Zhen Lin

Gene expression profiles of human chondrocytes during passaged monolayer cultivation

Chondrocyte phenotype has been shown to dedifferentiate during passaged monolayer cultivation. Hence, we have investigated the expression profile of 27 chondrocyte‐associated genes from both osteoarthritic cartilage tissue and healthy passaged human articular chondrocytes by quantitative real‐time PCR. Our results indicate that the gene expression levels of matrix proteins and proteases in chondrocytes from monolayer culture decrease compared with those from cartilage tissue, while monolayer cultured chondrocytes from normal and osteoarthritic cartilage exhibit similar gene expression patterns. However, chondrocytic gene expression profiles were differentially altered at various stages of passage. The expression of the matrix proteins aggrecan, type II collagen, and fibromodulin inversely correlated with increasing passage number, while fibronectin and link protein exhibited a marked increase with passage. The expression of matrix proteinases MMP‐3/9/13 and ADAMTS‐4/5 decreased with passage, whereas proteinase inhibitors TIMP‐2/3 were elevated. The cytokine IL‐1 also showed increased expression with monolayer chondrocyte culture, while IGF‐1 expression levels were diminished. No significant changes in TGF‐β, or the chondrogenic transcription factors Sox‐9, c‐fos, or c‐jun were observed. Our data indicates that cultured chondrocytes undergo dedifferentiation during monolayer culture, although the gene expression level of transcription factors necessary for chondrogenesis remains unchanged. This data may prove important for the future development of more specific and efficacious cultivation techniques for human articular chondrocyte‐based therapies.

V-ATPases in osteoclasts: Structure, function and potential inhibitors of bone resorption

The vacuolar-type H(+)-ATPase (V-ATPase) proton pump is a macromolecular complex composed of at least 14 subunits organized into two functional domains, V(1) and V(0). The complex is located on the ruffled border plasma membrane of bone-resorbing osteoclasts, mediating extracellular acidification for bone demineralization during bone resorption. Genetic studies from mice to man implicate a critical role for V-ATPase subunits in osteoclast-related diseases including osteopetrosis and osteoporosis. Thus, the V-ATPase complex is a potential molecular target for the development of novel anti-resorptive agents useful for the treatment of osteolytic diseases. Here, we review the current structure and function of V-ATPase subunits, emphasizing their exquisite roles in osteoclastic function. In addition, we compare several distinct classes of V-ATPase inhibitors with specific inhibitory effects on osteoclasts. Understanding the structure-function relationship of the osteoclast V-ATPase may lead to the development of osteoclast-specific V-ATPase inhibitors that may serve as alternative therapies for the treatment of osteolytic diseases.

Prevention of Wear Particle-Induced Osteolysis by a Novel V-ATPase Inhibitor Saliphenylhalamide through Inhibition of Osteoclast Bone Resorption

Wear particle-induced peri-implant loosening (Aseptic prosthetic loosening) is one of the most common causes of total joint arthroplasty. It is well established that extensive bone destruction (osteolysis) by osteoclasts is responsible for wear particle-induced peri-implant loosening. Thus, inhibition of osteoclastic bone resorption should prevent wear particle induced osteolysis and may serve as a potential therapeutic avenue for prosthetic loosening. Here, we demonstrate for the first time that saliphenylhalamide, a new V-ATPase inhibitor attenuates wear particle-induced osteolysis in a mouse calvarial model. In vitro biochemical and morphological assays revealed that the inhibition of osteolysis is partially attributed to a disruption in osteoclast acidification and polarization, both a prerequisite for osteoclast bone resorption. Interestingly, the V-ATPase inhibitor also impaired osteoclast differentiation via the inhibition of RANKL-induced NF-κB and ERK signaling pathways. In conclusion, we showed that saliphenylhalamide affected multiple physiological processes including osteoclast differentiation, acidification and polarization, leading to inhibition of osteoclast bone resorption in vitro and wear particle-induced osteolysis in vivo. The results of the study provide proof that the new generation V-ATPase inhibitors, such as saliphenylhalamide, are potential anti-resorptive agents for treatment of peri-implant osteolysis.

