Efficient in vivo bone formation by BMP-2 engineered human mesenchymal stem cells encapsulated in a projection stereolithographically fabricated hydrogel scaffold

Abstract

Background: Stem cell-based bone tissue engineering shows promise for bone repair but faces some challenges, such as insufficient osteogenesis and limited architecture flexibility of the cell-delivery scaffold.

Methods: In this study, we first used lentiviral constructs to transduce ex vivo human bone marrow-derived stem cells with human bone morphogenetic protein-2 (BMP-2) gene (BMP-hBMSCs). We then introduced these cells into a hydrogel scaffold using an advanced visible light-based projection stereolithography (VL-PSL) technology, which is compatible with concomitant cell encapsulation and amenable to computer-aided architectural design, to fabricate scaffolds fitting local physical and structural variations in different bones and defects.

Results: The results showed that the BMP-hBMSCs encapsulated within the scaffolds had high viability with sustained BMP-2 gene expression and differentiated toward an osteogenic lineage without the supplement of additional BMP-2 protein. In vivo bone formation efficacy was further assessed using an intramuscular implantation model in severe combined immunodeficiency (SCID) mice. Microcomputed tomography (micro-CT) imaging indicated rapid bone formation by the BMP-hBMSC-laden constructs as early as 14 days post-implantation. Histological examination revealed a mature trabecular bone structure with considerable vascularization. Through tracking of the implanted cells, we also found that BMP-hBMSC were directly involved in the new bone formation.

Conclusions: The robust, self-driven osteogenic capability and computer-designed architecture of the construct developed in this study should have potential applications for customized clinical repair of large bone defects or non-unions.

Keywords: 3D bioprinting; Bone formation; Bone tissue engineering; Ex vivo gene transduction; Gene therapy; Osteogenesis.

Fig. 1

a Fabrication scheme of bone scaffold using VL-PLS, with simultaneous incorporation of lenti-BMP-2 vector-transduced hBMSCs. b Structure of the BMP-2 lentiviral construct used in this study. CMV pro., cytomegalovirus promoter; HIV, human immunodeficiency virus; LTR, long-terminal repeat. eGFP was used as the reporter

Fig. 2

Bioactive BMP-2 produced by encapsulated BMP-hBMSCs promoted hBMSC osteogenesis. a BMP-2 concentration in the medium from the culture with the constructs from the Protein and Gene groups. Day 8 and Day 56 were the medium collection times. (n = 3). b Relative osteogenic gene expression in hBMSCs in the Protein and Gene groups. All data were normalized to the Protein group. (n = 3). c Compressive moduli of constructs from different groups at different time. (n = 4). **p < 0.05

Fig. 3

Scaffolds encapsulating BMP-hBMSCs showed much higher ALP activity and OCN deposition in the matrix. a ALP staining of the constructs in the Protein and Gene groups after 14 days of culture. The purple staining indicated the presence of ALP. b OCN deposition was detected by IHC in both groups after 14 days of culture. The brown staining, indicated by arrows, indicates OCN-positive deposition. Bar = 100 μm

Fig. 4

The micro-CT images and the bone volume and density of constructs at days 14, 28 and 56 days after implantation. a Representative reconstituted 3D micro-CT imaging from two groups at different time points. b Direct bone volume and mean bone density of new bone tissues in the Gene group at days 14, 28, and 56. (n = 4)

Fig. 5

Constructs laden with lentiviral BMP-2-transduced hBMSCs were stiffer and had more calcium deposition after 56 days of implantation in vivo. a Macroscopic images showed new tissue formation in the Gene group construct. Scale = 1 mm, whereas those from the Protein Group remained morphologically unchanged. b Stiffness test. Maximum peak force was considerably higher in the Gene group constructs. (n = 3). c Total calcium content in the constructs. Again, the Gene group showed considerably higher calcium accumulation. (n = 3)

Fig. 6

BMP-2-transduced hBMSCs promote bone formation in vivo after 56 days of implantation in vivo. a H&E staining. No bone tissue was observed in the Protein group; in contrast, in the Gene group, bone tissues were observed at the margin and in the center area, suggesting uniform bone formation throughout the scaffolds. b OCN IHC. Bone formation in the Gene group was further confirmed by strong OCN deposition (brown), indicated by arrows. Bar = 200 μm. I, implanted scaffolds; M, muscle tissue

Fig. 7

BMP-2-transduced hBMSCs were directly involved in new bone formation. GFP immunostaining was used to track the BMP-hBMSCs. a Fourteen days after implantation, massive engraftment of GFP-positive cells was seen not only on the edge of the new bone but also at the center. b At day 28, more bone tissue and increased GFP-positive area were observed. GFP-negative bone tissues (indicated by an asterisk) were also noticed, suggesting their origin from host cells. Bar = 200 μm