The effects of bone morphogenetic protein 2 and thrombopoietin treatment on angiogenic properties of endothelial cells derived from the lung and bone marrow of young and aged, male and female mice.

With an aging world population, there is an increased risk of fracture and impaired healing. One contributing factor may be aging-associated decreases in vascular function; thus, enhancing angiogenesis could improve fracture healing. Both bone morphogenetic protein 2 (BMP-2) and thrombopoietin (TPO) have pro-angiogenic effects. The aim of this study was to investigate the effects of treatment with BMP-2 or TPO on the in vitro angiogenic and proliferative potential of endothelial cells (ECs) isolated from lungs (LECs) or bone marrow (BMECs) of young (3-4 months) and old (22-24 months), male and female, C57BL/6J mice. Cell proliferation, vessel-like structure formation, migration, and gene expression were used to evaluate angiogenic properties. In vitro characterization of ECs generally showed impaired vessel-like structure formation and proliferation in old ECs compared to young ECs, but improved migration characteristics in old BMECs. Differential sex-based angiogenic responses were observed, especially with respect to drug treatments and gene expression. Importantly, these studies suggest that NTN1, ROBO2, and SLIT3, along with angiogenic markers (CD31, FLT-1, ANGPT1, and ANGP2) differentially regulate EC proliferation and functional outcomes based on treatment, sex, and age. Furthermore, treatment of old ECs with TPO typically improved vessel-like structure parameters, but impaired migration. Thus, TPO may serve as an alternative treatment to BMP-2 for fracture healing in aging owing to improved angiogenesis and fracture healing, and the lack of side effects associated with BMP-2.

Understanding Early-Stage Posttraumatic Osteoarthritis for Future Prospects of Diagnosis: from Knee to Temporomandibular Joint.

PURPOSE OF REVIEW: Many mechanical load-bearing joints of the body are prone to posttraumatic osteoarthritis (PTOA), including the knee joint and temporomandibular joint (TMJ). Early detection of PTOA can be beneficial in prevention or alleviating further progression of the disease. RECENT FINDINGS: Various mouse models, similar to those used in development of novel diagnosis strategies for early stages of OA, have been proposed to study early PTOA. While many studies have focused on OA and PTOA in the knee joint, early diagnostic methods for OA and PTOA of the TMJ are still not well established. Previously, we showed that fluorescent near-infrared imaging can diagnose inflammation and cartilage damage in mouse models of knee PTOA. Here we propose that the same approach can be used for early diagnosis of TMJ-PTOA. In this review, we present a brief overview of PTOA, application of relevant mouse models, current imaging methods available to examine TMJ-PTOA, and the prospects of near-infrared optical imaging to diagnose early-stage TMJ-OA.

The Effects of SRT1720 Treatment on Endothelial Cells Derived from the Lung and Bone Marrow of Young and Aged, Male and Female Mice.

Angiogenesis is critical for successful fracture healing. Age-related alterations in endothelial cells (ECs) may cause impaired bone healing. Therefore, examining therapeutic treatments to improve angiogenesis in aging may enhance bone healing. Sirtuin 1 (SIRT1) is highly expressed in ECs and its activation is known to counteract aging. Here, we examined the effects of SRT1720 treatment (SIRT1 activator) on the growth and function of bone marrow and lung ECs (BMECs and LECs, respectively), derived from young (3-4 month) and old (20-24 month) mice. While aging did not alter EC proliferation, treatment with SRT1720 significantly increased proliferation of all LECs. However, SRT1720 only increased proliferation of old female BMECs. Vessel-like tube assays showed similar vessel-like structures between young and old LECs and BMECs from both male and female mice. SRT1720 significantly improved vessel-like structures in all LECs. No age, sex, or treatment differences were found in migration related parameters of LECs. In males, old BMECs had greater migration rates than young BMECs, whereas in females, old BMECs had lower migration rates than young BMECs. Collectively, our data suggest that treatment with SRT1720 appears to enhance the angiogenic potential of LECs irrespective of age or sex. However, its role in BMECs is sex- and age-dependent.

