Correction to: Green tea polyphenol treatment is chondroprotective, anti-inflammatory and palliative in a mouse posttraumatic osteoarthritis model.

Following publication of the original article [1], the authors reported an error in Figs. 2C and 5C.

Procyanidins Mitigate Osteoarthritis Pathogenesis by, at Least in Part, Suppressing Vascular Endothelial Growth Factor Signaling.

Procyanidins are a family of plant metabolites that have been suggested to mitigate osteoarthritis pathogenesis in mice. However, the underlying mechanism is largely unknown. This study aimed to determine whether procyanidins mitigate traumatic injury-induced osteoarthritis (OA) disease progression, and whether procyanidins exert a chondroprotective effect by, at least in part, suppressing vascular endothelial growth factor signaling. Procyanidins (extracts from pine bark), orally administered to mice subjected to surgery for destabilization of the medial meniscus, significantly slowed OA disease progression. Real-time polymerase chain reaction revealed that procyanidin treatment reduced expression of vascular endothelial growth factor and effectors in OA pathogenesis that are regulated by vascular endothelial growth factor. Procyanidin-suppressed vascular endothelial growth factor expression was correlated with reduced phosphorylation of vascular endothelial growth factor receptor 2 in human OA primary chondrocytes. Moreover, components of procyanidins, procyanidin B2 and procyanidin B3 exerted effects similar to those of total procyanidins in mitigating the OA-related gene expression profile in the primary culture of human OA chondrocytes in the presence of vascular endothelial growth factor. Together, these findings suggest procyanidins mitigate OA pathogenesis, which is mediated, at least in part, by suppressing vascular endothelial growth factor signaling.

Mesenchymal stem cells in tendon repair and regeneration: basic understanding and translational challenges.

Tendon injuries are common and present a clinical challenge because they often respond poorly to treatment and require prolonged rehabilitation. Current treatments often do not completely repair or regenerate the injured or diseased tendon to its native composition, structure, and mechanical properties. Stem cell-based therapies have brought new hope for tissue repair and regeneration, including that for tendon rupture and tendinopathy. Despite tremendous effort and progress, the success of stem cell-based studies on tendon repair and regeneration has mainly been limited to preclinical studies with few clinical applications. In this concise review, we discuss basic understanding and translational challenges of using mesenchymal stem cells (MSCs) for tendon repair and regeneration, with a focus on (1) tendon stem/progenitor cells (TSPCs) and therapeutic approaches using TSPCs and other MSCs, (2) regulation of fate determination in MSCs for tendon-lineage differentiation, (3) pretreatment and condition of stem/progenitor cells for transplantation, and (4) a treatment approach that involves stimulating endogenous stem cells to enhance tendon healing. The review concludes with discussion on future directions.

Curcumin slows osteoarthritis progression and relieves osteoarthritis-associated pain symptoms in a post-traumatic osteoarthritis mouse model.

BACKGROUND: Curcumin has been shown to have chondroprotective potential in vitro. However, its effect on disease and symptom modification in osteoarthritis (OA) is largely unknown. This study aimed to determine whether curcumin could slow progression of OA and relieve OA-related pain in a mouse model of destabilization of the medial meniscus (DMM). METHODS: Expression of selected cartilage degradative-associated genes was evaluated in human primary chondrocytes treated with curcumin and curcumin nanoparticles and assayed by real-time PCR. The mice subjected to DMM surgery were orally administered curcumin or topically administered curcumin nanoparticles for 8 weeks. Cartilage integrity was evaluated by Safranin O staining and Osteoarthritis Research Society International (OARSI) score, and by immunohistochemical staining of cleaved aggrecan and type II collagen, and levels of matrix metalloproteinase (MMP)-13 and ADAMTS5. Synovitis and subchondral bone thickness were scored based on histologic images. OA-associated pain and symptoms were evaluated by von Frey assay, and locomotor behavior including distance traveled and rearing. RESULTS: Both curcumin and nanoparticles encapsulating curcumin suppressed mRNA expression of pro-inflammatory mediators IL-1ß and TNF-α, MMPs 1, 3, and 13, and aggrecanase ADAMTS5, and upregulated the chondroprotective transcriptional regulator CITED2, in primary cultured chondrocytes in the absence or presence of IL-1ß. Oral administration of curcumin significantly reduced OA disease progression, but showed no significant effect on OA pain relief. Curcumin was detected in the infrapatellar fat pad (IPFP) following topical administration of curcumin nanoparticles on the skin of the injured mouse knee. Compared to vehicle-treated controls, topical treatment led to: (1) reduced proteoglycan loss and cartilage erosion and lower OARSI scores, (2) reduced synovitis and subchondral plate thickness, (3) reduced immunochemical staining of type II collagen and aggrecan cleavage epitopes and numbers of chondrocytes positive for MMP-13 and ADAMTS5 in the articular cartilage, and (4) reduced expression of adipokines and pro-inflammatory mediators in the IPFP. In contrast to oral curcumin, topical application of curcumin nanoparticles relieved OA-related pain as indicated by reduced tactile hypersensitivity and improved locomotor behavior. CONCLUSION: This study provides the first evidence that curcumin significantly slows OA disease progression and exerts a palliative effect in an OA mouse model.

