Using poly(lactic-co-glycolic acid) microspheres to encapsulate plasmid of bone morphogenetic protein 2/polyethylenimine nanoparticles to promote bone formation in vitro and in vivo

Abstract

Repair of large bone defects is a major challenge, requiring sustained stimulation to continually promote bone formation locally. Bone morphogenetic protein 2 (BMP-2) plays an important role in bone development. In an attempt to overcome this difficulty of bone repair, we created a delivery system to slowly release human BMP-2 cDNA plasmid locally, efficiently transfecting local target cells and secreting functional human BMP-2 protein. For transfection, we used polyethylenimine (PEI) to create pBMP-2/PEI nanoparticles, and to ensure slow release we used poly(lactic-co-glycolic acid) (PLGA) to create microsphere encapsulated pBMP-2/PEI nanoparticles, PLGA@pBMP-2/PEI. We demonstrated that pBMP-2/PEI nanoparticles could slowly release from the PLGA@pBMP-2/PEI microspheres for a long period of time. The 3-15 μm diameter of the PLGA@pBMP-2/PEI further supported this slow release ability of the PLGA@pBMP-2/PEI. In vitro transfection assays demonstrated that pBMP-2/PEI released from PLGA@pBMP-2/PEI could efficiently transfect MC3T3-E1 cells, causing MC3T3-E1 cells to secrete human BMP-2 protein, increase calcium deposition and gene expressions of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), SP7 and I type collagen (COLL I), and finally induce MC3T3-E1 cell differentiation. Importantly, in vivo data from micro-computed tomography (micro-CT) and histological staining demonstrated that the human BMP-2 released from PLGA@pBMP-2/PEI had a long-term effect locally and efficiently promoted bone formation in the bone defect area compared to control animals. All our data suggest that our PLGA-nanoparticle delivery system efficiently and functionally delivers the human BMP-2 cDNA and has potential clinical application in the future after further modification.

Keywords: biodegradable polymer; bone regeneration; gene therapy; human BMP-2.

Figure 1

Diagram of preparation of gelatin sponge loaded with PLGA@pBMP-2/PEI. Abbreviations: pBMP2/PEI, plasmid of bone morphogenetic protein 2/polyethlenimine; PLGA, poly(lactic-co-glycolic acid); PVA, polyvinyl alcohol.

Figure 2

Physical characteristics of pBMP-2/PEI nanoparticles, morphology of PLGA@pBMP-2/PEI, and release assay of pBMP-2. (A) Size distribution of BMP-2/PEI nanoparticles; (B) Zeta potential of BMP-2/PEI nanoparticles; (C) Scanning electron microscope image of PLGA@pBMP-2/PEI; (D) Fluorescence image of PLGA@pBMP-2/PEI/FITC; (E) pBMP-2 release profile from PLGA@pBMP-2/PEI in PBS; (F) PCR assays to confirm pBMP-2 releasing into the PBS buffer. Lane 1: DNA marker. Lane 2: PCR amplicon of original pBMP-2 plasmid. Lane 3: PCR amplicon from day 7 PBS buffer Abbreviations: PLGA@pBMP-2/PEI/FITC, poly(lactic-co-glycolic acid)@plasmid of bone morphogenetic protein 2/polyethlenimine/fluorescein isothiocyanate; bp, base pair; PCR, polymerase chain reaction; PBS, phosphate buffered salin.

Figure 3

Evaluate properties of PLGA@pBMP-2/PEI in vitro. (A) Transfection ability of PLGA@pBMP-2/PEI/FITC in MC3T3-E1 cells at day 3; (B) Transfection ability of PLGA@ pBMP-2/PEI/FITC in MC3T3 cells at day 7; (C) PCR assays to confirm pBMP-2 in MC3T3 cells. Lane 1: DNA marker; Lane 2: PCR amplicon from PLGA@pBMP-2/PEI transfected MC3T3 cells; Lane 3: PCR from MC3T3 cells; (D) Human BMP-2 ELISA assays to confirm human BMP-2 secretion from transfected MC3T3 cells. (E) Calcium deposition analysis of PLGA@PEI group on day 14 using Alizarin red staining; (F) Calcium deposition analysis of PLGA@pBMP-2/PEI group on day 14 using Alizarin red staining. Note: *significant difference (p<0.01). Abbreviations: PLGA@PEI, poly(lactic-co-glycolic acid)@polyethlenimine; PLGA@pBMP-2/PEI, poly(lactic-co-glycolic acid)@plasmid of bone morphogenetic protein 2/polyethlenimine; PLGA@pBMP-2/PEI/FITC, poly(lactic-co-glycolic acid)@plasmid of bone morphogenetic protein 2/polyethlenimine/fluorescein isothiocyanate; bp, base pair; PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay.

