Ablating hedgehog signaling in tenocytes during development impairs biomechanics and matrix organization of the adult murine patellar tendon enthesis

Restoring the native structure of the tendon enthesis, where collagen fibers of the midsubstance are integrated within a fibrocartilaginous structure, is problematic following injury. As current surgical methods fail to restore this region adequately, engineers, biologists, and clinicians are working to understand how this structure forms as a prerequisite to improving repair outcomes. We recently reported on the role of Indian hedgehog (Ihh), a novel enthesis marker, in regulating early postnatal enthesis formation. Here, we investigate how inactivating the Hh pathway in tendon cells affects adult (12‐week) murine patellar tendon (PT) enthesis mechanics, fibrocartilage morphology, and collagen fiber organization. We show that ablating Hh signaling resulted in greater than 100% increased failure insertion strain (0.10 v. 0.05 mm/mm, p<0.01) as well as sub‐failure biomechanical deficiencies. Although collagen fiber orientation appears overtly normal in the midsubstance, ablating Hh signaling reduces mineralized fibrocartilage by 32%, leading to less collagen embedded within mineralized tissue. Ablating Hh signaling also caused collagen fibers to coalesce at the insertion, which may explain in part the increased strains. These results indicate that Ihh signaling plays a critical role in the mineralization process of fibrocartilaginous entheses and may be a novel therapeutic to promote tendon‐to‐bone healing. © 2015 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 33:1142–1151, 2015.     

mTOR signaling plays a critical role in the defects observed in muscle‐derived stem/progenitor cells isolated from a murine model of accelerated aging

Mice expressing reduced levels of ERCC1‐XPF (Ercc1 ^−/Δ mice) demonstrate premature onset of age‐related changes due to decreased repair of DNA damage. Muscle‐derived stem/progenitor cells (MDSPCs) isolated from Ercc1 ^−/Δ mice have an impaired capacity for cell differentiation. The mammalian target of rapamycin (mTOR) is a critical regulator of cell growth in response to nutrient, hormone, and oxygen levels. Inhibition of the mTOR pathway extends the lifespan of several species. Here, we examined the role of mTOR in regulating the MDSPC dysfunction that occurs with accelerated aging. We show that mTOR signaling pathways are activated in Ercc1 ^−/Δ MDSPCs compared with wild‐type (WT) MDSPCs. Additionally, inhibiting mTOR with rapamycin promoted autophagy and improved the myogenic differentiation capacity of the Ercc1 ^−/Δ MDSPCs. The percent of apoptotic and senescent cells in Ercc1 ^−/Δ MDSPC cultures was decreased upon mTOR inhibition. These results establish that mTOR signaling contributes to stem cell dysfunction and cell fate decisions in response to endogenous DNA damage. Therefore, mTOR represents a potential therapeutic target for improving defective, aged stem cells. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 35:1375–1382, 2017.

     

Indian Hedgehog信号通路在软骨细胞成熟及转分化过程中的调控作用

目的 探究Indian Hedgehog（IHH）信号通路对软骨内成骨过程中软骨细胞成熟以及转分化的影响。方法 取10日龄野生型小鼠的胫骨组织,采用原位杂交和免疫组织化学染色检测生长板区域IHH信号通路相关分子Ihh、Ptch1和Gli1的表达水平。构建肥大软骨细胞特异性Ihh基因敲除小鼠（Col10a1^Cre/+; Ihh^null/C）,并采用影像学检查和阿利新蓝染色评估该小鼠的骨骼发育状况。构建肥大软骨细胞IHH信号通路持续激活小鼠（Col10a1^Cre/+; R26Smo^M2/M2和 Col10a1^Cre/+; Ptch1^LacZ/C）,采用HE染色、原位杂交和TUNEL染色分别对受精15.5天胎鼠胫骨组织形态结构、Ihh（肥大软骨细胞分子标志物）和Col1a1（成骨细胞分子标志物）以及肥大软骨细胞凋亡水平进行检测;另外应用HE染色对10日龄小鼠的胫骨组织进行组织学分析。结果 肥大软骨细胞合成分泌IHH,但不表达Ptch1和Gli1。抑制肥大软骨细胞合成IHH蛋白会导致出生后小鼠出现侏儒症;X线检查结果显示小鼠出现严重的骨骼发育不良,包括胸廓狭小、球形头骨以及椎骨发育异常等表现。持续启动IHH信号通路时,胚胎早期软骨细胞成熟分化过程虽未见异常,但是出生后小鼠的骨小梁、骨内膜以及皮质骨等结构均出现一定的异常表现。结论 IHH信号通路虽然不参与肥大软骨细胞的终末分化过程,但在软骨细胞转分化的过程中起到了重要的调控作用。     

