Bone microbial decontamination agents in osseous grafting: an in vitro study with fresh human explants.
BACKGROUND
Establishing a safe prophylactic antimicrobial protocol in bone grafting may enhance osseous volume outcomes. The purpose of this in vitro study is to assess human osteoblast response and safety after explant antimicrobial exposure.


METHODS
Fresh human bone explants were exposed to three antimicrobials: povidone-iodine (PovI; 0.05%, 1%, and 5%), chlorhexidine (CHX; 0.2% and 1%), and sodium hypochlorite (NaOCl; 2.5%, 4.5%, and 5.25%) at different times (15, 30, 45, and 60 seconds) and concentrations to assess cellular toxicity. Explants were washed three times with saline after exposure. Controls, explants cultured in the absence of antimicrobials, were performed for all experimental situations tested. Trials were conducted in triplicate. Particle size influence on osteoblast growth was determined between bone fragments with a diameter <2 and ≥2 to 5 mm. Test and control groups were monitored by light microscopy to evaluate cellular growth. Osteoblast differentiation and morphology was assessed by alkaline phosphatase activity and scanning electron microscopy (SEM).


RESULTS
Osteoblast growth was similar for particles <2 and ≥2 to 5 mm. Alkaline phosphatase control reference values were not significantly different from test groups (0.35 mU/mL ± 0.004 versus 0.34 mU/mL ± 0.009; P >0.05). Light microscopy showed on average 97% osteoblastic growth for bone particles exposed to PovI 5% and CHX 0.2% for all times and CHX 1% up to 30 seconds. The odds ratio of positive osteoblastic growth after a 30-second 2.5% NaOCl exposure was 2.4 times higher than after 5.25%. On average, one of two replicas yielded positive growth with 2.5% NaOCl and one of three with 5.25%. After 60-second explant exposure, positive osteoblastic growth was 7.7 times more likely to occur with 5% PovI or 0.2% CHX than with 5.25% NaOCl (P <0.05). SEM analysis confirmed light microscopy similar cellular adhesion and osteoblast phenotypic features between test and control groups.


CONCLUSIONS
Best osteoblastic growth occurred after bone PovI exposure and CHX 0.2%. Cellular toxicity seems to be influenced by the type of antimicrobial, concentration, and exposure time. SEM analysis confirmed absence of osteoblast phenotypic alterations after exposure. Decontamination agents can safely be used in bone transplantation using up to 5% PovI and 0.2% CHX for 1 minute and CHX 1% for 30 seconds.
VA Hospital, Greater Los Angeles, Healthcare System, Los Angeles, CA, USA. fverdugo@perio.org
Fernando
F
Verdugo
Aranzazu
A
Sáez-Rosón
Agurne
A
Uribarri
Rafael
R
Martínez-Conde
Jonathan
J
Cabezas-Olcoz
María D
MD
Moragues
José
J
Pontón
D000328:Adult; D000368:Aged; D000891:Anti-Infective Agents, Local / Q000008:administration & dosage / Q000633:toxicity*; D001419:Bacteria / Q000187:drug effects; D016025:Bone Transplantation; D002448:Cell Adhesion / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D016009:Chi-Square Distribution; D002710:Chlorhexidine / Q000008:administration & dosage / Q000633:toxicity; D003666:Decontamination / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D005260:Female; D006801:Humans; D008297:Male; D008855:Microscopy, Electron, Scanning; D008875:Middle Aged; D016017:Odds Ratio; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism; D010316:Particle Size; D011206:Povidone-Iodine / Q000008:administration & dosage / Q000633:toxicity; D012973:Sodium Hypochlorite / Q000008:administration & dosage / Q000633:toxicity; D018709:Statistics, Nonparametric
D000891:Anti-Infective Agents, Local; D011206:Povidone-Iodine; D012973:Sodium Hypochlorite; D002710:Chlorhexidine