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In vivo and in vitro evaluation of an Acetobacter xylinum synthesized microbial cellulose membrane intended for guided tissue repair.

Mendes PN, Rahal SC, Pereira-Junior OC, Fabris VE, Lenharo SL, de Lima-Neto JF, da Cruz Landim-Alvarenga F - Acta Vet. Scand. (2009)

Bottom Line: Scanning electron microscopy of the cellulose membrane surface showed absence of pores.The in vitro evaluation of the interaction between cells and biomaterial was performed through viability staining analysis of the cells over the biomaterial, which showed that 95% of the mesenchymal stem cells aggregating to the cellulose membrane were alive and that 5% were necrotic.Scanning electron microscopy showed mesenchymal stem cells with normal morphology and attached to the cellulose membrane surface.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Surgery and Anesthesiology, São Paulo State University, Botucatu, SP, Brazil. periclesmendes@hotmail.com

ABSTRACT

Background: Barrier materials as cellulose membranes are used for guided tissue repair. However, it is essential that the surrounding tissues accept the device. The present study histologically evaluated tissue reaction to a microbial cellulose membrane after subcutaneous implantation in mice. Furthermore, the interaction between mesenchymal stem cells and the biomaterial was studied in vitro to evaluate its ability to act as cellular scaffold for tissue engineering.

Methods: Twenty-five Swiss Albino mice were used. A 10 x 10 mm cellulose membrane obtained through biosynthesis using Acetobacter xylinum bacteria was implanted into the lumbar subcutaneous tissue of each mouse. The mice were euthanatized at seven, 15, 30, 60, and 90 days, and the membrane and surrounding tissues were collected and examined by histology.

Results: A mild inflammatory response without foreign body reaction was observed until 30 days post-surgery around the implanted membrane. Polarized microscopy revealed that the membrane remained intact at all evaluation points. Scanning electron microscopy of the cellulose membrane surface showed absence of pores. The in vitro evaluation of the interaction between cells and biomaterial was performed through viability staining analysis of the cells over the biomaterial, which showed that 95% of the mesenchymal stem cells aggregating to the cellulose membrane were alive and that 5% were necrotic. Scanning electron microscopy showed mesenchymal stem cells with normal morphology and attached to the cellulose membrane surface.

Conclusion: The microbial cellulose membrane evaluated was found to be nonresorbable, induced a mild inflammatory response and may prove useful as a scaffold for mesenchymal stem cells.

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Histomorphology of a microbial cellulose membrane implanted subcutaneously in mice and surrounding tissue reaction 7(a), 15(b), 30(c), 60(d) and 90(e) days postoperatively. Observe the presence of the intact membrane (*) surrounded by immature granulation tissue and newly formed vessels and capillaries (a). At 15 days post-surgery, a reduction in inflammatory infiltrate, especially of lymphocytes, is observed (b). At 30 days postoperatively observe the collagen fibers commencing orientation parallel to the implant's surface (c). No inflammatory infiltrate is observed and the connective tissue surrounding the membrane is mature at 60 (d) and 90 (e) days post-surgery. (HE, Obj. ×10).
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Figure 3: Histomorphology of a microbial cellulose membrane implanted subcutaneously in mice and surrounding tissue reaction 7(a), 15(b), 30(c), 60(d) and 90(e) days postoperatively. Observe the presence of the intact membrane (*) surrounded by immature granulation tissue and newly formed vessels and capillaries (a). At 15 days post-surgery, a reduction in inflammatory infiltrate, especially of lymphocytes, is observed (b). At 30 days postoperatively observe the collagen fibers commencing orientation parallel to the implant's surface (c). No inflammatory infiltrate is observed and the connective tissue surrounding the membrane is mature at 60 (d) and 90 (e) days post-surgery. (HE, Obj. ×10).

Mentions: Descriptive analysis of the histological sections by light microscopy on day seven post surgery demonstrated an intact membrane surrounded by a mild inflammatory infiltrate of mainly polymorphonuclear cells and lymphocytes (Fig. 3a). Immature granulation tissue was evinced by intense presence of newly formed vessels and capillaries close to the membrane and mononuclear cells. Examination of mice 15 days post-surgery showed a reduced inflammatory infiltrate, especially due to lower numbers of lymphocytes (Fig. 3b). Granulation tissue appeared similar to that observed at day 7 post surgery. The membrane showed no signs of resorption. No polymorphonuclear cells and only a few lymphocytes were observed at 30 days post-surgery. There was a reduced number of newly formed vessels and collagen fibers began to be oriented parallel to the implant's surface that was apparently intact (Fig. 3c). At 60 and 90 days post-surgery, no inflammatory infiltrate was observed. Angiogenesis was markedly reduced and the connective tissue surrounding the membrane was mature. The membrane was still present with no signs of resorption (Figs 3d and 3e). Foreign body reaction or connective tissue cells penetrating the membrane were not observed at any time point throughout the study period. Polarized microscopy revealed that the membrane remained intact at all evaluation points (Fig. 4). Table 1 summarizes the mean score values (semi-quantitative analysis) regarding the intensity of inflammatory infiltrate, polymorphonuclear cells, multinucleated giant cells, lymphocytes, angiogenesis, and fibrosis. No significant differences were observed among the time points regarding presence of polymorphonuclear cells, multinucleated giant cells, and lymphocytes (P > 0.05). Angiogenesis and fibrosis decreased throughout the evaluation periods (P < 0.05).