Anti-inflammatory role of TPCA-1 encapsulated nanosomes in porcine chondrocytes against TNF-α stimulation.

In this study, we evaluated the hypothesis that immunonanosomes carrying the drug [5-(p-Fluorophenyl)-2-ureido]thiophene-3-carboxamide (TPCA-1) will help in reducing nuclear factor-kappaB (NF-κB)-associated inflammation in porcine chondrocytes against tumor necrosis factor-alpha (TNF-α)-induced stress. The nanosomes were tagged with monoclonal anti-type II collagen (MabCII) antibody to specifically target the exposed type II collagen in cartilage matrix. TPCA-1 at a concentration of 10 µM significantly reduced expression of the matrix-degrading enzyme, Matrix metalloproteinase-13 (MMP-13) and blocked the p65 nuclear translocation. In comparison to the TPCA-1 solution alone, the TPCA-1 nanosomes were found to be more effective in reducing the cellular toxicity, oxidative stress and inflammation in chondrocytes treated with TNF-α. In addition, TPCA-1 nanosomes were more effective in reducing the gene expression of hypoxia-inducible factor-2alpha (HIF-2α) that in turn is associated with the regulation of MMP-13 gene. TPCA-1 nanosomes significantly reduced expression of both these genes. The data also showed that TPCA-1 did not attenuate the down-regulated gene expression levels of anabolic genes aggrecan (ACAN) and collagen type II alpha (COL2A1). In conclusion, this study showed that TPCA-1 nanosomes carrying a dose of 10 µM TPCA-1 can effectively increase the survival of cultured porcine chondrocytes against TNF-α-induced stress. The findings of this study could be used to develop nanosome-based drug delivery systems (DDSs) for animal model of OA. Moreover, the approach presented here can be further utilized in other studies for targeted delivery of the drug of interest at a cellular level.

Cytoprotective role of vitamin E in porcine adipose-tissue-derived mesenchymal stem cells against hydrogen-peroxide-induced oxidative stress.

Survival of mesenchymal stem cells (MSCs) against oxidative stress and inflammation is vital for effective stem cell therapy. The reactive oxygen species (ROS) result in apoptosis and release of inflammatory mediators. Adipose-derived stem cells (ASCs) have shown promise for stem cell therapy owing to their anti-inflammatory and anti-oxidant activity. Previously, we showed the benefits of vitamin E against hydrogen peroxide (H2O2)-induced oxidative stress in rat bone marrow-derived MSCs. In this study, we aim to evaluate the effect of vitamin E treatment on porcine adipose-derived mesenchymal stem cells (pASCs) against H2O2-induced oxidative stress. The oxidative stress was induced by treating pASCs with 500 µM H2O2 with or without vitamin E. Viability of pASCs is enhanced after vitamin E treatment. In addition, reduced cellular toxicity, total NO level, PGE2 production and caspase-3 activity were observed after vitamin E treatment. Gene expression analysis of vitamin E-treated pASCs showed down-regulated expression for the genes associated with oxidative stress and apoptosis, viz., NOS2, Casp3, p53, BAX, MDM2, NFκB, HIF1α and VEGF-A genes. On the other hand, expression of anti-apoptotic and survival genes was up-regulated, viz., BCL2, BCL2L1 and MCL1. Furthermore, phosphorylation of Akt was attenuated following vitamin E treatment. The findings of this study may help in developing effective stem cell therapy for the diseases characterized by the oxidative stress and inflammation.

Cementogenic genes in human periodontal ligament stem cells are downregulated in response to osteogenic stimulation while upregulated by vitamin C treatment.

