0.23±0.01)mm]; Tb. Sp at 3 weeks after fracture [(0.39±0.07)mm] was significantly higher than that in the control group [(0.30±0.03)mm](P<0.05). The levels of serum BALP in experimental group at 2 and 3 weeks after fracture [(2.57±0.14)ng/ml, (3.47±0.26)ng/ml] were significantly higher than those of control group [(2.07±0.19)ng/ml, (2.77±0.29)ng/ml]; the PINP at 1, 2, and 3 weeks after fracture in the experimental group [(2 216.50±96.68)pg/ml, (2 692.33±136.76)pg/ml, and (3 196.75±221. 90)pg/ml] were significantly higher than those of the control group[(1 969.50+ 89.34)pg/ml, (2 241.33±107.86)pg/ml, and(2 603.25±361.60)pg/ml]; the levels of TRACP5b [(11.58±0.47)ng/ml] and CTX [(8.02±0.40)ng/ml] at 1 week after fracture were higher than those of the control group [(10.33±0.53)ng/ml, (7.11±0.36)ng/ml](P<0.05). Conclusion Gefitinib can promote the maturation of osteoblasts and the early reabsorption function of osteoclasts to induce bone formation in advance and accelerate the fracture healing. Key words: Receptor, epidermal growth factor; Fracture healing; Gefitinib

22 Comparing Three Extenders: Hashi, Green Buffer and INRA 96, for Chilled Storage of Bactrian Camel Semen

Semen preservation remains challenging in the camel industry. The objective of the present study was to compare 3 different extenders for chilled storage of Bactrian camel semen. Semen (n = 9 ejaculates) was collected from camel bulls (n = 2) manually using artificial vagina. Good neat semen, as far as mass vibration concerns, was equally distributed into 3 double-wall vessels filled with 35°C water. The 3 extenders used in the present study were Hashi, Green buffer (IMV, L’Aigle, France), and INRA 96 (IMV). The Hashi extender consisted of Tris, 2.6%; citric acid, 1.35%; glucose, 0.9%; fructose, 0.9%; penicillin G sodium, 1000 IU mL−1; streptomycin sulfate, 1000 mg mL−1 supplemented with 20% plasma egg yolk and 20% camel skim milk; osmolality of 330 and pH of 6.9). Green buffer was supplemented with 20% plasma egg yolk (osmolality of 335 and pH of 6.9). The osmolality and pH of INRA 96 were 310 and 7, respectively. Extenders at a ratio of 1:3 were added to semen followed by pipetting 10 times with an automatic pipettor. The water-jacketed extended specimen was covered with foam and transferred to individual vaccine carrier equipped with 4 ice packs. This system of cooling not only allows the specimen to cool down slowly and reach 4°C after 7 h, but also reduces the viscosity of camel semen. The assessment was carried out 7 and 24 h after semen dilution, when the specimen reached 4°C. Semen viability parameters were assessed after short-term semen preservation in different extenders. Total motility and progressive forward motility were examined subjectively by single operator using Sperm Track (ISAS, Proiser, Spain) after diluting the specimen to achieve 25 × 106 sperm mL−1. Live percentage of sperm was estimated using Eosin B Fast Green staining method. Plasma membrane integrity was assessed using the hypo-osmotic swelling (HOS) test. Following arcsin transformation, data were analysed by GLM procedure followed by Tukey test in SAS (SAS Institute Inc., Cary, NC, USA). At 7 and 24 h, there were no differences among the 3 extenders in total motility of sperm (Hashi: 73 and 67.4%; Green buffer: 71.6 and 62.1%; INRA 96: 70 and 66.2%; P &gt; 0.05), live percentage of sperm (Hashi: 76 and 73%; Green buffer: 70.5 and 65.6%; INRA 96: 77.8 and 70.7%; P &gt; 0.05), or HOS test (Hashi: 52.4 and 45.2%; Green buffer: 49.6 and 40.6%; INRA 96: 57.3 and 51.1%; P &gt; 0.05). However, at the same times, progressive forward motility was similar between Hashi (47.7 and 28.6%) and Green buffer (40 and 23.5%; P &gt; 0.05) but was different between Hashi and INRA 96 (23.6 and 16.7%; P &lt; 0.05). In conclusion, Hashi and Green buffer could be considered suitable extenders to preserve Bactrian camel semen under chilled condition. Further studies with a larger number of bulls and ejaculates are warranted.

