mscs can be extracted from adult fat and bone marrow , as well as peripheral blood,8 
 9 and can be induced to differentiate into various 
 mesenchymal tissues including bone , cartilage , and muscle.10 
 11 in vitro induction can be accomplished by growth factor 
 supplementation and creating culturing conditions that are favorable for the preferred 
 differentiation . specifically , adipogenic differentiation from mscs has been successfully 
 accomplished in dulbecco 's modified eagle medium supplemented with isobutylmethylxanthine , 
 indomethacin , and either dexamethasone and insulin or with hydrocortisone.11 
 12 successful induction is then verified by the 
 identification of lipid vacuoles within the cell and various other adipose cell markers . in 
 comparison , in vitro chondrogenic differentiation has been induced by centrifuging the mscs 
 into micromass pellets and culturing in a medium containing dexamethasone and transforming 
 growth factor -3.13 chrondrocytes are then detected by the 
 presence of secreted extracellular matrix components such as type ii collagen and aggrecan , 
 among others.14 osteogenic differentiation may be induced in 
 a culture of dexamethasone , ascorbic acid phosphate , -glycerol phosphate;11 
 12 
 14 
 15 successful differentiation is identified by the 
 expression of alkaline phosphate and using cell - specific antibodies . successful models used 
 to expand the msc population and induce osteogenic differentiation have also included 
 substances such as transforming growth factor- and fibroblast growth factor-2.16 
 17 bone morphogenetic protein has also been shown to 
 successfully expand the osteogenic cell population;7 
 18 however , limitations exist regarding cost and safety . the 
 authors refer the interested reader to some of the early studies inducing msc 
 differentiation for additional explanations as to the nature and function of the specific 
 growth factor supplements.11 
 12 
 13 
 14 
 19 
 20 
 21 in vivo induction occurs through site - specific 
 differentiation and is often implanted within scaffolds.7 
 10 
  in addition to requiring sufficient numbers of mscs implanted for bone regeneration,22 an osteoconductive matrix is also necessary for osseous 
 growth.18 the scaffold provides a structural support for 
 cell  cell interactions , extracellular matrix formation , and new tissue formation . the 
 osteoconductive scaffold occupies the site of the fusion , but also provides an environment 
 conducive for the osteoinductive factors and tissue growth . various biomaterials have been 
 investigated as scaffolds including hydroxyapatite , tricalcium phosphate , calcium sulfate , 
 metals , and biodegradable polymers.23 serving as the initial 
 structure for tissue growth and blood vessel formation , the biodegradable types of scaffolds 
 eventually resorb as new bone is formed.24 
  isolating human mscs has traditionally been accomplished by using their selective adherence 
 to plastic surfaces , which the hematopoietic cells less commonly do.25 this method , however , typically leads to heterogeneous cell isolation , in which 
 only ~30% of the cells are multipotent mscs.26 although the 
 mscs do not express the cell surface markers normally found on hematopoietic cells , such as 
 cd34 and cd45 , there are cell markers that are unique to the mscs.27 stro-1 was one of the earlier monoclonal antibodies developed to isolate 
 mscs,28 and this was followed by additional cell - surface 
 markers such as cd146 , cd200 , and cd271.29 
 30 
 31 use of these markers alone may not be sufficient to 
 isolate pure samples of mscs . gronthos and colleagues28 
 described a method whereby they used stro-1 monoclonal antibodies and then isolated the 
 cells using immunomagnetic cell sorting ( macs ) . this resulted in a milieu of cells with 
 varying intensities of stro-1 fluorescence intensity , which was then further purified for 
 the high - intensity fluorescing cells using fluorescence - activated cell sorting ( facs ) . further purification was accomplished using dual - color facs to isolate those cells with the 
 surface markers vam-1 , which are also expressed on mscs.32 
 another recent study utilized macs to separate cells that were cd45 ( as this 
 marker is not expressed on mscs ) and then subsequently used facs to isolate cells that were 
 cd146.30 the most effect cell identification 
 technique seems to be a combination of facs and macs , though the cell surface markers of 
 choice vary between laboratories . in an effort to identify the most efficient marker of 