COMP‐Ang1 promotes chondrogenic and osteogenic differentiation of multipotent mesenchymal stem cells through the Ang1/Tie2 signaling pathway

Mesenchymal stem cells (MSCs) are pleiotrophic cells that differentiate to chondrocytes, osteoblasts, or adipocytes, as a result of crosstalk by specific signaling pathways including MAPK pathway. Recently cartilage oligomeric matrix protein angiopoietin1 (COMP‐Ang1), an Ang1 variant which is more potent than native Ang1 in phosphorylating Tie2 receptor was developed. The Ang1/Tie2 signaling system not only plays a pivotal role in vessel growth, remodeling, and maturation, but also protective and recruit effect on MSCs. Thus, the aim of the present study was to investigate the differentiate effect of Ang1/Tie2 signaling on MSCs in the presence of chondrogenic, osteogenic and adipogenic induction medium, and to determine the possible mechanisms. Our results clearly demonstrated that MSCs cultured in each induction medium with COMP‐Ang1 revealed strongly chondrogenic and osteogenic morphological change (3.5‐ and 2‐fold, respectively) as well as up‐regulate each gene, except for adipogenic differentiation. Accordingly, we found that phosphorylation of Tie2 expression lead to phosphorylation of p38 and AKT and then accelerating each differentiation of MSCs to chondrocytes and osteoblasts. Therefore, our findings suggest that COMP‐Ang1 present a portal to promote MSCs differentiation to chondrocytes and osteoblasts through Ang1/Tie2 signaling pathway and provide insights into novel therapies for bone diseases. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1920–1928, 2013     

Laser irradiation promotes the proliferation of mouse pre-osteoblast cell line MC3T3-E1 through hedgehog signaling pathway

Low-level laser could promote osteoblast proliferation, and it has been applied in clinical practice to promote wound healing and tissue regeneration. However, the mechanism related to laser irradiation remains unclear. This study aimed to investigate the effects of low-level laser irradiation on the cell proliferation and the expressions of hedgehog signaling molecules Indian hedgehog (Ihh), Ptch, and Gli in vitro. In our present study, the MTT method was used to evaluate the effect on cell proliferation of laser irradiation on MC3T3-E1 cells. And cell cycle was examined by flow cytometry. Gene and protein expressions of hedgehog signaling molecules, including Ihh, Ptch, Smoothened (Smo), and Gli, were examined by qRT-PCR and western blot analysis. The results showed that laser irradiation at dosage of 3.75 J/cm² enhances the proliferation of MC3T3-E1 cells compared with control groups (p = 0.00). Moreover, laser irradiation (3.75 J/cm²) increased the cell amount at S phase (p = 0.00). In addition, the expressions of Ihh, Ptch, Smo, and Gli were significantly increased compared to the control during laser irradiation (3.75 J/cm²)-induced MC3T3-E1 osteoblast proliferation. After adding the hedgehog signaling inhibitor CY (cyclopamine), cell proliferation and Ihh, Ptch, Smo, and Gli expressions were inhibited (p = 0.00), and the cell amount at S phase was reduced compared with combination groups (p = 0.00). These results indicated that laser irradiation promotes proliferation of MC3T3-E1 cells through hedgehog signaling pathway. Our findings provide insights into the mechanistic link between laser irradiation-induced osteogenesis and hedgehog signaling pathway.     

Functional genomics in osteoarthritis: Past,present, and future

Osteoarthritis (OA) is a common complex disease of high public health burden. OA is characterized by the degeneration of affected joints leading to pain and reduced mobility. Over the last few years, several studies have focused on the genomic changes underpinning OA. Here, we provide a comprehensive overview of genome‐wide, non‐hypothesis‐driven functional genomics (methylation, gene, and protein expression) studies of knee and hip OA in humans. Individual studies have generally been limited in sample size and hence power, and have differed in their approaches; nonetheless, some common themes have started to emerge, notably the role played by biological processes related to the extracellular matrix, immune response, the WNT pathway, angiogenesis, and skeletal development. Larger‐scale studies and streamlined, robust methodologies will be needed to further elucidate the biological etiology of OA going forward. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 34:1105–1110, 2016.     

