In Vivo Evaluation of Permeable and Impermeable Membranes for Guided Bone Regeneration

Abstract

Background: The degree of biodegradation and the inflammatory response of membranes employed for guided bone regeneration directly impact the outcome of this technique. This study aimed to evaluate four different experimental versions of Poly (L-lactate-co-Trimethylene Carbonate) (PTMC) + Poly (L-lactate-co-glycolate) (PLGA) membranes, implanted in mouse subcutaneous tissue, compared to a commercially available membrane and a Sham group.

Methods: Sixty Balb-C mice were randomly divided into six experimental groups and subdivided into 1, 3, 6 and 12 weeks (n = 5 groups/period). The membranes (1 cm²) were implanted in the subcutaneous back tissue of the animals. The samples were obtained for descriptive and semiquantitative histological evaluation (ISO 10993-6).

Results: G1 and G4 allowed tissue adhesion and the permeation of inflammatory cells over time and showed greater phagocytic activity and permeability. G2 and G3 detached from the tissue in one and three weeks; however, in the more extended periods, they presented a rectilinear and homogeneous aspect and were not absorbed. G2 had a major inflammatory reaction. G5 was almost completely absorbed after 12 weeks.

Conclusions: The membranes are considered biocompatible. G5 showed a higher degree of biosorption, followed by G1 and G4. G2 and G3 are considered non-absorbable in the studied periods.

Keywords: PLGA; PTMC; biocompatibility; membranes; mice; subcutaneous.

Figure 1

Graphical abstract of the experimental design. This figure was created with Biorender.com.

Figure 2

Macroscopic images of the membranes used in the study according to the experimental groups. (A): Group 1; (B): Group 2; (C): Group 3; (D): Group 4; (E): Group 5.

Figure 3

Scanning electron microscopy (SEM) micrographs according to the experimental groups. (A,B): Group 1; (C,D): Group 2; (E,F): Group 3; (G,H): Group 4; (I,J): Group 5. (A,C,E,G,I): 150× magnification; (B,D,F,H,J): 1000× magnification.

Figure 4

Fourier transform infrared (FTIR) spectrums. The FTIR spectrum showed vibrational modes typical of PTMC + PLGA (Groups 1–4) and PLG + PTMC (Group 5).

Figure 5

Macroscopic aspect of the tissue response to different membranes after 12 weeks of implantation. (A): Group 1; (B): Group 2; (C): Group 3; (D): Group 4; (E): Group 5; (F): Sham group. The dotted area corresponds to the membrane region. The black arrow indicates the granulation reaction present in Group 5, where the membrane was not visualized.

Figure 6

(A,B) Group 1; (C,D) Group 2; (E,F) Group 3; (G,H) Group 4; (I,J) Group 5 (K,L). Group sham. Membrane (M); connective tissue (CT); multinucleated giant cells (black arrows); adipose tissue (AT); empty space (*); muscle tissue (TM); hair follicles (HF); mesenchymal cells (green arrows); mesenchymal cells surrounding the membrane spheres (red arrows); hyperplastic stratified squamous epithelium (SSE). Scale bar: (A,C,E,G,I,K): 100 µm and (B,D,F,H,J,L): 400 µm. Staining: Hematoxylin and Eosin.

Figure 7

(A,B) Group 1; (C,D) Group 2; (E,F) Group 3; (G,H) Group 4; (I,J) Group 5; (K,L) Sham group. Membrane (M); loose connective tissue (LCT); multinucleated giant cells (black arrows); adipose tissue (AT); hair follicles (HF); muscle tissue (TM); mesenchymal cells (green arrows); connective tissue (CT); mononuclear cells inside the membrane (blue arrows); mesenchymal cells surrounding the membrane spheres (red arrows); orthokeratinized stratified squamous epithelium (SSE); area of incision (Star). Scale bar: (A,C,E,G,I,K): 100 µm and (B,D,F,H,J,L): 400 µm. Staining: Hematoxylin and Eosin.

Figure 8

(A,B) Group 1; (C,D) Group 2; (E,F) Group 3; (G,H) Group 4; (I,J) Group 5; (K,L) Sham group. Membrane (M); loose connective tissue (LCT); multinucleated giant cells (black arrows); adipose tissue (AT); hair follicles (HF); muscle tissue (TM); granulation reaction (GR); mononuclear cells inside the membrane (blue arrows); mesenchymal cells (green arrows); connective tissue (CT); Glandular tissue (orange arrows). Scale bar: (A,C,E,G,I,K): 100 µm and (B,D,F,H,J,L): 400 µm. Staining: Hematoxylin and Eosin.

Figure 9

(A,B) Group 1; (C,D) Group 2; (E,F) Group 3; (G,H) Group 4; (I,J) Group 5; (K,L) Sham group. Membrane (M); loose connective tissue (LCT); multinucleated giant cells (black arrows); connective tissue (CT); adipose tissue (AT); hair follicles (HF); muscle tissue (TM); mesenchymal cells (green arrows); granulation reaction (GR); mononuclear cells inside the membrane (blue arrows). Scale bar: (A,C,E,G,I,K): 100 µm and (B,D,F,H,J,L): 400 µm. Staining: Hematoxylin and Eosin.

Figure 10

Inflammatory cell response and overall tissue reaction (A–F) after experimental periods of 1, 3, 6, and 12 weeks. The values are presented as median ± confidence interval. The horizontal bars represent significant differences between different groups at the same experimental time point (Kruskal–Wallis and Dunn’s post hoc tests; p < 0.05). The letters represent significant differences between time points with the same treatment. (a) The significant difference compared to 1 week; (b) significant difference compared to 3 weeks (c) significant difference compared to 6 weeks (Kruskal–Wallis and Dunn’s post hoc tests; p < 0.05). (* p = 0.01–0.04; ** p = 0.001–0.009; *** p = 0.0001–0.0006; **** p < 0.0001).