 mscs , delorme and colleagues31 used microarrays and flow 
 cytometry to culture a pure sample of mscs that expressed 113 transcripts and 17 proteins 
 not found on other hematopoietic cells . they found that cd146 and cd200 were among the most 
 efficient markers to purify mscs . following isolation , mscs can be cultured in either fetal calf serum or human serum , which 
 show no difference in their effects on the cells to proliferate and differentiate.33 for bone formation , mscs are then directed to differentiate 
 into osteoblast lineage cells via the aforementioned factors ( e.g. , transforming growth 
 factor- ) or via selective genetic expression ( e.g. , osx , zip1).34 
 35 allogeneic transplantation of mscs can be done in the 
 site of fusion , due to their hypoimmunogenic and even immunosuppressive nature . flow 
 cytometry experiments have shown that mscs express intermediate levels of hla class i and 
 little to no hla class ii or costimulatory molecules ( e.g. , cd40).11 
 36 neither undifferentiated nor differentiated major 
 histocompatibility complexes elicit lymphocyte proliferation when transplanted,36 
 37 and in fact , they tend to alter the cytokine profile to 
 an anti - inflammatory state by decreasing tumor necrosis factor- and interferon - gamma and 
 increasing interleukin-10 , interleukin-4 , and regulatory t cells.38 mscs have even been used for their immunosuppressive actions in treating acute 
 graft - versus - host disease.39 for these reasons , 
 host - versus - graft disease , or graft rejection , does not appear to be a problem with msc use 
 in spine fusion . the use of msc therapy in bone regeneration has been and is currently investigated both in 
 animal models and in the clinical setting . this is being done through both local 
 implantation of the stem cells and via gene therapy , as well as through autologous transfer 
 of engineered extracted mscs . following the original discoveries of mscs by friedenstein et al40 
 41 and then of owen in 1988 that mscs could differentiate 
 into bone,42 
 43 several groups demonstrated successful autologous 
 transfer of mscs in healing long bone lesions in various animal models.44 
 45 
 46 for example , arinzeh and colleagues used allogeneic mscs 
 loaded in a ceramic hydroxyapatite - tricalcium phosphate scaffold to treat large femur 
 defects in adult dogs . they showed that not only did a callus of lamellar bone fill the 
 lesion within 8 weeks , with complete new bone form by 16 weeks , histologically , there was no 
 immune response detected.46 
  in a rat posterior spinal fusion model , cui and colleagues showed that cloned 
 osteoprogenitor cells implanted in the fusion bed led to successful spine fusion in all 
 animals , compared with only 50% fusion success in animals that were implanted with mixed 
 marrow stromal cells.47 similarly , muschler et al , in a 
 canine model of dorsal spinal fusion , demonstrated the superiority of the marrow - derived 
 osteoblastic progenitors in promoting spine fusion versus the growth factor and cellular 
 milieu found in a bone marrow clot.48 the effectiveness of 
 mscs in promoting spinal fusion has been shown in progressively larger animals , including 
 rabbit,49 
 50 
 51 
 52 
 53 ovine,54 
 55 
 56 
 57 and primate models.58 
 59 specifically , wang and colleagues58 performed anterior lumbar interbody fusions in nine rhesus monkeys , with two 
 fusion sites each , that either utilized autologous bone - marrow derived mscs on a calcium 
 phosphate scaffold , icbg , or a control ceramic graft treatment . they found that the msc 
 group had equivalent biomechanical strength as compared with the icbg group , and that they 
 were both biomechanically and histologically superior to the control ceramic graft group . orii et al59 performed posterolateral lumbar spine fusions 
 in nine macaque monkeys , which received either marrow - derived mscs with a -tricalcium 
 phosphate graft , autogenous bone , or a control tricalcium phosphate graft treatment . using 
 both x - ray and manual palpation to identify fusion status , they found that the group 
 receiving the mscs had the highest fusion rate ( 83.3% ) compared with the autogenous bone 
 group ( 66.7% ) and the control group ( 0% fused ) . the aforementioned studies used bone 
 marrow - derived mscs ; however , another approach with potentially fewer complications and less 
 morbidity may be the use of adipose - derived mscs ( ascs ) . ascs were first identified in 2001 by zuk and colleagues60 
 61 who showed that ascs can differentiate into multiple cell 
