Guenther Schuldt Filho

Conditioned Medium of Demineralized Freeze-Dried Bone Activates Gene Expression in Periodontal Fibroblasts In Vitro

BACKGROUND Demineralized bone matrix (DBM) is used for the treatment of osseous defects. Conditioned medium from native bone chips can activate transforming growth factor (TGF)-β signaling in mesenchymal cells. The aim of this study is to determine whether processing of native bone into DBM affects the activity of the conditioned medium. METHODS Porcine cortical bone blocks were subjected to defatting, different concentrations of hydrochloric acid, and various temperatures. DBM was lyophilized, ground, and placed into culture medium. Human gingiva and periodontal fibroblasts were exposed to the respective conditioned medium obtained from DBM (DBCM). Changes in the expression of TGF-β target genes were determined. RESULTS DBCM altered the expression of TGF-β target genes (e.g., adrenomedullin, pentraxin 3, KN motif and ankyrin repeat domains 4, interleukin 11, NADPH oxidase 4, and BTB [POZ] domain containing 11) by at least five-fold. The response was observed in fibroblasts from both sources. Defatting lowered the activity of DBCM. The TGF-β receptor type I kinase inhibitor SB431542 [4-(4-(benzo[d][1,3]dioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl)benzamide] but not the inhibitor of bone morphogenetic protein receptor dorsomorphin, blocked the effects of DBCM on gene expression. Moreover, conditioned medium obtained from commercial human DBM modulated the expression of TGF-β target genes. CONCLUSION The findings suggest that the DBCM can activate TGF-β signaling in oral fibroblasts.

Bone Conditioned Medium: Preparation and Bioassay.

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing bone, they also support the complex process of bone regeneration. This favorable behavior of autografts is attributed to the three characteristics: osteoconductivity, osteogenicity, and osteoinductivity. However, there is another aspect: Bone grafts release a myriad of molecules, including growth factors, which can target mesenchymal cells involved in bone regeneration. The paracrine properties of bone grafts can be studied in vitro by the use of bone-conditioned medium (BCM). Here we present a protocol on how to prepare bone-conditioned medium from native pig cortical bone, and bone that underwent thermal processing or demineralization. Cells can be directly exposed to BCM or seeded onto biomaterials, such as collagen membranes, previously soaked with BCM. We give examples for in vitro bioassays with mesenchymal cells on the expression of TGF-β regulated genes. The presented protocols should encourage to further reveal the paracrine effects of bone grafts during bone regeneration and open a path for translational research in the broad field of reconstructive surgery.

Biofilm Analysis of Retrieved Dental Implants after Different Peri-Implantitis Treatments

The aim of the current study was to analyse the planktonic growth of Streptococcus mutans on the surfaces of three implants retrieved after three different peri-implantitis treatments. Three implants from a male patient with high levels of bone loss were treated by mechanical debridement, chemical decontamination, and implantoplasty. After 4 months of follow-up, the implants were removed. The growth and biofilm formation were measured by spectrophotometry (OD630 nm) and scanning electron microscopy (SEM), after 48 hours of incubation. Results showed an average of Streptococcus mutans planktonic growth over the implants of 0.21 nm (mechanical debridement), 0.16 nm (chemical decontamination), and 0.15 nm (implantoplasty). Data were analysed by ANOVA and Tukeys test (p < 0.05 for chemical decontamination and implantoplasty). Implantoplasty and chemical decontamination showed the lowest levels of planktonic growth, indicating a possible influence of the modification procedures on the titanium surface on the initial biofilm attachment.