Fracture healing physiology and the quest for therapies for delayed healing and nonunion

Delayed healing and nonunion of fractures represent enormous burdens to patients and healthcare systems. There are currently no approved pharmacological agents for the treatment of established nonunions, or for the acceleration of fracture healing, and no pharmacological agents are approved for promoting the healing of closed fractures. Yet several pharmacologic agents have the potential to enhance some aspects of fracture healing. In preclinical studies, various agents working across a broad spectrum of molecular pathways can produce larger, denser and stronger fracture calluses. However, untreated control animals in most of these studies also demonstrate robust structural and biomechanical healing, leaving unclear how these interventions might alter the healing of recalcitrant fractures in humans. This review describes the physiology of fracture healing, with a focus on aspects of natural repair that may be pharmacologically augmented to prevent or treat delayed or nonunion fractures (collectively referred to as DNFs). The agents covered in this review include recombinant BMPs, PTH/PTHrP receptor agonists, activators of Wnt/β‐catenin signaling, and recombinant FGF‐2. Agents from these therapeutic classes have undergone extensive preclinical testing and progressed to clinical fracture healing trials. Each can promote bone formation, which is important for the stability of bridged calluses, and some but not all can also promote cartilage formation, which may be critical for the initial bridging and subsequent stabilization of fractures. Appropriately timed stimulation of chondrogenesis and osteogenesis in the fracture callus may be a more effective approach for preventing or treating DNFs compared with stimulation of osteogenesis alone. © 2016 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 35:213–223, 2017.

     

Effects of Sprifermin,IGF1, IGF2, BMP7, or CNP on Bovine Chondrocytes in Monolayer and 3D Culture

One possible approach to treat osteoarthritis (OA) is to counteract cartilage degeneration with anabolic compounds that stimulate chondrocyte proliferation and/or extracellular matrix (ECM) production. Several molecules including sprifermin (recombinant human fibroblast growth factor [FGF18]), insulin-like growth factor-1 [IGF1] and -2 [IGF2], C-type natriuretic peptide [CNP], and bone metamorphic protein 7 [BMP7] have been shown to have these characteristics both in vitro and in vivo. However, it is not known how these molecules compare each other regarding their effect on phenotype and stimulation of ECM production in primary chondrocytes. The effects of sprifermin, IGF1, IGF2, CNP, and BMP7 were evaluated on bovine articular chondrocytes, first in monolayer to determine their effective concentrations, and then in three-dimensional (3D) culture at concentrations of 100 ng/ml for sprifermin; 300 ng/ml for IGF1, IGF2, and BMP7; and 10 nM for CNP. In 3D culture, the effects of a permanent exposure or a cyclic exposure consisting of 24 h incubation per week with the compounds were evaluated. All growth factors increased ECM production and cell proliferation to a similar extent but CNP had almost no effect on bovine chondrocytes. Sprifermin was more effective with cyclic exposure, IGF1, and IGF2 with permanent exposure, and BMP7 showed similar results with both exposures. Regarding the cell phenotype, sprifermin appeared to be the only compound favoring the chondrocyte phenotype; it decreased type I collagen expression and had no hypertrophic effect. Together, these results confirmed that sprifermin is a promising disease-modifying OA drug. © 2019 The Authors. Journal of Orthopaedic Research^® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 38:653–662, 2020     

Neuropeptide Y modulates fracture healing through Y1 receptor signaling

Neuropeptide Y acting via it's Y₁ receptor represents a powerful pathway in the control of bone mass. The global or osteoblast‐specific Y₁ receptor deletion induces pronounced bone anabolic effects in mice. However, the contribution of Y₁ receptor deletion in bone repair/healing remained to be clarified. Therefore, in this study we characterized the role of Y₁ receptor deletion in fracture healing. Closed tibial fractures were generated in germline (Y₁^?/?) and osteoblastic‐specific Y₁ receptor knockout mice. The progression of tibial repair monitored from 1‐ until 6‐weeks post‐fracture demonstrated that in Y₁^?/? mice there is a delay in fracture repair, as seen by a decrease in bone callus volume and callus strength. Moreover, the histological features included elevated avascular and cartilage area and consequently delayed cartilage removal, and hence impaired union. Interestingly, this delay in bone repair was not related directly to Y₁ receptors expressed by mature osteoblasts. These findings suggest that the global absence of the Y₁ receptor delays fracture healing, through impairing the early phases of fracture repair to achieve bony union. The data acquired on the role of Y₁ receptor signaling disruption in bone regeneration is critical for the design of future therapeutic strategies. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1570–1578, 2013

     

Understanding the tissue effects of tribo‐corrosion: Uptake,distribution, and speciation of cobalt and chromium in human bone cells