 types including osteogenic and chondrogenic cells , thereby providing an potential 
 therapeutic avenue for bone regeneration . these cell types were subsequently shown to be 
 capable of adhering to a bioengineered scaffold , as well as remaining viable , proliferating , 
 and differentiating under various conditions.62 rodbell and 
 jones standardized the first protocol for asc isolation.63 
 64 
 65 cowan et al66 and later 
 levi et al67 used ascs to regenerate bone in large mouse 
 calvarial defects . lopez and colleagues68 performed 
 dorsolateral spinal fusions in 56 fischer rats , with either no graft , only a scaffold , a 
 scaffold with allogeneic ascs , or a scaffold with syngeneic ascs . similar to the studies 
 investigating marrow - derived mscs , they found that when ascs were used there were fewer 
 inflammatory infiltrates compared with the control groups , as well as superior bone 
 formation and fusion when using the ascs . clinical trials are currently investigating ascs 
 in treatments ranging from type i diabetes mellitus to liver disease69 
 70 and may soon be used for spine fusion treatments as 
 well . although there has been much success demonstrated in the animal models , there remain 
 barriers prior to this therapy 's translation into the clinical setting . this includes 
 identification of the optimal number and concentration of mscs , as well as the ideal 
 preparation and implantation techniques needed.71 minamide 
 and colleagues50 compared the use of differing number of 
 marrow - derived mscs in a rabbit posterolateral spine fusion model . the low dose contained 
 one million cells per milliliter and resulted in the fusion of zero of seven spines . in 
 contrast , the high dose was one hundred million cells per milliliter and led to the fusion 
 of five of seven rabbit spines . perhaps an even greater concentration would have led to 100% 
 fusion . wang et al58 compared bone marrow - derived mscs to 
 autogenous icbgs in rhesus monkey spine fusion models and found that although the fusions 
 were equivalent in stiffness , the autograft produced greater bone volume . this can be 
 explained by the fact that only three million cells per milliliter were used for the 
 msc - treated group , in contrast to the 100 million that was used successfully in the study by 
 minamide et al . finally , gan and colleagues,72 used 
 bone - marrow - derived mscs for posterior spine fusion in 41 patients . using a slightly 
 different quantification method , they recovered and implanted 16.1 million nucleated bone 
 marrow cells per milliliter , of which they measured an average of 213 mscs per milliliter 
 ( mscs were defined as colony - forming units expressing alkaline phosphatase ) . despite the 
 relatively small number of implanted cells , 95% of their patients achieved complete fusion 
 by 34 months ' follow - up . it is clear that we have yet to determine the optimal number of 
 mscs needed for complete fusion prior to the translation of this technology to the clinical 
 setting . although some experimental data suggest that increased cell concentrations are 
 required to repair bony defects,58 
 73 others have suggested that the cell concentration is not 
 sufficient but rather the delivery type and biological environment of the graft will 
 significantly affect success of the fusion.48 for example , 
 the study by minamide et al50 used a three - dimensional 
 culture , as compared with the more traditional two - dimensional monolayer culture , as they 
 believed that it enhanced proliferation and differentiation . however , 
 there is literature on the osteogenic potential of mscs in various other skeletal defects as 
 well as ongoing clinical investigations of mscs in spine surgery with various commercial 
 products such as neofuse ( mesoblast ltd . , melbourne , austrialia ) and 
 osteocel ( nuvasive , san diego , ca the earliest clinical studies involving mscs involved small case studies of autologous mscs 
 used for bone regeneration . quarto et al74 showed successful 
 and abundant callus formation in three patients with tibial , ulnar , or humeral fractures 
 using autologous mscs ; lendeckel and colleagues75 reported a 
 case study where autologous ascs were successfully used to treat a large calvarial injury . subsequent trials , including some larger ones , involved autologous bone marrow successfully 
 used to promote bone fusion in tibial nonunions,76 
 77 autologous mscs for femoral head osteonecrosis,78 
 79 
 80 
 81 and allogeneic mscs to treat osteogenesis imperfect,82 all of which showed the clinical feasibility of therapeutic 
