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

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.

Additive Manufacturing for Guided Bone Regeneration: A Perspective for Alveolar Ridge Augmentation

“…SLA is a great option for highly detailed scaffolds, requiring tight tolerances and smooth surfaces, such as molds, patterns, and functional parts, as it can achieve a spatial resolution of approximately 50 μm [ 25 ]. Various techniques that fall under SLA, include: two-photon polymerization (2PP) [ 25 ], holography [ 27 ], and visible light-based [ 28 ] techniques. The various SLA techniques offer different possibilities, such as improved resolutions of up to 200 nm with 2PP or the use of visible light to avoid the negative impact of UV light on cells.…”

Enhancement of osteogenesis of rabbit bone marrow derived mesenchymal stem cells by transfection of human BMP‑2 and EGFP recombinant adenovirus via Wnt signaling pathway

“…In recent years, the researches of stem cells have provided new ideas for bone tissue engineering ( 27 , 28 ). It has been confirmed that BMSCs has significant differentiation ability of osteoblasts in vitro in the treatment of specific chemical substances including β-phosphoglycerol, dexamethasone and vitamin C; cytokines and mechanical mechanics stimulation ( 28 , 29 ). Many factors including cytokines can induce BMSCs to differentiate into osteoblasts.…”

“…Many factors including cytokines can induce BMSCs to differentiate into osteoblasts. The most important one of the cytokines are BMPs ( 28 , 30 ). BMP is a major regulatory factor for the differentiation of BMSCs into osteoblasts among a number of factors that activate or inhibit the osteoblastic signaling pathways.…”

Natural Polymeric Scaffolds in Bone Regeneration

“…In fact, the cationic nature of CS elicits an efficient complexation with DNA molecules making it an ideal candidate for gene delivery (Di Martino et al, 2005). Successful CS-pDNA complexation and sustained transfection of MSCs makes CS a promising vector also for gene activated matrices (GAMs), which are scaffolds engineered to provide a direct and sustained delivery of nucleic acids ensuring efficient and durable cell transfection in situ (Raisin et al, 2016;D'Mello et al, 2017;Lin et al, 2017). In particular, the encapsulation of plasmids into nano or micro-particulate CS-systems to be loaded within scaffolds could offer significant spatiotemporal control on the activity of the encoded biofactors (Peng et al, 2009).…”