Ultrastructural change of the subchondral bone increases the severity of cartilage damage in osteoporotic osteoarthritis of the knee in rabbits

Osteoporotic osteoarthritis is a phenotype of osteoarthritis (OA) manifested as fragile and osteoporotic subchondral bone. However, the ultrastructural features of subchondral bone in osteoporosis OA have not been determined. The study was aimed to investigate the ultrastructural dynamic changes of subchondral bone in osteoporotic OA model and how the ultrastructural damage in the subchondral bone caused by osteoporosis deteriorated the cartilage damage in OA. Eighteen rabbits were equally randomized to three groups, including the control, the OA and the osteoporotic OA groups. The structural changes of cartilage were evaluated by HE and safranin-O fast green staining, the Mankin’s grading system was used to assess the stage of OA progression. And microstructural or ultrastructural changes in subchondral bone were assessed by micro-computed tomography or by scanning electron microscopy. According to the changes of cartilage histopathology, the OA group was in the early pathological stage of OA while the osteoporotic OA group was in the middle stage of OA based on Mankin’s grading system. In addition, the damage of cartilage surface, reduction in the number of chondrocytes and the matrix staining were more increased in the osteoporotic OA group compared to the OA group. Compared to the OA group, the subchondral bone in the microstructure and ultrastructure in the osteoporotic OA group showed more microfracture changes in trabecular bone with more destructions of the tree-like mesh. Moreover, the collagen fibers were random rough with a fewer amount of bone lacunae in subchondral cortical plate in the osteoporotic OA group compared to the OA group. These findings indicated that the subchondral bone ultrastructure in the osteoporotic OA model was characterized by the destruction of the network structure and collagen fibers. The subchondral bone ultrastructural damage caused by osteoporosis may change mechanical properties of the upper cartilage and aggravate OA cartilage. Therefore, early diagnosis and treatment of osteoporosis is of great significance to prevent early OA from further developing osteoporotic OA.

Chondral Flaps in Patients with Femoral Acetabular Impingement Cam Type are not Suitable for Reattachment and Preservation

According to our results the chondral flap remains viable, but with poor quality tissue. The proposed chondral flap reattachment is not a suitable option. Further studies are required to judge the clinical benefit of chondral flap repair. Francisco Rodriguez Fontan, MD1; Jorge Chahla, MD2; Abigail Richards1; Michelle Sauque3; Karin Payne PhD3; Cecilia Pascual-Garrido, MD1 1 Sports Medicine, Hip Preservation, 3 Regenerative Orthopedics Laboratory, Orthopedics Department, University of Colorado Denver, Aurora, CO; 2 Steadman Philippon Research lnstitute, Vail, CO. Chondrolabral damage is commonly observed in patients with FAI (femoroacetabular impingement) Cam type. Acetabular chondral flaps are normally treated with flap removal and subsequent microfracture. Evaluation of the viability and tissue quality of the chondral flap may raise the need to develop a chondral flap reattachment preservation technique. To evaluate the viability and tissue quality of chondral flaps obtained from patients undergoing hip arthroscopy secondary to FAI Cam type. The study included nine male patients with an average age of 36.3 years old (range 30 - 55). All patients were consented and IRB was approved for the proposed study. All patients underwent hip arthroscopy for FAI cam type impingement and chondral flap removal. All flaps were classified according to Beck’s classification. The resected flap was sent to the lab and evaluated immediately after collection. A control sample was obtained from the cartilage next to the fovea and evaluated concurrently with the study sample. The viability of the samples was evaluated using live-dead staining with Calcein AM and Ethidium homodimer-1. Images were composed using Nikon software. Percent of living cells was evaluated using the Image J software. The number of live cells was then divided by the total number of cells and multiplied by 100% as previously described. Histological analysis was performed using Safranin-O and Fast green staining, along with Hematoxylin and Eosin solution staining. Evaluation of the staining was performed using the OARSI grading system. Compositions of the tissue samples were evaluated using EVOS microscope program. All chondral flaps appeared macroscopically normal. However, the average cellular viability of the chondral flap specimens was reduced (52.1% +- 25.85 SD). In addition, the chondral flap tissue showed microscopic degeneration and loss of ECM. Control samples had 35.6% (+- 30.28 SD) viability and minimal signs of degeneration as well (p>.05). Overall, both study and control samples did show some degenerative changes, with an OARSI grading of 3.8 (+- 1.30 SD) for the study sample and 3.5 (+- 1.38 SD) for the control sample (p>.05). This study shows that acetabular chondral flaps encountered during hip arthroscopy for FAI remain viable. However, there is degeneration of the chondral flap with suboptimal tissue quality. Thus, we do not encourage reattachment of the chondral flap. Unexpectedly, control sample appeared to have degeneration as well. This could be secondary to either the posterior hip impingement that is also generated in patients with FAI Cam lesion or a possible “unhealthy or pre- osteoarthritis” joint phase that patients with FAI Cam lesions may have. According to our results the chondral flap remains viable, but with poor quality tissue. The proposed chondral flap reattachment is not a suitable option. Further studies are required to judge the clinical benefit of chondral flap repair.