Andrographolide suppresses RANKL-induced osteoclastogenesis in vitro and prevents inflammatory bone loss in vivo.

Osteoclasts play a pivotal role in diseases such as osteoporosis, rheumatoid arthritis and tumour bone metastasis. Thus, searching for natural compounds that may suppress osteoclast formation and/or function is promising for the treatment of osteoclast‐related diseases. Here, we examined changes in osteoclastogenesis and LPS‐induced osteolysis in response to andrographolide (AP), a diterpenoid lactone isolated from the traditional Chinese and Indian medicinal plant Andrographis paniculata.

Versatile Roles of V-ATPases Accessory Subunit Ac45 in Osteoclast Formation and Function

Vacuolar-type H+-ATPases (V-ATPases) are macromolecular proton pumps that acidify intracellular cargos and deliver protons across the plasma membrane of a variety of specialized cells, including bone-resorbing osteoclasts. Extracellular acidification is crucial for osteoclastic bone resorption, a process that initiates the dissolution of mineralized bone matrix. While the importance of V-ATPases in osteoclastic resorptive function is well-defined, whether V-ATPases facilitate additional aspects of osteoclast function and/or formation remains largely obscure. Here we report that the V-ATPase accessory subunit Ac45 participates in both osteoclast formation and function. Using a siRNA-based approach, we show that targeted suppression of Ac45 impairs intracellular acidification and endocytosis, both are prerequisite for osteoclastic bone resorptive function in vitro. Interestingly, we find that knockdown of Ac45 also attenuates osteoclastogenesis owing to a reduced fusion capacity of osteoclastic precursor cells. Finally, in an effort to gain more detailed insights into the functional role of Ac45 in osteoclasts, we attempted to generate osteoclast-specific Ac45 conditional knockout mice using a Cathepsin K-Cre-LoxP system. Surprisingly, however, insertion of the neomycin cassette in the Ac45-FloxNeo mice resulted in marked disturbances in CNS development and ensuing embryonic lethality thus precluding functional assessment of Ac45 in osteoclasts and peripheral bone tissues. Based on these unexpected findings we propose that, in addition to its canonical function in V-ATPase-mediated acidification, Ac45 plays versatile roles during osteoclast formation and function.

Autologous Tenocyte Injection for the Treatment of Severe, Chronic Resistant Lateral Epicondylitis A Pilot Study

Background: Severe chronic lateral epicondylitis (LE) is associated with degenerative tendon changes, extracellular matrix breakdown, and tendon cell loss. On the basis of positive outcomes from preclinical studies, this study is the first clinical trial of autologous tenocyte injection (ATI) on severe tendinopathy associated with chronic LE. Hypothesis: Autologous tenocyte injection is a safe and effective procedure that enables a reduction in pain and improvement in function in resistant LE. Study Design: Case series; Level of evidence, 4. Methods: Patients with severe refractory LE underwent clinical evaluation and magnetic resonance imaging (MRI) before intervention. A patellar tendon needle biopsy was performed under local anesthetic, and tendon cells were expanded by in vitro culture. Tenocytes used for the injection were characterized by flow cytometry and real-time polymerase chain reaction. Autologous tenocytes were injected into the site of tendinopathy identified at the origin of the extensor carpi radialis brevis tendon under ultrasound guidance on a single occasion. Patients underwent serial clinical evaluations and repeat MRI at 12 months after intervention. Results: A total of 20 consecutive patients were included in the study. Three patients withdrew consent after enrollment and before ATI. No adverse event was reported at either biopsy or injection sites. Furthermore, no infection or excessive fibroblastic reaction was found in any patient at the injection site. Clinical evaluation revealed an improvement in mean visual analog scale scores, for a maximum pain score from 5.94 at the initial assessment to 0.76 at 12 months (P < .001). Mean quick Disabilities of the Arm, Shoulder and Hand (QuickDASH) and grip strength scores also significantly improved over the 12-month follow-up (QuickDASH score, 45.88 [baseline] to 3.84; grip strength, 20.17 kg [baseline] to 37.38 kg; P < .001). With use of a validated MRI scoring system, the grade of tendinopathy at the common extensor origin improved significantly by 12 months (P < .001). One patient elected to proceed to surgery 3 months after ATI following a reinjury at work. Conclusion: In this study, patients with chronic LE who had previously undergone an unsuccessful full course of nonoperative treatment showed significantly improved clinical function and structural repair at the origin of the common extensor tendon after ATI. This novel treatment is encouraging for the treatment of tendinopathy and warrants further evaluation.