The Role of Scleraxis in Fate Determination of Mesenchymal Stem Cells for Tenocyte Differentiation.

Mesenchymal stem cells (MSCs) are pluripotent cells that primarily differentiate into osteocytes, chondrocytes, and adipocytes. Recent studies indicate that MSCs can also be induced to generate tenocyte-like cells; moreover, MSCs have been suggested to have great therapeutic potential for tendon pathologies. Yet the precise molecular cascades governing tenogenic differentiation of MSCs remain unclear. We demonstrate scleraxis, a transcription factor critically involved in embryonic tendon development and formation, plays a pivotal role in the fate determination of MSC towards tenocyte differentiation. Using murine C3H10T1/2 pluripotent stem cells as a model system, we show scleraxis is extensively expressed in the early phase of bone morphogenetic protein (BMP)-12-triggered tenocytic differentiation. Once induced, scleraxis directly transactivates tendon lineage-related genes such as tenomodulin and suppresses osteogenic, chondrogenic, and adipogenic capabilities, thus committing C3H10T1/2 cells to differentiate into the specific tenocyte-like lineage, while eliminating plasticity for other lineages. We also reveal that mechanical loading-mediated tenocytic differentiation follows a similar pathway and that BMP-12 and cyclic uniaxial strain act in an additive fashion to augment the maximal response by activating signal transducer Smad8. These results provide critical insights into the determination of multipotent stem cells to the tenocyte lineage induced by both chemical and physical signals.

Green tea polyphenol treatment is chondroprotective, anti-inflammatory and palliative in a mouse post-traumatic osteoarthritis model.

INTRODUCTION: Epigallocatechin 3-gallate (EGCG), a polyphenol present in green tea, was shown to exert chondroprotective effects in vitro. In this study, we used a post-traumatic osteoarthritis (OA) mouse model to test whether EGCG could slow the progression of OA and relieve OA-associated pain. METHODS: C57BL/6 mice were subjected to surgical destabilization of the medial meniscus (DMM) or sham surgery. EGCG (25 mg/kg) or vehicle control was administered daily for four or eight weeks by intraperitoneal injection starting on the day of surgery. OA severity was evaluated by Safranin O staining and Osteoarthritis Research Society International (OARSI) score, and by immunohistochemical analysis to detect cleaved aggrecan and type II collagen, and expression of proteolytic enzymes matrix metalloproteinase (MMP)-13 and A Disintegrin And Metalloproteinase with Thrombospondin Motifs (ADAMTS5). Real-time polymerase chain reaction (PCR) was performed to characterize the expression of genes critical for articular cartilage homeostasis. During the course of the experiments, tactile sensitivity testing (von Frey test) and open field assays were used to evaluate pain behaviors associated with OA, and expression of pain expression markers and inflammatory cytokines in the dorsal root ganglion (DRG) were determined by real-time PCR. RESULTS: Four and eight weeks after DMM surgery, the cartilage in EGCG-treated mice exhibited less Safranin O loss and cartilage erosion, and lower OARSI scores compared to vehicle-treated controls, which was associated with reduced staining for aggrecan and type II collagen cleavage epitopes, and reduced staining for MMP-13 and ADAMTS5 in the articular cartilage. Articular cartilage in the EGCG-treated mice also exhibited reduced levels of MMP-1, -3, -8, -13, ADAMTS5, interleukin (IL)-1ß, and tumor necrosis factor (TNF)-α mRNA and elevated gene expression of the MMP regulator Cbp/p300 Interacting Transactivator 2 (CITED2). Compared to vehicle controls, mice treated with EGCG exhibited reduced OA-associated pain, as indicated by higher locomotor behavior (i.e. distance traveled). Moreover, expression of chemokine receptor (CCR2), and pro-inflammatory cytokines IL-1ß and TNF-α in the DRG were significantly reduced to levels similar to sham-operated animals. CONCLUSIONS: This study provides the first evidence in an OA animal model that EGCG significantly slows OA disease progression and exerts a palliative effect.