Halofuginone attenuates articular cartilage degeneration by inhibition of elevated TGF‑β1 signaling in articular cartilage in a rodent osteoarthritis model.

Osteoarthritis (OA) is the most common degenerative condition of the weight‑bearing joints worldwide without effective medical therapy. In order to investigate whether administration of halofuginone (HF) may attenuate OA, the present study allocated 3‑month‑old male mice into Sham group, vehicle‑treated anterior cruciate ligament transection (ACLT) group and HF‑treated ACLT group. The present study determined that HF treatment reduced the expression of matrix metallopeptidase‑13 and collagen X in articular cartilage. Additionally, it lowered the Osteoarthritis Research Society International‑Modified Mankin score and prevented the loss of articular cartilage from Safranin O and Fast Green staining. HF reduced the progression of osteoarthritis by downregulating abnormally elevated TGF‑β1 activity in articular cartilage. Administration of HF may be a potential preventive therapy for OA.

Analysis of the mechanism by which nerve growth factor promotes callus formation in mice with tibial fracture

The aim of the present study was to analyze the mechanism by which nerve growth factor (NGF) promotes callus formation in mice with tibial fracture. NGF transgenic homozygotic mice and NGF wild homozygotic mice were selected to construct non-stabilized fracture model of tibia. The mice were sacrificed on days 7, 14 and 21, respectively, and each group had a sample with 8 mice at each point in time. X-ray radiography and safranin fast green were used to observe fracture healing and in situ hybridization was used to examine the NGF mRNA expression of tibia at each phase of fracture healing. Tartrate-resistant acid phosphatase (TRAP) staining of callus tissue and the expression level of TRAP mRNA were combined to observe osteoclast formation. COL2A1, a chondrocyte differentiation-related gene in callus, and the mRNA level of SOX9 were combined to observe chondrocyte differentiation. It was found that under X-ray radiography, the fracture of NGF transgenic homozygotic mice healed in advance (P<0.05). Cartilage and bone tissue were identified by safranin and fast green staining. The residual cartilage on the callus tissue of NGF transgenic homozygotic mice had decreased significantly (P<0.05). The NGF mRNA expression level in each phase of callus formation of NGF transgenic homozygotic mice was significantly higher than that of the wild group (P<0.05). The number of positive cells in NGF-TRAP staining at each time point after fracture and the NGF mRNA expression level was markedly higher than that of the wild group, and the expression levels of COL2A1 and SOX9 mRNA were distinctively higher than that of the wild group. In conclusion, NGF potentially improves the healing of tibial fracture by osteoclast formation. Additionally, an increase in the number of osteoblasts in the NGF transgenic homozygotic mice compared with the wild-type mice may be achieved by cartilage differentiation due to NGF increasing the COL2A1 and SOX9 mRNA expression levels.

Chondromodulin‑I expression and correlation with angiogenesis in human osteoarthritic cartilage.