Regeneration of periodontal tissues, particularly cementum, is key to regaining periodontal attachment and health. Human periodontal ligament stem cells (hPDLSCs) have been shown to be a good cell source to regenerate periodontal tissues. However, their subpopulations and the differentiation induction in relation to cementogenic lineages is unclear. Thus, we aim to examine the expression of cementum-associated genes in PDLSC subpopulations and determine the effect of broadly used osteogenic stimulus or vitamin C (VC) on the expression of cementogenic and osteogenic genes in PDLSCs. Our real-time quantitative polymerase chain reaction (qPCR) analysis showed that cementogenic marker cementum attachment protein (CAP) expressed only slightly higher in STRO-1+/CD146+, STRO-1-/CD146+ and STRO-1-/CD146- subpopulations than in the original cell pool, while cementum protein 1 (CEMP1) expression in these subpopulations was not different from the original pool. Notably, under the stimulation with osteogenic differentiation medium, CAP and CEMP1 were downregulated while osteogenic markers bone sialoprotein (BSP) and osteocalcin (OCN) were upregulated. Both CAP and CEMP1 were upregulated by VC treatment. Transplantation of VC-treated PDLSCs into immunocompromised mice resulted in forming significantly more ectopic cementum- and bone-like mineral tissues in vivo. Immunohistochemical analysis of the ectopic growth showed that CAP and CEMP1 were mainly expressed in the mineral tissue and in some cells of the fibrous tissues. We conclude that osteogenic stimulation is not inductive but appears to be inhibitory of cementogenic pathways, whereas VC induces cementogenic lineage commitment by PDLSCs and may be a useful stimulus for cementogenesis in periodontal regeneration.

Adipose stem cells differentiated chondrocytes regenerate damaged cartilage in rat model of osteoarthritis.

Transplantation of mesenchymal stem cells (MSCs) or autologous chondrocytes has been shown to repair damages to articular cartilage due to osteoarthritis (OA). However, survival of transplanted cells is considerably reduced in the osteoarthritic environment and it affects successful outcome of the transplantation of the cells. Differentiated chrondroytes derived from adipose stem cells have been proposed as an alternative source and our study investigated this possibility in rats. We investigated the regenerative potential of ADSCs and DCs in osteoarthritic environment in the repair of cartilage in rats. We found that ADSCs maintained fibroblast morphology in vitro and also expressed CD90 and CD29. Furthermore, ADSCs differentiated into chondrocytes, accompanied by increased level of proteoglycans and expression of chondrocytes specific genes, such as, Acan, and Col2a1. Histological examination of transplanted knee joints showed regeneration of cartilage tissue compared to control OA knee joints. Increase in gene expression for Acan, Col2a1 with concomitant decrease in the expression of Col1a1 suggested formation of hyaline like cartilage. A significant increase in differentiation index was observed in DCs and ADSCs transplanted knee joints (P = 0.0110 vs. P = 0.0429) when compared to that in OA control knee joints. Furthermore, transplanted DCs showed increased proliferation along with reduction in apoptosis as compared to untreated control. In conclusion, DCs showed better survival and regeneration potential as compared with ADSCs in rat model of OA and thus may serve a better option for regeneration of osteoarthritic cartilage.

In Vivo Dual Fluorescence Imaging to Detect Joint Destruction.

Diagnosis of cartilage damage in early stages of arthritis is vital to impede the progression of disease. In this regard, considerable progress has been made in near-infrared fluorescence (NIRF) optical imaging technique. Arthritis can develop due to various mechanisms but one of the main contributors is the production of matrix metalloproteinases (MMPs), enzymes that can degrade components of the extracellular matrix. Especially, MMP-1 and MMP-13 have main roles in rheumatoid arthritis and osteoarthritis because they enhance collagen degradation in the process of arthritis. We present here a novel NIRF imaging strategy that can be used to determine the activity of MMPs and cartilage damage simultaneously by detection of exposed type II collagen in cartilage tissue. In this study, retro-orbital injection of mixed fluorescent dyes, MMPSense 750 FAST (MMP750) dye and Alexa Fluor 680 conjugated monoclonal mouse antibody immune-reactive to type II collagen, was administered in the arthritic mice. Both dyes were detected with different intensity according to degree of joint destruction in the animal. Thus, our dual fluorescence imaging method can be used to detect cartilage damage as well as MMP activity simultaneously in early stage arthritis.

Lovastatin protects chondrocytes derived from Wharton's jelly of human cord against hydrogen-peroxide-induced in vitro injury.

