Autologous solution protects bovine cartilage explants from IL‐1α‐ and TNFα‐induced cartilage degradation

Osteoarthritis (OA) is characterized by deterioration of articular cartilage driven by an imbalance of pro‐ and anti‐inflammatory cytokines. To address the cartilage deterioration observed in OA, an autologous protein solution (APS) has been developed which has been shown to inhibit the production of destructive proteases and inflammatory cytokines from chondrocytes and monocytes, respectively. The purpose of this study was to determine the chondroprotective effect of APS on IL‐1α‐ or TNFα‐challenged bovine articular cartilage explants. Cartilage explants were cultured in the presence or absence of recombinant inflammatory cytokines, IL‐1α and TNFα. Explants under equivalent inflammatory conditions were pretreated with recombinant antagonists IL‐1ra, sTNF‐RI, or APS to measure their inhibition of matrix degradation. Explants were further evaluated with Safranin‐O, Masson's Trichrome, and Hematoxylin and Eosin histological staining. APS was more effective than recombinant antagonists in preventing cartilage matrix degradation and inhibited any measurable IL‐1α‐induced collagen release over a 21‐day culture period. APS treatment reduced the degree of Safranin‐O staining loss when cartilage explants were cultured with IL‐1α or TNFα. Micrographs of APS treated cartilage explants showed an increase in observed cellularity and apparent cell division. APS may have the potential to prevent cartilage loss associated with early OA. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1929–1935, 2013     

Anti‐inflammatory effect of platelet‐rich plasma on nucleus pulposus cells with response of TNF‐α and IL‐1

The purpose of this study was to investigate the anti‐inflammatory effect of platelet‐rich plasma (PRP) with collagen matrix on human nucleus pulposus (NP) cell in response to pro‐inflammatory cytokines such as tumor necrosis factor‐alpha (TNF‐α) and interleukin‐1 (IL‐1). NP cells from human disks were cultured in a monolayer and maintained in the collagen matrix prior to the addition of recombinant human IL‐1 and TNF‐α. After applying IL‐1 and TNF‐α, PRP prepared by using a commercially available platelet concentration system was added. The response was investigated using real‐time PCR for mRNA expression of type II collagen, aggrecan, matrix metalloproteinase‐3 (MMP‐3), and cyclooxygenase‐2 (COX‐2). The combination of IL‐1β and TNF‐α led to decrease of matrix synthesis gene expression such as collagen type II and aggrecan and increase of the degradation gene expression of COX‐2 and MMP‐3, compared to the control. Consecutive PRP exposure significantly recovered the down‐regulated gene expression of collagen type II and aggrecan and significantly reduced the increased MMP‐3 and COX‐2 gene expression, compared to that of control groups with pro‐inflammatory cytokines. The administration of PRP with collagen matrix markedly suppressed cytokine‐induced pro‐inflammatory degrading enzymes and mediators in the NP cell. It also rescued gene expression concerning matrix synthesis, thereby stabilizing NP cell differentiation. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:551–556, 2014.     

Autologous protein solution prepared from the blood of osteoarthritic patients contains an enhanced profile of anti‐inflammatory cytokines and anabolic growth factors

The objective of this clinical study was to test if blood from osteoarthritis (OA) patients (n = 105) could be processed by a device system to form an autologous protein solution (APS) with preferentially increased concentrations of anti‐inflammatory cytokines compared to inflammatory cytokines. To address this objective, APS was prepared from patients exhibiting radiographic evidence of knee OA. Patient metrics were collected including: demographic information, medical history, medication records, and Knee Injury and Osteoarthritis Outcome Score (KOOS) surveys. Cytokine and growth factor concentrations in whole blood and APS were measured using enzyme‐linked immunosorbent assays. Statistical analyses were used to identify relationships between OA patient metrics and cytokines. The results of this study indicated that anti‐inflammatory cytokines were preferentially increased compared to inflammatory cytokines in APS from 98% of OA patients. APS contained high concentrations of anti‐inflammatory proteins including 39,000 ± 20,000 pg/ml IL‐1ra, 21,000 ± 5,000 pg/ml sIL‐1RII, 2,100 ± 570 pg/ml sTNF‐RI, and 4,200 ± 1,500 pg/ml sTNF‐RII. Analysis of the 82 patient metrics indicated that no single patient metric was strongly correlated (R² > 0.7) with the key cytokine concentrations in APS. Therefore, APS can be prepared from a broad range of OA patients. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1349–1355, 2014.     