The present study aimed to evaluate the expression and localization of chondromodulin‑I (ChM‑I) in human osteoarthritic cartilage and its correlation with vascular invasion during osteoarthritis (OA) progression. Osteochondral specimens were collected from patients with OA, as well as from young and aged donors without joint diseases. The grade and the number of vascular channels terminating in non‑calcified cartilage of these collected specimens were assessed by Safranin‑O/Fast green staining. ChM‑I expression in articular cartilage was examined by immunohistochemistry, western blotting and reverse transcription‑quantitative polymerase chain reaction analyses. ChM‑I protein and mRNA levels in articular cartilage appeared to be consistent between the normal young and aged groups (P>0.05). In mildly degene-rated cartilage, ChM‑I expression decreased in the extracellular matrix (ECM) of the superficial zone and in the cytoplasm of the superficial and middle zone compared with normal cartilage (P<0.05). In moderately degenerated cartilage, ChM‑I protein expression was reduced in the ECM of all zones of articular cartilage, but the immunostaining intensity in the cytoplasm was increased. In severely degenerated cartilage, ChM‑I expression was detected primarily in the cytoplasm of the cluster‑forming chondrocytes. The density of vascular channels was correlated with the ChM‑I expression levels in cartilage ECM. ChM‑I expression was reduced in OA cartilage matrix, compared with normal cartilage (both young and aged), and correlated with angiogenesis, indicating that loss of ChM‑I may promote angiogenesis in OA cartilage. Expression of ChM‑I protein in the cytoplasm was decreased in mildly degenerated cartilage, whereas ChM‑I expression increased in moderately degenerated cartilage accompanied by chondrocyte proliferation. These findings suggested that attenuation of ChM‑I in the cartilage ECM may be due to decreased expression of ChM‑I in cytoplasm of early stage OA and increased depletion of ChM‑I in the ECM of advanced stage OA.

Exosomes derived from miR-140-5p-overexpressing human synovial mesenchymal stem cells enhance cartilage tissue regeneration and prevent osteoarthritis of the knee in a rat model.

OBJECTIVES: Osteoarthritis (OA) is the most common joint disease throughout the world. Exosomes derived from miR-140-5p-overexpressing synovial mesenchymal stem cells (SMSC-140s) may be effective in treating OA. We hypothesized that exosomes derived from SMSC-140 (SMSC-140-Exos) would enhance the proliferation and migration abilities of articular chondrocytes (ACs) without harming extracellular matrix (ECM) secretion.METHODS: SMSCs were transfected with or without miR-140-5p. Exosomes derived from SMSCs or SMSC-140s (SMSC-Exos or SMSC-140-Exos) were isolated and identified. Proliferation, migration and ECM secretion were measured in vitro and compared between groups. The mechanism involving alternative Wnt signalling and activation of Yes-associated protein (YAP) was investigated using lentivirus, oligonucleotides or chemical drugs. The preventative effect of exosomes in vivo was measured using Safranin-O and Fast green staining and immunohistochemical staining.RESULTS: Wnt5a and Wnt5b carried by exosomes activated YAP via the alternative Wnt signalling pathway and enhanced proliferation and migration of chondrocytes with the side-effect of significantly decreasing ECM secretion. Highly-expressed miR-140-5p blocked this side-effect via RalA. SMSC-140-Exos enhanced the proliferation and migration of ACs without damaging ECM secretion in vitro, while in vivo, SMSC-140-Exos successfully prevented OA in a rat model.CONCLUSIONS: These findings highlight the promising potential of SMSC-140-Exos in preventing OA. We first found a potential source of exosomes and studied their merits and shortcomings. Based on our understanding of the molecular mechanism, we overcame the shortcomings by modifying the exosomes. Such exosomes derived from modified cells hold potential as future therapeutic strategies.

Biomechanical and Endplate Effects on Nutrient Transport in the Intervertebral Disc.

