title: Matrix metalloproteinase-9 expression in alveolar extraction sockets of Zoledronic acid-treated rats.
abstract: OBJECTIVE
  The use of nitrogen-containing bisphosphonates (n-bis) is associated with necrosis of the jaws, also known as bisphosphonate-related osteonecrosis of the jaws (BRONJ); however, the pathophysiology is unknown. Matrix metalloproteinase-9 (MMP-9) expression is essential for normal bone healing and is also required for angiogenesis. N-bis alters MMP-9 expression in vitro and in vivo; therefore, we hypothesized that n-bis alters MMP-9 expression during oral wound healing after tooth extraction.
  
  
  METHODS
  A total accumulated dose of 2.25 mg/kg (n = 20) of Zoledronic acid (ZA) Zometa or saline (control, n = 20) was administered to Sprague-Dawley male rats. Next, both groups had maxillary molar teeth extracted. Rats were sacrificed at postoperative day 1, 3, 7, or 21. Western blotting or multiplex ELISA was used to evaluate proteins of interest. Real-time polymerase chain reaction was used to assess the relative quantities of target gene mRNA. MMP-9 enzymatic activity was assessed by zymography.
  
  
  RESULTS
  The ZA group showed a statistically significant reduction in bone mineralization rate 21 days after tooth extraction compared with the control group (Student t test, P = .005). Moreover, ZA-treated animals showed a statistically significant increase in MMP-9-specific mRNA at postoperative days 3 (P = .003), 7 (P < .0001), and 21 (P < .0001) and protein on postoperative days 3 (P = .005) and 7 (P < .0001). MMP-9 enzymatic activity was also increased in ZA-treated rats compared with control animals (Student t test, P = .014). We also evaluated the extraction sockets for the presence of tissue inhibitor of MMP-1 (TIMP1), which is an inhibitor of MMP-9 enzymatic activity. TIMP1-specific mRNA and protein were not significantly altered by ZA treatment at the times tested (P > .05). Receptor of NF-κB ligand (RANKL) is known to regulate the expression of MMP-9; we therefore assessed the RANKL expression in our experimental oral wound-healing model. The ZA-treated animals had significantly increased RANKL mRNA at postoperative days 3 (P = .02) and 21 (P = .004), while the protein expression was significantly increased at postoperative days 1 (P < .0001), 7 (P = .02), and 21 (P = .03) compared with the control group.
  
  
  CONCLUSIONS
  ZA reduced bone mineralization within tooth extraction sockets, suggesting aberrant bone healing. ZA increases the amount and enzymatic activity of MMP-9, while apparently not altering the amount of TIMP1 within extraction sockets. RANKL is increased in ZA-treated rats, which suggests that increased MMP-9 expression is due, in part, to augmented RANKL expression.
authors.affiliation: Division of Oral and Maxillofacial Surgery, Department of Developmental and Surgical Science, University of Minnesota Dental School, Minneapolis, MN 55455, USA. basix001@umn.edu
authors.forename: David L
authors.initials: DL
authors.lastname: Basi
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Hughes
authors.forename: Vivek
authors.initials: V
authors.lastname: Thumbigere-Math
authors.forename: Ma'ann
authors.initials: M
authors.lastname: Sabino
authors.forename: Ami
authors.initials: A
authors.lastname: Mariash
authors.forename: Scott A
authors.initials: SA
authors.lastname: Lunos
authors.forename: Eric
authors.initials: E
authors.lastname: Jensen
authors.forename: Rajaram
authors.initials: R
authors.lastname: Gopalakrishnan
mesh_terms: D000539:Alveolar Process / Q000187:drug effects / Q000201:enzymology*; D000818:Animals; D015153:Blotting, Western; D050071:Bone Density Conservation Agents / Q000627:therapeutic use*; D002113:Calcification, Physiologic / Q000187:drug effects; D004164:Diphosphonates / Q000627:therapeutic use*; D005456:Fluorescent Dyes; D007093:Imidazoles / Q000627:therapeutic use*; D015850:Interleukin-6 / Q000032:analysis; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000032:analysis / Q000187:drug effects; D020780:Matrix Metalloproteinase 9 / Q000032:analysis / Q000187:drug effects*; D008437:Maxilla / Q000601:surgery; D008963:Molar / Q000601:surgery; D053245:RANK Ligand / Q000032:analysis / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D013997:Time Factors; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000032:analysis / Q000187:drug effects; D014081:Tooth Extraction; D020390:Tooth Socket / Q000187:drug effects / Q000201:enzymology*; D014945:Wound Healing / Q000502:physiology; D000077211:Zoledronic Acid
chemical_list: D050071:Bone Density Conservation Agents; D004164:Diphosphonates; D005456:Fluorescent Dyes; D007093:Imidazoles; D015850:Interleukin-6; D053245:RANK Ligand; D019715:Tissue Inhibitor of Metalloproteinase-1; D000077211:Zoledronic Acid; D020778:Matrix Metalloproteinase 2; C522363:Mmp2 protein, rat; D020780:Matrix Metalloproteinase 9