Extracellular Matrix Mediates BMP-2 in a Model of Temporomandibular Joint Osteoarthritis

Abstract

Temporomandibular joint (TMJ) osteoarthritis (OA) is a complex disease that affects both cartilage and subchondral bone. It is accompanied by loss of extracellular matrix (ECM) and may be controlled by bone morphogenetic protein-2 (BMP-2). We analyzed the effect of BMP-2 in both cartilage and subchondral bone in a TMJ-OA animal model that is deficient in biglycan (Bgn) and fibromodulin (Fmod) (Bgn-/-Fmod-/-). Whole mandibles were dissected from 3-week-old wild-type (WT) and Bgn-/-Fmod-/- mice and incubated with and without 250 µg/mL BMP-2 for 2 days using an explant culture system. Condyle growth was measured by microCT and the expression levels of cartilage and bone-related genes were analyzed using RT-PCR or by immunohistochemistry from condyles that contained an intact cartilage/subchondral bone interface. Osteoclast activity was estimated by tartrate-resistant acid phosphatase (TRAP) staining and by TRAP, Rankl, and Adamts4 mRNA expression levels. Our results showed that most parameters examined were slightly up-regulated in WT samples treated with BMP-2, and this up-regulation was significantly enhanced in the Bgn-/-Fmod-/- mice. The up-regulation of both catabolic and anabolic agents did not appear to positively affect the overall growth of Bgn-/-Fmod-/- condyles compared to WT controls. In summary, the up-regulation of both anabolic and catabolic genes in the WT and Bgn-/-Fmod-/- TMJs treated with BMP-2 suggests that BMP increases matrix turnover in the condyle, and, further, that Bgn and Fmod could have protective roles in regulating this process.

Keywords: Biglycan; Fibromodulin; Growth factors; Mineralization; Osteoclasts; Turnover.

Figure 1

A) Representative microCT image of a 3 week-old mouse condyles after explant culture indicating the growth regions that were measured. B) Height of the condyles in WT or Bgn^−/−Fmod^−/− tissue with our without BMP-treatment.

Figure 2

A) Safranin-O staining of WT (left panels) and Bgn^−/−Fmod^−/− condyles (right panels) treated without (top panel) or with (bottom panels) BMP-2. B) Histomorphometic quantitation of safranin-O positive area/total area. C) Immunohistochemistry for aggrecan (Acan) in WT (left panels) and Bgn^−/−Fmod^−/− condyles (right panels) treated without (top panel) or with (bottom panels) BMP-2 D) histomorphic evaluation of Agan positive cells. E) Relative expression of Acan mRNA in WT vs. Bgn^−/−Fmod^−/− with or without BMP-2. *p<0.05, #p<0.01.

Figure 3

Real-time PCR of cartilage expressed genes in WT and Bgn^−/−Fmod^−/− treated without or with BMP-2. A) Type II collagen (Col2), B) Type X collagen (Col10) C) Sox9, D) ihh. #p<0.01.

Figure 4

Immunohistochemistry (IHC) and real-time PCR of genes expressed in fibrocartilage and bone. A) IHC of type I collagen in WT (left panels) and bgn ^−/−, fmod^−/− deficient condyles (right panels) treated without (top panel) or with (bottom panels) BMP-2. B) Real-time PCR of type I collagen (Col1), C) Runx2 D) osterix (Osx) and E) Wisp1 mRNA in WT and Bgn^−/−Fmod^−/− condyles treated with or without BMP-2. *p<0.05, #p<0.01

Figure 5

Assessment of bone resorption A) Tartrate Resistant Acid Phosphatase (TRAP) staining of osteoclasts in WT (left panels) and Bgn^−/−Fmod^−/− deficient condyles (right panels) treated without (top panel) or with (bottom panels) BMP-2. Real-time PCR to measure levels of B) TRAP (Acp5) C) Rankl D) and AdamTS4 mRNA.