Physical data are lacking on nutrient transport in human intervertebral discs (IVDs), which support regeneration. Our objective was to study nutrient transport in porcine IVDs to determine the effects of biomechanical and physiological factors. Invitro testing of whole porcine IVDs was performed under different loading conditions. Fifty cervical, thoracic, and lumbar discs with attached end plates were removed from 4 Yorkshire pigs (90-150 lbs). Discs were placed in Safranin O or Fast Green FCF histological stains in diffusion or diurnal compression-tested groups. The end plate was studied by the use of polyurethane to block it. Traction was studied with a mechanical testing frame. Discs were cut transversely and photographed. Images were analyzed for depth of annulus fibrosus (AF) stained. The nucleus pulposus (NP) was assigned a staining score. Results showed no difference in AF staining between the 2 stains (P= 0.60). The depth of AF staining did not increase (P= 0.60) due to convection or disc height change via diurnal loading. The NP in all open end plate samples was stained completely by day 3. NP staining was decreased in blocked end plate samples (P= 0.07) and AF staining was significantly less in traction samples than in diffusion-only samples (P= 0.04). This method showed that most small molecule nutrient transport occurs via the end plate. Compressive load was a negligible benefit or hindrance to transport. Traction hindered transport in the short term. This method can be used to study strategies for increasing nutrient transport in IVDs.

Icariin Promotes Tendon-Bone Healing during Repair of Rotator Cuff Tears: A Biomechanical and Histological Study.

To investigate whether the systematic administration of icariin (ICA) promotes tendon-bone healing after rotator cuff reconstruction in vivo, a total of 64 male Sprague Dawley rats were used in a rotator cuff injury model and underwent rotator cuff reconstruction (bone tunnel suture fixation). Rats from the ICA group (n = 32) were gavage-fed daily with ICA at 0.125 mg/g, while rats in the control group (n = 32) received saline only. Micro-computed tomography, biomechanical tests, serum ELISA (calcium; Ca, alkaline phosphatase; AP, osteocalcin; OCN) and histological examinations (Safranin O and Fast Green staining, type I, II and III collagen (Col1, Col2, and Col3), CD31, and vascular endothelial growth factor (VEGF)) were analyzed two and four weeks after surgery. In the ICA group, the serum levels of AP and OCN were higher than in the control group. More Col1-, Col2-, CD31-, and VEGF-positive cells, together with a greater degree of osteogenesis, were detected in the ICA group compared with the control group. During mechanical testing, the ICA group showed a significantly higher ultimate failure load than the control group at both two and four weeks. Our results indicate that the systematic administration of ICA could promote angiogenesis and tendon-bone healing after rotator cuff reconstruction, with superior mechanical strength compared with the controls. Treatment for rotator cuff injury using systematically-administered ICA could be a promising strategy.

Atrial Fibrillation Associated Genetic Variants and Left Atrial Histology: Evaluation for Molecular Sub-Phenotypes.

Genome wide association studies have identified several single nucleotide polymorphisms (SNPs) associated with atrial fibrillation (AF), but the mechanisms underlying these relationships have not yet been elucidated. Inflammation and fibrosis have been posited as important mechanisms responsible for AF. We sought to investigate the impact of SNP carrier status on inflammation and fibrosis in left atrial appendage tissue. Carrier status of 10 AF-associated SNPs was evaluated on DNA extracted from left atrial appendage tissue in 176 individuals (120 with AF). The presence of inflammation was evaluated through visual quantification of leukocyte infiltration following hematoxylin and eosin staining, while fibrosis was quantified using picrosirius red with fast green staining. Unadjusted and adjusted linear and logistic regression models were utilized to evaluate for an association between SNP carrier status and inflammation and fibrosis. On adjusted logistic regression analysis, the rs7164883 SNP (intronic within HCN4) was associated with a reduced odds of inflammation (odds ratio: 0.42; 95% CI: 0.22-0.81, P = 0.01), and was not associated with fibrosis on adjusted linear regression analysis (β-coefficient: -0.31; 95% CI: -1.03-0.40, P = 0.40). None of the remaining SNPs exhibited significant associations with left atrial inflammation or fibrosis. Among 10 AF-associated SNPs, a single genetic variant was associated with reduced left atrial inflammation, while no histologic differences were observed in the remaining 9. The known AF-associated SNPs do not appear to predispose to the development of pro-inflammatory or pro-fibrotic AF sub-phenotypes.

