Gene-Activated Matrix with Self-Assembly Anionic Nano-Device Containing Plasmid DNAs for Rat Cranial Bone Augmentation.

Masahito Hara,Takunori Ogaeri,Yukinobu Kodama,Izumi Asahina,Rena Shido,Yoshinori Sumita,Mayumi Iwatake,Hitoshi Sasaki,Shun Narahara,Hideyuki Yamamoto

doi:10.3390/ma14227097

Masahito Hara, Takunori Ogaeri + Show 8 more

Open Access

https://doi.org/10.3390/ma14227097

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Abstract

We have developed nanoballs, a biocompatible self-assembly nano-vector based on electrostatic interactions that arrange anionic macromolecules to polymeric nanomaterials to create nucleic acid carriers. Nanoballs exhibit low cytotoxicity and high transfection efficiently in vivo. This study investigated whether a gene-activated matrix (GAM) composed of nanoballs containing plasmid (p) DNAs encoding bone morphogenetic protein 4 (pBMP4) could promote bone augmentation with a small amount of DNA compared to that composed of naked pDNAs. We prepared nanoballs (BMP4-nanoballs) constructed with pBMP4 and dendrigraft poly-L-lysine (DGL, a cationic polymer) coated by γ-polyglutamic acid (γ-PGA; an anionic polymer), and determined their biological functions in vitro and in vivo. Next, GAMs were manufactured by mixing nanoballs with 2% atelocollagen and β-tricalcium phosphate (β-TCP) granules and lyophilizing them for bone augmentation. The GAMs were then transplanted to rat cranial bone surfaces under the periosteum. From the initial stage, infiltrated macrophages and mesenchymal progenitor cells took up the nanoballs, and their anti-inflammatory and osteoblastic differentiations were promoted over time. Subsequently, bone augmentation was clearly recognized for up to 8 weeks in transplanted GAMs containing BMP4-nanoballs. Notably, only 1 μg of BMP4-nanoballs induced a sufficient volume of new bone, while 1000 μg of naked pDNAs were required to induce the same level of bone augmentation. These data suggest that applying this anionic vector to the appropriate matrices can facilitate GAM-based bone engineering.

Highlights

We investigated whether atelocollagen-based gene-activated matrix (GAM) containing nanoball vectors encoding pBMP4 (BMP4-nanoballs) could facilitate rat cranial bone augmentation, which is a definitive model of regenerative therapy for jawbone atrophy
MRNA expression was significantly upregulated in macrophages, MSCs, and fibroblasts via transfection with BMP4-nanoballs compared with that in cells transfected with GFPnanoballs (Figure 2C)
The positive outcomes of this study were as follows: (1) after in vitro transfection, nanoball vectors containing pBMP4 reliably induced BMP4 protein production in cells that would migrate to the bone formation site; (2) this vector was gradually taken up by cells that infiltrated into the transplanted GAMs, leading to the promotion of osteogenic gene expression over time; and (3) the resultant bone augmentation was eventually promoted when 1–10 μg of BMP4-nanoballs was incorporated into atelocollagenbased GAMs

Summary

Introduction

Autogenous bone grafts, known as autografts, are still widely considered to be the “gold standard” augmentation technique for facial and orbital reconstruction, especially for the purpose of regenerating bony defects in the craniofacial region [1,2]. There are some difficulties in controlling their bioactivities at the transplanted site due to several disadvantages, such as early inactivation with short half-lives and potential side effects [7,8]. Among such morphogens and growth factors, bone morphogenetic proteins (BMPs), such as recombinant (r) BMP2 or rBMP4, have been reported to induce bone formation in various settings [4,9,10]. To advance bone engineering, the development of a more efficient and controlled delivery system is needed [6]

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