Transforming growth factor‐β1 suppresses the up‐regulation of matrix metalloproteinase‐2 by lung fibroblasts in response to tumor necrosis factor‐α

Exposed to inflammatory factors or cytokines, fibroblasts appear to play additional roles beyond the deposition of extracellular matrix. It has been reported that tumor necrosis factor‐α (TNF‐α) induces the production of matrix metalloproteinase‐2 (MMP‐2) and transforming growth factor‐β1 (TGF‐β1) in fibroblasts. In this study, we demonstrated that the active MMP‐2 secreted by lung fibroblasts reached the peak level at 12 hours after TNF‐α treatment, whereas, by adding anti‐TGF‐β1 antibody in the culture medium, the MMP‐2 production in response to TNF‐α was maintained at high levels after 24 hours of treatment. We also confirmed that TNF‐α induced up‐regulation of active TGF‐β1 and exogenous TGF‐β1 induced down‐regulation of MMP‐2 synthesis in lung fibroblasts. Moreover, an increased MMP‐2 level was observed in a rat model with pulmonary inflammation and fibrosis induced by bleomycin‐A5. This revealed that MMP‐2 in the lung reached the peak level when TNF‐α reached the peak level at the 7th day, and then MMP‐2 decreased along with an increase in the TGF‐β1 level. Taken together, our results demonstrate that TNF‐α induced an increase of MMP‐2 and TGF‐β1 in lung fibroblasts, and the TGF‐β1 attenuated the up‐regulation of MMP‐2. This suggests that MMP‐2 secreted from fibroblasts modulated by TNF‐α/TGF‐β1 might play an important role in pulmonary inflammation and fibrosis.     

Combined effects of TNF‐α, IL‐1β, and HIF‐1α on MMP‐2 production in ACL fibroblasts under mechanical stretch: An in vitro study

The dynamics between inflammatory factors, mechanical stress, and healing factors, in an intra‐articular joint, are very complex after injury. Injury to intra‐articular tissue [anterior cruciate ligament (ACL), synovium] results in hypoxia, accumulation of various pro‐inflammatory factors, cytokines, and metalloproteases. Although the presence of increased amounts of matrix‐metalloproteinases (MMP) in the joint fluid after knee injury is considered the key factor for ACL poor healing ability; however, the exact role of collective participants of the joint fluid on MMP‐2 activity and production has not been fully studied yet. To investigate the combined effects of mechanical injury, inflammation and hypoxia induced factor‐1α (HIF‐1α) on induction of MMP‐2; we mimicked the microenvironment of joint cavity after ACL injury. The results show that TNF‐α and IL‐1β elevate the activity of MMP‐2 in a dose‐ and time‐dependent manner. In addition, mechanical stretch further enhances the MMP‐2 protein levels with TNF‐α, IL‐1β, and their mixture. CoCl₂‐induced HIF‐1α (100 and 500 µM) also increases the levels and activity of MMP‐2. Mechanical stretch has a strong additional effect on MMP‐2 production with HIF‐1α. Our results conclude that mechanical injury, HIF‐1α and inflammatory factors collectively induce increased MMP‐2 production in ACL fibroblasts, which was inhibited by NF‐κB pathway inhibitor (Bay‐11‐7082). © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1008–1014, 2011     

Ethylene carbodiimide‐fixed donor splenocytes combined with α‐1 antitrypsin induce indefinite donor‐specific protection to mice cardiac allografts

Peritransplant infusion of ethylene carbodiimide‐fixed donor splenocytes (ECDI‐SPs) induces protection of islet and cardiac allografts. However, pro‐inflammatory cytokine production during the peritransplantation period may negate the effect of ECDI‐SPs. Therefore, we hypothesized that blocking pro‐inflammatory cytokine secretion while increasing levels of anti‐inflammatory cytokines would enhance the tolerance‐induced efficacy of ECDI‐SPs. The objective of this study was to determine the effectiveness of using ECDI‐SPs combined with a short course of α1‐antitrypsin (AAT) for induction of tolerance. Using a mice cardiac transplant model, we demonstrated that ECDI‐SPs + AAT effectively induced indefinite mice cardiac allograft protection in a donor‐specific fashion. This effect was accompanied by modulation of cytokines through decreasing levels of pro‐inflammatory cytokines (including IFN‐γ, TNF‐α, IL‐1β, IL‐6, IL‐17, and IL‐23) and increasing levels of anti‐inflammatory cytokines (including IL‐10, IL‐13, and TGF‐β), and by inhibition of effector T cells (Teff) and expansion of regulatory T cells (Tregs). Therefore, we concluded that combined ECDI‐SPs and AAT appeared to modulate the expression of cytokines and regulate the Teff:Treg balance to create a support milieu for graft protection. Our strategy of combining ECDI‐SPs and AAT provides a promising approach for inducing donor‐specific transplant tolerance.     

