3.1. Structure and Characteristics of the PLGA Membrane
shows the synthetic membrane of PLGA. The membranes exhibited a micro-fibrous appearance that indicates guided bone regeneration, as demonstrated in Sanaei-Rad et al.’s study [16
] in which this fibrous architecture mimics the extra-cellular matrix and was shown to be suitable for cell attachment, proliferation and differentiation. The micro-fibrous surface exhibited a highly porous structure with interlaced non-woven fibers. The diameters of the fibers ranged from 0.2 to 2 μm. The micro-fibrous appearance can be seen in the different thicknesses of membranes in Figure 2
The XRD evaluation showed two crystalline mineral phases present in the membranes (HA and β-TCP), as well as the proportions of HA (54.2%) and β-TCP phases (45.8%) (Figure 3
). Table 1
shows the mean values for Carbon (39.4%), Hydrogen (4.5%) and Nitrogen (0.1%). The carbon, hydrogen and nitrogen contents of the membrane with 500 µm were quantified in duplicate using the organic elemental analyzer.
When analyzing the FTIR spectra of the membrane, the PLGA polymer and the calcium phosphates used for its preparation, we observed no peaks above 3500 cm−1
on the membrane, which could indicate degradation of the polymer by hydrolysis, according to Tan et al. [17
]. The membrane spectrum corresponds to a junction of the characteristic peaks of BTCP and HAP (peaks identical in FTIR) with the characteristic peaks of PLGA (Figure 4
3.2. In Vivo Implantation
Overall, the animals tolerated well the anesthesia, pre-surgery and post-surgery, without complications or setbacks. Biological effects after the implantation of the different experimental membranes were evaluated according to the criteria established by ISO 10993-6: 2016/Part 6/Annex E and the descriptive analysis of the tissue response to the membranes was evaluated as a function of tissue disposition in the different membranes: bioabsorption, the presence of inflammatory cells, vascular neoformation and the presence of fibrosis. The degree of inflammation was evaluated and quantified manually according to the number and distribution of inflammatory cells present at the membrane-tissue interface: that is, polymorphonuclear cells, lymphocytes, plasma cells, macrophages and giant cells. In addition, the degree of degeneration (debris) was determined by morphological alterations due to necrosis extension.
For the 7-day experimental period, small vascular neoformation with minimal capillary proliferation (presence of 1 to 3 bulbs per examined field) was observed in the Sham Group. Also found in the Sham Group were a cicatricial process with an absence of multinucleated giant cells and macrophages and an abundance of lymphocytes and plasmacytes, in addition to the moderate presence of polymorphonuclear cells. In Group 2 (200 μm), a moderate infiltration of lymphocytes, macrophages and multinucleated giant cells was observed, along with a slight presence of polymorphonuclear cells. Moderate vascular neoformation with capillary proliferation was also present (presence of a wide range of capillaries per examined field).
In Group 3 (500 μm), a moderate infiltration of lymphocytes and macrophages and a discrete infiltration of plasmacytes and multinucleated giant cells were observed, alongside mild vascular neoformation with a proliferation of capillaries. Group 4 (700 μm) presented integral membranes with a moderate presence of lymphocytes, plasm cells and macrophages and a discrete polymorphonuclear infiltration. Local neovascularization with small capillary proliferation was also observed in this group. The control Group (Pratix ®
) did not show the membrane in any cuts performed; therefore, the cellular response tissue of the periphery membrane was evaluated. There was a moderate presence of lymphocytes, macrophages and polymorphonuclear infiltration. Also observed in this group was moderate local neovascularization with small capillary proliferation (Figure 5
In the 30-day experimental period, a discrete presence of lymphocytes and plasm cells and a moderate presence of macrophages were observed in the Sham Group. Regarding tissue response, a discrete presence of vascular neoformation with few vascular proliferations characterized this sample. In Group 2 (200 μm), membranes began to exhibit fragmentation with tissue invasion. There was a moderate presence of multinucleated giant cells and macrophages, a discrete presence of lymphocytes with an absence of polymorphonuclear cells and mild local neovascularization.
The membranes of the 500-μm and 700-μm Groups showed no changes in integrity after 30 days. Group 3 showed a discrete presence of lymphocytes, plasma cells, macrophages and a few multinucleated giant cells, with a moderate tissue response of local neovascularization. Group 4 had a discrete presence of lymphocytes and plasm cells, a moderate presence of multinucleated giant cells and few polymorphonuclear cells. Regarding tissue response, there was moderate vascular neoformation with proliferation of more than 7 shoots per field. The Pratix®
membranes used as a control group did not adhere to the subcutaneous tissue, so during removal of the samples they were detached from the tissue and the tissue adjacent to the implantation site of this membrane was observed, which revealed discrete lymphoplasmacytic infiltration and a moderate infiltration of macrophages (Figure 6
After 60 days, the discrete presence of lymphocytes, plasm cells and macrophages was observed in the Sham Group. There were no multinucleated giant cells or polymorphonuclear cells and there was a slight presence of vascular neoformation shoots. In Group 2, there was a moderate presence of lymphocytes, macrophages and multinucleated giant cells, a discrete presence of plasm cells and an absence of polymorphonuclear cells. Groups 3 and 4 presented a discrete/moderate presence of lymphocytes, macrophages, multinucleated giant cells and plasm cells. However, while Group 3 presented mild vascular neoformation, Groups 2, 4 and 5 showed moderate neovascularization. Group 5 was macroscopically detached from the tissue and unconnected to the subcutaneous tissue. The presence of multinucleated and polymorphonuclear giant cells was not observed and there was only a moderate presence of lymphocytes and macrophages (Figure 7
After the 90-day period, Group 1 showed discrete lymphocyte and macrophages infiltration and an absence of multinucleated giant cells and polymorphonuclear cells. The 200-μm group showed a moderate presence of macrophages, lymphocytes and multinucleated giant cells and a discrete presence of plasma cells. In the 500-μm and 700-μm groups, there was no presence of polymorphonuclear cells or plasmocytes and a moderate presence of lymphocytes, multinucleated giant cells and macrophages. The control group had a discrete presence of polymorphonuclear cells and a moderate presence of macrophages, multinucleated giant cells, lymphocytes and plasma cells. All presented moderate vascular neoformation, with the exception of Group 1, which had discrete neoformation (Figure 8
There was no presence of necrosis, fibrosis or fatty infiltration for all experimental periods.
According to ISO, at the end of the evaluation, test samples may present scores ranging from 0.0 to 2.9 (absence of tissue reaction), 3.0 to 8.9 (discrete tissue reaction), 9.0 to 15 (moderate tissue reaction) and above 15.1 (severe tissue reaction). Thus, after evaluation of each cell type and tissue within the conditions of this study, following ISO 10993-6/2016, it was observed that the 200-μm test sample showed a slight tissue reaction (4) compared to the control sample after 7 days of implantation, whereas the 500-μm (2.6) and 700-μm (0) test samples showed no tissue reactions after 7 days of deployment. After 30 days, the tested 700-μm membrane showed a slight tissue reaction (4.4) when compared to the control sample, whereas the 200-μm (1.2) and 500-μm (1.8) samples did not present tissue reactions. After 60 days, the 200-μm membrane presented a slight tissue reaction (7) when compared to the control group. There was an absence of tissue reactions for the 500-μm (2.6) and 700-μm (3.2) Membranes. After 90 days of implantation, the 700-μm membrane showed tissue reaction (3.8), while the 200-μm membrane obtained a score of 0.6 and the 500-μm membrane obtained a score of 0, characterizing an absence of tissue reaction.