Influence of Periostin on Synovial Fibroblasts in Knee Osteoarthritis

Research Article

Chronic Dis Int. 2015;2(1): 1013.

Influence of Periostin on Synovial Fibroblasts in Knee Osteoarthritis

Ishikawa S¹*, Asano K1,2, Kusayanagi H¹, Takashima M1,3, Yoshida N1,3, Yamasaki E¹ and Hisamitsu T¹

1Department of Physiology, School of Medicine, Showa University, Japan

2Division of Physiology, School of Nursing and Rehabilitation Sciences, Showa University, Japan

3Orthopedic Surgery, Showa University, Fujigaoka Hospital, Japan

*Corresponding author: Ishikawa S, Department of Physiology, School of Medicine, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan

Received: December 25, 2014; Accepted: April 07, 2015; Published: April 08, 2015


Osteoarthritis (OA) is a slowly progressive degenerative joint disease characterized by joint space narrowing, osteophyte formation, and subchondral sclerosis. Despite extensive efforts, actual breakthroughs in the identification of biochemical biomarkers of OA have been limited. Therefore, we investigated in vivo periostin production in knee synovial fluid of OA patients and assessed its influence on the extracellular matrix using synovial fibroblasts in vitro. The study population included 40 OA patients (mean age, 75.3±6.6 years) who were classified according to the Kellgren–Lawrence system. Our results showed that periostin and IL-13 levels were up regulated along with progression of OA. A second round of in vitro experiments using human fibroblast-like synoviocytes suggested that elevated periostin mediated an increase in matrix metalloproteinase-9, which is an important molecule in bone turnover. Taken together, these observations indicate that periostin may be a useful diagnostic and/or prognostic marker of OA.

Keywords: Periostin; knee osteoarthritis; Synovial fibroblast; Bone turnover


HFLSs: Human Fibroblast-Like Synoviocytes; IL: Interleukin; MMP-9: Matrix Metalloproteinase-9; OA: Osteoarthritis; RPMI: Roswell Park Memorial Institute; TGF-β: Transforming Growth Factor Beta; TIMP-1: Tissue Inhibitor of Metalloproteinase-1


The number of patients with painful knee Osteoarthritis (OA) continues to dramatically increase with the aging of society: the estimated number of patients exceeds 25 million in the United States [1] and 8 million in Japan [2]. OA refers to the clinical syndrome of joint pain characterized by varying degrees of functional limitation and impaired quality of life. It is the most common form of arthritis and one of the leading causes of pain and disability worldwide, which most commonly affects the peripheral joints, especially the knee. OA is a complex chronic progressive disease attacked by biological and mechanical factors, and as a result from the anabolic and catabolic imbalance in chondrocytes, subchondral bone and extracellular matrix. The degradation and destruction of collagen caused by Matrix Metalloproteinases (MMPs) are considered as the core factor in the occurrence and development of OA for remodeling disorder [3].

Periostin is a member of the fasciclin family of proteins based on its homology to fasciclin I, which was initially identified in insects [4]. Periostin, also termed osteoblast-specific factor 2, is a 93.3-kDa, secreted, vitamin K-dependent, glutamate-containing matricellular protein, originally isolated from a mouse osteoblast cell line [5,6], with known functions in osteology, tissue repair, oncology, cardiovascular and respiratory systems, and various inflammatory settings. Periostin is regulated by Interleukin (IL)-4, IL-13 or Transforming Growth Factor Beta (TGF-β) produced in inflammation, which has a role for remodeling [7].

Periostin is considered an important structural mediator, balancing appropriate versus inappropriate tissue adaption in response to insult/injury. However, the paracrine effect of periostin in OA-synovial fibroblast biology remains poorly understood. Therefore, we investigated the in vivo production of periostin in synovial fluid of the knee of OA patients. Furthermore, we evaluated expression levels of Matrix Metalloproteinase-9 (MMP-9) and Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) in synovial fluid from the knees of OA patients [8,9] and examined the influence of these proteins as extracellular matrix modulators of periostin using OAsynovial fibroblasts in vitro.

Patients and Methods

Patient selection

Synovial fluid was collected from 40 OA patients (mean age, 75.3±6.6 years; age range, 59-82 years) who underwent medical examinations at Showa University Fujigaoka Hospital (Yokohama, Japan). The Institutional Review Board of our teaching hospital approved the study protocol (authorization number: 2013027) and signed informed consent was obtained from all subjects before study participation. Radiographs were reviewed to determine the size and stage of progression of the OA lesions. Radiographic findings were classified according to the Kellgren–Lawrence system [10], as follows: grade 0 = no radiographic features of OA; grade 1 = doubtful Joint Space Narrowing (JSN) and possible osteophytic lipping; grade 2 = the presence of definite osteophyte and possible JSN on anteroposterior weight-bearing radiograph; grade 3 = multiple osteophyte, definite JSN, sclerosis, possible bony deformities; and grade 4 = large osteophyte, marked JSN, severe sclerosis and definite bony deformities.


Periostin (recombinant human periostin/OSF-2, CF) was purchased from R&D systems, Inc. (Minneapolis, MN, USA) and dissolved in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich Corporation, St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal calf serum (RPMI-FCS; Nihon Bio- Supply Center, Tokyo, Japan), sterilized by passing through 0.2-μm pore filters, and stored at 4°C until use.

Culture of fibroblasts

Human Fibroblast-Like Synoviocytes (HFLSs) were purchased from Cell Applications, Inc. (San Diego, CA, USA) and resuspended at a density of 5×105 cells/mL in RPMI-FCS and cultured with different concentration of periostin in 24-well plates in triplicate. After 24 h, culture supernatants were obtained and stored at -80°C until use.

Collection of synovial fluid

Synovial fluid was collected from the OA patients using an18- gauge needle and then stored at -80°C until assayed.

Assay for biologically active substance

Periostin content in synovial fluid was measured using a commercially available Enzyme-Linked Immuno Sorbent Assay (ELISA) kit (catalog no.: EK-074-41; Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA). Concentration of the inflammatory cytokines IL-13 and TGF-β in synovial fluid were measured using commercially available ELISA test kits (catalog nos.: D1300B and DLAP00; R&D Systems, Inc.) according to the manufacturer’s recommendations. Additionally, MMP-9 and TIMP-1 concentrations in culture supernatants were measured with commercially available ELISA test kits (catalog nos.: RPN2614 and RPN2611; GE Healthcare, Ltd., Chalfont St Giles, Buckinghamshire, UK) according to the manufacturer’s recommendations. The minimum detectable level of these ELISA kits was 0.14 ng/mL for human periostin, 57.4 pg/mL for human IL-13, 3.4 pg/mL for human TGF-β, 0.6 ng/mL for human MMP-9, and 1.25 ng/mL for human TIMP-1.

Statistical analysis

Data are expressed as means ± standard deviations. All assays were repeated three times to ensure reproducibility. Statistical significance between the control and experimental groups was analyzed by oneway analysis of variance followed by the Scheffe test. A probability (p) value <0.05 was considered statistically significant.


Analysis of biologically active substances in synovial fluid

We conducted analysis of synovial fluid samples to identify correlations between OA grade and periostin concentration. As shown in Figure 1, periostin concentration significantly increased with OA. Next, we examined levels of cytokines and periostin production in synovial fluid samples. As shown in Figure 2A, IL-13 levels were significantly increased in the samples along with progression of knee OA. As shown in Figure 2B, no significant difference was found in TGF-β content between each stage of OA.