The overexpression of SIRT1 inhibited osteoarthritic gene expression changes induced by interleukin‐1β in human chondrocytes

In this study, we examined the effects of overexpression of SIRT1 on IL‐1β‐induced gene expression changes in human chondrocytes to explore a protective role of SIRT1 in human chondrocytes. SIRT1 was overexpressed in human chondrocytes by expression plasmid under stimulation with IL‐1β. SIRT1 was also inhibited by siRNA under stimulation with IL‐1β. Gene expression changes were examined by real‐time PCR. The interaction of SIRT1 and p65 (NF‐κB) were examined by Western blotting. SIRT1, MMP‐13, and ADAMTS‐5 expressions in human cartilage were examined by immunohistochemistry. IL‐1β stimulation significantly up‐regulated MMP‐1, 2, 9, and 13 and ADAMTS‐5. Overexpression of SIRT1 significantly inhibited the up‐regulation of those genes caused by IL‐1β while the inhibition of SIRT1 further increased them. In addition, the overexpression of SIRT1 markedly reduced the IL‐1β‐induced acetylation of p65. SIRT1 expression was clearly detected in the non‐OA cartilage while MMP‐13 and ADAMTS‐5 were undetectable. In contrast, in the OA cartilage, SIRT1 expression was decreased while MMP‐13 and ADAMTS‐5 were increased. Our observations suggested that SIRT1 can play a protective role by suppressing IL‐1β‐induced expressions of cartilage‐degrading enzymes partially through the modulation of the NF‐κB pathway. SIRT1 overexpression might be a new therapeutic approach for OA. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 531–537, 2013     

Interleukin‐1β stimulates stromal‐derived factor‐1α expression in human subacromial bursa

Chemokines produced by synoviocytes of the subacromial bursa are up‐regulated in subacromial bursitis and rotator cuff disease. We hypothesized that SDF‐1α production in bursal synoviocytes may be induced by local cytokines such as interleukin IL‐1β and IL‐6. Subacromial bursa specimens were obtained from patients undergoing shoulder surgery. Bursal specimens were stained with anti‐human antibodies to IL‐1, IL‐6, and SDF‐1α by immunohistochemistry and compared to normal and rheumatoid controls. Bursal cells were also isolated from specimens and cultured. Early passaged cells were then treated with cytokines (IL‐1β and IL‐6) and SDF‐1α expression was measured by ELISA and RT‐PCR. SDF‐1α, IL‐1β, and IL‐6 were expressed at high levels in bursitis specimens from human subacromial bursa compared to normal controls. In cultured bursal synoviocytes, there was a dose‐dependent increase in SDF‐1α production in the supernatants of cells treated with IL‐1β. SDF‐1α mRNA expression was also increased in bursal cells treated with IL‐1β. IL‐6 caused a minimal but not statistically significant increase in SDF‐1α expression. SDF‐1α, IL‐1β, and IL‐6 are expressed in the inflamed human subacromial bursal tissues in patients with subacromial bursitis. In cultured bursal synoviocytes, SDF‐1α gene expression and protein production are stimulated by IL‐1β. IL‐1β produced by bursal syvoviocytes and inflammatory cells in the human subacromial bursa is an important signal in the inflammatory response that occurs in subacromial bursitis and rotator cuff disease. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1695–1699, 2011     

Regulation of the friction coefficient of articular cartilage by TGF‐β1 and IL‐1β