Programmable mechanical stimulation influences tendon homeostasis in a bioreactor system

Identification of functional programmable mechanical stimulation (PMS) on tendon not only provides the insight of the tendon homeostasis under physical/pathological condition, but also guides a better engineering strategy for tendon regeneration. The aims of the study are to design a bioreactor system with PMS to mimic the in vivo loading conditions, and to define the impact of different cyclic tensile strain on tendon. Rabbit Achilles tendons were loaded in the bioreactor with/without cyclic tensile loading (0.25 Hz for 8 h/day, 0–9% for 6 days). Tendons without loading lost its structure integrity as evidenced by disorientated collagen fiber, increased type III collagen expression, and increased cell apoptosis. Tendons with 3% of cyclic tensile loading had moderate matrix deterioration and elevated expression levels of MMP‐1, 3, and 12, whilst exceeded loading regime of 9% caused massive rupture of collagen bundle. However, 6% of cyclic tensile strain was able to maintain the structural integrity and cellular function. Our data indicated that an optimal PMS is required to maintain the tendon homeostasis and there is only a narrow range of tensile strain that can induce the anabolic action. The clinical impact of this study is that optimized eccentric training program is needed to achieve maximum beneficial effects on chronic tendinopathy management. Biotechnol. Bioeng. 2013; 110: 1495–1507.

Sanguinarine inhibits osteoclast formation and bone resorption via suppressing RANKL-induced activation of NF-κB and ERK signaling pathways

Sanguinarine is a natural plant extract that has been supplemented in a number of gingival health products to suppress the growth of dental plaque. However, whether sanguinarine has any effect on teeth and alveolar bone health is still unclear. In this study, we demonstrated for the first time that sanguinarine could suppress osteoclastic bone resorption and osteoclast formation in a dose-dependent manner. Sanguinarine diminished the expression of osteoclast marker genes, including TRAP, cathepsin K, calcitonin receptor, DC-STAMP, V-ATPase d2, NFATc1 and c-fos. Further investigation revealed that sanguinarine attenuated RANKL-mediated IκBα phosphorylation and degradation, leading to the impairment of NF-κB signaling pathway during osteoclast differentiation. In addition, sanguinarine also affected the ERK signaling pathway by inhibiting RANKL-induced ERK phosphorylation. Collectively, this study suggested that sanguinarine has protective effects on teeth and alveolar bone health.

Cyclic mechanical stimulation rescues achilles tendon from degeneration in a bioreactor system

Physiotherapy is one of the effective treatments for tendinopathy, whereby symptoms are relieved by changing the biomechanical environment of the pathological tendon. However, the underlying mechanism remains unclear. In this study, we first established a model of progressive tendinopathy‐like degeneration in the rabbit Achilles. Following ex vivo loading deprivation culture in a bioreactor system for 6 and 12 days, tendons exhibited progressive degenerative changes, abnormal collagen type III production, increased cell apoptosis, and weakened mechanical properties. When intervention was applied at day 7 for another 6 days by using cyclic tensile mechanical stimulation (6% strain, 0.25 Hz, 8 h/day) in a bioreactor, the pathological changes and mechanical properties were almost restored to levels seen in healthy tendon. Our results indicated that a proper biomechanical environment was able to rescue early‐stage pathological changes by increased collagen type I production, decreased collagen degradation and cell apoptosis. The ex vivo model developed in this study allows systematic study on the effect of mechanical stimulation on tendon biology.