Figure 4

Cell differentiation analysis of MC3T3-E1 cells after 1, 3, 7, and 14 days post-transfection using qPCR assays. (A) Alkaline phosphatase gene expression; (B) runt-related transcription factor 2 gene expression; (C) SP7 gene expression; (D) I type collagen gene expression. All assays were repeated three times. Data shown are mean values ± SD. Notes: *P < 0.05; **P < 0.01. Abbreviations: PLGA@PEI, poly(lactic-co-glycolic acid)@polyethlenimine; PLGA@pBMP-2/PEI, poly(lactic-co-glycolic acid)@plasmid of bone morphogenetic protein 2/polyethlenimine; qPCR, Real-Time quantitative polymerase chain reaction; SD, standard deviation.

Figure 5

SEM image of gelatin sponge with or without PLGA@pBMP-2/PEI and representative photos of bone defect with or without treatment. (A) SEM image of gelatin sponge without PLGA@pBMP-2/PEI; (B) SEM image of gelatin sponge with PLGA@pBMP-2/PEI; (C) Cranial bone defect with gelatin sponge loaded PLGA@pBMP-2/PEI treatment; (D) Cranial bone defect without treatment (5 mm diameter). Abbreviations: SEM, Scanning Electron Microscopy; PLGA@pBMP-2/PEI, poly(lactic-co-glycolic acid)@plasmid of bone morphogenetic protein 2/polyethlenimine.

Figure 6

Micro-CT images of rat cranial bone after 8 weeks post-implantation. (A) Negative control; (B) Gelatin sponge loaded with PLGA@PEI; (C) Gelatin sponge loaded with PLGA@pBMP-2/PEI. Abbreviation: PLGA@PEI, poly(lactic-co-glycolic acid)@polyethlenimine; PLGA@ pBMP-2/PEI, poly(lactic-co-glycolic acid)@plasmid of bone morphogenetic protein 2/polyethlenimine; Micro-CT, micro-computed tomography.

Figure 7

H&E staining and immunohistochemistry staining after 4 or 8 weeks postimplantation. (A) H&E staining of non-implant control after 4 weeks (×40); (B) H&E staining of non-implant control after 8 weeks (×40); (C) H&E staining of PLGA@ PEI group after 4 weeks (×40); (D) H&E staining of PLGA@PEI group after 8 weeks (×40); (E) H&E staining of PLGA@pBMP-2/PEI group after 4 weeks (×40), red arrow indicates new bone; (F) H&E staining of PLGA/pBMP-2/PEI group after 8 weeks (×40), red arrows indicate new bone; (G) Enlarged image of new bone area from Figure 6E. Red arrows indicate osteoblasts and yellow arrow indicates a macrophages phagocytized foreign materials; (H) Enlarged image of new bone area from Figure 6F. Red arrow indicates osteoblasts, and yellow arrow indicates a macrophages phagocytized foreign materials; (I) H&E staining of PLGA@pBMP-2/PEI group after 4 weeks (×200). OB: original bone (or lamellar bone); NB: new bone (or woven bone); Yellow arrow indicates the edge of the defect area; (J) H&E staining of PLGA@pBMP-2/PEI group after 8 weeks (×200). Yellow arrow indicates the edge of the defect area; (K) Human BMP-2 immunohistochemistry staining of PLGA@PEI group; (L) Human BMP-2 immunohistochemistry staining of PLGA/pBMP-2/PEI group. Abbreviations: H&E, hematoxylin and eosin stain; PLGA@PEI, poly(lactic-co-glycolic acid)@polyethlenimine; PLGA@pBMP-2/PEI, poly(lactic-co-glycolic acid)@plasmid of bone morphogenetic protein 2/polyethlenimine;