Nutraceuticals: potential for chondroprotection and molecular targeting of osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease and a leading cause of adult disability. There is no cure for OA, and no effective treatments which arrest or slow its progression. Current pharmacologic treatments such as analgesics may improve pain relief but do not alter OA disease progression. Prolonged consumption of these drugs can result in severe adverse effects. Given the nature of OA, life-long treatment will likely be required to arrest or slow its progression. Consequently, there is an urgent need for OA disease-modifying therapies which also improve symptoms and are safe for clinical use over long periods of time. Nutraceuticals-food or food products that provide medical or health benefits, including the prevention and/or treatment of a disease-offer not only favorable safety profiles, but may exert disease- and symptom-modification effects in OA. Forty-seven percent of OA patients use alternative medications, including nutraceuticals. This review will overview the efficacy and mechanism of action of commonly used nutraceuticals, discuss recent experimental and clinical data on the effects of select nutraceuticals, such as phytoflavonoids, polyphenols, and bioflavonoids on OA, and highlight their known molecular actions and limitations of their current use. We will conclude with a proposed novel nutraceutical-based molecular targeting strategy for chondroprotection and OA treatment.

Acid ceramidase maintains the chondrogenic phenotype of expanded primary chondrocytes and improves the chondrogenic differentiation of bone marrow-derived mesenchymal stem cells.

Acid ceramidase is required to maintain the metabolic balance of several important bioactive lipids, including ceramide, sphingosine and sphingosine-1-phosphate. Here we show that addition of recombinant acid ceramidase (rAC) to primary chondrocyte culture media maintained low levels of ceramide and led to elevated sphingosine by 48 hours. Surprisingly, after three weeks of expansion the chondrogenic phenotype of these cells also was markedly improved, as assessed by a combination of histochemical staining (Alcian Blue and Safranin-O), western blotting (e.g., Sox9, aggrecan, collagen 2A1), and/or qPCR. The same effects were evident in rat, equine and human cells, and were observed in monolayer and 3-D cultures. rAC also reduced the number of apoptotic cells in some culture conditions, contributing to overall improved cell quality. In addition to these effects on primary chondrocytes, when rAC was added to freshly harvested rat, equine or feline bone marrow cultures an ~2-fold enrichment of mesenchymal stem cells (MSCs) was observed by one week. rAC also improved the chondrogenic differentiation of MSCs, as revealed by histochemical and immunostaining. These latter effects were synergistic with TGF-beta1. Based on these results we propose that rAC could be used to improve the outcome of cell-based cartilage repair by maintaining the quality of the expanded cells, and also might be useful in vivo to induce endogenous cartilage repair in combination with other techniques. The results also suggest that short-term changes in sphingolipid metabolism may lead to longer-term effects on the chondrogenic phenotype.

Activation of resorption in fatigue-loaded bone involves both apoptosis and active pro-osteoclastogenic signaling by distinct osteocyte populations.

Osteocyte apoptosis is required to initiate osteoclastic bone resorption following fatigue-induced microdamage in vivo; however, it is unclear whether apoptotic osteocytes also produce the signals that induce osteoclast differentiation. We determined the spatial and temporal patterns of osteocyte apoptosis and expression of pro-osteoclastogenic signaling molecules in vivo. Ulnae from female Sprague-Dawley rats (16-18weeks old) were cyclically loaded to a single fatigue level, and tissues were analyzed 3 and 7days later (prior to the first appearance of osteoclasts). Expression of genes associated with osteoclastogenesis (RANKL, OPG, VEGF) and apoptosis (caspase-3) were assessed by qPCR using RNA isolated from 6mm segments of ulnar mid-diaphysis, with confirmation and spatial localization of gene expression performed by immunohistochemistry. A novel double staining immunohistochemistry method permitted simultaneous localization of apoptotic osteocytes and osteocytes expressing pro-osteoclastogenic signals relative to microdamage sites. Osteocyte staining for caspase-3 and osteoclast regulatory signals exhibited different spatial distributions, with apoptotic (caspase 3-positive) cells highest in the damage region and declining to control levels within several hundred microns of the microdamage focus. Cells expressing RANKL or VEGF peaked between 100 and 300µm from the damage site, then returned to control levels beyond this distance. Conversely, osteocytes in non-fatigued control bones expressed OPG. However, OPG staining was reduced markedly in osteocytes immediately surrounding microdamage. These results demonstrate that while osteocyte apoptosis triggers the bone remodeling response to microdamage, the neighboring non-apoptotic osteocytes are the major source of pro-osteoclastogenic signals. Moreover, both the apoptotic and osteoclast-signaling osteocyte populations are localized in a spatially and temporally restricted pattern consistent with the targeted nature of this remodeling response.