Our aim was to improve the survival and reduce the apoptosis of chondrocytes derived from mesenchymal stem cells from Wharton's jelly of human umbilical cord (WJMSCs) by Lovastatin supplementation under hydrogen-peroxide-induced injury conditions to simulate the osteoarthritic micro-environment. Chondrocytes were differentiated in vitro from WJMSCs. The cultured WJMSCs expressed CD90 (84.07%), CD105 (80.84%), OCT4 (26.90%), CD45 (0.42%) and CD34 (0.48%) as determined by flow cytometry. Increased aggregation of proteoglycans observed by Safranin-O staining accompanied by increased expression of COL2A1, ACAN, SOX9 and BGN shown by immunocytochemistry and reverse transcription with the polymerase chain reaction (PCR) confirmed the chondrogenic differentiation of the WJMSCs. The in vitro differentiated chondrocytes were subjected to oxidative stress by exposure to 200 µM hydrogen peroxide, either in the presence or absence of Lovastatin (2 µM) for 5 h. Lovastatin treatment resulted in decreased apoptosis, senescence and LDH release and in increased viability and proliferation of WJMSC-derived chondrocytes. Real time PCR analysis showed markedly up-regulated expression of prosurvival, proliferation and chondrogenic genes (BCL2L1, BCL2, AKT, PCNA, COL2A1, ACAN, SOX9 and BGN) and significantly down-regulated expression of pro-apoptotic genes (BAX, FADD) in the Lovastatin-treated group in comparison with injured cells. The reduced expression of VEGF and p53 as determined by enzyme-linked immunosorbent assay and PCR suggests the suitability of the use of Lovastatin in adjunct to WJMSC-derived chondrocytes for the treatment of osteoarthritis. We conclude that Lovastatin protects WJMSC-derived chondrocytes from hydrogen-peroxide-induced in vitro injury.

Vitamin E protects chondrocytes against hydrogen peroxide-induced oxidative stress in vitro.

OBJECTIVE AND DESIGN: This study evaluated the effect of an antioxidant, Vitamin E, on cultured chondrocytes against H2O2-induced damage in vitro. MATERIAL: Rat chondrocytes isolated from articular cartilage. TREATMENT: Chondrocytes were pretreated with either 50 or 100 µM Vitamin E or serum-free medium for 24 h followed by their exposure to 200 µM H2O2 for 3 h. Chondrocytes without exposure to H2O2 served as control group. METHODS: The effect of Vitamin E pretreatment was evaluated by examining proteoglycan contents, nitrite levels, viability, apoptosis, and senescence of cultured chondrocytes. RESULTS: Proteoglycan contents increased in groups treated with Vitamin E. Semi-quantitative real-time PCR data also correlated with these results and demonstrated that Vitamin E up-regulated expression of Agc1, Col2a1, and PCNA genes along with down-regulation in the expression of Col1a1 and Casp3 genes. The differentiation index improved after Vitamin E pretreatment. Nitrite levels were reduced with a corresponding increase in cell viability. Reduction in apoptosis and senescence was also observed after Vitamin E pretreatment. Moreover, a dose-dependent effect of Vitamin E was seen. In contrast to 50 µM Vitamin E, 100 µM was more potent in inducing protection of chondrocytes from H2O2-induced oxidative damage. CONCLUSION: Vitamin E reversed the oxidant-induced alterations in chondrocytes and may be a good option to pretreat chondrocytes before transplantation.

Characterization of Non-Invasively Induced Post-Traumatic Osteoarthritis in Mice.

