Enhancing BMSCs functions is a critical step in optimizing stem cell mediated bone repair. Firstly, increased proliferative capacity enables MSCs to be expanded in vitro to sufficient numbers for clinical transplantation[22]. Secondly, after being engrafted, it is of utmost importance for BMSCs to continuously proliferate and migrate to injury sites[23, 24], and either differentiate into osteoblasts[25], or secrete trophic factors to stimulate targeted cells[26, 27]. However, various factors such as aging and pathological conditions might affect MSCs survival or functions after transplantation and therefore reduce their therapeutic effects[6, 28]. Hence, we investigated mitochondrial transfer as a novel strategy to overcome these limitations.
To our knowledge, this study is the first to transfer autologous mitochondria into BMSCs and evaluate its effects on functional cellular changes. Our results conclusively demonstrated that mitochondria transfer could significantly increase proliferation, osteogenesis and migration of BMSCs in vitro. OXPHOS activity and mitochondrial ATP production were found to be upregulated after mitochondria transfer. Furthermore, we transplanted the mitochondria-recipient BMSCs into rat cranial bone defect sites and found that mitochondria transfer could accelerate the bone defect healing process mediated by BMSCs.
The safety issues of genetic modification techniques have always been an intractable challenge in stem-cell based tissue engineering[5, 14, 29–31]. For example, viral vector used in gene therapy has genotoxicity issues[32], and the off-target mutations or effects of techniques like CRISPR-Cas9 (Clustered regularly interspaced short palindromic repeats, associated RNA guided endonuclease Cas9)[33, 34] or RNA interference[35] might exert detrimental toxic effects and induce unwanted phenotypes. Alternatively, the functions of BMSCs may be enhanced by treatment with growth factors or small molecule drugs, but these also have intrinsic drawbacks such as unclear safe dosage range, possible side effects or ectopic influences[6]. For instance, even BMP-2 (Bone morphogenetic protein-2), the only current FDA (Food and Drug Administration)-approved osteo-inductive growth factor has been reported to exert numerous side effects that can result in potentially devastating complications such as ectopic bone formation, osteoclast-mediated bone resorption, and inappropriate adipogenesis, etc., which tend to manifest at higher concentration[36, 37]. Hence, the various aforementioned drawbacks impede the clinical translation of these potentially-useful therapeutic tools.
However, mitochondria transfer can circumvent biosafety concerns due to the following reasons. First and foremost, mitochondria are intrinsic cellular organelles that are ubiquitously present in all eukaryocytes[38]. In our study, we transferred autologous mitochondria isolated from the same batch of cells, since all available scientific data have shown that autologous mitochondria transplantation does not provoke any auto-immune responses, thus indicating that it is immunologically safe[39]. Moreover, transfer of mitochondria is believed not to involve any transfer of nuclear materials, which would thus allay safety concerns relating to nuclear genomic modification[40]. More importantly, mitochondria transfer can modulate BMSCs function without any changes to the extracellular microenvironment, unlike treatment with drugs, growth factors or biomaterials, thus avoiding any possible safety concerns pertaining to cytotoxicity or biocompatibility. Therefore, mitochondria transfer should be considered a rather safe technique for modulating BMSCs function. In our study, mitochondria isolated from the same batch of cells was demonstrated to exert the strongest effects. The procedure of isolating and transferring mitochondria have been proven to be simple and not too time-consuming, with relatively high success rates. As compared to other enhancement strategies, mitochondria transfer is more easily controllable, stable and effective.
It has been observed in our study, as well as other studies that the acquisition of additional mitochondria during transfer results in an increase in OXPHOS activity and ATP production of mitochondria-recipient MSCs[41, 42]. The increased aerobic metabolic levels of BMSCs might then contribute to the enhancement of proliferation, osteogenic and migratory functions. There are several possible explanations for these observed changes in cellular function. During the process of proliferation and colony formation in vitro, which usually occurs under normoxic conditions (around 20% O2 tension), MSCs rely more on OXPHOS for energy supply rather than glycolysis[43], and the proliferative process of cells, particularly cell-cycle entry requires increased oxygen consumption and ATP generation[44]. Cell differentiation is also associated with an increase in mitochondrial content and activity, according to previous studies[45–47]. The activation of mitochondrial OXPHOS in BMSCs is known to trigger osteogenic differentiation via acetylation and activation of β-catenin signaling[48]. The relationship between BMSC migration and cellular energy metabolism has yet to be investigated. However, cancer cells were found to be expending energy via the dephosphorylation of ATP into ADP during the metastatic process[49]. In migrating ovarian cancer cells, mitochondria actively infiltrate the leading edge of the lamellipodia, increasing the local mitochondrial mass and relative ATP concentration[50]. Thus, it can be hypothesized that mitochondria transfer enhanced BMSCs functions through the up-regulation of aerobic respiratory levels. In order to validate our hypothesis, we utilized Oligomycin, an ATP synthase (mitochondria respiratory chain complex V) inhibitor to attenuate OXPHOS and ATP production in BMSCs, and found that any enhancement of proliferation, differentiation and migration by mitochondria transfer was eliminated. This finding thus proved that mitochondria transfer enhanced BMSCs proliferation, osteogenic differentiation and migration through up-regulation of OXPHOS activity and ATP production.
Nevertheless, there are still a number of limitations to our study. Firstly, although transplantation of mitochondria-recipient BMSCs resulted in stronger bone regeneration efficacy compared to transplantation of normal BMSCs, the underlying mechanisms still remain unclear. Because an increasing number of reports emphasized the paracrine effects of MSCs on tissue regeneration, further investigations of the crosstalk between mitochondria-recipient BMSCs and other cell types (e.g. macrophages or endothelial cells) after transplantation need be performed. Secondly, although our data demonstrated the key role of increased aerobic metabolism in regulating BMSCs function after mitochondria transfer, other mechanisms that elicit functional modification of BMSCs also need to be further investigated. Thirdly, there is an obvious limit to the number of BMSCs that can be isolated from each individual patient, which could in turn impede the clinical application of autogenous mitochondria transfer between BMSCs from the same patient. Hence, our future studies would investigate mitochondria transfer between different patient and tissue sources. For example, autogenic mitochondria transfer between adipose MSCs and BMSCs from the same patient, or even allogeneic mitochondria transfer from the BMSCs of younger patients to that of older patients.