 application of mscs to promote bone growth . the 2011 study by goldschlager and colleagues,55 which 
 demonstrated superior bone formation in ewes with the use of allogeneic ovine mscs 
 ( mesoblast limited , melbourne , australia ) compared with autograft or stand - alone scaffold , 
 was the precursor study to the ongoing clinical trials ( nct01290367 , nct00996073 , 
 nct01097486 , nct00810212 , nct00549913 , nct01106417 ) using this msc allograft product in both 
 cervical and lumbar spine fusions . results on safety and preliminary efficacy in patients 
 will likely be revealed in the coming years . much of the preliminary research done in animal models has shown potential for the use of 
 mscs , both bone marrow and adipose derived , for bone regeneration . although there have been 
 limited systematic clinical trials , small case studies have shown that msc use in humans may 
 have successful bone growth and long - term durability.74 some 
 current limitations include decreased bone growth compared with autograft,58 weaker mechanical stability of the implanted graft and poor 
 resorption of the bioceramic constructs,74 and ambiguity 
 surrounding the optimal cell concentration and delivery method . more studies examining 
 optimal msc concentrations are needed in larger animals , which are more comparable to 
 humans , considering the fact that there is decreased potential for bone growth as compared 
 with smaller animals ( e.g. , rabbits , rats , etc.).58 
 83 
 84 this also demonstrates the need for methods to maximize 
 the number of mscs collected , as well as techniques that can be feasible in the operating 
 room setting.85 other options can include obtaining somatic 
 cells and converting them into pluripotent cells,86 using 
 minimally invasive approaches to collect and culture bone marrow- or adipose - derived mscs 
 prior to surgery , and potentially even using recombinant forms of mscs . the present hurdles 
 to clinical use include optimization of osteoinductive and osteoconductive properties of 
 mscs in bone grafts . vascularization of the implant and integration of the vasculature with 
 the host will prove to be important ; additionally the long - term mechanical strength and 
 durability , particularly at the load - bearing sites such as the lower lumbar spine regions 
 will need to be comparable to native bone.87 allogeneic msc therapy for spine fusion and other skeletal treatments is still in its 
 infancy . there has been a surge of interest in the various msc formulations commercially 
 available for clinical use in spinal surgery . this enthusiasm , and clinical use , must be 
 tempered with the understanding that there are no clinical data that had defined the 
 efficacy or safety profiles in spine surgery patients . therefore , it is imperative that the 
 spine surgery community carefully evaluate the use of msc in spine fusion through 
 well - designed and executed studies . although more than a decade of preclinical animal 
 research that has shown promising results , the safety and efficacy of these products in 
 randomized controlled trials must be ascertained . with the rapidly growing number of spine 
 fusion surgeries performed annually , further study into fusion - enhancing compounds becomes 
 increasingly necessary .
more than 50% of patients complain of postoperative donor site morbidity following 
 iliac crest bone graft harvest , and recent discoveries have identified adverse outcomes 
 following bone morphogenetic protein use in spine fusion . 
 this has led the spine 
 community to turn toward alternative methods to promote fusion following spine surgery . 
 the present article reviews numerous studies that have shown the osteogenic potential of 
 mesenchymal stem cells ( mscs ) . 
 mscs have been used with both in vitro and in vivo models 
 and have involved animal studies ranging from rats to macaque monkeys to successfully 
 induce bone regeneration in lesions of the tibia and spine . 
 there is no fear of graft 
 rejection , as there may be with other allograft materials , because neither 
 undifferentiated nor differentiated mscs elicit lymphocyte response when transplanted ; 
 they tend to alter the cytokine profile to an anti - inflammatory state . 
 early clinical 
 trials are underway with various commercially available msc formulations . 
 although there 
 is much enthusiasm , it is integral that the spine surgery community carefully evaluate 
 the use of mscs in spine fusion through well - designed and executed studies to determine 
 the efficacy and safety profiles in spine surgery patients .