BMP6-Engineered MSCs Induce Vertebral Bone Repair in a Pig Model: A Pilot Study

Abstract

Osteoporotic patients, incapacitated due to vertebral compression fractures (VCF), suffer grave financial and clinical burden. Current clinical treatments focus on symptoms' management but do not combat the issue at the source. In this pilot study, allogeneic, porcine mesenchymal stem cells, overexpressing the BMP6 gene (MSC-BMP6), were suspended in fibrin gel and implanted into a vertebral defect to investigate their effect on bone regeneration in a clinically relevant, large animal pig model. To check the effect of the BMP6-modified cells on bone regeneration, a fibrin gel only construct was used for comparison. Bone healing was evaluated in vivo at 6 and 12 weeks and ex vivo at 6 months. In vivo CT showed bone regeneration within 6 weeks of implantation in the MSC-BMP6 group while only minor bone formation was seen in the defect site of the control group. After 6 months, ex vivo analysis demonstrated enhanced bone regeneration in the BMP6-MSC group, as compared to control. This preclinical study presents an innovative, potentially minimally invasive, technique that can be used to induce bone regeneration using allogeneic gene modified MSCs and therefore revolutionize current treatment of challenging conditions, such as osteoporosis-related VCFs.

Figure 1

Vertebral bone defect: surgical procedure. Cylindrical bone defects were generated in the lumbar vertebrae of minipigs and treated with either BMP6-MSCs or fibrin gel only. A surgical drill was used to perform the defect in the lumbar vertebral body (a). A 4 mm in diameter, 15-mm deep, cylindrical defect was generated and prepared for construct implantation (b). BMP6-MSCs suspended in fibrin gel or fibrin gel only (control) constructs were implanted in the defect site (c). White arrows point to the location of the defect.

Figure 2

pBM-MSCs nucleofected with rhBMP-6 secrete BMP6 in vitro. Porcine BM-MSCs were nucleofected with cDNA3-pCMV-rhBMP-6 and cultured in vitro. The media were changed after 24 hours and 48 hours after nucleofection the secretion of the BMP6 protein was evaluated using quantitative protein immunoassay (ELISA). The amount of BMP6 secreted was normalized to cell number and compared to the cells that were nucleofected with GFP; bars indicate SE, n = 3.

Figure 3

BMP6-MSCs induce vertebral bone repair. Bone regeneration in lumbar vertebral defects was monitored using clinical CT imaging on 6 ((a) and (e)) and 12 weeks after surgery ((b) and (f)). Animals were sacrificed on week 24 (i.e., 6 months) after surgery and excised vertebrae were subjected to μCT imaging. Bone formation was quantified based on μCT scans (analyzed region is highlighted in red, (c) and (g)). Marked differences in bone regeneration can be seen in defects treated with BMP6-MSCs ((g) and (h)) versus fibrin gel only ((c) and (d)).

Figure 4

Quantification of bone regeneration in lumbar vertebral defects. Bone volume (BV) and connectivity density (Conn-Dense) parameters were higher in lumbar defects treated with BMP6-MSCs compared to defects treated with fibrin gel only. Statistical significance difference was not reached, probably due to the small sample size (bars indicate SE, n = 3).

Figure 5

Histological sections of vertebral bone defects. Treated vertebrae were harvested and processed for histology. Sections were stained with H&E and Masson's Trichrome and imaged using a light microscope; representative images showing advanced defect closure in the BMP6-MSC group versus the control (fibrin gel only) group.