In vitro evaluation of demineralized freeze-dried bone allograft in combination with enamel matrix derivative.
BACKGROUND
Preclinical and clinical studies suggest that a combination of enamel matrix derivative (EMD) with demineralized freeze-dried bone allograft (DFDBA) may improve periodontal wound healing and regeneration. To date, no single study has characterized the effects of this combination on in vitro cell behavior. The aim of this study is to test the ability of EMD to adsorb to the surface of DFDBA particles and determine the effect of EMD coating on downstream cellular pathways such as adhesion, proliferation, and differentiation of primary human osteoblasts and periodontal ligament (PDL) cells.


METHODS
DFDBA particles were precoated with EMD or human blood and analyzed for protein adsorption patterns via scanning electron microscopy. Cell attachment and proliferation were quantified using a commercial assay. Cell differentiation was analyzed using real-time polymerase chain reaction for genes encoding Runx2, alkaline phosphatase, osteocalcin, and collagen 1α1, and mineralization was assessed using alizarinred staining.


RESULTS
Analysis of cell attachment revealed no significant differences among control, blood-coated, and EMD-coated DFDBA particles. EMD significantly increased cell proliferation at 3 and 5 days after seeding for both osteoblasts and PDL cells compared to control and blood-coated samples. Moreover, there were significantly higher messenger ribonucleic acid levels of osteogenic differentiation markers, including collagen 1α1, alkaline phosphatase, and osteocalcin, in osteoblasts and PDL cells cultured on EMD-coated DFDBA particles at 3, 7, and 14 days.


CONCLUSIONS
The results suggest that the addition of EMD to DFDBA particles may influence periodontal regeneration by stimulating PDL cell and osteoblast proliferation and differentiation.
Department of Periodontology, School of Dental Medicine, University of Bern, Bern, Switzerland.
Richard J
RJ
Miron
Dieter D
DD
Bosshardt
Oliver
O
Laugisch
Michel
M
Dard
Anja C
AC
Gemperli
Daniel
D
Buser
Reinhard
R
Gruber
Anton
A
Sculean
D000293:Adolescent; D000327:Adsorption; D000328:Adult; D000469:Alkaline Phosphatase / Q000032:analysis; D064591:Allografts / Q000033:anatomy & histology / Q000737:chemistry*; D001769:Blood; D016025:Bone Transplantation; D001842:Bone and Bones / Q000033:anatomy & histology / Q000737:chemistry*; D002113:Calcification, Physiologic / Q000502:physiology; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D002452:Cell Count; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D020099:Coated Materials, Biocompatible / Q000737:chemistry* / Q000494:pharmacology; D024042:Collagen Type I / Q000032:analysis; D000090602:Collagen Type I, alpha 1 Chain; D050659:Core Binding Factor Alpha 1 Subunit / Q000032:analysis; D003746:Dental Enamel Proteins / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D008297:Male; D008855:Microscopy, Electron, Scanning; D010006:Osteoblasts / Q000187:drug effects / Q000502:physiology; D015675:Osteocalcin / Q000032:analysis; D010513:Periodontal Ligament / Q000166:cytology / Q000187:drug effects; D013997:Time Factors; D055815:Young Adult
D020099:Coated Materials, Biocompatible; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D050659:Core Binding Factor Alpha 1 Subunit; D003746:Dental Enamel Proteins; C493709:RUNX2 protein, human; C109069:enamel matrix proteins; D015675:Osteocalcin; D000469:Alkaline Phosphatase