Session VI - Basic Science / Injury Prevention / Spine

Sat., 10/10/09 Basic Sci./Injury Prevent./Spine, Paper #65, 10:16 am OTA-2009

Expression of Bone Morphogenetic Protein Antagonists during Fracture Healing

D. Brian Dean, MD1 (n); J. Tracy Watson, MD1 (3-DePuy, A Johnson &Johnson Company, Wright Medical Technology; 3, 5B, 7- Smith &Nephew);Wu Jin, BS1 (n);
Charles Peters, BS2 (n);  Tyler Enders, BS2 (n);
Berton R. Moed, MD1 (7-DePuy Research Grant; 3-DePuy);
Zijun Zhang, PhD1, 2(7-Orthopaedic Trauma Foundation, St. Louis University Department of Orthopaedic Surgery)
1Department of Orthopaedic Surgery,
2Anatomical Science Program, Saint Louis University, Saint Louis, Missouri, USA

Purpose: One of the most significant advances in bone biology has been the discovery of the bone morphogenetic protein (BMP) family of growth factors and their role in fracture healing. These factors are now being regularly used clinically as adjunct therapy for fracture nonunions and severe acute fractures. By defining the specific roles played by BMP antagonists during fracture healing, the potential exists to create an environment in which BMP therapy is more efficacious. In the present study, the temporal and spatial dynamics of gene and protein expression of several BMPs, BMP receptors, and BMP antagonists were investigated.

Methods: Cohorts of 8 mice were anesthetized and an open, transverse femur fracture was created. Animals were sacrificed at 1, 3, 7, 14, and 21 days after fracture, when tissue/bone blocks were collected from the injured and uninjured side (for use as controls). Real-time polymerase chain reaction was used to quantify gene expression of BMP-2, -4, -6, and -7; BMPR-1A and -2; and BMP antagonists noggin, DAN, CHRD, BAMBI, PRODC, SOST, Smad6, Smad7, Cerberus, and Grem1. Selected tissue sections from each time point were stained with hematoxylin and eosin for tissue structure. The primary antibodies for immunohistochemistry were mouse antirabbit noggin, BAMBI, and DAN. One-way analysis of variance was performed for multiple comparison of gene expression among different time points, followed by post hoc t test.

Results: Radiographs demonstrated normal fracture healing in all animals. Gene expression quantification demonstrated a gradual increase in BMP-2 and -7 after the fracture, but the descent of BMP-7 expression was slower than BMP-2. BMP-4 levels reached a twofold increase at weeks 2 and 3, which was statistically significant. The expression of BMPR-1A was not significantly altered in the first 2 weeks of fracture but was downregulated after week 2. BMPR-2 expression was unchanged. BMP antagonists PRDC, SOST, Smad7, Grem1 and Cerberus as a group had their expression downregulated during the time course. Noggin was upregulated from day 1 through day 7, and substantially downregulated at weeks 2 and 3. DAN expression increased steadily to the final time point of the study, a 50-fold increase compared to the control at week 3. CHRD and BAMBI had increased expression only after day 7 and CHRD was downregulated at week 3.

Conclusions: At the fracture site, PRDC, SOST, Smad7, Grem1 and Cerberus were expressed at levels below that seen in the nonfractured bone, suggesting that they may have contributed to bone formation by minimizing BMP antagonism. Noggin was upregulated early in the time course, similar to previous studies. The increase of BAMBI expression during fracture healing may indicate this factor is functioning as a negative feedback inhibitor to BMP activity. Chordin demonstrated a peak at day 14, while DAN was the most upregulated BMP antagonist in the study. Late upregulation of this group of BMP antagonists suggests their roles in the bone remodeling stage of fracture healing. BMP antagonists are dynamically involved in the initiation and modulation of fracture healing in mouse femur fractures. For the purpose of enhancing bone formation, they could be targets to suppress and thus amplify BMP signaling for bone formation. When the molecular function of these factors is fully characterized, a more effective BMP application for augmentation of fracture healing may be possible. As well, this study has implications for timing and dosage of these BMP adjuvants, which are unknown and currently delivered empirically in clinical practice.

Disclosure: (n=Respondent answered 'No' to all items indicating no conflicts; 1=Board member/owner/officer/committee appointments; 2=Medical/Orthopaedic Publications; 3=Royalties; 4=Speakers bureau/paid presentations; 5A=Paid consultant or employee; 5B=Unpaid consultant; 6=Research or institutional support from a publisher; 7=Research or institutional support from a company or supplier; 8=Stock or Stock Options; 9=Other financial/material support from a publisher; 10=Other financial/material support from a company or supplier).

• The FDA has not cleared this drug and/or medical device for the use described in this presentation   (i.e., the drug or medical device is being discussed for an “off label” use).  ◆FDA information not available at time of printing. Δ OTA Grant