Cobalt and chromium species are released in the local tissues as a result of tribo‐corrosion, and affect bone cell survival and function. However we have little understanding of the mechanisms of cellular entry, intracellular distribution, and speciation of the metals that result in impaired bone health. Here we used synchrotron based X‐ray fluorescence (XRF), X‐ray absorption spectroscopy (XAS), and fluorescent‐probing approaches of candidate receptors P2X7R and divalent metal transporter‐1 (DMT‐1), to better understand the entry, intra‐cellular distribution and speciation of cobalt (Co) and chromium (Cr) in human osteoblasts and primary human osteoclasts. We found that both Co and Cr were most highly localized at nuclear and perinuclear sites in osteoblasts, suggesting uptake through cell membrane transporters, and supported by a finding that P2X7 receptor blockade reduced cellular entry of Co. In contrast, metal species were present at discrete sites corresponding to the basolateral membrane in osteoclasts, suggesting cell entry by endocytosis and trafficking through a functional secretory domain. An intracellular reduction of Cr⁶⁺ to Cr³⁺ was the only redox change observed in cells treated with Co²⁺, Cr³⁺, and Cr⁶⁺. Our data suggest that the cellular uptake and processing of Co and Cr differs between osteoblasts and osteoclasts. © 2014 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 33:114–121, 2015.     

Endostatin Inhibits Callus Remodeling during Fracture Healing in Mice

Information on the impact of endogenous anti‐angiogenic factors on bone repair is limited. The hypothesis of the present study was endostatin, an endogenous inhibitor of angiogenesis, disturbs fracture healing. We evaluated this hypothesis in a closed femoral fracture model studying two groups of mice, one that was treated by a daily injection of 10 µg recombinant endostatin subcutaneously (n = 38) and a second one that received the vehicle for control (n = 37). Histomorphometric analysis showed a significantly increased callus formation in endostatin‐treated animals at 2 and 5 weeks post‐fracture. This was associated with a significantly higher callus tissue fraction of cartilage and fibrous tissue at 2 weeks and a significantly higher fraction of bone at 5 weeks post‐fracture. Biomechanical testing revealed a significantly higher torsional stiffness in the endostatin group at 2 weeks. For both groups, we could demonstrate the expression of the endostatin receptor unit integrin alpha5 in endothelial cells, osteoblasts, osteoclasts, and chondrocytes at 2 weeks. Immunohistochemical fluorescence staining of CD31 showed a lower number of blood vessels in endostatin‐treated animals compared to controls. The results of the present study indicate endostatin promotes soft callus formation but inhibits callus remodeling during fracture healing most probably by an inhibition of angiogenesis. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1579–1584, 2013.     

Prostaglandin E2 inhibits BMP signaling and delays chondrocyte maturation

While cyclooxygenases are important in endochondral bone formation during fracture healing, mechanisms involved in prostaglandin E2 (PGE2) regulation of chondrocyte maturation are incompletely understood. The present study was undertaken to determine if PGE2 effects on chondrocyte differentiation are related to modulation of the bone morphogenetic protein (BMP) signaling pathway. In primary murine sternal chondrocytes, PGE2 differentially regulated genes involved in differentiation. PGE2 induced type II collagen and MMP-13, had minimal effects on alkaline phosphatase, and inhibited the expression of the maturational marker, type X collagen. In BMP-2–treated cultures, PGE2 blocked the induction of type X collagen. All four EP receptors were expressed in chondrocytes and tended to be inhibited by BMP-2 treatment. RCJ3.1C5.18 chondrocytes transfected with the protein kinase A (PKA) responsive reporter, CRE-luciferase, showed luciferase induction following exposure to PGE2, consistent with activation of PKA signaling and the presence of the EP2 and EP4 receptors. Both PGE2 and the PKA agonist, dibutyryl cAMP, blocked the induction of the BMP-responsive reporter, 12XSBE, by BMP-2 in RCJ3.1C5.18 chondrocytes. In contrast, PGE2 increased the ability of TGF-β to activate the TGF-β-responsive reporter, 4XSBE. Finally, PGE2 down-regulated BMP-mediated phosphorylation of Smads 1, 5, and 8 in RCJ3.1C5.18 cells and in primary murine sternal chondrocytes. Altogether, the findings show that PGE2 regulates chondrocyte maturation in part by targeting BMP/Smad signaling and suggest an important role for PGE2 in endochondral bone formation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 785–792, 2009     

Genetic inactivation of ERK1 and ERK2 in chondrocytes promotes bone growth and enlarges the spinal canal