Articular cartilage functions to provide a low‐friction surface for joint movement for many decades of life. Superficial zone protein (SZP) is a glycoprotein secreted by chondrocytes in the superficial layer of articular cartilage that contributes to effective boundary lubrication. In both cell and explant cultures, TGF‐β1 and IL‐1β have been demonstrated to, respectively, upregulate and downregulate SZP protein levels. It was hypothesized that the friction coefficient of articular cartilage could also be modulated by these cytokines through SZP regulation. The friction coefficient between cartilage explants (both untreated and treated with TGF‐β1 or IL‐1β) and a smooth glass surface due to sliding in the boundary lubrication regime was measured with a pin‐on‐disk tribometer. SZP was quantified using an enzyme‐linked immunosorbant assay and localized by immunohistochemistry. Both TGF‐β1 and IL‐1β treatments resulted in the decrease of the friction coefficient of articular cartilage in a location‐ and time‐dependent manner. Changes in the friction coefficient due to the TGF‐β1 treatment corresponded to increased depth of SZP staining within the superficial zone, while friction coefficient changes due to the IL‐1β treatment were independent of SZP depth of staining. However, the changes induced by the IL‐1β treatment corresponded to changes in surface roughness, determined from the analysis of surface images obtained with an atomic force microscope. These findings demonstrate that the low friction of articular cartilage can be modified by TGF‐β1 and IL‐1β treatment and that the friction coefficient depends on multiple factors, including SZP localization and surface roughness. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:249–256, 2009     

TNFα From Classically Activated Macrophages Accentuates Epithelial to Mesenchymal Transition in Obliterative Bronchiolitis

Bronchiolitis obliterans syndrome is characterized by fibrotic obliteration of small airways which severely impairs graft function and survival after lung transplantation. Bronchial epithelial cells from the transplanted lung can undergo epithelial to mesenchymal transition and this can be accentuated by activated macrophages. Macrophages demonstrate significant plasticity and change phenotype in response to their microenvironment. In this study we aimed to identify secretory products from macrophages that might be therapeutic targets for limiting the inflammatory accentuation of epithelial to mesenchymal transition in bronchiolitis obliterans syndrome. TNFα, IL‐1β and IL‐8 are elevated in bronchoalveolar lavage from lung transplant patients prior to diagnosis of bronchiolitis obliterans syndrome. Classically activated macrophages secrete more TNFα and IL‐1β than alternatively activated macrophages and dramatically accentuate TGF‐β1‐driven epithelial to mesenchymal transition in bronchial epithelial cells isolated from lung transplant patients. Blocking TNFα, but not IL‐1β, inhibits the accentuation of epithelial to mesenchymal transition. In a pilot unblinded therapeutic intervention in five patients with progressive bronchiolitis obliterans syndrome, anti‐TNFα treatment improved forced expiratory volume in 1 second and 6‐min walk distances in four patients. Our data identify TNFα as a potential new therapeutic target in bronchiolitis obliterans syndrome deserving of a randomized placebo controlled clinical trial.     

Effect of pro‐inflammatory and immunoregulatory cytokines on human tenocytes

Tendon injury induces a local inflammatory response, characterized by the induction of pro‐inflammatory cytokines. The aim of the present study was to analyze the effects of TNFα, IL‐6 and IL‐10 on key parameters of tendon homeostasis. Cultured primary human tenocytes were treated with the recombinant cytokines IL‐6, IL‐10, TNFα, or combinations of TNFα with IL‐6 and IL‐10 (10 ng/mL, 6, 24 h). Expression of type I collagen, elastin, MMP‐1, TNFα, IL‐1β, IL‐6, IL‐10, and suppressors of cytokine signaling (SOCS1, 3) was analyzed with the use of RTD‐PCR, immunocytochemistry, and Western blot analysis. In response to TNFα, tenocytes reduced their type I collagen deposition but increased their elastin gene expression and highly upregulated their expression for MMP‐1, pro‐inflammatory (TNFα, IL‐1β) and immunoregulatory (IL‐6, IL‐10) cytokines. TNFα stimulation augmented SOCS1, whereas SOCS3 expression in tenocytes was also induced by IL‐6. The treatment of tenocytes with IL‐6 and IL‐10 had no effect on cytokine expression. Neither IL‐6 nor IL‐10 modulated the observed effects of TNFα significantly. These results indicate that TNFα strongly activates the tenocytes to amplify their own TNFα expression and, subsequently, that of other regulatory cytokines and matrix degrading enzymes. However, the impact of IL‐6 and IL‐10 on tenocytes remains unclear. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1071–1077, 2010     

Interleukin‐1α, ‐6, and ‐8 decrease Cdc42 activity resulting in loss of articular chondrocyte phenotype