Estrogen promotes spontaneous repair of osteochondral defect via inhibition of osteoclast activity

Objective To investigate the role of estrogen in spontaneous repair of osteochondral defect, so as to provide guidance for the application of estrogen in tissue engineering. Methods Seventy-two healthy adult female SD rats (4-month-old) were assigned to negative control group (group A, n=24), ovariectomized group (group B, n=24) and ovariectomized + estrogen treatment group (group C, n=24) according to the random number table. Rats in group C were intraperitoneally injected 0.1 mg/kg estradiol benzoate once per week after operation. An osteochondral defect model (diameter: 2 mm, height: 1.5 mm) was established in femoral trochlea of rats 4 weeks after operation. At 2, 10 and 20 weeks after the modeling, 8 rats from each group were sacrificed. Femoral condyles were collected to measure bone volume fraction(BVF), number of bone trabeculae(Tb.N), trabecular thickness(Tb.Th)and trabecular spacing(Tb.Sp)in the osteochondral defect area by micro-CT scanning. Morphological changes were observed by HE staining, histomorphological changes and cartilage regeneration by safranine O- fast green staining, osteoclast count by tartrate-resistant acid phosphatase (TRAP) staining, distribution of matrix metalloproteinase-9 (MMP-9) by immunohistochemistry and expressions of osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL) and MMP-9 by real-time-PCR. Results Compared to group B, increased BVF and Tb.N and decreased Tb.Sp were observed in groups A and C at each time point. More bone matrix formations were observed in groups A and C than in group B at 2 weeks, cysts (also known as cystic cavities) in regenerated subchondral bone and cracks in subchondral bone plate were observed in group B at 10 and 20 weeks, and proliferation of osteoclasts was active within lesions in group B at 10 and 20 weeks. Compared to group B, lowered levels of RANKL and MMP-9 (P<0.05) and significantly increased level of OPG (P<0.01) were observed in groups A and C at each time point, and osteoclast count in groups A and C was lowered at 10 and 20 weeks (P<0.05). No regeneration of cartilage layer was observed in all groups. Conclusions Excessive proliferation of osteoclasts results in formation of cystic cavities and cracks. Estrogen improves the speed and quality of bone repair by regulating activities of osteoblasts and osteoclasts, so it can be used to treat osteochondral defect in tissue engineering. Key words: Estrogens; Osteoclasts; Cysts; Osteochondral defect

Chondrocyte-Specific Ablation of AMPKα1 Does Not Affect Bone Development or Pathogenesis of Osteoarthritis in Mice.

AMP-activated protein kinase (AMPK) acts as an intracellular sensor that modulates the energy balance within the cell. AMPKα1 is the dominant catalytic isoform expressed in the bone, but the significance of AMPKα1 in articular cartilage has not been well studied. In this study, we aimed to assess the in vivo function of AMPKα1 in chondrocytes. We created chondrocyte-specific AMPKα1 conditional knockout (KO) mice using Col2α1-Cre and analyzed and compared growth characteristics, HE staining, and AMPKα gene expression between wild-type (WT) mice and AMPKα1 conditional KO mice under normal physiological conditions or following activation of AMPK by metformin intake or treadmill exercise. Microcomputed tomography and safranin O-fast green staining were compared between WT and KO mice after induction of experimental osteoarthritis (OA). Our data showed that there was no somatic difference between WT mice and KO mice of the same age. Metformin intake and treadmill exercise did not alter the phenotype of KO mice, and no difference in cartilage degradation was observed in WT mice or in KO mice after induction of traumatic arthritis. We thought that chondrocyte-specific ablation of AMPKα1 had no effect on bone growth or on pathogenesis of OA in mice, probably because the feedback overexpression of AMPKα2 compensated for loss of AMPKα1 and maintained the combination of AMPKα subunits.

Collagen density gradient on poly(epsilon-caprolactone) surface for the design of 3D printed osteochondral

