Projection Stereolithographic Fabrication of BMP-2 Gene-activated Matrix for Bone Tissue Engineering

Hang Lin,Thomas P Lozito,Nicholas Oyster,Bing Wang,Robert B Kang,Ying Tang,Rocky S Tuan,Madalyn R Fritch

doi:10.1038/s41598-017-11051-0

Abstract

Currently, sustained in vivo delivery of active bone morphogenetic protein-2 (BMP-2) protein to responsive target cells, such as bone marrow-derived mesenchymal stem cells (BMSCs), remains challenging. Ex vivo gene transfer method, while efficient, requires additional operation for cell culture and therefore, is not compatible with point-of-care treatment. In this study, two lentiviral gene constructs – (1) Lv-BMP/GFP, containing human BMP-2 and green fluorescent protein (GFP) gene (BMP group); or (2) Lv-GFP, containing GFP gene (GFP group) – were incorporated with human BMSCs into a solution of photocrosslinkable gelatin, which was then subjected to visible light-based projection stereolithographic printing to form a scaffold with desired architectures. Upon in vitro culture, compared to the GFP group, cells from BMP group showed >1,000-fold higher BMP-2 release, and the majority of them stained intensely for alkaline phosphatase activity. Real-time RT-PCR also showed dramatically increased expression of osteogenesis marker genes only in the BMP group. 3.5 months post-implantation into SCID mice, the micro-computed tomography imaging showed detectable mineralized areas only in the BMP group, which was restricted within the scaffolds. Alizarin red staining and immunohistochemistry of GFP and osteocalcin further indicated that the grafted hBMSCs, not host cells, contributed primarily to the newly formed bone.

Highlights

Tissue engineering, an emerging biomedical technology that aims to produce replacement tissues in vitro, offers exciting promise to overcome the obstacles of using native tissues
We hypothesized that the simultaneous packaging of regenerative bone morphogenetic protein-2 (BMP-2) gene and human bone marrow MSCs (hBMSCs) would result in localized gene transduction in situ, BMP-2 production, robust osteogenesis, and efficient bone regeneration
Using a conventional ex vivo transduction procedure on 2D cultures of hBMSCs, green fluorescence was observed as early as 48 hours in either Lv-green fluorescent protein (GFP) or Lv-BMP/GFP transduced cells

Summary

Introduction

An emerging biomedical technology that aims to produce replacement tissues in vitro, offers exciting promise to overcome the obstacles of using native tissues. Without a second intervention, up to 12 mg of BMP-2 is needed to heal tibial fractures[6], which has resulted in unpredictable, severe side effects in some cases, including high rates of nerve root irritation and ectopic bone formation[8] Such potential risks necessitate the controlled release of BMP-2 or other delivery strategy, such as direct gene transfer of BMP-2 gene. In 1996, Fang et al.[10] reported the use of collagen sponge impregnated with plasmid DNA carrying BMP-4 gene for the repair of rat bone nonunion, and showed that some of repair cells were transduced by BMP-4 gene, inducing enhanced bone growth. We hypothesized that the simultaneous packaging of regenerative BMP-2 gene and hBMSCs would result in localized gene transduction in situ, BMP-2 production, robust osteogenesis, and efficient bone regeneration

Methods

Results

Discussion

Conclusion