Small GTPase proteins mediate changes in cellular morphology and other cellular functions. The aim of this study was to examine signaling of the small GTPase Cdc42 by stimulating chondrocytes grown in monolayer with long‐ (96 h) or short‐ (2 and 30 min) term exposure to interleukin‐1α (IL‐1α), IL‐6, or IL‐8. Quantitative PCR was used to determine changes in collagen type IIB (COL2A1), aggrecan (AGG), and matrix metalloproteinase‐13 (MMP‐13) gene expression after prolonged cytokine exposure. Effects of short‐term treatment with IL‐α, IL‐6, or IL‐8 on endogenous GTP‐bound Cdc42 levels were assessed using an affinity assay, and on actin filament organization using confocal microscopy. Cytokine treatments significantly decreased COL2A1 and AGG expression and increased MMP‐13 expression. Short exposure to IL‐1α, IL‐6, or IL‐8 decreased endogenous GTP‐Cdc42 and increased stress fibers, which were reversed with cytochalasin D treatment. These results show that IL‐mediated Cdc42 signaling modifies chondrocyte phenotype and morphology. This may lend insight into the altered chondrocyte phenotype in catabolic conditions such as osteoarthritis. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:246–251, 2012     

Inflammation and Epithelial to Mesenchymal Transition in Lung Transplant Recipients: Role in Dysregulated Epithelial Wound Repair

Epithelial to mesenchymal transition (EMT) has been implicated in the pathogenesis of obliterative bronchiolitis (OB) after lung transplant. Although TNF‐α accentuates TGF‐β1 driven EMT in primary human bronchial epithelial cells (PBECs), we hypothesized that other acute pro‐inflammatory cytokines elevated in the airways of patients with OB may also accentuate EMT and contribute to dysregulated epithelial wound repair. PBECs from lung transplant recipients were stimulated with TGF‐β1 ± IL‐1β, IL‐8, TNF‐α or activated macrophages in co‐culture and EMT assessed. The quality and rate of wound closure in a standardized model of lung epithelial injury was assessed in response to above stimuli. Co‐treatment with TGF‐β1 + TNF‐α or IL‐1β significantly accentuates phenotypic and some functional features of EMT compared to TGF‐β1 alone. Co‐treatment with TGF‐β1 + TNF‐α or IL‐1β accelerates epithelial wound closure however the quality of repair is highly dysregulated. Co‐treatment with TGF‐β1 + IL‐8 has no significant effect on EMT or the speed or quality of wound healing. Activated macrophages dramatically accentuate TGF‐β1‐driven EMT and cause dysregulated wound repair. Crosstalk between macrophage‐derived acute inflammation in the airway and elevated TGF‐β1 may favor dysregulated airway epithelial repair and fibrosis in the lung allograft via EMT.     

The small GTPase Rho mediates articular chondrocyte phenotype and morphology in response to interleukin‐1α and insulin‐like growth factor‐I

Small GTPases regulate the cytoskeleton and numerous other cellular functions. In this study, the role of Rho GTPase was examined in articular chondrocytes. Chondrocytes grown in monolayer were treated with interleukin‐1α (IL‐1α), insulin‐like growth factor‐I (IGF‐I), C3 Transferase, Y27632, or transfected with Rho wild type or two constitutively active mutants of Rho (Q63L and G14V). Quantitative PCR was used to determine changes in matrix metalloproteinase‐13 (MMP‐13), collagen types IIB (COL2A1) and type I (COL1A1), aggrecan (AGG), and SOX‐9 gene expression. Affinity assays were performed to measure endogenous GTP‐bound Rho, and confocal microscopy was used to assess changes in organization of the actin cytoskeleton. IL‐1α and RhoG14V increased cytoplasmic actin stress fiber formation, which was blocked by C3 Transferase, and Y27632. IL‐1α treatment also increased Rho activity. Conversely, IGF‐I lead to formation of a cortical rim of actin and decreased Rho activity. Inhibition of Rho signaling with C3 Transferase significantly decreased Rho activity and returned IL‐1α‐induced Rho activity to a level not different from control. C3 Transferase treatment also increased mRNA expression of AGG, COL2A1, and SOX‐9, and decreased expression of MMP‐13. Expression of RhoQ63L or RhoG14V resulted in increased MMP‐13 expression; however, inhibition of Rho with Y27632 was unable to inhibit IL‐1α‐induced MMP‐13 expression. Together, these results indicate a role for increased Rho activity in mediation of chondrocyte catabolic signaling pathways. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:58–64, 2009     