Activating mutations in FGFR3 cause the most common forms of human dwarfism: achondroplasia and thanatophoric dysplasia. In mouse models of achondroplasia, recent studies have implicated the ERK MAPK pathway, a pathway activated by FGFR3, in creating reduced bone growth. Our recent studies have indicated that increased Fgfr3 and ERK MAPK signaling in chondrocytes also causes premature synchondrosis closure in the cranial base and vertebrae, accounting for the sometimes fatal stenosis of the foramen magnum and spinal canal in achondroplasia. Conversely, whether the decrease—or inactivation—of ERK1 and ERK2 promotes bone growth and delays synchondrosis closure remains to be investigated. In this study, we inactivated ERK2 in the chondrocytes of ERK1‐null mice using the Col2a1‐Cre and Col2a1‐CreER transgenes. We found that the genetic inactivation of ERK1 and ERK2 in chondrocytes enhances the growth of cartilaginous skeletal elements. We also found that the postnatal inactivation of ERK1 and ERK2 in chondrocytes delays synchondrosis closure and enlarges the spinal canal. These observations make ERK1 and ERK2 an attractive target for the treatment of achondroplasia and other FGFR3‐related skeletal syndromes. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:375–379, 2011     

Odanacatib increases mineralized callus during fracture healing in a rabbit ulnar osteotomy model

The effects of the cathepsin K inhibitor odanacatib (ODN) on fracture healing were monitored for ～6 and 15 weeks post‐fracture in two separate studies using the unilateral transverse mid‐ulnar osteotomy model in skeletally mature female rabbits. Rabbits were pre‐treated for 3–4 weeks with vehicle (Veh), ODN (2 mg/kg, po, daily), or alendronate (ALN) (0.3 mg/kg, sc, twice‐weekly) prior to osteotomy. In Study 1, the animals were maintained on the same respective treatment for ～6 weeks. In Study 2, the animals were also continued on the same therapy or switched from Veh to ODN or ODN to Veh for 15 weeks. No treatment‐related impairment of fracture union was seen by qualitative histological assessments in the first study. Cartilage retention was detected in the calluses of ALN‐treated rabbits at week‐6, while calluses in the ODN and Veh groups contained bony tissue with significantly less residual cartilage. ODN treatment also markedly increased the number of cathepsin K‐(+) osteoclasts in the callus, indicating enhanced callus remodeling. From the second study, ex vivo DXA and pQCT confirmed that ODN treatment pre‐ and post‐osteotomy increased callus bone mineral content and bone mineral density (BMD) versus Veh (p < 0.001) and discontinuation of ODN post‐surgery returned callus BMD to Veh. Peak load of ODN‐ or ALN‐treated calluses were comparable to Veh. ODN increased callus yield load (20%, p = 0.056) and stiffness (26%, p < 0.05) versus Veh. These studies demonstrated that ODN increased mineralized callus during the early phase of fracture repair without impairing callus formation or biomechanical integrity at the fracture site. © 2015 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 34:72–80, 2016.     

PTEN regulates matrix synthesis in adult human chondrocytes under oxidative stress

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was identified as an important tumor suppressor gene. PTEN functions as a negative regulator of phosphoinositol‐3‐kinase (PI3K)‐Akt and MEK/ERK signaling. The PI3K‐Akt pathway is critical for cell survival, differentiation, and matrix synthesis. Oxidative stress is considered a critical factor in the onset and progression of osteoarthritis (OA). Therefore, we investigated the function of PTEN in OA chondrocytes under oxidative stress. Chondrocytes were treated with insulin‐like growth factor‐1 (IGF‐1) and/or tert‐butyl hydroperoxide (tBHP), which causes oxidative stress. The expression levels of type2 collagen (Col2a1) and aggrecan were analyzed by real‐time PCR, and phosphorylation of Akt and ERK1/2 was analyzed by Western blotting. Chondrocytes were treated with PTEN‐specific small interfering RNA (siRNA), as well as IGF‐1 and/or tBHP. PTEN and IGF‐1 expressions in OA chondrocytes were increased. The downregulation of PTEN expression increased the expression levels of Col2a1 and aggrecan, and increased proteoglycan synthesis under oxidative stress. Oxidative stress decreased the phosphorylation of Akt and increased that of ERK1/2. The downregulation of PTEN expression increased Akt phosphorylation, but did not increase that of ERK 1/2. Our results suggest that PTEN regulates matrix synthesis via the PI3K‐Akt pathway under oxidative stress. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:231–237, 2014.