Event Abstract Back to Event Collagen density gradient on poly(epsilon-caprolactone) surface for the design of 3D printed osteochondral Ugo D'Amora1, Matteo D'Este2, David Eglin2, Antonio Gloria1, Roberto De Santis1, Mauro Alini2 and Luigi Ambrosio1, 3 1 National Research Council of Italy, Institute of Polymers, Composites and Biomaterials, Italy 2 AO Research Institute Davos, Switzerland 3 National Research Council of Italy, Chemical Science & Materials Technology Department, Italy Introduction: The ability to engineer scaffolds which resemble the transition between tissues, like cartilage and bone, has yet to be achieved. Indeed, such constructs should present several continuous gradients in terms of geometry, stiffness and biochemical composition to mimic tissue organization. Although the introduction of fused deposition modeling (FDM) techniques allows tailoring of mechanical, mass transport and morphological properties, the possibility to additionally functionalize such structures by introducing biochemical signal gradients has not been fully explored. Thus, a two-step functionalization method in which collagen was covalently grafted to a polyester surface is presented. A continuous collagen gradient was created along the length of a polymer surface which was characterized for collagen content, topography and cell adhesion. Materials and Methods: Poly(ε-caprolactone) (PCL) surfaces were produced by a melting/molding technique. First, the PCL surfaces were modified by immersing the substrates in a 1,6-hexanediamine/isopropanol solution. An amino-group (NH2) density gradient was achieved along the length of the substrate via the controlled surface exposure into the reactive solution[1]. In the second step, lyophilized collagen type I was grafted using a carbodiimide reaction. Ninhydrin and hydroxyproline assays, contact angle measurements, confocal laser scanning microscopy (CLSM), Fourier transform infrared spectroscopy (FTIR), fast green and picrosirius red staining were performed to characterize the surfaces and gradients. Results and Discussion: The NH2 concentration varied between 5*10-7 mol/cm2 (15 min processing) and 15*10-7 mol/cm2 (45 min processing). (Fig. 1). Figure 1. NH2 concentration as function of the reaction times and of the position along the PCL surface. The PCL-NH2 substrates were treated with Rhodamine B Isothiocyanate to visualize the gradient by using CLSM. The fluorescence intensity decreased from 80 (a.u.) for the region treated for 45 minutes to 10 (a.u.) for the PCL zone. The region treated for 45 minutes showed a greater roughness, probably due to surface erosion of the material. (Fig. 2). Figure 2. Representative images of PCL-NH2-Rhod surfaces functionalized after right) 45; middle) 30; and left) 15 minutes of treatment. Static water contact angle decreased by about 15° whilst collagen density increased with increased NH2 concentration. The introduction of collagen improved the hydrophilicity of the surfaces. Microscopy imaging/analysis visualized the collagen distribution onto the substrates whilst FTIR confirmed collagen grafting and preservation of its helical structure. Conclusion: A two-step functionalization procedure protein was developed. A continuous collagen gradient along the length of a PCL substrate was confirmed by morphological and physico-chemical analysis. Future studies will apply the methodology to FDM PCL-based scaffolds for the repair/regeneration of focal osteochondral defects. The authors would like to thank FIRB MERIT project (GAE: P0000570) for providing financial support

Effect of advanced oxidation protein products on articular cartilage and synovium in a rabbit osteoarthritis model.

Advanced oxidation protein products (AOPPs), a marker of oxidative stress, are prevalent in many kinds of disorders. Osteoarthritis (OA), mainly resulting from the regression of cartilage, chronic inflammation of the synovium and the subchondral bone remodeling. Although the inflammatory response of AOPPs on fibroblast-like synoviocytes (FLSs) were reported, the effect of AOPPs on cartilage and synovial in vivo remains unclear. Therefore, our study aims to investigate whether AOPPs have an effect on the articular cartilage and synovial in a rabbit model of OA. OA model were created by anterior cruciate ligament transection and medial meniscus resection (ACLT + MMx). Forty-eight male New Zealand rabbits were randomly divided into 3 groups: sham-operated group, AOPPs/ACLT + MMx group, and phosphate buffered saline (PBS)/ACLT + MMx group. In sham-operated group, the anterior cruciate ligament was just exposed without transection, and then the incision was sutured. Then intra-articular injection of AOPPs or PBS was performed in the other two groups. Through four weeks and eight weeks of treatment, rabbits in each group were sacrificed. Both hind legs were removed. India ink staining and Safranin O and fast green staining were used to evaluate the macroscopic and microscopic cartilage morphology. The protein expression of matrix metalloproteinases (MMP)-3, MMP-13 in synovium was measured by Western blot. The India ink score and Mankin score of AOPPs/ACLT + MMx group were both higher than the other two groups at the two time points. Western blot have revealed that intra-articular injection of AOPPs upregulated the protein expression of MMP-3 and MMP-13 in synovium. AOPPs participated in the occurrence and development of OA by upregulating the protein expression of MMP-3 and MMP-13 in synovium.