Mechanical loading: bone remodeling and cartilage maintenance.

Bone remodeling and cartilage maintenance are strongly influenced by biomechanical signals generated by mechanical loading. Although moderate loading is required to maintain bone mass and cartilage homeostasis, loading can cause deleterious effects such as bone fracture and cartilage degradation. Because a tight coupling exists between cartilage and bone, alterations in one tissue can affect the other. Bone marrow lesions are often associated with an increased risk of developing cartilage defects, and changes in the articular cartilage integrity are linked to remodeling responses in the underlying bone. Although mechanisms regulating the maintenance of these two tissues are different, compelling evidence indicates that the signal pathways crosstalk, particularly with the Wnt pathway. A better understanding of the complex tempero-spatial interplay between bone remodeling and cartilage degeneration will help develop a therapeutic loading strategy that prevents bone loss and cartilage degeneration.

BMP-12 treatment of adult mesenchymal stem cells in vitro augments tendon-like tissue formation and defect repair in vivo.

We characterized the differentiation of rat bone marrow-derived mesenchymal stem cells (BM-MSCs) into tenocyte-like cells in response to bone morphogenetic protein-12 (BMP-12). BM-MSCs were prepared from Sprague-Dawley rats and cultured as monolayers. Recombinant BMP-12 treatment (10 ng/ml) of BM-MSCs for 12 hours in vitro markedly increased expression of the tenocyte lineage markers scleraxis (Scx) and tenomodulin (Tnmd) over 14 days. Treatment with BMP-12 for a further 12-hour period had no additional effect. Colony formation assays revealed that ~80% of treated cells and their progeny were Scx- and Tnmd-positive. BM-MSCs seeded in collagen scaffolds and similarly treated with a single dose of BMP-12 also expressed high levels of Scx and Tnmd, as well as type I collagen and tenascin-c. Furthermore, when the treated BM-MSC-seeded scaffolds were implanted into surgically created tendon defects in vivo, robust formation of tendon-like tissue was observed after 21 days as evidenced by increased cell number, elongation and alignment along the tensile axis, greater matrix deposition and the elevated expression of tendon markers. These results indicate that brief stimulation with BMP-12 in vitro is sufficient to induce BM-MSC differentiation into tenocytes, and that this phenotype is sustained in vivo. This strategy of pretreating BM-MSCs with BMP-12 prior to in vivo transplantation may be useful in MSC-based tendon reconstruction or tissue engineering.

Physiological loading of joints prevents cartilage degradation through CITED2.

Both overuse and disuse of joints up-regulate matrix metalloproteinases (MMPs) in articular cartilage and cause tissue degradation; however, moderate (physiological) loading maintains cartilage integrity. Here, we test whether CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2), a mechanosensitive transcriptional coregulator, mediates this chondroprotective effect of moderate mechanical loading. In vivo, hind-limb immobilization of Sprague-Dawley rats up-regulates MMP-1 and causes rapid, histologically detectable articular cartilage degradation. One hour of daily passive joint motion prevents these changes and up-regulates articular cartilage CITED2. In vitro, moderate (2.5 MPa, 1 Hz) intermittent hydrostatic pressure (IHP) treatment suppresses basal MMP-1 expression and up-regulates CITED2 in human chondrocytes, whereas high IHP (10 MPa) down-regulates CITED2 and increases MMP-1. Competitive binding and transcription assays demonstrate that CITED2 suppresses MMP-1 expression by competing with MMP transactivator, Ets-1 for its coactivator p300. Furthermore, CITED2 up-regulation in vitro requires the p38δ isoform, which is specifically phosphorylated by moderate IHP. Together, these studies identify a novel regulatory pathway involving CITED2 and p38δ, which may be critical for the maintenance of articular cartilage integrity under normal physical activity levels.

