MSCs are being extensively investigated for use in tissue regeneration. In the presence of a potent osteogenic inducer, such as BMP, MSCs can accelerate bone formation and lead to spine fusion. This can be achieved either by culturing MSCs in the presence of BMP and then transplanting them35
or by overexpressing the gene encoding for BMP-2 in the implanted cells. The latter approach has the advantage of a sustained secretion of BMP in vivo
, affecting both host and the implanted cells, in a synergistic autocrine/paracrine mechanism.36
We have previously shown that BMP gene-modified MSCs secrete physiological quantities (nanograms) of BMP28,34
over a period of few weeks compared to the mega doses used in rhBMP therapy (milligrams). Thus, the use of gene-modified MSCs might prevent recently described toxic side effects and inflammatory response induced by high doses of BMP11,12
. In addition, gene modification could be applied to freshly isolated, noncultured MSCs37
without the need for a cell culture phase. The use of noncultured MSCs could facilitate the translation of this therapeutic approach to the clinical arena. The present study marks the first time that the biomechanical properties of spinal fusion induced by injected genetically engineered MSCs have been evaluated. Before this study, we have only evaluated the morphometric and histological properties of the newly formed bone28,38
and the nanomechanical and nanostructural properties of the engineered tissue.26
The biomechanical tests that we performed represent a novel quantitative investigation of fusion integrity that enabled us to evaluate the quality of the spinal fusion and its clinical relevance.
Our findings demonstrate that the rigidity of spines fused using genetically engineered cells is similar to that of spines fused using bilateral stainless steel fixation across the vertebral processes; the MSC/+DOX and FG Only control groups exhibited significantly less rigidity, similar to those found in the Native Spine group. These findings show that the spinal fusion achieved using injections of genetically engineered MSCs can provide segmental stabilization of vertebrae without the need for an invasive surgical operation.
The structural properties of de novo
bone tissue formation induced by genetically engineered MSCs were quantified using μCT. 3D μCT reconstructions of the spines demonstrated a bridge spanning 3.1
0.6 vertebrae per mouse (n
9) and 2.4
0.4 vertebrae per injection (n
9), although the bone tissue formed along the paraspinal muscle. The localization of spinal fusion in desired segments may be easier in a large animal model, because the choice of injection site in such a model can be more accurately controlled. In addition, we envision that in a large animal, multiple injections of stem cells will induce incremental bone tissue formation, increasing control over the site of tissue formation and spinal fusion.
In this study we also evaluated the relative contribution of the fused spine segments to the rigidity of the entire spine. Our findings demonstrated that the largest contribution came from the fused L3–L5 segment, corresponding to the site of the MSC injection and correlating to stainless steel pin-based fusion. When we examined the structural properties of the new bone formation, which we quantified using μCT, we found that the newly formed bone had a lower BVD and BMD than the posterolateral compartment of intact vertebra. These findings were probably due to the relatively short period of tissue formation, which is insufficient to create mature bone that can hold up to the same parameters as intact bone, which was used as a control (Native Spine group) in this study. On the other hand, when it comes to the structural parameters of the newly formed bone mass, such as DA and average BT, we found values similar to that of native vertebra. Further, the Conn-Den of new bone mass was greater than that in the control vertebra (p
0.05), demonstrating that although not completely matured, the new bone has a branched structure that is sufficiently robust to enable a stable fusion.
Histological analyses of bone mass performed using standard H&E and Masson trichrome stains confirmed that the newly formed mass has typical morphological characteristics of bone, including cortex, trabeculae, and bone marrow. In addition, several cartilage-like islands were found inside the new bone formation, which probably represent cells that have not yet fully differentiated but will do so later and will contribute to the BVD and the strength of the tissue and fusion.
An immunohistochemical analysis was performed to detect engineered MSCs within the new bone mass. The assay showed that the newly formed bone contained cells that expressed β-Gal
. Most of the positively stained cells were hypertrophic chondrocytes, whereas others were osteocytes embedded in new bone. Yet, it is hard to determine precisely using this method how much of the bone tissue was formed by the implanted cells compared to the contribution of host cells recruited to the region. However, it is safe to assume that both donor and host cells contributed to the new bone formation, given the paracrine action of the rhBMP-2 expressed by the MSCs, which would activate nearby host cells to differentiate into osteogenic cells.28
All these findings support and reinforce the value of our MSC-based treatment as a method of stabilizing vertebrae. The biomechanical data represent an essential proof of principle with regard to the functional stability of the fused vertebrae. Although biomechanical stabilization has been demonstrated, it is important to note that this approach involves heretrotopic ossification, which is not the current practice in the clinic today. Therefore, further examination of this model is needed in larger animal models. These future experiments will eventually allow us to advance the method to clinical trials and further evaluate the quality of MSC induced spinal fusion as a potential noninvasive technique for fixture of spinal vertebrae. We envision that in the future, the clinical application of this model to posterior and anterior spinal fusion will include fresh immunoisolated human MSCs37
and that overexpression of BMP will be achieved via nonviral gene delivery21,24
of MSCs shortly after their isolation. In summary, we presented here an injectable therapy for spine fusion that does not involve complex surgical procedures. In addition, the use of potent osteogenic cells could potentially replace the use of autografts that result in comorbidity at the donor site or allografts that have limited osteoinduction properties.