title: Topical cutaneous CO2 application by means of a novel hydrogel accelerates fracture repair in rats.
abstract: BACKGROUND
  We previously demonstrated that topical cutaneous application of CO2, by means of a hydrogel in which the CO2 readily dissolves, increases blood flow and oxygen dissociation from hemoglobin in the soft tissues surrounding bone. In the present study, we utilized a rat fracture model to test the hypothesis that application of this treatment to fractured limbs would accelerate fracture repair.
  
  
  METHODS
  A closed femoral shaft fracture was created in each rat. Topical cutaneous application of CO2 by means of a hydrogel was performed five times a week for up to four weeks in the CO2/hydrogel group (n = 60). Sham treatments were performed in the control group (n = 60). Radiographic, histological, immunohistochemical, laser Doppler perfusion imaging, real-time polymerase chain reaction, and biomechanical assessments were performed.
  
  
  RESULTS
  Radiographic fracture union was evident at week 3 in twelve (86%) of fourteen animals in the CO2/hydrogel group compared with five (36%) of fourteen in the control group (p < 0.05; 95% CI [confidence interval] for the difference in union rate, 2.26% to 99.64%). Histological assessment revealed promotion of endochondral ossification in the CO2/hydrogel group. Immunohistochemical assessment at week 2 showed significantly greater capillary density in the CO2/hydrogel group (p < 0.05; 95% CI for the difference, 161 to 258 per mm(2)). Laser Doppler perfusion imaging demonstrated that the blood flow in the fractured limb was significantly greater at weeks 2 and 3 in the CO2/hydrogel group (p < 0.05; 95% CI for the difference, 8.4% to 22.4% and 6.7% to 19.0%, respectively). Gene expression of chondrogenic, osteogenic, and angiogenic markers was significantly greater in the CO2/hydrogel group at several time points. Ultimate stress, extrinsic stiffness, and failure energy (relative to the contralateral limb) were significantly greater in the CO2/hydrogel group at week 3 (p < 0.05; 95% CI for the difference, 24.8% to 67.5%, 4.0 % to 22.7%, and 9.6% to 58.8%, respectively). There were no significant differences between the groups with respect to any outcome measure at week 4.
  
  
  CONCLUSIONS
  Topical cutaneous application of CO2 by means of a hydrogel accelerated fracture repair in association with the promotion of angiogenesis, blood flow, and endochondral ossification. .
authors.affiliation: Department of Orthopaedic Surgery (T.K., T.N., S.Y.L., E.O., T.I., M.M., R.K., and M.K.) and Division of Rehabilitation Medicine (T.U. and Y.S.), Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail address for T. Niikura: tniikura@med.kobe-u.ac.jp.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Koga
authors.affiliation: Department of Orthopaedic Surgery (T.K., T.N., S.Y.L., E.O., T.I., M.M., R.K., and M.K.) and Division of Rehabilitation Medicine (T.U. and Y.S.), Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail address for T. Niikura: tniikura@med.kobe-u.ac.jp.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Niikura
authors.affiliation: Department of Orthopaedic Surgery (T.K., T.N., S.Y.L., E.O., T.I., M.M., R.K., and M.K.) and Division of Rehabilitation Medicine (T.U. and Y.S.), Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail address for T. Niikura: tniikura@med.kobe-u.ac.jp.
authors.forename: Sang Yang
authors.initials: SY
authors.lastname: Lee
authors.affiliation: Department of Orthopaedic Surgery (T.K., T.N., S.Y.L., E.O., T.I., M.M., R.K., and M.K.) and Division of Rehabilitation Medicine (T.U. and Y.S.), Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail address for T. Niikura: tniikura@med.kobe-u.ac.jp.
authors.forename: Etsuko
authors.initials: E
authors.lastname: Okumachi
authors.affiliation: Department of Orthopaedic Surgery (T.K., T.N., S.Y.L., E.O., T.I., M.M., R.K., and M.K.) and Division of Rehabilitation Medicine (T.U. and Y.S.), Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail address for T. Niikura: tniikura@med.kobe-u.ac.jp.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Ueha
authors.affiliation: Department of Orthopaedic Surgery (T.K., T.N., S.Y.L., E.O., T.I., M.M., R.K., and M.K.) and Division of Rehabilitation Medicine (T.U. and Y.S.), Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail address for T. Niikura: tniikura@med.kobe-u.ac.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Iwakura
authors.affiliation: Department of Orthopaedic Surgery (T.K., T.N., S.Y.L., E.O., T.I., M.M., R.K., and M.K.) and Division of Rehabilitation Medicine (T.U. and Y.S.), Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail address for T. Niikura: tniikura@med.kobe-u.ac.jp.
authors.forename: Yoshitada
authors.initials: Y
authors.lastname: Sakai
authors.affiliation: Department of Orthopaedic Surgery (T.K., T.N., S.Y.L., E.O., T.I., M.M., R.K., and M.K.) and Division of Rehabilitation Medicine (T.U. and Y.S.), Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail address for T. Niikura: tniikura@med.kobe-u.ac.jp.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Miwa
authors.affiliation: Department of Orthopaedic Surgery (T.K., T.N., S.Y.L., E.O., T.I., M.M., R.K., and M.K.) and Division of Rehabilitation Medicine (T.U. and Y.S.), Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail address for T. Niikura: tniikura@med.kobe-u.ac.jp.
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Kuroda
authors.affiliation: Department of Orthopaedic Surgery (T.K., T.N., S.Y.L., E.O., T.I., M.M., R.K., and M.K.) and Division of Rehabilitation Medicine (T.U. and Y.S.), Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail address for T. Niikura: tniikura@med.kobe-u.ac.jp.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kurosaka
mesh_terms: D000279:Administration, Cutaneous; D000818:Animals; D002245:Carbon Dioxide / Q000008:administration & dosage / Q000627:therapeutic use*; D005264:Femoral Fractures / Q000188:drug therapy*; D017102:Fracture Healing / Q000187:drug effects*; D020100:Hydrogels / Q000008:administration & dosage / Q000627:therapeutic use*; D051381:Rats; D016896:Treatment Outcome
chemical_list: D020100:Hydrogels; D002245:Carbon Dioxide