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Decellularization and Delipidation Protocols of Bovine Bone and Pericardium for Bone Grafting and Guided Bone Regeneration Procedures.

Gardin C, Ricci S, Ferroni L, Guazzo R, Sbricoli L, De Benedictis G, Finotti L, Isola M, Bressan E, Zavan B - PLoS ONE (2015)

Bottom Line: This protocol is more effective in removal of cellular materials, and shows superior biocompatibility compared to other three methods tested in this study.In the second part of this study, two methods of bovine pericardium decellularization are compared.The osmotic shock-based protocol gives better results in terms of removal of cell components, biocompatibility, maintenance of native ECM structure, and host tissue reaction, in respect to the freeze/thaw method.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, University of Padova, Padova, Italy.

ABSTRACT
The combination of bone grafting materials with guided bone regeneration (GBR) membranes seems to provide promising results to restore bone defects in dental clinical practice. In the first part of this work, a novel protocol for decellularization and delipidation of bovine bone, based on multiple steps of thermal shock, washes with detergent and dehydration with alcohol, is described. This protocol is more effective in removal of cellular materials, and shows superior biocompatibility compared to other three methods tested in this study. Furthermore, histological and morphological analyses confirm the maintenance of an intact bone extracellular matrix (ECM). In vitro and in vivo experiments evidence osteoinductive and osteoconductive properties of the produced scaffold, respectively. In the second part of this study, two methods of bovine pericardium decellularization are compared. The osmotic shock-based protocol gives better results in terms of removal of cell components, biocompatibility, maintenance of native ECM structure, and host tissue reaction, in respect to the freeze/thaw method. Overall, the results of this study demonstrate the characterization of a novel protocol for the decellularization of bovine bone to be used as bone graft, and the acquisition of a method to produce a pericardium membrane suitable for GBR applications.

No MeSH data available.


Related in: MedlinePlus

Lipids quantification in decellularized bovine bone.Quantification analysis of residual lipid content in bone samples DBS1, DBS2, DBS3, and DBS4 compared to NBS, estimated with Oil red O quantification. Values are expressed as mean ± standard deviation (n = 3 per group). Statistically significant differences are indicated as *P<0.05 and compared with NBS.
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pone.0132344.g003: Lipids quantification in decellularized bovine bone.Quantification analysis of residual lipid content in bone samples DBS1, DBS2, DBS3, and DBS4 compared to NBS, estimated with Oil red O quantification. Values are expressed as mean ± standard deviation (n = 3 per group). Statistically significant differences are indicated as *P<0.05 and compared with NBS.

Mentions: Oil red O staining was then used to evaluate the residual lipid content in bone samples. As an azo dye, Oil Red O can combine with triacylglycerol to give out jacinth lipid droplets, which can be spectrophotometrically quantified [34]. As evident from Fig 3, protocols B#1 and B#3 produced a significant (P<0.05) removal of lipids from bone granules. On the contrary, bovine bone samples processed with protocols B#2 and B#4 did not achieve an appropriate lipid elimination. It is reasonable to think that treatment with ethanol positively contributed to removal of lipids from bone. This result should be taken strongly into account, since delipidation is an important procedure for the preparation of bone grafts because residual lipids negatively affect the osseointegration [11].


Decellularization and Delipidation Protocols of Bovine Bone and Pericardium for Bone Grafting and Guided Bone Regeneration Procedures.

Gardin C, Ricci S, Ferroni L, Guazzo R, Sbricoli L, De Benedictis G, Finotti L, Isola M, Bressan E, Zavan B - PLoS ONE (2015)

Lipids quantification in decellularized bovine bone.Quantification analysis of residual lipid content in bone samples DBS1, DBS2, DBS3, and DBS4 compared to NBS, estimated with Oil red O quantification. Values are expressed as mean ± standard deviation (n = 3 per group). Statistically significant differences are indicated as *P<0.05 and compared with NBS.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4507977&req=5

pone.0132344.g003: Lipids quantification in decellularized bovine bone.Quantification analysis of residual lipid content in bone samples DBS1, DBS2, DBS3, and DBS4 compared to NBS, estimated with Oil red O quantification. Values are expressed as mean ± standard deviation (n = 3 per group). Statistically significant differences are indicated as *P<0.05 and compared with NBS.
Mentions: Oil red O staining was then used to evaluate the residual lipid content in bone samples. As an azo dye, Oil Red O can combine with triacylglycerol to give out jacinth lipid droplets, which can be spectrophotometrically quantified [34]. As evident from Fig 3, protocols B#1 and B#3 produced a significant (P<0.05) removal of lipids from bone granules. On the contrary, bovine bone samples processed with protocols B#2 and B#4 did not achieve an appropriate lipid elimination. It is reasonable to think that treatment with ethanol positively contributed to removal of lipids from bone. This result should be taken strongly into account, since delipidation is an important procedure for the preparation of bone grafts because residual lipids negatively affect the osseointegration [11].

Bottom Line: This protocol is more effective in removal of cellular materials, and shows superior biocompatibility compared to other three methods tested in this study.In the second part of this study, two methods of bovine pericardium decellularization are compared.The osmotic shock-based protocol gives better results in terms of removal of cell components, biocompatibility, maintenance of native ECM structure, and host tissue reaction, in respect to the freeze/thaw method.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, University of Padova, Padova, Italy.

ABSTRACT
The combination of bone grafting materials with guided bone regeneration (GBR) membranes seems to provide promising results to restore bone defects in dental clinical practice. In the first part of this work, a novel protocol for decellularization and delipidation of bovine bone, based on multiple steps of thermal shock, washes with detergent and dehydration with alcohol, is described. This protocol is more effective in removal of cellular materials, and shows superior biocompatibility compared to other three methods tested in this study. Furthermore, histological and morphological analyses confirm the maintenance of an intact bone extracellular matrix (ECM). In vitro and in vivo experiments evidence osteoinductive and osteoconductive properties of the produced scaffold, respectively. In the second part of this study, two methods of bovine pericardium decellularization are compared. The osmotic shock-based protocol gives better results in terms of removal of cell components, biocompatibility, maintenance of native ECM structure, and host tissue reaction, in respect to the freeze/thaw method. Overall, the results of this study demonstrate the characterization of a novel protocol for the decellularization of bovine bone to be used as bone graft, and the acquisition of a method to produce a pericardium membrane suitable for GBR applications.

No MeSH data available.


Related in: MedlinePlus