Induction of porcine‐specific regulatory T cells with high specificity and expression of IL‐10 and TGF‐β1 using baboon‐derived tolerogenic dendritic cells

Background

Regulatory T cells (Treg) play an important role in maintenance of homeostasis in vivo. Treg application to alleviate allo‐organ rejection is being studied extensively. However, natural Treg (nTreg) expansion in vitro is laborious and expensive. Antigen‐specific Treg are more effective and require lower cell numbers than use of nTreg for immune control. The baboon, as a non‐human primate experimental animal model, is widely used in xenotransplantation research. An effective method to generate baboon xeno‐specific Treg would benefit research on immune tolerance in xenotransplantation using this model system.

Method

Baboon tolerogenic dendritic cells (tolDC) were generated in 3 days from monocytes isolated from baboon peripheral blood mononuclear cells in medium supplemented with anti‐inflammatory cytokines. After loading with porcine‐specific (PS) in vitro‐transcribed RNA (ivtRNA), tolDC were used to induce CD4⁺ T cells to become porcine‐specific Treg (PSTreg) in cocultures supplemented with IL‐2 and rapamycin for 10 days. Anti‐inflammatory and inflammatory cytokine expression was evaluated at the mRNA and protein levels in both baboon tolDC and PSTreg. Functional assays, suppression of activation markers on porcine‐specific effector T cells (PSTeff) and inhibition of PSTeff proliferation, were used to test PSTreg specificity.

Results

TolDC generated with this method exhibited a tolerogenic phenotype, expressed CCR7 and produced high levels of IL‐10 and TGF‐β1, whereas IL‐12p40 and IFN‐γ were not expressed. PSTreg were successfully generated in cocultures of CD4⁺ T cells and PS ivtRNA‐loaded tolDC. They exhibited a CD3⁺ CD4⁺ CD25⁺ CD127^low/? CD45RA^low Foxp3⁺ phenotype and were characterized by high expression of IL‐10 and TGF‐β1 mRNA and protein. They showed upregulated expression of EBI3 and GARP mRNA. PSTreg exhibited highly suppressive effects toward PSTeff, secreting high amounts of IL‐10 and TGF‐β1 cytokine upon interaction with PSTeff and suppressing IFN‐γ expression on PSTeff.

Conclusion

In this study, a fast 3‐day method to generate baboon‐derived tolDC is provided that allows subsequent induction of PSTreg displaying high porcine‐antigen specificity and expression of IL‐10 and TGF‐β1. Porcine‐specific baboon Treg can be used in porcine solid organ or cell xenotransplantation studies through adoptive cell transfer into host baboons.     

Hyaluronan inhibits matrix metalloproteinase‐13 in human arthritic chondrocytes via CD44 and P38

We investigated the effects of hyaluronan (HA) on interleukin‐1β (IL‐1β)‐stimulated matrix metalloproteinase (MMP)‐13 production in human chondrocytes from patients with osteoarthritis (OA) or rheumatoid arthritis (RA). Secreted levels of MMP‐13 in conditioned media were detected by immunoblotting, while intracellular MMP‐13 synthesis in articular cartilage was evaluated by immunofluorescence microscopic analysis. Mitogen‐activated protein kinases (MAPKs), p38, extracellular signal‐regulated kinases (ERK), and c‐jun NH2‐terminal kinase (JNK) were assessed by Western blotting. IL‐1β (2 ng/ml) stimulates the secretion of MMP‐13 in both OA and RA chondrocytes. Inhibition studies using specific MAPK inhibitors revealed that IL‐1β induced MMP‐13 via p38 in both OA and RA chondrocytes. HA down‐regulates IL‐1β‐stimulated MMP‐13 and phosphorylated p38 (p‐p38) in a dose‐dependent manner (0.1, 1, 2, and 4 mg/ml). When used at 4 mg/ml, HA inhibits p‐p38 phosphorylation by more than 60%. In response to IL‐1β, RA chondrocytes express a higher level of p‐p38 than that of OA chondrocytes. Inhibition of CD44, using a blocking antibody, significantly reversed the inhibitory effect of HA on both MMP‐13 and p‐p38. Our study clearly shows that HA inhibits IL‐1β‐induced MMP‐13 via its principal receptor, CD44, and subsequent intracellular p38 MAPK signaling in OA and RA chondrocytes. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:258–264, 2011     