In vivo and in vitro evaluation of an Acetobacter xylinum synthesized microbial cellulose membrane intended for guided tissue repair.

Mendes PN, Rahal SC, Pereira-Junior OC, Fabris VE, Lenharo SL, de Lima-Neto JF, da Cruz Landim-Alvarenga F - Acta Vet. Scand. (2009)

Histomorphology of a microbial cellulose membrane implanted subcutaneously in mice and surrounding tissue reaction 7(a), 15(b), 30(c), 60(d) and 90(e) days postoperatively. Observe the presence of the intact membrane (*) surrounded by immature granulation tissue and newly formed vessels and capillaries (a). At 15 days post-surgery, a reduction in inflammatory infiltrate, especially of lymphocytes, is observed (b). At 30 days postoperatively observe the collagen fibers commencing orientation parallel to the implant's surface (c). No inflammatory infiltrate is observed and the connective tissue surrounding the membrane is mature at 60 (d) and 90 (e) days post-surgery. (HE, Obj. ×10).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667521&req=5

Figure 3: Histomorphology of a microbial cellulose membrane implanted subcutaneously in mice and surrounding tissue reaction 7(a), 15(b), 30(c), 60(d) and 90(e) days postoperatively. Observe the presence of the intact membrane (*) surrounded by immature granulation tissue and newly formed vessels and capillaries (a). At 15 days post-surgery, a reduction in inflammatory infiltrate, especially of lymphocytes, is observed (b). At 30 days postoperatively observe the collagen fibers commencing orientation parallel to the implant's surface (c). No inflammatory infiltrate is observed and the connective tissue surrounding the membrane is mature at 60 (d) and 90 (e) days post-surgery. (HE, Obj. ×10).
Mentions: Descriptive analysis of the histological sections by light microscopy on day seven post surgery demonstrated an intact membrane surrounded by a mild inflammatory infiltrate of mainly polymorphonuclear cells and lymphocytes (Fig. 3a). Immature granulation tissue was evinced by intense presence of newly formed vessels and capillaries close to the membrane and mononuclear cells. Examination of mice 15 days post-surgery showed a reduced inflammatory infiltrate, especially due to lower numbers of lymphocytes (Fig. 3b). Granulation tissue appeared similar to that observed at day 7 post surgery. The membrane showed no signs of resorption. No polymorphonuclear cells and only a few lymphocytes were observed at 30 days post-surgery. There was a reduced number of newly formed vessels and collagen fibers began to be oriented parallel to the implant's surface that was apparently intact (Fig. 3c). At 60 and 90 days post-surgery, no inflammatory infiltrate was observed. Angiogenesis was markedly reduced and the connective tissue surrounding the membrane was mature. The membrane was still present with no signs of resorption (Figs 3d and 3e). Foreign body reaction or connective tissue cells penetrating the membrane were not observed at any time point throughout the study period. Polarized microscopy revealed that the membrane remained intact at all evaluation points (Fig. 4). Table 1 summarizes the mean score values (semi-quantitative analysis) regarding the intensity of inflammatory infiltrate, polymorphonuclear cells, multinucleated giant cells, lymphocytes, angiogenesis, and fibrosis. No significant differences were observed among the time points regarding presence of polymorphonuclear cells, multinucleated giant cells, and lymphocytes (P > 0.05). Angiogenesis and fibrosis decreased throughout the evaluation periods (P < 0.05).

Bottom Line: Scanning electron microscopy of the cellulose membrane surface showed absence of pores.The in vitro evaluation of the interaction between cells and biomaterial was performed through viability staining analysis of the cells over the biomaterial, which showed that 95% of the mesenchymal stem cells aggregating to the cellulose membrane were alive and that 5% were necrotic.Scanning electron microscopy showed mesenchymal stem cells with normal morphology and attached to the cellulose membrane surface.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Surgery and Anesthesiology, São Paulo State University, Botucatu, SP, Brazil. periclesmendes@hotmail.com

ABSTRACT

Background: Barrier materials as cellulose membranes are used for guided tissue repair. However, it is essential that the surrounding tissues accept the device. The present study histologically evaluated tissue reaction to a microbial cellulose membrane after subcutaneous implantation in mice. Furthermore, the interaction between mesenchymal stem cells and the biomaterial was studied in vitro to evaluate its ability to act as cellular scaffold for tissue engineering.

Methods: Twenty-five Swiss Albino mice were used. A 10 x 10 mm cellulose membrane obtained through biosynthesis using Acetobacter xylinum bacteria was implanted into the lumbar subcutaneous tissue of each mouse. The mice were euthanatized at seven, 15, 30, 60, and 90 days, and the membrane and surrounding tissues were collected and examined by histology.

Results: A mild inflammatory response without foreign body reaction was observed until 30 days post-surgery around the implanted membrane. Polarized microscopy revealed that the membrane remained intact at all evaluation points. Scanning electron microscopy of the cellulose membrane surface showed absence of pores. The in vitro evaluation of the interaction between cells and biomaterial was performed through viability staining analysis of the cells over the biomaterial, which showed that 95% of the mesenchymal stem cells aggregating to the cellulose membrane were alive and that 5% were necrotic. Scanning electron microscopy showed mesenchymal stem cells with normal morphology and attached to the cellulose membrane surface.

Conclusion: The microbial cellulose membrane evaluated was found to be nonresorbable, induced a mild inflammatory response and may prove useful as a scaffold for mesenchymal stem cells.

Show MeSH
Related in: MedlinePlus