PDGF-metronidazole-encapsulated nanofibrous functional layers on collagen membrane promote alveolar ridge regeneration

Abstract

This study aimed to develop a functionally graded membrane (FGM) to prevent infection and promote tissue regeneration. Poly(l-lactide-co-d,l-lactide) encapsulating platelet-derived growth factor (PDLLA-PDGF) or metronidazole (PDLLA-MTZ) was electrospun to form a nanofibrous layer on the inner or outer surface of a clinically available collagen membrane, respectively. The membrane was characterized for the morphology, molecule release profile, in vitro and in vivo biocompatibility, and preclinical efficiency for alveolar ridge regeneration. The PDLLA-MTZ and PDLLA-PDGF nanofibers were 800-900 nm in diameter, and the thicknesses of the functional layers were 20-30 μm, with sustained molecule release over 28 days. All of the membranes tested were compatible with cell survival in vitro and showed good tissue integration with minimal fibrous capsule formation or inflammation. Cell proliferation was especially prominent on the PDLLA-PDGF layer in vivo. On the alveolar ridge, all FGMs reduced wound dehiscence compared with the control collagen membrane, and the FGM with PDLLA-PDGF promoted osteogenesis significantly. In conclusion, the FGMs with PDLLA-PDGF and PDLLA-MTZ showed high biocompatibility and facilitated wound healing compared with conventional membrane, and the FGM with PDLLA-PDGF enhanced alveolar ridge regeneration in vivo. The design represents a beneficial modification, which may be easily adapted for future clinical use.

Keywords: alveolar process; animal models; metronidazole; platelet-derived growth factor; tissue engineering.

Figure 1

The design and characterization of the FGM. Notes: (A) The schematic diagram of the FGM. (B) The SEM image of the PDLLA-BSA functional layer, surface scan. Magnification: 1,000×. (C) The SEM image of the PDLLA-MTZ functional layer, surface scan. Magnification: 1,000×. (D) The SEM image of the membrane with PDLLA-BSA (on the left surface) and PDLLA-MTZ (on the right surface) functional layers, cross-sectional scan. Magnification: 150×. (E) The in vitro release profile of PDGF and MTZ from the respective nanofibrous layer. Abbreviations: BSA, bovine serum albumin; FGM, functionally graded membrane; MTZ, metronidazole; PDGF, platelet-derived growth factor; PDLLA, poly(l-lactide-co-d,l-lactide); SEM, scanning electron microscopy.

Figure 2

The in vitro assessments of FGM biocompatibility. Notes: (A) Cell metabolic activity measured by the Alamar Blue assay. (B) Vital cell density determined by a DAPI assay. (C) Immunofluorescence images of MSCs seeded on the control and PDLLA nanofiber-coated cell culture dishes after 1 day. F-actin was labeled by rhodamine-conjugated phalloidin (red), and nuclei was labeled by DAPI (blue). Magnification: 400×. Scale bar: 50 μm (*P<0.05, **P<0.01). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; FGM, functionally graded membrane; MSCs, mesenchymal stem cells; MTZ, metronidazole; PDGF, platelet-derived growth factor; PDLLA, poly(l-lactide-co-d,l-lactide).

Figure 3

The preclinical assessments of FGM biocompatibility in vivo. Notes: (A) Histology from the interface of the membrane and the surrounding subcutaneous tissue after 2 weeks of implantation. Asterisks indicate the matrix of the membrane, and red arrows indicate the fibrous encapsulation on the surface of the membrane. Magnification: 100×. Scale bar: 20 μm. (B) Quantification of the thickness of fibrous encapsulation. (C) Immunohistochemistry showing PCNA-positive cells (cells with brown nucleus staining) on the membrane surfaces. Asterisks indicate the matrix of the collagen core of the membrane. Magnification: 100×. Scale bar: 20 μm. Abbreviations: FGM, functionally graded membrane; MTZ, metronidazole; PCNA, proliferating cell nuclear antigen; PDGF, platelet-derived growth factor; PDLLA, poly(l-lactide-co-d,l-lactide).

Figure 4

The extent of wound dehiscence 1 week after FGM application on the alveolar ridge. Note: The results are presented as percentages of the mesiodistal length of the opened wound, with 0% indicating complete wound closure and 100% indicating completely open wound. Abbreviations: FGM, functionally graded membrane; CT, no membrane control; CL, membrane with the core layer only; MT, membrane with PDLLA-MTZ layer; PD, membrane with the PDLLA-PDGF layer; MP, membrane with both PDLLA-MTZ and PDLLA-PDGF layers.

Figure 5

The micro-CT assessments of the FGM on the alveolar ridge on days 14 and 28. Notes: (A) The representative sagittal sections from each group on days 14 and 28. Dash boxes indicate the osseous defects. (B) The bone volume fraction, defined as the ratio of bone volume to the entire volume of defect (BV/TV). (C) The mean trabecular thickness (Tb.Th). (D) The mean trabecular number (Tb.N) per square millimeter. *P<0.05. Abbreviations: BV/TV, bone volume fraction; FGM, functionally graded membrane; Tb.N, trabecular number; Tb.Th, trabecular thickness; CT, no membrane control; CL, membrane with the core layer only; MT, membrane with PDLLA-MTZ layer; PD, membrane with the PDLLA-PDGF layer; MP, membrane with both PDLLA-MTZ and PDLLA-PDGF layers.

Figure 6

The histologic assessments of the FGM on the alveolar ridge on day 28. Notes: (A) The representative sagittal section of each group. Dash boxes indicate the osseous defects. Magnification: 40×. Scale bar: 200 μm. (B) Tissue surrounding the residual membrane in Groups CL (upper panel) and PD (lower panel). Arrowheads indicate foreign-body giant cells. Magnification: 400×. Scale bar: 100 μm. (C) Histomorphometric assessment for the defect fill, which was defined as the percentage of defect area occupied with mineralized tissue (*P<0.05, **P<0.01). Abbreviations: FGM, functionally graded membrane; CT, no membrane control; CL, membrane with the core layer only; MT, membrane with PDLLA-MTZ layer; PD, membrane with the PDLLA-PDGF layer; MP, membrane with both PDLLA-MTZ and PDLLA-PDGF layers.