Osteoblast‐Derived TGF‐β1 Stimulates IL‐8 Release Through AP‐1 and NF‐κB in Human Cancer Cells

Introduction: The bone marrow microenvironment is further enriched by growth factors released during osteoclastic bone resorption. It has been reported that the chemokine interleukin (IL)‐8 is a potent and direct activator of osteoclastic differentiation and bone resorption. However, the effect of bone‐derived growth factors on the IL‐8 production in human cancer cells and the promotion of osteoclastogenesis are largely unknown. The aim of this study was to investigate whether osteoblast‐derived TGF‐β1 is associated with osteolytic bone diseases. Materials and Methods: IL‐8 mRNA levels were measured using RT‐PCR analysis. MAPK phosphorylation was examined using the Western blot method. siRNA was used to inhibit the expression of TGF‐β1, BMP‐2, and IGF‐1. DNA affinity protein‐binding assay and chromatin immunoprecipitation assays were used to study in vitro and in vivo binding of c‐fos, c‐jun, p65, and p50 to the IL‐8 promoter. A transient transfection protocol was used to examine IL‐8, NF‐κB, and activator protein (AP)‐1 activity. Results: Osteoblast conditioned medium (OBCM) induced activation of IL‐8, AP‐1, and NF‐κB promoter in human cancer cells. Osteoblasts were transfected with TGF‐β1, BMP‐2, or IGF‐1 small interfering RNA, and the medium was collected after 48 h. TGF‐β1 but not BMP‐2 or IGF‐1 siRNA inhibited OBCM‐induced IL‐8 release in human cancer cells. In addition, TGF‐β1 also directly induced IL‐8 release in human cancer cells. Activation of AP‐1 and NF‐κB DNA‐protein binding and MAPKs after TGF‐β1 treatment was shown, and TGF‐β1–induced IL‐8 promoter activity was inhibited by the specific inhibitors of MAPK cascades. Conclusions: In this study, we provide evidence to show that the osteoblasts release growth factors, including TGF‐β1, BMP‐2, and IGF‐1. TGF‐β1 is the major contributor to the activation of extracellular signal‐related kinase (ERK), p38, and c‐Jun N‐terminal kinase (JNK), leading to the activation of AP‐1 and NF‐κB on the IL‐8 promoter and initiation of IL‐8 mRNA and protein release, thereby promoting osteoclastogenesis.     

Temporal growth factor release from platelet‐rich plasma,trehalose lyophilized platelets,and bone marrow aspirate and their effect on tendon and ligament gene expression

Platelet‐rich plasma (PRP) has generated substantial interest for tendon and ligament regeneration because of the high concentrations of growth factors in platelet α‐granules. This study compared the temporal release of growth factors from bone marrow aspirate (BMA), PRP, and lyophilized platelet product (PP), and measured their effects on tendon and ligament gene expression. Blood and BMA were collected and processed to yield PRP and plasma. Flexor digitorum superficialis tendon (FDS) and suspensory ligament (SL) explants were cultured in 10% plasma in DMEM (control), BMA, PRP, or PP. TGF‐β1 and PDGF‐BB concentrations were determined at 0, 24, and 96 h of culture using ELISA. Quantitative RT‐PCR for collagen types I and III (COL1A1, COL3A1), cartilage oligomeric matrix protein (COMP), decorin, and matrix metalloproteinases‐3 and 13 (MMP‐3, MMP‐13) was performed. TGF‐β1 and PDGF‐BB concentrations were highest in PRP and PP. Growth factor quantity was unchanged in BMA, increased in PRP, and decreased in PP over 4 days. TGF‐β1 and platelet concentrations were positively correlated. Lyophilized PP and PRP resulted in increased COL1A1:COL3A1 ratio, increased COMP, and decreased MMP‐13 expression. BMA resulted in decreased COMP and increased MMP‐3 and MMP‐13 gene expression. Platelet concentration was positively correlated with COL1A1, ratio of COL1A1:COL3A1, and COMP, and negatively correlated with COL3A1, MMP‐13, and MMP‐3. White blood cell concentration was positively correlated with COL3A1, MMP3, and MMP13, and negatively correlated with a ratio of COL1A1:COL3A1, COMP, and decorin. These findings support further in vivo investigation of PRP and PP for treatment of tendonitis and desmitis. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 1033–1042, 2009