The pathophysiology of post-traumatic arthritis (PTOA) is not fully understood. This study used non-invasive repetitive mechanical loading (ML) mouse models to study biochemical, biomechanical, and pain-related behavioral changes induced in mice. Mouse models reflected the effects of the early stages of PTOA in humans. For the PTOA model, cyclic comprehensive loading (9N) was applied to each mouse's left knee joint. ML-induced biochemical and molecular changes were analyzed after loading completion. Cartilage samples were examined using gene expression analysis. Tissue sections were used in subsequent OA severity scoring. Biomechanical features and pain-related behavior were studied after 24 h and three weeks post-ML sessions to examine the development of PTOA. The loaded left knee joint showed a greater ROS/RNS signal than the right knee, which was not loaded. There was a significant increase in cartilage damage and MMP activity in the mechanically loaded joints relative to non-loaded control knee joints. Similarly, we found a difference in the viscoelastic tangent, which highlights significant changes in mechanical properties. Biochemical analyses revealed significant increases in total NO, caspase-3 activity, H2O2, and PGE2 levels. Gene expression analysis highlighted increased catabolism (MMP-13, IL-1ß, TNF-α) with a concomitant decrease in anabolism (ACAN, COL2A1). Histopathology scores clearly indicated increases in OA progression and synovitis. The gait pattern was significantly altered, suggesting signs of joint damage. This study showed that biomechanical, biochemical, and behavioral characteristics of the murine PTOA groups are significantly different from the control group. These results confirm that the current mouse model can be considered for translational PTOA studies.

The effects of high fat diet, bone healing, and BMP-2 treatment on endothelial cell growth and function.

Angiogenesis is a vital process during the regeneration of bone tissue. The aim of this study was to investigate angiogenesis at the fracture site as well as at distal locations from obesity-induced type 2 diabetic mice that were treated with bone morphogenetic protein-2 (BMP-2, local administration at the time of surgery) to heal a femoral critical sized defect (CSD) or saline as a control. Mice were fed a high fat diet (HFD) to induce a type 2 diabetic-like phenotype while low fat diet (LFD) animals served as controls. Endothelial cells (ECs) were isolated from the lungs (LECs) and bone marrow (BMECs) 3 weeks post-surgery, and the fractured femurs were also examined. Our studies demonstrate that local administration of BMP-2 at the fracture site in a CSD model results in complete bone healing within 3 weeks for all HFD mice and 66.7% of LFD mice, whereas those treated with saline remain unhealed. At the fracture site, vessel parameters and adipocyte numbers were significantly increased in BMP-2 treated femurs, irrespective of diet. At distal sites, LEC and BMEC proliferation was not altered by diet or BMP-2 treatment. HFD increased the tube formation ability of both LECs and BMECs. Interestingly, BMP-2 treatment at the time of surgery reduced tube formation in LECs and humeri BMECs. However, migration of BMECs from HFD mice treated with BMP-2 was increased compared to BMECs from HFD mice treated with saline. BMP-2 treatment significantly increased the expression of CD31, FLT-1, and ANGPT2 in LECs and BMECs in LFD mice, but reduced the expression of these same genes in HFD mice. To date, this is the first study that depicts the systemic influence of fracture surgery and local BMP-2 treatment on the proliferation and angiogenic potential of ECs derived from the bone marrow and lungs.

Nanosome-Mediated Delivery Of Protein Kinase D Inhibitor Protects Chondrocytes From Interleukin-1ß-Induced Stress And Apoptotic Death.

BACKGROUND: Inflammatory stress caused by protein kinase D (PKD) plays a critical role in damaging chondrocytes and extracellular matrix (ECM) during osteoarthritis (OA). The PKD inhibitor (PKDi) (CRT0066101) has been used to overcome inflammation in different cell types. However, the efficacy of a therapeutic drug can be limited due to off-target distribution, slow cellular internalization, and limited lysosomal escape. In order to overcome this issue, we developed nanosomes carrying CRT0066101 (PKDi-Nano) and tested their efficacy in vitro in chondrocytes. METHODS: Chondrocytes were subjected to IL-1ß-induced inflammatory stress treated with either PKDi or PKDi-Nano. Effects of treatment were measured in terms of cytotoxicity, cellular morphology, viability, apoptosis, phosphorylation of protein kinase B (Akt), and anabolic/catabolic gene expression analyses related to cartilage tissue. RESULTS AND DISCUSSION: The effects of PKDi-Nano treatment were more pronounced as compared to PKDi treatment. Cytotoxicity and apoptosis were significantly reduced following PKDi-Nano treatment (P < 0.001). Cellular morphology was also restored to normal size and shape. The viability of chondrocytes was significantly enhanced in PKDi-Nano-treated cells (P < 0.001). The data indicated that PKDi-Nano acted independently of the Akt pathway. Gene expression analyses revealed significant increases in the expression levels of anabolic genes with concomitant decreases in the level of catabolic genes. Our results indicate that PKDi-Nano attenuated the effects of IL-1ß via the nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) pathway. CONCLUSION: Taken together, these results suggest that PKDi-Nano can be used as a successful strategy to reduce IL1ß-induced inflammatory stress in chondrocytes.