Mechanical loading, cartilage degradation, and arthritis.

Joint tissues are exquisitely sensitive to their mechanical environment, and mechanical loading may be the most important external factor regulating the development and long-term maintenance of joint tissues. Moderate mechanical loading maintains the integrity of articular cartilage; however, both disuse and overuse can result in cartilage degradation. The irreversible destruction of cartilage is the hallmark of osteoarthritis and rheumatoid arthritis. In these instances of cartilage breakdown, inflammatory cytokines such as interleukin-1 beta and tumor necrosis factor-alpha stimulate the production of matrix metalloproteinases (MMPs) and aggrecanases (ADAMTSs), enzymes that can degrade components of the cartilage extracellular matrix. In order to prevent cartilage destruction, tremendous effort has been expended to design inhibitors of MMP/ADAMTS activity and/or synthesis. To date, however, no effective clinical inhibitors exist. Accumulating evidence suggests that physiologic joint loading helps maintain cartilage integrity; however, the mechanisms by which these mechanical stimuli regulate joint homeostasis are still being elucidated. Identifying mechanosensitive chondroprotective pathways may reveal novel targets or therapeutic strategies in preventing cartilage destruction in joint disease.

Identification of CITED2 as a negative regulator of fracture healing.

The transcription regulator CITED2 (CBP/p300-Interacting-Transactivator-with-ED-rich-tail-2) is known to suppress genes mediating angiogenesis and extracellular matrix (ECM) remodeling. However, it is unclear whether CITED2 has a role in controlling skeletal repair or remodeling. We tested the hypothesis that CITED2 functions in bone fracture healing by suppressing the expression of genes critical to ECM remodeling, angiogenesis and osteogenesis, importantly the matrix metalloproteinases (MMPs). Three hours following mandibular osteotomy or sham surgery of adult rats, osteotomy fronts were harvested and the expression of CITED2 and genes associated with fracture healing was ascertained by quantitative PCR. In parallel, gain-of-function studies examined the effect of overexpressing CITED2 on the expression and activity of several MMPs. In the fractured mandible, CITED2 expression was inversely related to the expression of MMP-2, -3, -9, -13, VEGF, HIF-1alpha, M-CSF, RANK-L, and OPG. Consistent with this, the over-expression of CITED2 in osteoblasts inhibited the expression and activity of MMP-2, -3, -9, and -13. Taken together, the studies suggest that CITED2 is a critical upstream regulator of fracture healing. The suppression of CITED2 early after fracture may allow an optimal initiation of the healing response.

SU5416 is a potent inhibitor of hepatocyte growth factor receptor (c-Met) and blocks HGF-induced invasiveness of human HepG2 hepatoma cells.

BACKGROUND/AIMS: SU5416 is a potent inhibitor of receptor tyrosine kinases, including those of the vascular endothelial growth factor receptor, stem cell factor receptor, and platelet-derived growth factor receptor. Because of the overwhelming evidence favoring the role of aberrant hepatocyte growth factor (HGF)/Met signaling in the pathogenesis of various human cancers, various inhibitor strategies have been employed to therapeutically target this receptor. METHODS: Cell proliferation was determined by incorporation of [(3)H] thymidine. Invasiveness was assayed in Boyden Chambers with 8 microm Matrigel coated filters. Phosphorylation of ERK1/2, Akt by HGF stimulation was detected by Western blotting. RESULTS: We found that SU5416 inhibited motility scattering and the invasive activity of a hepatocellular carcinoma cell line HepG2 in vitro and growth in primary cultured hepatocytes induced by HGF. Consequently, tyrosine autophosphorylation of the c-met induced by HGF was inhibited in these cells by SU5416 in a dose-dependent manner. Furthermore, ERK1/2 and Akt phosphorylation, the signaling events down-stream of c-met activation were reduced. Moreover, SU5416 caused reversion in NIH3T3 fibroblasts transformed by the oncogenic form of the receptor, Tpr-Met. CONCLUSIONS: Inhibition of various solid tumors growth and metastasis by SU5416 may be partially attributed to blocking activation of the hepatocyte growth factor receptor.