A Rationale for the Use of Clotted Vertebral Bone Marrow to Aid Tissue Regeneration Following Spinal Surgery

Abstract

Vertebral body bone marrow aspirate (V-BMA), easily accessible simultaneously with the preparation of the site for pedicle screw insertion during spinal procedures, is becoming an increasingly used cell therapy approach in spinal surgery. However, the main drawbacks for V-BMA use are the lack of a standardized procedure and of a structural texture with the possibility of diffusion away from the implant site. The aim of this study was to evaluate, characterize and compare the biological characteristics of MSCs from clotted V-BMA and MSCs from whole and concentrate V-BMAs. MSCs from clotted V-BMA showed the highest cell viability and growth factors expression (TGF-β, VEGF-A, FGF2), the greatest colony forming unit (CFU) potency, cellular homogeneity, ability to differentiate towards the osteogenic (COL1AI, TNFRSF11B, BGLAP) and chondrogenic phenotype (SOX9) and the lowest ability to differentiate toward the adipogenic lineage (ADIPOQ) in comparison to all the other culture conditions. Additionally, results revealed that MSCs, differently isolated, expressed different level of HOX and TALE signatures and that PBX1 and MEIS3 were down-regulated in MSCs from clotted V-BMA in comparison to concentrated one. The study demonstrated for the first time that the cellular source inside the clotted V-BMA showed the best biological properties, representing an alternative and advanced cell therapy approach for patients undergoing spinal surgery.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Cells morphology after 10 days of culture of MSCs from (a) whole BMA, (b) concentrated BMA and (c) clotted BMA. Magnification 4×.

Figure 2

Cell viability by Alamar blue dye performed on MSCs derived from whole BMA (dark bar), concentrated BMA (grey bar) and clotted BMA (light bar) after 3, 7 and 14 days of culture (***p vs. concentrated BMA; ***clotted BMA vs. whole BMA.

Figure 3

Colony forming units (CFUs) count of MSCs derived from whole, concentrated and clotted BMAs after 10 days of culture in basal medium. The graphs indicate the number of positive colonies/well by toluidine blue staining. *Clotted BMA vs. whole BMA; ***Clotted BMA vs. concentrated BMA. *p<0.05; ***p < 0.0005. Representative images of CFUs observed by toluidine blue staining after10 days of culture, magnification 4×.

Figure 4

Representative images of in vitro osteogenic (Left: Alizarin Red S staining, magnification 4×; Right: LIVE/DEAD fluorescent staining, magnification 10×), adipogenic (Left: Oil Red O, magnification 40×; Right: LIVE/DEAD fluorescent staining, magnification 10×) and chondrogenic (Left: Alcian Blue/Nuclear Fast Red staining, magnification 80×; Right: Collagen Type II magnification 80×) differentiation of MSCs from (a) whole, (b) concentrated and (c) clotted BMAs. Black arrows: chodrocytes; Red arrows: extracellular matrix.

Figure 5

Gene expression measured by quantitative reverse transcriptase polymerase chain reaction of TGF-β, VEGF and EGF in MSCs from whole, concentrated and clotted BMAs. TGF-β, VEGF-A: *MSCs from clotted V-BMA vs. MSCs from concentrated V-BMA and MSCs from whole V-BMA; EGF2: *MSCs from clotted V-BMA vs. MSCs from whole V-BMA; VEGF-A, FGF2: *MSCs from concentrated V-BMA vs. MSCs from whole V-BMA. *p < 0.05.

Figure 6

Gene expression measured by quantitative reverse transcriptase polymerase chain reaction of osteogenic markers comparing the differentiation potential of MSCs from whole BMA, concentrated BMA, clotted BMA after 21 days of culture. (a) COL1A1: ***clotted BMA vs. concentrated BMA; **clotted BMA vs. whole BMA; *whole BMA vs. concentrated BMA. TNFRSF11B: ***clotted BMA vs. concentrated BMA; **clotted BMA vs. whole BMA. BGLAP: *clotted BMA vs. concentrated BMA; *clotted BMA vs. whole BMA. RUNX2: ***clotted BMA vs. concentrated BMA. ALP: ***clotted BMA vs. concentrated BMA. *p < 0.05; **p < 0.005; ***p < 0.0005.

Figure 7

Gene expression measured by quantitative reverse transcriptase polymerase chain reaction of adipogenic markers comparing the differentiation potential of MSCs from whole BMA, concentrated BMA, clotted BMA after 21 days of culture. ADIPOQ: *concentrated BMA vs. whole BMA and clotted BMA; *whole BMA vs. clotted BMA.*p < 0.05.

Figure 8

Gene expression measured by quantitative reverse transcriptase polymerase chain reaction of chondrogenic markers comparing the differentiation potential of MSCs from whole BMA, concentrated BMA, clotted BMA after 30 days of culture. SOX9: *clotted BMA vs. whole and concentrated BMA; *whole BMA vs. concentrated BMA. *p < 0.05.

Figure 9

Gene expression measured by quantitative reverse transcriptase polymerase chain reaction of HOX and TALE signatures for MSCs from whole BMA, concentrated BMA, clotted BMA after 21 days of osteogenic induction. HOXB8: *whole BMA vs. concentrated BMA and clotted BMA; PBX1 and MEIS3: ***concentrated BMA vs. clotted BMA. *p < 0.05; ***p < 0.0005.

Figure 10

Surgical feasibility of V-BMA clot procedure in the clinical theatre.

Figure 11

Study experimental set-up. Human BMA harvest from vertebrae divided in 3 equal parts: two parts placed into test tubes containing heparin as anticoagulant for MSCs isolation from whole and concentrated BMAs, one part placed in test tube without any anticoagulant for MSCs isolation from clotted BMA.