The effects of spaceflight and fracture healing on distant skeletal sites.

Spaceflight results in reduced mechanical loading of the skeleton, which leads to dramatic bone loss. Low bone mass is associated with increased fracture risk, and this combination may compromise future, long-term, spaceflight missions. Here, we examined the systemic effects of spaceflight and fracture surgery/healing on several non-injured bones within the axial and appendicular skeleton. Forty C57BL/6, male mice were randomized into the following groups: (1) Sham surgery mice housed on the earth (Ground + Sham); (2) Femoral segmental bone defect surgery mice housed on the earth (Ground + Surgery); (3) Sham surgery mice housed in spaceflight (Flight + Sham); and (4) Femoral segmental bone defect surgery mice housed in spaceflight (Flight + Surgery). Mice were 9 weeks old at the time of launch and were euthanized approximately 4 weeks after launch. Micro-computed tomography (µCT) was used to evaluate standard bone parameters in the tibia, humerus, sternebra, vertebrae, ribs, calvarium, mandible, and incisor. One intriguing finding was that both spaceflight and surgery resulted in virtually identical losses in tibial trabecular bone volume fraction, BV/TV (24-28% reduction). Another important finding was that surgery markedly changed tibial cortical bone geometry. Understanding how spaceflight, surgery, and their combination impact non-injured bones will improve treatment strategies for astronauts and terrestrial humans alike.

Non-invasive dual fluorescence in vivo imaging for detection of macrophage infiltration and matrix metalloproteinase (MMP) activity in inflammatory arthritic joints.

Detection and intervention at an early stage is a critical factor to impede arthritis progress. Here we present a non-invasive method to detect inflammatory changes in joints of arthritic mice. Inflammation was monitored by dual fluorescence optical imaging for near-infrared fluorescent (750F) matrix-metalloproteinase activatable agent and allophycocyanin-conjugated anti-mouse CD11b. Increased intensity of allophycocyanin (indication of macrophage accumulation) and 750F (indication of matrix-metalloproteinase activity) showed a biological relationship with the arthritis severity score and the histopathology score of arthritic joints. Our results demonstrate that this method can be used to detect early stages of arthritis with minimum intervention in small animal models.

Differential Properties of Human ALP+ Periodontal Ligament Stem Cells vs Their ALP- Counterparts.

Characterizing subpopulations of stem cells is important to understand stem cell properties. Tissue-nonspecific alkaline phosphatase (ALP) is associated with mineral tissue forming cells as well as stem cells. Information regarding ALP subpopulation of human periodontal ligament stem cells (hPDLSCs) is limited. In the present study, we examined ALP+ and ALP- hPDLSC subpopulations, their surface markers STRO-1 and CD146, and the expression of stemness genes at various cell passages. We found that ALP+ subpopulation had higher levels of STRO-1 (30.6 ± 5.6%) and CD146 (90.4 ± 3.3%) compared to ALP- (STRO-1: 0.5 ± 0.1%; CD146: 75.3 ± 7.2%). ALP+ cells expressed significantly higher levels of stemness associated genes, NANOG, OCT4 and SOX than ALP- cells at low cell passages of 2-3 (p<0.05). ALP+ and ALP- cells had similar osteogenic, chondrogenic and neurogenic potential while ALP-, not ALP+ cells, lacked adipogenic potential. Upon continuous culturing and passaging, ALP+ continued to express higher stemness genes and STRO-1 and CD146 than ALP- cells at ≥passage 19. Under conditions (over-confluence and vitamin C treatment) when ALP+ subpopulation was increased, the stemness gene levels of ALP+ was no longer significantly higher than those in ALP- cells. In conclusion, ALP+ hPDLSCs possess differential properties from their ALP- counterparts.