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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 vitro and in vivo experiments evidence osteoinductive and osteoconductive properties of the produced scaffold, respectively.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

Nucleic acids quantification in decellularized bovine bone.Quantification analysis of amount of residual (a) DNA and (b) RNA in decellularized bone samples DBS1, DBS2, DBS3, and DBS4 compared to that of NBS. Content of residual DNA and RNA was normalized by dry weight of each specimen. Values are expressed as mean ± standard deviation (n = 3 per group). Statistically significant differences are indicated as *P<0.05 and **P<0.01 and compared with NBS.
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pone.0132344.g002: Nucleic acids quantification in decellularized bovine bone.Quantification analysis of amount of residual (a) DNA and (b) RNA in decellularized bone samples DBS1, DBS2, DBS3, and DBS4 compared to that of NBS. Content of residual DNA and RNA was normalized by dry weight of each specimen. Values are expressed as mean ± standard deviation (n = 3 per group). Statistically significant differences are indicated as *P<0.05 and **P<0.01 and compared with NBS.

Mentions: The quantification of nucleic acids in all the four samples is shown in Fig 2. In detail, the average DNA content was: 0.037 ± 0.005 ng/mg for DBS1; 0.031 ± 0.006 ng/mg for DBS2; 0.011 ± 0.003 ng/mg for DBS3; 0.019 ± 0.002 ng/mg for DBS4. The average RNA content was: 0.098 ± 0.004 ng/mg for DBS1; 0.093 ± 0.007 ng/mg for DBS2; 0.029 ± 0.003 ng/mg for DBS3; 0.036 ± 0.004 ng/mg for DBS4. The reduction in both DNA and RNA content was significant (P<0.05 and P<0.01, respectively) and higher than 90% compared to that of native bone (0.639 ± 0.048 ng DNA/mg; 1.678 ± 0.058 ng RNA/mg), indicating that cells and their nuclear materials have been effectively removed. This result is particularly important because it would ensure that the bone tissue is essentially devoid of immunogenic active molecules [33].


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)

Nucleic acids quantification in decellularized bovine bone.Quantification analysis of amount of residual (a) DNA and (b) RNA in decellularized bone samples DBS1, DBS2, DBS3, and DBS4 compared to that of NBS. Content of residual DNA and RNA was normalized by dry weight of each specimen. Values are expressed as mean ± standard deviation (n = 3 per group). Statistically significant differences are indicated as *P<0.05 and **P<0.01 and compared with NBS.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132344.g002: Nucleic acids quantification in decellularized bovine bone.Quantification analysis of amount of residual (a) DNA and (b) RNA in decellularized bone samples DBS1, DBS2, DBS3, and DBS4 compared to that of NBS. Content of residual DNA and RNA was normalized by dry weight of each specimen. Values are expressed as mean ± standard deviation (n = 3 per group). Statistically significant differences are indicated as *P<0.05 and **P<0.01 and compared with NBS.
Mentions: The quantification of nucleic acids in all the four samples is shown in Fig 2. In detail, the average DNA content was: 0.037 ± 0.005 ng/mg for DBS1; 0.031 ± 0.006 ng/mg for DBS2; 0.011 ± 0.003 ng/mg for DBS3; 0.019 ± 0.002 ng/mg for DBS4. The average RNA content was: 0.098 ± 0.004 ng/mg for DBS1; 0.093 ± 0.007 ng/mg for DBS2; 0.029 ± 0.003 ng/mg for DBS3; 0.036 ± 0.004 ng/mg for DBS4. The reduction in both DNA and RNA content was significant (P<0.05 and P<0.01, respectively) and higher than 90% compared to that of native bone (0.639 ± 0.048 ng DNA/mg; 1.678 ± 0.058 ng RNA/mg), indicating that cells and their nuclear materials have been effectively removed. This result is particularly important because it would ensure that the bone tissue is essentially devoid of immunogenic active molecules [33].

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 vitro and in vivo experiments evidence osteoinductive and osteoconductive properties of the produced scaffold, respectively.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